Rodriguez Lucena Patricia
Rodriguez Lucena Patricia
Rodriguez Lucena Patricia
TESIS DOCTORAL
TESIS DOCTORAL
DIRECTORES:
I
que he tenido la suerte de compartir todos estos años, por tu buen humor y tu amistad
incondicional. A mis niñas, que habéis estado siempre ahí para escucharme. A Paloma,
por transmitirme tu entusiasmo por todo esto cuando yo no encontraba el mío. A
Sandra, que tanto me has recordado que no hay que desesperar porque todo llega. A
Diana, por todo lo que me has enseñado. A las pequeñitas, Anita y Cova, que sois tan
grandes a la vez. A Edu, por apoyarme en todo momento. A Sonia, por tu amistad y por
enseñarme tanto desde hace tanto tiempo. A Cris, por ponerle música a todo esto. A
Tere y Carlos, que con vuestra sonrisa perpetua me alegráis tantos momentos. A Rebe
por aguantar mis bromas día a día. A Bea, que ya se va acercando a la recta final. A las
chicas de la 6ª planta, Yoli y Vane. A todos los demás, María Prieto, Iván, Marcos, Silvia,
Yasna, Edgar, José, Guillermo, Sheila, Laura, Elena, Saúl y María Villén.
A mis niñas. A Ana, por todo lo que me has dado desde los lejanos días lioneses. A
Celia, por preocuparte tanto, y transmitirme tu optimismo y alegría. A Carmen, porque
sé que de haber podido habrías escrito esta tesis por mí. A las tres, por cuidarme tanto,
en especial durante estos últimos meses.
A mis ambientólogas. A Silvia, Alo, Laura, Vicky y Rebe, que con tanta paciencia
me habéis esperado estos años, perdonándome las llamadas no devueltas y todos los
momentos importantes que me he perdido. A las demás, Andrea, Ali y Nuria, por
vuestra fuerza y vuestro apoyo, que me han acompañado durante todo este tiempo. Y a
Cris, que es casi una ambientóloga más.
A Patri, gracias una y mil veces, porque sin ti no habría llegado al final de todo
esto. A Antonio, por respetar este proyecto a pesar de todo. A José, por tu amistad, que
es uno de mis pequeños tesoros. A Carmen, que ha llegado casi al final, por todo lo que
me has ayudado estos meses. A Juanfra, por el cariño que siempre me demuestras. A
Sara y Luis, por seguir ahí desde hace tantos años.
Por último, a mí familia. A mis tíos, Pepe y Emi, y mis primos, Alberto y Marta,
por quererme tanto y tan bien. A mi tía Mari Carmen, a la que tanto quiero, por
transmitirme su fuerza y por su ayuda en los momentos difíciles que ahora parecen tan
lejanos.
A los más importantes, mis padres a los que quiero más de lo que muchas veces les
demuestro, para los que sé que esta Tesis es un orgullo que no todos pueden
comprender, y a los que nunca podré agradecer lo suficiente todo lo que me dieron y me
enseñaron, lo que me dan y me enseñan cada día. A mí madre, por su paciencia y
comprensión, que no siempre merezco. A mí padre, por hacerme siempre volar un poco
más alto y recordarme que mis límites son sólo los que yo me imponga. A los dos, por
respetarme y apoyarme, por transmitirme vuestro orgullo por mi trabajo, por ser la red
bajo mis pies que me ayuda a saltar sin miedo y por vuestro amor que me hace tan
fuerte. Por todo eso y por mucho más, esta Tesis es sobre todo vuestra.
A todos, porque todos sois coautores de este trabajo.
II
III
La presente memoria se ha realizado a través de la financión de los siguientes
proyectos:
IV
Rodríguez-Lucena P, Hernández-Apaolaza L y Lucena J J. 2008. Foliar
application of commercial Fe chelates and complexes to correct iron chlorosis in
soybean plants. Proc. 17th Intern. Symp. of CIEC, pp. 305-311. NCR. ISBN: 977-
5041-60-017. NCR (National Research Center), Eds. El-Zaiem Press, Giza,
Egipto.
V
Prólogo
PRÓLOGO
El Capítulo III recoge un artículo ya aceptado (Journal of Plant Nutrition and Soil
Science) en el cual se evalúa la capacidad de distintos quelatos biodegradables y
complejos de Fe, aplicados por vía foliar o a la disolución nutritiva, para solventar
deficiencias de Fe en plantas de soja desarrolladas en hidroponía pura. Se compararon
ambos tipos de productos (quelatos y complejos), intentando dilucidar si la fracción de
Fe quelado/complejado es un buen indicador de la eficacia de los mismos, así como si la
aplicación de quelatos biodegradables y complejos naturales puede constituir una buena
alternativa a otros quelatos tradicionalmente empleados para corregir la clorosis férrica.
VI
Prólogo
En este Capítulo se incluyen tres trabajos, uno de los cuales ya ha sido publicado
en la revista internacional Plant and Soil, mientras que los otros dos se encuentran
actualmente en revisión (Journal of Agricultural and Food Chemistry). Se ha estudiado la
capacidad de ambos sub-productos para complejar Fe, la estabilidad de los complejos
formados, y su eficacia al ser aplicados por vía foliar o a la disolución nutritiva. En el
caso concreto de los lignosulfonatos, se compararon productos con distintas
concentraciones de grupos funcionales capaces de complejar Fe y se aplicó el marcaje
isotópico al estudio de la absorción y redistribución del Fe aportado por los complejos
Fe-Lignosulfonato.
VII
Preface
PREFACE
The chapters in which the experimental results are reported and discussed are
briefly summarized below.
The assays and results described in this chapter are presented in the form of a
scientific publication, although they have not been submitted for publication. The
effectiveness of biodegradable IDHA/Fe3+ to provide Fe soybean plants with different
degrees of Fe deficiency was tested in two experiments under controlled conditions in a
VIII
Preface
Chapter V. Fe complexes formed from pulp and paper industry and olive oil
extraction process by-products as Fe chlorosis correctors in soybean, cucumber
and tomato plants. Use of isotopes to study Fe uptake and redistribution.
This chapter includes three works. One of them is already published in Plant and
Soil, while the others are currently under revision in Journal of Agricultural and Food
Chemistry. The ability of these by-products to form stable complexes has been studied,
as well as their performance when applied as foliar sprays or to nutrient solution to
soybean, cucumber and tomato plants grown in hydroponics. Their main structural
features were also elucidated by FITR. In the case of pulp and paper by-products
(lignosulfonates) isotopes were employed to study Fe uptake and redistribution when
supplied as Fe-lignosulfonate complexes.
IX
Indice de General
INDICE DE FIGURAS................................................................................ XI
ABREVIATURAS........................................................................................... XVII
I
RESUMEN....................................................................................................... 1
CAPÍTULO I. Introducción.........................................................................5
1. LA CLOROSIS FÉRRICA..................................................................................7
2. BIODISPONIBILIDAD DEL Fe....................................................................8
3. FUNCIONES DEL Fe EN LAS PLANTAS................................................ 10
4. LOCALIZACIÓN DEL Fe EN LA PLANTA..............................................11
5. RESPUESTAS DE LA RAÍCES ANTE LA DEFICIENCIA DE Fe... 11
5.1. Estrategia I.....................................................................................................12
5.1.1. Acidificación de la rizosfera........................................................... 13
5.1.2. Inducción de la actividad reductasa................................................14
5.1.3. Transporte del Fe3+ al interior de las células de la raíz.................15
5.1.4. Otras respuestas................................................................................ 15
5.2. Estrategia II.................................................................................................. 18
6. TRANSPORTE DEL Fe EN LA PLANTA................................................. 19
7. CORRECIÓN DE LA CLOROSIS FÉRRICA............................................21
7.1. Mejora genética y selección de cultivares resistentes.......................22
7.2. Prácticas agrícolas adecuadas................................................................. 22
7.3. Mejora de las condiciones del suelo...................................................... 23
7.3.1. Adición de materia orgánica...........................................................23
7.3.2. Adición de productos acidificantes.................................................23
7.4. Adición de fertilizantes.............................................................................. 24
7.4.1. Sales inorgánicas................................................................................24
7.4.2. Quelatos sintéticos............................................................................ 24
7.4.3. Complejos de Fe................................................................................27
7.4.3.1. Lignosulfonatos...................................................................... 28
7.4.3.2. Sustancias húmicas (humatos y fulvatos)..................................31
7.4.3.3. Otros agentes complejantes.......................................................34
7.5. Fertilización foliar....................................................................................... 36
7.5.1. Adquisición de solutos por vía foliar..............................................39
7.5.2. Factores que condicionan la eficacia de la aplicación foliar de
fertilizantes de Fe........................................................................................ 40
X
Indice General
XI
Indice de Figuras
ÍNDICE DE FIGURAS
CAPÍTULO I. Introducción
Figura 10. Estructura de un ácido fúlvico propuesta por Schnitzer y Khan (1972).
XII
Indice de Figuras
XIII
Indice de Figuras
Figura 1. Cantidad de 59Fe eluida a través de una columna de suelo (Eutric cambisol) al
ser aplicado como 59Fe-LS o 59Fe-EDDHA. El eluyente es una disolución nutritiva (A,
tamponada con HEPES-KOH pH 7.5 10 mM) sin Fe. Los datos son la media ± ET de
dos experimentos independientes con tres replicados.
Figura 2. Espectro FTIR ed los lignosulfonatos y de los complejos Fe-LS (A- LS, B-
complejos LS/Fe2+, C- complejos LS/Fe3+).
XIV
Indice de Tablas
ÍNDICE DE TABLAS
Tabla 3. Efecto de los quelatos y complejos aplicados por vía foliar (Experimento II) en
las variaciones de Índice SPAD medido en las hojas formadas tras las aplicación de los
tratamientos, el peso seco de las plantas y la concentración foliar de Fe de las plantas de
soja crecidas en hidroponía.
XV
Indice de Tablas
Tabla 3. Análisis mediante ANOVA de dos factores del peso seco de las plantas, la
concentración foliar de Fe y la relación Fe/Mn en hoja, tras la aplicación de los
complejos y quelatos de Fe a la disolución nutritiva
Tabla 5. Efecto de los diferentes complejos y quelatos férricos aplicados por vía foliar a
la disolución nutritiva en el peso seco, la concentración de Fe en raíz y la relación Fe/Mn
en raíz de las plantas de soja a 22 días.
XVI
Indice de Tablas
Tabla 3. 59Fe eluido a través de una columna de suelo (Eutric cambisol), cuando el 59Fe
59
se añdió como FeLS. El eluyente es una disolución nutritiva (A, tamponada con
HEPES-KOH 10mM, pH 7.5) sin Fe. Los datos corresponden a la media ± ET de dos
experimentos independientes con cuatro replicados. También se muestra el porcentaje de
59
Fe eluido respecto a los 5 nmoles de 59Fe añadidos en la parte superior de la columna.
La cantidad de 59Fe eluida al añadir 59Fe-EDDHA fue 3162 ± 87 pmoles (63.2% de la
cantidad total de 59Fe añadida).
XVII
Indice de Tablas
XVIII
Abreviaturas
ABREVIATURAS
XIX
Abreviaturas
XX
Resumen
RESUMEN
-1-
Resumen
-2-
Abstract
ABSTRACT
Iron chlorosis is a common disorder in calcareous regions, where many crops can
be affected by this deficiency. Fe chelates applied to soils are the most efficient way to
overcome iron chlorosis. However, due to their high price these chelates are only
suitable for cash crops. On the other hand, their degradability is scarce in most of cases,
and contamination of underground waters can occur du to their washing after rain or
irrigation. Thus, in this dissertation the ability of several biodegradable chelates and
complexes with different origins but always derived from natural products, to overcome
Fe chlorosis has been studied. Since the stability of these products is usually low in
calcareous soils, their application has been tested as foliar sprays or through their
application to the nutrient solution of crops grown in hydroponics.
Complexes include products with different origins, often derived from the
transformation by industrial processes of natural products such as lignin. In this
dissertation the efficacy as Fe fertilizers of a modified olive mill waste and of different
lignosulfonates has been studied. Both type of products have exhibited a good ability to
provide Fe to chlorotic plants as foliar sprays or through the nutrient solution, but since
their stability under calcareous soils conditions is very low, their application to soils is
nor recommended. Several lignosulfonates derived from eucalyptus and spruce wood
and modified to increase the percentage of functional groups (carboxylic and sulfonic)
capable to complex Fe have also been compared, being concluded that the modifications
3
Abstract
4
Capítulo I:
Introducción
CAPÍTULO I Introducción
INTRODUCCIÓN GENERAL
1. LA CLOROSIS FÉRRICA
Las plantas deficientes en Fe muestran diversos síntomas, entre los cuales el más
característico es el amarillamiento de las hojas jóvenes (Figura 1) debido a una
disminución de la concentración de pigmentos fotosintéticos, fundamentalmente
clorofilas (Abadía y Abadía, 1993). Aparece, generalmente, en la zona intervenal de las
hojas jóvenes, mientras que los nervios permanecen verdes debido a que el Fe es
distribuido escasamente hacia las zonas en crecimiento. En casos extremos, la hoja
adquiere un color blanco y puede llegar a manifestar necrosis (Chaney, 1984). Cuando la
clorosis es grave se produce incluso la
muerte de la planta.
7
CAPÍTULO I Introducción
2. BIODISPONIBILIDAD DEL Fe
-4
-6
-8 Fe(OH)2+
-10 Fe(OH)3º
log actividad
-12
Fe(OH)4+ Fe(OH)2+
-14
Fe3+
-16
-18 Fe2(OH)24+
-20
-22
3 4 5 6 7 8 9
pH
8
CAPÍTULO I Introducción
Las condiciones que causan una baja solubilidad y/o movilidad del Fe en la
solución del suelo son una de las causas principales de la poca disponibilidad de Fe para
las plantas (Wallace, 1982). Un pH elevado, altas cantidades de arcillas y carbonatos
(Kashirad y Marschner, 1974; Kolesch et al, 1987 a,b), elevada humedad, alta salinidad
(Award et al., 1988) y altas cantidades de fosfatos (Lindsay y Schwab, 1982) puede ser
desencadenantes de la deficiencia de Fe.
9
CAPÍTULO I Introducción
10
CAPÍTULO I Introducción
En las plantas, el 80% del Fe está localizado en el cloroplasto (Terry y Low, 1982),
y aproximadamente un 63% del Fe en la hoja está asociado a proteínas (Price, 1968). El
tipo de proteínas en las que se incorpora el Fe se puede clasificar en:
Proteínas con grupo hemo. Este grupo comprende todas las enzimas que contienen
un grupo hemo, Fe-porfirina, como grupo prostético. Lo integran proteínas
como los citocromos, catalasas y peroxidasas, representando cerca del 10% del
Fe en la hoja.
Proteínas Fe-S. En este grupo el Fe está coordinado con el grupo tiol de una
cisteína. Este conjunto lo integran proteínas como la ferredoxina, superóxido
dismutasa y la aconitasa. Constituyen aproximadamente un 20% del hierro en la
hoja.
Fitoferritina. Se trata de una proteína de reserva de Fe, y representa cerca de un
35% del Fe en la hoja, llegando a contener hasta el 80% del hierro de los
cloroplastos (Tiffin y Brown, 1962).
11
CAPÍTULO I Introducción
Estrategia I
Eficientes: Disminución del pH
Liberación reductores
Incremento reducción
Fe-disolución Deficiencia de Fe
Estrategia II
Fitosideróforos
Fe-suelo
No eficientes: Permanecen indiferentes
Kaplan, 2002). En la actualidad no se sabe con certeza si las plantas poseen ambos
sistemas de transporte de Fe (Bauer y Hell, 2005).
5.1. Estrategia I
12
CAPÍTULO I Introducción
Estrategia I Estrategia II
A. Quelante
NADH
Fe(III)-quelato R NAD+ Fitosideróforo (FS) E
Fe(III)
ATP
A Mn (II)
H+ H+ Cu (II)
ADP + Pi Fe(III)-FS
Fe(III) TR Fe(III)-FS
Reductores X Mn(II)-FS ? Cu (II)
Quelantes Zn(II)-FSCu(II)-FS Zn (II)
Mn (II)
13
CAPÍTULO I Introducción
La reducción del Fe3+ a Fe2+ es un paso necesario para la toma de Fe por las
plantas de Estrategia I (Chaney et al., 1972). La capacidad de la raíces de reducir Fe3+ del
medio aumenta en condiciones de deficiencia de Fe y puede llegar a ser superior en 10-
20 veces a los valores control (Moog y Brüggemann, 1994; Susín et al., 1996). Este
aumento de actividad se ha observado en todas las plantas de Estrategia I investigadas
(Schmidt, 2005). La actividad reductasa se localiza generalmente en la superficie de las
partes subapicales de las raíces que muestran engrosamiento, así como en los pelos
radiculares (Moog and Brüggemann, 1994).
14
CAPÍTULO I Introducción
Además de estas tres reacciones, las plantas de Estrategia I pueden poseen otros
mecanismos para aumentar la capacidad de absorción de Fe del suelo (López-Millán,
2000).
15
CAPÍTULO I Introducción
Excreción de compuestos de bajo peso molecular. Las raíces de las plantas son capaces de
excretar activa o pasivamente gran variedad de compuestos orgánicos, entre los que se
encuentran azúcares reductores, aminoácidos y ácidos orgánicos. El tipo de exudados
radiculares está determinado genéticamente, aunque existen factores medioambientales
que pueden causar una alteración en la composición de estos exudados. La tasa de
exudación y su composición dependen del pH, temperatura y tipo de suelo, intensidad
de la luz, así como de la edad y el estado nutricional de la planta, y de la presencia de
microorganismos (Jones, 1998). La exudación de estos compuestos está estimulada en
condiciones de deficiencia de Fe.
16
CAPÍTULO I Introducción
17
CAPÍTULO I Introducción
5.2. Estrategia II
18
CAPÍTULO I Introducción
la planta (Kawai y Alam, 2005). Ambos muestran diferente absorción, sugiriendo que las
células de la raíz podrían diferenciar entre el FS libre y el complejado, de manera que l FS
libre podría ser excretado de nuevo a la rizosfera.
Una de las características más llamativas de la clorosis férrica es que en las hojas
cloróticas de especies cultivadas en el campo la concentración de Fe total puede ser
superior al de las hojas verdes (Morales et al., 1998). Igualmente, la concentración de Fe
en las raíces de plantas cloróticas puede ser varias veces superior a la presente en las
hojas de estas mismas plantas (Mengel, 1994). Este fenómeno, conocido como “paradoja
de la clorosis férrica” (Morales et al., 1998; Römheld, 2000; Nikolic y Römheld, 2002),
sugiere que la clorosis férrica es más un problema fisiológico relacionado con los
procesos metabólicos en raíces y hojas, que un problema de disponibilidad de Fe en la
disolución del suelo. El recorrido del Fe desde la raíz hasta el cloroplasto presenta varias
etapas críticas que pueden originar la clorosis férrica.
Los iones Fe2+ libres son muy reactivos y deben ser protegidos para evitar su
precipitación, así como su toxicidad potencial debido a que pueden catalizar la
transferencia de electrones a especies de oxígeno, para producir finalmente radicales
19
CAPÍTULO I Introducción
hidroxilo (Guerinot y Yi, 1994; Hell y Stephan, 2003; Kim y Guerinot, 2007). Esto hace
suponer que el Fe en el citoplasma está unido a algún tipo de compuesto que evita estos
efectos. Distintos autores (Scholz et al., 1988; Pitch et al., 1997) han propuesto la
nicotiamina como principal complejante del Fe libre en las células, siempre que no esté
formando parte de proteínas o como ferritina. El transporte del Fe desde las células
epidérmicas de la raíz hasta el xilema se realiza a través del simplasto en forma de
complejo NA/Fe2+. Una vez allí, es oxidado a Fe3+ y transportado a larga distancia a las
partes superiores de la planta como citrato/Fe3+ (Tiffin, 1966a,b; Chaney, 1989; López-
Millán et al., 2001; López-Millán et al., 2009), o bien formando complejos con aniones
orgánicos como el malato o el malonato (Brown, 1963; Chaney, 1989) e incluso con otro
tipo de sustancias orgánicas (Cataldo et al., 1988). El complejo malato/Fe3+ se vería
favorecido cuando los niveles de citrato fuesen bajos (Chaney, 1989). El tipo de
complejo con Fe3+ puede variar en función de la edad de la planta y en situaciones de
estrés, ya que la composición y el pH de la savia del xilema pueden verse afectados en
dichos casos.
20
CAPÍTULO I Introducción
Además del transporte mayoritario del Fe vía xilema, siguiendo la dirección del
flujo de masas creado por la transpiración, el Fe es transportado en parte vía floema
hasta los órganos en crecimiento. En estos órganos la intensidad de la transpiración es
menor debido a la estructura incompleta del xilema (Landsberg, 1994), por lo que es
necesario utilizar también la ruta del floema. El hierro libre tiene una movilidad
intermedia en el floema (Marschner, 1995), debido sobre todo a su alta afinidad por el
fosfato, el anión inorgánico más abundante en él y con el que forma fosfato férrico,
altamente insoluble (Hill, 1980). El transporte de Fe en el floema se realiza en forma de
uno o varios complejos de Fe (Grusak, 1995), principalmente como NA/Fe2+ (Kim y
Guerinot, 2007; Briat et al., 2007).
21
CAPÍTULO I Introducción
22
CAPÍTULO I Introducción
23
CAPÍTULO I Introducción
24
CAPÍTULO I Introducción
EDTA
o,o-EDDHA
HOOC
NH NH OH HO
HOOC COOH O O
NH HN
IDHA HO
OH
HOOC COOH
H
N
HOOC COOH o,p-EDDHA
OH HO
O O
EDDS
NH HN
HOOC COOH
N NH OH
HOOC COOH
OH
(Figura 5), ya que sólo posee cinco grupos funcionales capaces de quelar el Fe.
Precisamente esta característica es la que parece determinar la eficacia de este agente
quelante, ya que si bien su estabilidad es menor a la de aquellos que cuentan con seis
25
CAPÍTULO I Introducción
26
CAPÍTULO I Introducción
7.4.3. Complejos de Fe
Al igual que los quelatos, los complejos son moléculas formadas por un ión
metálico que es un aceptor de electrones y por una sustancia orgánica capaz de compartir
los electrones con el metal. Mientras que en la terminología química un quelato es un
tipo de complejo en el que la sustancia comparte dos o más pares de electrones, en la
terminología agrícola la diferencia entre quelatos y complejos está basada en la fortaleza
del enlace y se encuentra recogida en el Reglamento (CE) nº 2003/2003 (D.O.C.E. 21 de
noviembre de 2003). En dicho Reglamento se definen los productos autorizados como
agentes quelantes, pero se deja la lista de agentes complejantes pendiente de elaboración.
27
CAPÍTULO I Introducción
7.4.3.1. Lignosulfonatos
28
CAPÍTULO I Introducción
además las llamadas “ligninas comerciales”. En este grupo se incluyen los lignosulfonatos
y las ligninas de Kraft.
C C
C C
C C
C C
CH3O OCH3
O C C O
C C
C C
CH3O OCH3
O C C O
C C
CH3O OCH3
O O
29
CAPÍTULO I Introducción
viscosa.
30
CAPÍTULO I Introducción
Otros autores (Raese y Staiff, 1988) han demostrado la eficacia de los LS para
aliviar la clorosis férrica en peral cuando los complejos eran aplicados por vía foliar. No
se observaron efectos fitotóxicos en el fruto derivados de las aplicaciones foliares.
31
CAPÍTULO I Introducción
HC O
(HC-OH)4
COOH COOH Azúcar H
COOH
C O O O
HO
R-CH H O
O COOH
O
O N
HO CH CH2
O COOH
OH OH O O CH
O
N
O NH
OH
R-CH O
C O
Péptido
NH
Metales esenciales para la planta pero que no forman parte de enlaces de coordinación: se trata
de todos los cationes monovalentes (K+, por ejemplo) y Ca2+ y Mg2+ como
cationes divalentes.
Metales esenciales para las plantas y que forman enlaces coordinados con ligandos orgánicos,
como Cu2+, Mn2+, Zn2+ y Fe3+.
Metales sin una función bioquímica conocida pero que, sin embargo, se acumulan en el
ambiente. Se incluyen en este grupo metales contaminantes como Cd2+, Pb2+ y
Hg2+.
32
CAPÍTULO I Introducción
carga positiva del catión. La formación de complejos se produce cuando las moléculas de
agua que rodean a los cationes metálicos son sustituidas por otras moléculas o cationes,
formándose el compuesto de coordinación. La habilidad de las sustancias húmicas para
formar complejos con cationes metálicos dependerá de su contenido en grupos
funcionales donantes de electrones (Stevenson, 1994). La complejación de los metales
O O OH
O OH O O
C
O
HO C C OH HO C C OH
HO C OH
O O O
O C OH C C
HO C HO HO OH
C OH
OH OH OH
O
O
O OH
O C C OH
O C OH O O
OH
HO C OH
HO C C OH OH
HO
O O OH OH
O O
C C
HO OH C C
C HO OH
OH C
OH
O
O
Figura 10. Estructura de un ácido fúlvico propuesta por Schnitzer y Khan (1972).
puede ocurrir de dos modos: en el primero y más importante, los metales se unen por los
grupos fenólicos y carboxílicos (Van Dijk, 1971) y en el segundo, sólo se unen a grupos
carboxílicos (Schnitzer, 1969 y Gamble, et al., 1970). También se pueden producir
interacciones a través de puentes de hidrógeno, enlace relativamente débil que sólo cobra
importancia para metales con una elevada energía de hidratación. El tipo de complejo
formado dependerá en última instancia del grado de saturación, ya que los enlaces
débiles jugarán un papel más importante cuando los sitios de los enlaces fuertes ya están
saturados. La formación de más de un enlace entre el metal y la molécula dará lugar a
complejos más estables. Esta estabilidad también se ve influida por el pH de la
disolución.
33
CAPÍTULO I Introducción
y las sustancias húmicas enriquecidas en Fe sirven como fuente de Fe para las plantas
(Lobartini y Orioli, 1988). Estas moléculas se han mostrado eficaces para recuperar
plantas de pepino (Estrategia I) y cebada (Estrategia II) deficientes en Fe (Cesco et al.,
2002). Asimismo, son capaces de solubilizar el Fe nativo de diferentes sustratos y suelos
(Cesco et al., 2000).
34
CAPÍTULO I Introducción
Ácidos orgánicos: Los ácidos orgánicos pueden definirse como compuestos de bajo
peso molecular que contienen uno o más grupos carboxílicos y que se encuentran en
todos los organismos. Los ácidos orgánicos poseen la capacidad de complejar metales en
disolución. El grado de complejación depende del ácido orgánico en particular (número
y proximidad de los grupos carboxílicos), de la concentración y tipo de metal y del pH de
la disolución del suelo. Por esta razón, están implicados en varios procesos como la
movilización y toma de nutrientes por las plantas y microorganismos, detoxificación de
metales como el Al, proliferación microbiana en la rizosfera, etc. La mayor parte se estos
proceden de los exudados de las raíces, de los restos de plantas muertas y de la
descomposición microbiana de estos productos. Los principales ácidos orgánicos
presentes en las raíces de las plantas son el lactato, acetato, oxalato, succinato, fumarato,
malato, citrato, isocitrato y aconitato. En la mayoría de los casos aquellos empleados en
agricultura se obtienen industrialmente de la fermentación de azúcares. Por ejemplo, el
ácido cítrico es obtenido de la fermentación de la sacarosa por Aspergillus Níger.
Los ácidos orgánicos con un sólo grupo carboxílico como el lactato o acetato,
poseen una baja capacidad de complejación. A partir de las constantes de formación
publicadas por Martell y Smith (1976-1989) se puede concluir que el malato, citrato y
oxalato presentan una elevada afinidad por metales trivalentes como el Al3+ y Fe3+. Sin
35
CAPÍTULO I Introducción
embargo, esto no implica que se produzca una buena complejación al pH de los suelos
(Mench y Martin, 1991). Por ejemplo, empleando el programa de especiación química
Geochem-PC (Parker et al., 1995), se puede predecir que la complejación del Fe por el
malato, citrato y oxalato es altamente dependiente del pH de la disolución del suelo con
una baja o nula complejación en suelos con pH elevados. Además, el citrato, malato y
oxalato tienen tendencia a precipitar en presencia de Ca2+.
Debido a su capacidad de complejar metales, los ácidos orgánicos participan en
biodisponibilidad y en el transporte de metales en los suelos. Pueden formar complejos
específicos con los grupos funcionales presentes en las superficies de los minerales
afectando a las características de la carga de estas superficies (Yao y Yeh, 1996) y
competir con otras sustancias retenidas por los lugares de adsorción (Geelhoed et al.,
1998, Grafe et al., 2002).
36
CAPÍTULO I Introducción
Desde comienzos del siglo pasado son muchos los trabajos centrados en la
fertilización foliar en condiciones de campo. Estos trabajos hacen referencia
principalmente a la fertilización de frutales con diferentes macro y micronutrientes como
Fe (Wallace, 1928; Burke, 1932; Guest y Chapman, 1949; Wallihan et al., 1964),
nitrógeno (Hamilton et al., 1942; Weinberger et al., 1949; Bullock et al., 1952; Cook y
Boyton, 1952) o Zn y Mn (McClung, 1954; Wallihan y Heymann-Herschberg, 1956;
Cook, 1957; Leyden y Toth, 1960; Labanauskas et al., 1963; Embleton et al., 1964;
Labanauskas y Puffer, 1964).
37
CAPÍTULO I Introducción
En los últimos años se han publicado diversos trabajos que intentan comparar la
eficacia de diferentes quelatos y complejos de Fe como correctores de la clorosis férrica,
muchos de ellos en condiciones controladas y empleando isótopos como trazadores, con
el fin de intentar reducir la enorme variabilidad de resultados observada en este tipo de
fertilización. Así, Nikolic et al. (2003) comprobaron que el EDTA/59Fe3+ era capaz de
aportar más Fe por vía foliar a plantas de girasol (desarrolladas con o sin deficiencia de
Fe) que un complejo férrico formado con la fracción hidrosoluble de una turba
(WEHS/59Fe3+). La aplicación foliar de diferentes complejos formados con sideróforos
también se ha mostrado eficaz aportando Fe por vía foliar. Se observó además que este
Fe era redistribuido a otros órganos tras su absorción por la hoja (Fernández et al.,
2004).
38
CAPÍTULO I Introducción
A diferencia de las raíces, las hojas de las plantas superiores se encuentran cubiertas
de una capa impermeable que evita la pérdida de agua hacia el exterior de la misma. Esta
capa se conoce con el nombre de cutícula. A pesar de ello, entre el 5% y el 10% del agua
transpirada por la hoja tiene lugar a través de la cutícula, lo cual prueba que ésta puede
ser excretada vía epidermis. Por tanto, es de esperar que también tenga lugar el proceso
inverso de entrada de agua y sustancias disueltas en ella.
Las ceras actúan como la interfase final que separa la hoja y el medio. La
composición química de las ceras es variable (n-alcanos, ésteres, alcoholes y ácidos
grasos, de cadena larga en las ceras epicuticulares y de cadena corta en las
intracuticulares) y varía entre los distintos grupos filogenéticos, en incluso dentro del
mismo grupo entre especies o hasta entre individuos. Las ceras, por su composición
química y su capacidad para formar sobre la cutina una capa externa, constituyen la
principal barrera a la pérdida de agua desde el interior al exterior de la hoja (Schönherr,
1976). Las ceras pueden formar estructuras cristalinas o amorfas, en todo caso estas
estructuras son muy variables. En su conjunto, la cutícula resulta una estructura tortuosa
y con múltiples irregularidades, debidas sobre todo a la presencia de las ceras.
39
CAPÍTULO I Introducción
Tradicionalmente se consideró que los estomas debían servir como vía de entrada
para la penetración de los compuestos aplicados foliarmente a la hoja. Sin embargo, esta
hipótesis fue prácticamente descartada tras la publicación del trabajo de Schönherr y
Bukovac (1972). Estos autores concluyeron que los estomas estaban protegidos frente a
la entrada de líquidos por la combinación de tres mecanismos: (i) la escasa mojabilidad
del estoma, al estar recubierto de una cutícula similar a la del resto de la hoja; (ii) la
elevada tensión superficial del agua y las soluciones acuosas; y (iii) la geometría específica
del estoma. En la última década se han retomado las investigaciones en torno a la posible
penetración de solutos a través del estoma, y varios estudios han sugerido que ésta
ocurre por difusión a través del poro estomático (Eichert y Goldbach, 2008; Eichert et
al., 2008).
Son muchos los factores que determinan la eficacia de las aplicaciones foliares, y se
relacionan con el ambiente en el cual se desarrolla la planta y en el cual tiene lugar el
rociado foliar, con las características de la especie vegetal y variedad tratada, y con el
producto aplicado
40
CAPÍTULO I Introducción
41
CAPÍTULO I Introducción
Por otra parte, las condiciones ambientales también influyen en las características
de la hoja a nivel morfológico y estructural (grosor de la cutícula, cantidad y composición
de las ceras cuticulares, etc.) (Currier y Dybing, 1959; Koch et al., 2004) y,
consecuentemente, en los procesos de absorción a través de la superficie de la hoja.
42
CAPÍTULO I Introducción
43
CAPÍTULO I Introducción
44
CAPÍTULO I Introducción
45
Capítulo II:
Objetivos
CAPÍTULO II Objetivos
OBJETIVOS
Objetivos Generales
Dado que el empleo de estos productos, más “eco-compatibles” que los quelatos
sintéticos y económicamente viables en función del tipo de cultivo tratado, no está
contemplado en la legislación vigente en la actualidad a nivel europeo (Reglamento Nº
2003/2003), se pretende justificar su empleo, al menos parcialmente, en base a criterios
de eficacia. Se intenta de este modo llenar las lagunas existentes en el ámbito de la
aplicabilidad de dichos productos, en especial en el caso de los agentes complejantes.
Puesto que dicha efectividad aparece estrechamente ligada al modo de aplicación
escogido y, sobre todo en el caso de los complejos, a sus características físico-químicas,
su estudio se ha abordado mediante el empleo de plantas modelo (pepino, soja y tomate)
y en condiciones controladas. Estas aportaciones contribuirán a crear un listado
específico de agentes complejantes, así como a ampliar el de agentes quelantes
autorizados a nivel europeo. Los trabajos incluidos en esta memoria se plantearon para
desarrollar los objetivos específicos mencionados a continuación.
Objetivos Específicos
49
CAPÍTULO II Objetivos
50
CAPÍTULO II Objectives
OBJECTIVES
General Objectives
Specific Objectives
51
CAPÍTULO II Objectives
Objective 3: Asses the ability of modified olive mill wastes and lignosulfonates
to improve Fe nutritional status of chlorotic plants grown in hydroponics, when
the complexes were applied to the nutrient solution or through foliar sprays.
Labeled Fe (59Fe y 57Fe) will be used to evaluate uptake and redistribution of the
Fe supplied as Fe-LS complexes.
52
Capítulo III:
Corrección de la clorosis
férrica en soja por distintos
quelatos y complejos de Fe.
Aplicación por vía foliar o en
disolución nutritiva.
53
54
CAPÍTULO III
RESUMEN
Los quelatos sintéticos son los correctores más eficaces de la clorosis férrica. Sin
embargo, son productos caros y generalmente poco degradables, por lo que
recientemente se han comenzado a estudiar nuevos quelatos biodegradables para su uso
como correctores de la clorosis férrica. Por otra parte, los complejos de Fe son
fertilizantes con un precio inferior al de los quelatos sintéticos y derivados
frecuentemente de productos naturales, que también se emplean en la corrección de la
clorosis férrica. En dos experimentos se estudió la eficacia de cinco quelatos sintéticos
(Fe-EDDS, Fe-IDHA y tres formulaciones comerciales de Fe-EDTA) y diez complejos
(humatos, lignosulfonatos, aminoácidos, glicoproteínas, polipéptidos, citrato y gluconato)
al ser aplicados a plantas de soja con deficiencias de Fe desarrolladas en hidroponía pura.
En el primer ensayo, los correctores fueron aplicados a la disolución nutritiva, mientras
que en el segundo la aplicación se realizó por vía foliar. En ambos ensayos se
determinaron el peso seco de las plantas, la concentración de Fe en raíz y en parte aérea,
y los valores SPAD, con el fin de evaluar la eficacia de los correctores utilizados. En las
aplicaciones a la disolución nutritiva los quelatos sintéticos favorecieron el crecimiento
de las plantas en mayor medida que los quelatos, así como el incremento en la
concentración de Fe y en el índice SPAD. Entre los complejos sólo la transferrina
mostró una eficacia similar a la de los quelatos. En cuanto a la aplicación foliar de los
productos, el mayor reverdecimiento de las hojas correspondió a las plantas rociadas con
quelatos sintéticos y aminoácidos, pero las translocación a raíz del Fe aplicado sólo fue
evidente en el caso del lignosulfonato. El Fe-EDDS y el Fe-EDTA mostraron eficacias
similares en ambos tipos de aplicaciones.
55
CAPÍTULO III
56
CAPÍTULO III
ABSTRACT
The application of synthetic chelates is the most efficient remedy for correcting
iron (Fe) chlorosis. However, chelates are usually expensive and non-degradable
products. Recently, new degradable chelates have been proposed for their use as Fe
fertilizers. Also, Fe complexes cheaper than synthetic chelates and derived from natural
products are also used to correct Fe deficiencies. Fifteen products, including five
different synthetic chelates (Fe-EDDS, Fe-IDHA and three Fe-EDTA formulations)
and ten natural complexes (humates, lignosulfonates, amino acids, glycoproteins,
polyamines, citrate and gluconate), have been compared when applied at low
concentration to soybean (Glycine max L.) chlorotic plants grown in hydroponics under
controlled conditions. In the first experiment, Fe compounds were applied to the
nutrient solution, while in the second trial Fe was foliar-supplied. Dry matter, Fe
concentration in shoots and roots, and SPAD values were used to evaluate the
effectiveness of the Fe in the different products. In the nutrient solution experiment,
synthetic chelates provided better plant growth, Fe concentration and SPAD values than
complexes. Among the Fe complexes, transferrin generally provided good plant
responses, similar to those obtained with synthetic chelates. After foliar application, the
highest re-greening was observed for plants treated with synthetic chelates and amino
acid complexes, but the translocation to roots only occurred for Fe lignosulfonate. Fe-
EDDS and Fe-EDTA performed in a similar way when applied in nutrient solution or as
foliar sprays.
57
CAPÍTULO III
1. INTRODUCTION
Iron (Fe) chlorosis is a common plant disorder throughout the world that occurs
mainly on calcareous and/or alkaline soils. Under these conditions, calcium carbonate
buffers soil solution pH in the range 7.5-8.5 (Lindsay and Schwab, 1982) and there is a
high concentration of bicarbonate in the soil solution (Lucena, 2000). Consequently, Fe
precipitates as Fe oxyhydroxides (Lindsay, 1979), and Fe availability is reduced. In the
Mediterranean area, it is estimated that 20-50% of fruit crops are affected by Fe
deficiency (Jaeger et al., 2000).
58
CAPÍTULO III
(i.e. crops grown under low Fe concentration that suffer only moderate chlorosis), non-
susceptible crops, fertigation or foliar sprays. They are cheaper than synthetic Fe
chelates, so their application to low-value crops can be a profitable and environmentally
friendly alternative. While the stability of chelates is a good index of their effectiveness,
this has not been demonstrated for complexes. Due to the large number of competing
reactions, with different impacts on the compounds studied, an effective theoretical
comparison of the stability of the chelates and complexes requires a speciation
calculation of the pFe (related to the binding strength between the metal and the ligand)
for each compound under different agronomic conditions. That would inform about the
different chemical species formed by the Fe complex and their availability to plants
depending on pH, Fe concentration and soil solution characteristics. While this has been
successfully undertaken for chelates (Yunta et al., 2003), in the case of Fe complexes it is
almost impossible. There are many different complexing sites with various stability
constants, so single stability constants cannot be obtained and the calculation of the
chemical species formed cannot be done. The relative stability of the complexes can be
related to their ability to maintain the element in solution at high pH, as shown by the
method presented by Villén et al. (2007a).
Foliar application of synthetic chelates, complexes and inorganic salts can be used
to overcome Fe chlorosis. These applications have been tested by several authors
(Álvarez-Fernández et al., 2004; Fernández et al., 2006; Fernández et al., 2008;
Rodríguez-Lucena et al., 2009) with variable results, as the factors controlling foliar
absorption are diverse (depending on treated leaf, type of compound applied, surfactant,
etc.) and optimum foliar fertilizer formulation is still difficult to specify (Fernández et al.,
2006).
The objective of this work is to compare the ability of Fe chelates and complexes
prepared from commercial sources to correct Fe deficiency in Fe-susceptible plants, i.e.
without mechanisms to overcome Fe chlorosis. Given the environmental risks associated
with the soil application of recalcitrant chelates, biodegradable Fe chelates (EDDS and
IDHA) and Fe complexes (humates, lignosulfonates, amino acids, polyamines, proteins
and organic acids) have been evaluated. Both nutrient solution and foliar applications
have been tested in separate experiments using soybean (Glycine max L.) as test plant. In
59
CAPÍTULO III
both assays, regreening effect, growing parameters and total Fe concentration in relation
to the application of the products were determined.
Fifteen Fe compounds with different chemical properties were compared (Tab. 1):
Five synthetic Fe chelates: EDDS, IDHA and three EDTA formed with
commercial sources of chelating agents and FeSO4. HPLC analysis revealed that
they were all Fe(III) chelates.
Three Fe amino acid complexes: one of them with glycine as main component
(Gly) and mixtures of glycine with glutamate (Gly/Glu) and arginine (Gly/Arg)
formed with commercial sources of amino acid extracts and FeSO4. Theoretical
speciation indicated that mainly Fe(II) complexes were formed, suggesting that
Fe(II) oxidation was limited when these complexes were formed.
Transferrin (TR): a commercial complex with a Fe(III) binding protein, but the
Fe bonded naturally is approximately 0.1%, and the commercial product contains
added Fe bonded to the amino acid chain. It can therefore be considered a
polypeptide complex.
One modified anionic polyamine complex (POL/Fe): a polyethyleneamine
acetate with FeSO4 added. Like in EDTA, Fe is bonded by carboxylate and
amine groups in the POL/Fe, so it is expected that it will oxidize to Fe(III).
Two humate complexes from leonardite: Fe-humate, comprising humic acid (H);
and Fe-humate/fulvate, a mixture of humic and fulvic acids (HF). Both were
formed using FeSO4, though oxidation to Fe(III)complexes was expected, at
least partially.
Two Fe organic acid complexes: citrate (CA) and gluconate (GA), also prepared
using FeSO4. According to Bechtold et al. (2002) gluconate forms mainly Fe(III)
complexes, with the same behavior being expected for citrate.
60
Table 1: Fe chelates and complexes studied, and percentage of chelated or complexed and water-soluble Fe of the commercial products.
Chelated or Fe chelated or
Soluble Fe
Treatments Chelating or complexing agent complexed Fe complexed fraction
(%)
(%) (%)
Chelates
EDDS EDDS 8.3 ± 0.1 8.3 ± 0.1 100.0 ± 0.1
IDHA IDHA 8.6 ± 0.1 8.6 ± 0.5 100.0 ± 0.3
EDTA1 EDTA 2.9 ± 0.1 12.5 ± 0.1 103.5 ± 0.1
EDTA2 EDTA 9.8 ± 0.1 12.6 ± 0.1 77.8 ± 0.1
EDTA3 EDTA 10.1 ± 0.2 12.8 ± 0.4 78.9 ± 0.3
Complexes
Amino acid (Glycine)
Gly Free amino acids: 24 % 0.3 ± 0.01 21.5 ± 0.5 1.4 ± 0.2
Polypeptides: 15 %
Amino acid (Glycine + Glutamate)
Gly/Glu Free amino acids: 19 % 0.02 ± 0.01 5.6 ± 0.2 0.4 ± 0.01
Polypeptides: 3 %
Amino acid (Glycine + Arginine)
Gly/Arg Free amino acids: 10 % 0.02 ± 0.01 4.3 ± 0.3 0.5 ± 0.1
Polypeptides: 14 %
POL Modified anionic polyamine 8.5 ± 1.1 8.8 ± 0.1 95.8 ± 2.3
TR Transferrin (polypeptides) 2.5 ± 0.1 2.6 ± 0.1 96.1 ± 0.6
H/ Humate 5.5 ± 0.3 5.3 ± 0.1 103.5 ± 9.3
Humate + Fulvate
HF Humic acids: 25 % 7.9 ± 0.2 7.8 ± 0.1 100.8 ± 2.1
Fulvic acids: 8 %
LS Lignosulfonate 6.9 ± 0.2 6.1 ± 1.4 113.8 ± 5.3
CA Citrate 12.7 ± 1.5 12.5 ± 0.1 101.1 ± 0.9
GA Gluconate 2.4 ± 1.1 12.9 ± 0.3 18.5 ± 2.8
Data are means ± standard error (SE) of three independent replicates. Chelated Fe has been determined using ion-pair chromatography. The
methodology by Lucena et al. (1996) was employed for EDTA and EDDS. IDHA was measured according to prEN 15950, methodology under
review by the CEN TC 260 WG 5. Complexed Fe was determined using the precipitation method described by Villén et al. (2007a).
61
CAPÍTULO III
CAPÍTULO III
All products were originally in solid form, except for TR, Gly/Glu and Gly/Arg,
which were liquid formulations.
The content of chelated Fe for EDTA and EDDS chelates was determined by
HPLC as described by Lucena et al. (1996). IDHA-chelated Fe was determined using
HPLC ion-pair chromatography, according to according to prEN 15950, methodology
under review by the CEN TC 260 WG 5. For Fe complexed by natural complexes the
precipitation method by Villén et al. (2007a) was used. This method can also be
considered as an index of the stability of complexes at high pH.
The soluble Fe content in all the products was measured according to the
European official method for fertilizers (EU Directive, 2003), based on dissolution of
the fertilizers in water and determination of soluble element after removal of organic
compounds to allow the assessment of the element by Atomic Absorption Spectroscopy
(AAS, Perkin-Elmer AAnalyst 800 Spectrophotometer).
Soybean plants (Glycine max L. cv. Stine 0480), were used in both experiments.
Seedlings were obtained in the growth chamber by standard growing procedure (Villén et
al, 2007b) after three days of germination, five days of growing in 1/5 diluted nutrient
solution and six days with complete micronutrient buffered nutrient solution without Fe.
Plants were then moved to the greenhouse and transferred to 2 L polyethylene pots
containing 2 L of full-strength nutrient solution. The stems of two plants were wrapped
together with foam, and placed in the pots (three holes in the lid, six plants per pot).
This nutrient solution had the same composition than the initial one, but was not
buffered on micronutrients. The pH was adjusted at 7.5 with 1.0 M KOH, and buffered
with 1.0 x 10-4 M HEPES and 0.4 g of solid CaCO3 per pot. Water was added every 2 d,
and the solution was renewed every week.
62
CAPÍTULO III
When the plants were moved to the greenhouse, Fe treatments (three replicates)
were applied with the applications being repeated every 7 d.
Experiment II (foliar application): Each pair of plants was sprayed with 2 mL of the
products using a nebulizer system, with a Fe concentration of 5.0 mM, calculated
according to the soluble Fe concentration (Tab. 1). Contamination of the nutrient
solution was avoided by placing a plastic sheet and a paper layer over the pot lid, below
the shoots, to collect any possible spill from the leaves. Leaf sprays were applied both on
the adaxial and abaxial leaf surface. All solutions were adjusted to pH 5.0 to avoid
altering the ion exchange properties of the cuticle (Fernández et al., 2005). Tween 80
(non-ionic surfactant; PROBUS, Barcelona, Spain) was added to the foliar solutions (rate
0.1 % (v/v)), just before leaf spraying. The same controls (+Fe and –Fe) were prepared
as in the nutrient solution application experiment.
2.5 Measurements
SPAD readings were taken with a chlorophyll meter (Minolta SPAD-502) for all
the leaf stages (average of three readings per leaf) every 2 or 3 d. Whole plants were
sampled 8 d after transplanting (DAT, two pairs of plants) and 22 DAT (one pair of
plants) after transplanting. The sampled roots, stems and leaves were separated and
washed as described by Álvarez-Fernández et al. (2001), dried at 65ºC for 3 d and
weighed to calculate dry matter production. After dry digestion micronutrients were
determined in the leaves using AAS.
63
CAPÍTULO III
Data for soluble and chelated or complexed Fe concentrations, as well as for the
chelated or complexed fractions (percentage of chelated or complexed Fe with respect to
soluble Fe) are shown in Tab. 1. The Fe chelated or complexed fraction is an index of
complex stability, which can be related to the efficiency of the Fe fertilizer. In the case of
pure synthetic chelates stability is closely related to stability constants (the higher log Kº,
the higher the stability of the chelate). Nevertheless, in the manufacturing process,
impurities that reduce the efficiency of the commercial chelates may be produced. For
the products studied in our experiments, Fe chelated or complexed fractions are high in
most cases (e.g. synthetic chelates, transferrin, polyamine, humates, lignosulfonate and
citrate). Since for EDTA2 and EDTA3 the chelated fraction is lower (77.8% and 78.9%,
respectively) than for EDTA1, IDHA/Fe and EDDS (100%), their stability and
effectiveness is expected to be lower. The high Fe complexed fraction of transferrin,
polyamine, humates, lignosulfonate and citrate (around 100%) is expected to favor the
performance of these products. For amino acids, this value is very low, so the ability of
the amino acid complex to deliver Fe to plants is questionable. In the case of the
gluconate complex, this percentage is also quite low.
Table 2 presents changes Tab. 2 presents the variations in SPAD values in the
second level of leaves 21 days after the application of the treatments to the nutrient
solution”.. In order to find differences between treatments, very low concentrations of
64
CAPÍTULO III
Fe were used in the nutrient solution (except for control +Fe) so changes in SPAD
readings were generally low. At the end of the experiment (21 d after transplanting), for
all Fe chelates increases in the SPAD values were recorded, especially for EDDS (similar
to control +Fe), while for complexes only TR and LS resulted in significantly higher
SPAD values. Table 2 also presents plant dry weight and leaf Fe concentration at the 2nd
sampling. Only the EDDS, IDHA and EDTA1 were statistically different to control –Fe
with regard to plant dry weight, while the highest leaf Fe concentrations (apart from
control +Fe) were found after application of synthetic chelates, POL, TR and GA.
Table 2: Effect of the Fe chelates and complexes applied to the nutrient solution (Experiment I) on
changes in SPAD values measured in the second level of leaves, plants dry weight and leaf Fe concentration
of soybean plants grown in hydroponics.
2nd Sampling
∆ SPAD (22 DAT)
Treatments
(from 0 to 21 DAT) Biomass Leaf Fe Concentration
(g plant-1, DW) (µg g-1 DW)
Control - Fe -2.8 ± 0.5 cd 1.1 ± 0.1 e 26.9 ± 1.3 i
Control + Fe 7.5 ± 4.9 a 3.8 ± 0.3 a 184.4 ± 1.8 a
EDDS 17.2 ± 3.5 a 2.9 ± 0.8 b 52.8 ± 4.2 c
IDHA 1.5 ± 0.4 bcd 2.4 ± 0.4 bcd 55.8 ± 0.7 c
EDTA1 2.0 ± 0.8 bc 2.6 ± 0.3 bc 65.9 ± 2.2 b
EDTA2 0.6 ± 0.2 bcd 1.9 ± 0.3 cde 51.5 ± 0.8 cd
EDTA3 4.1 ± 0.7 b 1.7 ± 0.4 cde 54.5 ± 3.7 c
Gly -1.8 ± 0.6 cd 1.3 ± 0.3 e 38.5 ± 3.1 efg
Gly/Glu -0.7 ± 0.3 bcd 1.3 ± 0.1 e 33.8 ± 0.1 ghi
Gly/Arg -2.6 ± 0.8 cd 1.6 ± 0.1 de 43.2 ± 0.3 def
POL -3.6 ± 0.5 cd 1.4 ± 0.1 de 54.3 ± 4.2 c
TR 4.1 ± 1.1 b 1.5 ± 0.2 de 54.5 ± 5.8 c
H -3.9 ± 0.8 cd 1.1 ± 0.1 e 29.1 ± 1.7 hi
HF -4.4 ± 1.7 d 1.1 ± 0.1 e 34.1 ± 1.3 ghi
LS 1.2 ± 0.1 bcd 1.2 ± 0.1 e 31.7 ± 2.5 ghi
CA -4.6 ± 0.7 cd 1.3 ± 0.2 e 28.1 ± 1.1 i
GA 0.4 ± 1.8 bcd 1.6 ± 0.6 de 47.4 ± 3.2 cde
Data are means ± standard error (SE) of three independent replicates. Two pairs of plants per pot (twelve
plants in total) were analyzed at 1st sampling time. The remaining pairs of plants were sampled at the end of
the assay (six plants in total).
Different letters in the same column denote significant differences between the treatments (P < 0.05, n = 3).
DW: dry weight.
One of the aims of this work was to compare the effectiveness of chelates and
complexes. In general, all the parameters studied (Tab. 2) were higher for synthetic
chelates than for natural complexes, indicating that in most cases chelates behaved more
efficiently than natural complexes. For commercial products, both the type of ligand and
the formulation of the product are relevant for their efficacy. A positive correlation was
65
CAPÍTULO III
expected between the Fe chelated or complexed fraction and plant dry weight, SPAD
value and plant Fe concentration. For synthetic chelates, Fe chelated fraction and
stability constants (log Kº) can explain their high efficiency. By applying Pearson
Correlation Analysis (P < 0.05), a positive and statistically significant correlation was
found between Fe chelated fraction and Fe concentration in leaves (R = 0.903), while for
plant dry weight and SPAD value the correlation was also high and positive, although
not statistically significant, possibly due to the low Fe concentrations used in the
experiment.
The chelates with the highest Fe chelated fraction (EDDS and EDTA1) showed
the best results. The lower effectiveness of EDTA2 and EDTA3 confirmed the
influence of Fe chelated fraction on the performance of the products. The stability of
the Fe chelates also influenced the effectiveness of the chelates. Log Kº is higher for
EDTA/Fe3+ (27.6; Lindsay, 1979) than for EDDS/Fe3+ (22.0; Tandy et al., 2004), so
EDTA is expected to be more capable of maintaining Fe in solution. However,
competition of other metals e.g. Ca2+, Mn2+ and Zn2+ for the chelating agents is also
important. For EDDS log Kº values for Ca, Mn and Zn (7.9, 8.6 and 13.0, respectively)
are lower than for EDTA (12.3, 15.5 and 18.1, respectively) reducing the competing
effect and stabilizing the EDDS/Fe3+ chelate similarly to the EDTA/Fe3+ (Lindsay,
1979; Orama et al., 2002).
The lower stability of IDHA/Fe3+ (log Kº 15.2; Tandy et al., 2004), related to the
presence of only five functional groups able to bind Fe, might explain the lower
effectiveness of the IDHA treatments with regard to the EDDS and the EDTA1. In
previous studies (Villén et al., 2007b), we found that pure IDHA was faster than pure
EDTA in delivering Fe to soybean plants grown in hydroponics. However, in the
experiment presented here, the plants treated with EDTA1 had a higher leaf Fe
concentration and presented higher SPAD values than those treated with IDHA at the
end of the experiment, while statistical differences between IDHA and EDTA2 or
EDTA3 did not appear. These results are consistent with those obtained in greenhouse
experiments (Lucena et al. 2008) applying Fe to green bean and tomato plants grown in
commercial hydroponics with rockwool as substrate. In the case of green bean, EDTA
was more efficient than IDHA in delivering Fe to plants, whereas no differences were
found when these products were applied to tomato plants.
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CAPÍTULO III
Fe complexed fraction for humates and lignosulfonates was also high, but these
compounds are polymers with different binding sites that can form Fe complexes of
variable stability (log Kº ranging from 2 to 12; García-Mina et al., 2004) depending on
the Fe/ligand ratio. If the ratio is high, complexes are of low stability while at low ratios
they are highly stable (Stevenson, 1994). If weakly bound Fe is released, the remaining Fe
will be strongly bound and may not be available for plant use. The Fe complexed
fraction was very low for the amino acids studied (Gly, Gly/Arg, Gly/Glut), and this
explains the low efficiency observed for these products. However, since the stimulating
effect of amino acids on plant development has been described previously (Breteler et
al., 1985; Ashmead, 1986), and this effect has also been observed when Fe chelates and
amino acids were simultaneously applied to the plant (Sánchez-Sánchez et al., 2002), the
combined application of Fe chelates and amino acids could be an alternative for
improving the efficiency of these products.
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CAPÍTULO III
Variations in SPAD values in the second level of leaves after foliar application are
shown in Tab. 3. In general, synthetic chelates were more efficient than complexes in
improving SPAD values. At the end of the trial most of the synthetic chelates (EDDS,
EDTA1, EDTA2 and EDTA3) did not show statistical differences compared with the
control +Fe treatment. In fact, at that time the highest SPAD values were found for
EDTA3. All natural complexes (except Gly, Gly/Arg and TR) showed statistically lower
SPAD values compared to control +Fe at the end of the experiment. However dark
spots were observed for all the treatments and the control –Fe at the end of the trial,
probably associated to the damage of the leaf surface after spraying the leaves.
Therefore, SPAD measurements have to be considered with caution. Regarding dry
weight no statistical differences were found comparing chelate/complex treatments with
the control –Fe at the end of the trial (22 DAT, Tab. 3), while data corresponding to leaf
Table 3: Effect of foliar applied Fe chelates and complexes (Experiment II) on changes in SPAD values
measured in the second level of leaves, plants dry weight and root Fe concentration of soybean plants grown in
hydroponics.
2nd Sampling
∆ SPAD (22 DAT)
Treatments
(from 0 to 21 DAT) Biomass Root Fe concentration
(g plant-1, DW) (µg g-1 DW)
Control - Fe -4.5 ± 0.7 g 0.8 ± 0.2 b 54.1 ± 1.7 cde
Control + Fe 16.7 ± 2.9 a 1.7 ± 0.1 a 193.2 ± 8.6 a
EDDS 9.5 ± 3.6 abc 1.1 ± 0.2 b 60.7 ± 4.1 cd
IDHA -1.3 ± 0.4 efg 1.1± 0.1 b 44.8 ± 6.9 de
EDTA1 14.3 ± 3.3 ab 1.0 ± 0.3 b 65.7 ± 6.5 cd
EDTA2 8.6 ± 1.3 abcd 0.8 ± 0.3 b 76.5 ± 10.6 c
EDTA3 17.9 ± 4.5 a 0.9 ± 0.2 b 66.9 ± 2.4 cd
Gly 9.5 ± 2.3 abc 1.1 ± 0.1 b 45.7 ± 5.1 de
Gly/Glu 6.9 ± 1.8 bcde 1.1 ± 0.1 b 72.2 ± 17.1 cd
Gly/Arg 12.4 ± 3.4 abc 0.9 ± 0.1 b 49.1 ± 6.5 cde
POL 4.5 ± 1.6 cdef 1.1 ± 0.1 b 47.6 ± 2.4 de
TR 14.3 ± 2.3 ab 1.1 ± 0.2 b 50.7 ± 11.5 cde
H 2.3 ± 1.1 defg 0.6 ± 0.1 b 55.1 ± 6.7 cde
HF -2.2 ± 0.8 fg 0.8 ± 0.1 b 58.2 ± 3.4 cde
LS 2.7 ± 0.6 cdefg 1.0 ± 0.1 b 129.5 ± 16.5 b
CA 2.9 ± 1.1 cdefg 1.0 ± 0.1 b 33.1 ± 1.1 de
GA 5.1 ± 3.1 cdefg 1.1 ± 0.3 b 48.1 ± 2.9 de
Data are means ± standard error (SE) of three independent replicates. Two pairs of plants per pot (twelve plants
in total) were analyzed at 1st sampling time. The remaining pairs of plants were sampled at the end of the assay
(six plants in total).
Different letters in the same column denote significant differences between the treatments (P < 0.05, n = 3).
DW: dry weight.
68
CAPÍTULO III
Fe concentration were not taken into consideration since it was not possible to assure
that Fe remaining on leaf surfaces could be removed after washing. With regard to root
Fe concentration, a significant increase was only found for the lignosulfonate treatment
compared with control –Fe.
Any attempt failed to correlate Fe chelated or complexed fraction with plant dry
weight, SPAD values or root Fe concentration. EDDS, EDTA compounds, amino acids
and transferrin were the most efficient treatments for re-greening leaves. Fernández et al.
(2006) tested the effectiveness of EDTA/Fe3+, IDHA/Fe3+ and other Fe-containing
compounds applied through foliar sprays to peach orchards, showing that EDTA/Fe3+
produced a higher increase in leaf chlorophyll concentration than IDHA/Fe3+. In our
experiment, an increase in SPAD values was not observed for IDHA. The type of
surfactant can have a major influence on the efficiency of IDHA/Fe3+ and EDTA/Fe3+
chelates (Fernández et al., 2008). This effect can be minimized by the use of non-ionic
surface-active agents such as the one employed in our experiment. Regarding the EDDS,
its foliar application to lettuce was more effective than EDTA/Fe3+ in regreening leaves
(Ylivainio et al., 2004), while in our trial both chelates behaved similarly.
Evidence for different absorption rates of synthetic chelates and complexes has
already been described. Rodríguez-Lucena et al. (2009) compared the effectiveness of
LS/59Fe3+ complexes foliar application and EDTA/59Fe3+ to chlorotic cucumber and
tomato plants and observed that a higher amount of 59Fe was absorbed and redistributed
to other plant organs when Fe was applied as EDTA/59Fe3+. This observation is not
consistent with the high root Fe concentration found in the LS treatment. The factors
that promoted the better behavior of LS with regard to the other treatments cannot be
identified from our data. However, since statistical differences among the other
treatments did not appear, even for those products with negligible Fe complexed
fraction (e.g. amino acids), this better behavior could be ascribed to a higher Fe uptake
when Fe was supplied as commercial LS/Fe sprays. In any case, this highest Fe
concentration did not imply a better Fe nutritional status. The low root Fe concentration
presented by the rest of products suggests that Fe uptake was very low, even for those
complexes with a high Fe complexed fraction (humates and citrate).
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CAPÍTULO III
4. CONCLUSIONS
Synthetic chelates have been more efficient than natural complexes at correcting
Fe chlorosis when applied in a nutrient solution or through foliar sprays The Fe chelated
fraction can be used as an index of the effectiveness of commercial chelates. When
applied at low concentrations to the nutrient solution, biodegradable EDDS was more
efficient than EDTA or IDHA, whereas EDDS behaved similarly to EDTA in foliar
applications. Therefore, EDDS may be an efficient and environmentally friendly
alternative to less degradable EDTA for curing Fe chlorosis in susceptible plants.
ACKNOWLEDGMENTS
This study was supported by the Spanish Ministry of Education and Science
(Projects AGL2004-07849-C02-01/AGR and AGL2007—63756) and by Tradecorp. P.
Rodríguez-Lucena was on a Spanish Ministry of Science and Education “FPI” pre-
doctoral contract co-financed by the European Social Fund. We thank Prof. R. J. Goos
(North Dakota State University, Fargo, USA) for supplying the soybean seeds and
Guillermo Esteban and Edgar Ropero for their technical support during the
development of these experiments.
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CAPÍTULO III
REFERENCES
Ashmead H D. 1986. The absorption mechanism of amino acids chelates by plant cells.
In.: Foliar Feeding of Plants with Amino Acid Chelates. Ashmead H D, Ashmead
H H, Miller G W, Hsu H. H. eds. Noyes Publications, New Jersey, pp. 219-235.
Chen Y and Barak P. 1982. Iron nutrition of plants in calcareous soils. Adv. Agron.
5:217.
Fernández V and Ebert G. 2005. Foliar Iron Fertilization: A Critical Review. J. Plant
Nutr. 28:2113-2124.
Fernández V, Río V, Abadía J and Abadía A. 2006. Foliar iron fertilization of peach
(Prunus persica (L.) Batsch): effects of iron compounds, surfactants and other
adjuvants. Plant Soil. 289:239-252.
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Jaeger B, Goldbach H and Sommer K. 2000. Release from lime induced iron
chlorosis by CULTAN in fruit trees and its characterisation by analysis. Acta Hort.
531:107–113.
Lindsay W L. 1979. Chemical Equilibria in Soils. John Wiley & sons. New York, pp.
129-149.
Lindsay W L and Schwab A P. 1982. The chemistry of iron in soils and its availability
to plants. J. Plant Nutr. 5:821-840.
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Stevenson F J. 1994. Humus Chemistry: Genesis, Composition, Reaction. John Wiley &
Sons, New York, pp. 428-405.
73
Capítulo IV:
Aplicación foliar de
IDHA/Fe3+ para corregir la
clorosis férrica en plantas de
soja. Influencia del estado
nutricional y del tipo de
surfactante.
CAPÍTULO IV
RESUMEN
77
CAPÍTULO IV
78
CAPÍTULO IV
ABSTRACT
79
CAPÍTULO IV
1. INTRODUCTION
Fe chelates applied to soils are the most efficient remedy to control Fe chlorosis.
Most of these chelates degrade very slowly in the environment, and the concern about
the environmental risk of its application (Hyvönen et al., 2003) has risen in the last
decades. Recently the biodegradable chelating agent N-(1,2-dicarboxyethyl)-D,L-aspartic
acid, commonly known as iminodisuccinic acid or IDHA (Figure 1), has been proposed
for its use in agriculture (Mitschker et al., 2004). The IDHA shares structural similarities
with EDTA (Figure 1), but only contains five functional groups able to complex Fe. Due
to this characteristic, the IDHA/Fe3+ presents lower stability than the EDTA/Fe3+ and
high reactivity in agronomic conditions. However, the IDHA/Fe3+ has been comparable
to EDTA/Fe3+ providing Fe through nutrient solution to cucumber and soybean plants,
cultivated in a growth chamber in hydroponics under calcareous soil conditions (Villén
et al., 2007). When applied to tomato or green bean plants grown under field conditions
or in commercial hydroponics, the IDHA/Fe3+ behaved similarly to EDTA/Fe3+ solving
Fe chlorosis (Lucena et al., 2008).
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CAPÍTULO IV
foliar sprays and can increase cuticular penetration, diffusion into the apoplast and
uptake by leaf cells (Schönherr and Baur, 1996). Nevertheless, as many adjuvants can
cross the cuticle and damage cells and cell membranes, those applied with foliar
fertilizers should not penetrate or penetrate very slowly. Moreover, they should be
biodegradable and non-phytotoxic products and behave efficiently when applied at low
concentration.
EDTA
HOOC COOH
NH NH
HOOC COOH
IDHA
HOOC COOH
H
N
HOOC COOH
o,o-EDDHA
OH HO
O O
NH HN
OH HO
81
CAPÍTULO IV
type of adjuvant used. In general, the EDTA/Fe3+ promoted higher leaf re-greening than
the IDHA/Fe3+, while leaf Fe concentration was more elevated in the trees sprayed with
IDHA/Fe3+ (Fernández et al., 2008).
Given the variability of the results obtained in field experiments, the aim of this
work was to asses the ability of IDHA/Fe3+ foliar sprays to overcome Fe chlorosis under
controlled conditions when applied to soybean (Glycine max.), a non-efficient model plant
for Fe nutrition. First, the effectiveness of the foliar application of IDHA/Fe3+ to
soybean plants with severe chlorosis was studied when the foliar sprays were the only
source used to provide Fe to plants, and different adjuvants (non-ionic or glycine based)
were applied in combination with the chelate. In a second experiment the efficiency of
the IDHA/Fe3+ applied with urea or with a non-ionic adjuvant to provide foliar Fe to
plants was tested, when applied to soybean mildly chlorotic plants which were also being
supplied with Fe at low concentrations (as EDTA/Fe3+) through the nutrient solution to
simulate the low availability of Fe in calcareous soils. For comparison EDTA/Fe3+,
frequently used in foliar applications in field, was evaluated in both experiments.
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2.2 Adjuvants
Different adjuvants were tested: two non-ionic products with ethylene oxide
groups as hydrophilic component (an alkylpolyglucoside (APG) and
polyoxyethylene(20)sorbitan monooleate (PS)), an anionic glycine based solution (GLY)
and an urea based adjuvant (U).
Soybean seeds (Glycine max L. cv Stine 0480, gently provided by Prof. R. J. Goos,
North Dakota State University, Fargo, United States) were germinated using a standard
seed growing procedure in sterilized trays. The seeds were washed with water during 30
minutes. After, seeds were placed in trays between two sheets of cellulose paper
moistened with distilled water. The trays were kept in darkness, at 28º C for three days in
a thermostatized stove. After germination, seedlings were transferred to a growth
chamber, where were grown under controlled climatic conditions: day/night
photoperiod, 16/8 h; temperature (day/night) 30/25 ºC; R.H. (day/night) 50/70 %.
Seedlings of similar development were placed on a holed plate, floating over containers
with continuously aerated 1/5 diluted nutrient solution for six days. Then, diluted
nutrient solution was changed by full strength solution with the following composition:
(macronutrients in mM) 1.0 Ca(NO3)2, 0.9 KNO3, 0.3 MgSO4, 0.1 KH2PO4; (cationic
micronutrients in µM, as buffered micronutrient solution) 2.5 MnSO4, 1.0 CuSO4, 10.0
ZnSO4, 1.0 NiCl2, 1.0 CoCl2, 5.0 Fe(NO3)3, 120.5 EDTANa2, 50.0 KOH; (anionic
micronutrients in µM) 35.0 NaCl, 10.0 H3BO3, 0.05 Na2MoO4. The pH was buffered
with HEPES 1.0 x 10-4 M and adjusted at 7.5 with KOH 1.0 M.. Seedlings were kept in
this solution for six days, until chlorotic symptoms were observed. After this time, the
stems of two plants were wrapped together with foam, and placed in 2 L polyethylene
vessels (three holes in the lid, six plants per pot) containing 2 L of a full strength nutrient
solution with the same composition than the initial one, except in the content of
micronutrients (not buffered micronutrient solution, in µM): 1.0 MnSO4, 0.5 M CuSO4,
0.5 ZnSO4, 0.1 NiCl2, 0.1 CoCl2. In the Experiment I Fe was not added in this nutrient
solution, while in the Experiment II 5.0 µM EDTA/Fe3+ was added to avoid severe
chlorosis. The pH was adjusted at 7.5 with KOH 1.0 M and buffered with HEPES 1.0 x
10-4 and 0.4 g -1
of solid CaCO3 per pot were added. Water was added every two days,
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CAPÍTULO IV
and the solution was renewed every week. Three replicates were prepared for each
treatment and for each control.
At this point, Fe treatments were applied (Table 1 for Experiment I and Table 2
for Experiment II), and applications were repeated after 8 and 15 days.
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2.4 Measurements
During the experiments, SPAD readings with a chlorophyll meter (Minolta SPAD-
502) were taken for all the leaf stages (average of three readings per leaf) periodically.
Whole plants were sampled 8 days (two pairs of plants) and 22 days (one pair of plants)
after the first application of treatments. Sampled roots, stems, treated leaves and non
treated leaves (only in the Experiment II) were separated, weighted, washed with 0.1 %
HCl and 0.01 % non-ionic detergent solution, and rinsed twice with ultrapure water.
Then, samples were dried in a forced air oven at 65 ºC for three days. Micronutrients
were determined in the leaves and roots after dry digestion procedure by Atomic
Absorption Spectroscopy (AAS, Perkin-Elmer Aanalyst 800).
Data were statistically evaluated using Analysis of Variance (ANOVA) with the
program SPSS 15.0 to asses the significance of the main factors and interactions. Means
were also compared using Duncan’s test at P ≤ 0.05 in order to find significant
differences between treatments.
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3. RESULTS
In this experiment, foliar sprays described in Table 1 were the only source of Fe
for plants, which showed severe chlorosis symptoms. The effect of the foliar application
of the treatments on plant dry weight, root Fe and Mn concentration, and Fe/Mn and
Fe/(Mn+Zn+Cu) was studied using one-way ANOVA. Statistical differences only
appeared for root Fe concentration (at P ≤ 0.05), and indicate that the combination of
FeIDHA+GLY was more effective than the other Fe containing compounds and
adjuvants tested.
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Table 3 : SPA D values, dry weight, root Fe and Mn concentrations, androot Fe/Mn and Fe/(Mn+ Zn + Cu) ratios in soybean plants sprayed
with the Fe chelates tested, at the end of the Experiment I.
Fe
Root DW Mn Con centr ation Fe/
SPAD Concentration Fe/Mn
(g plant- 1 DW) (µg g-1 DW) (Mn+ Zn+Cu )
(µg g-1 DW)
FeIDHA+APG 11. 3 ± 0.7 a 0.51 ± 0. 05 ns 61.1 ± 1. 4 ab 165. 7 ± 21. 4 b 0.38 ± 0. 04 a 0.20 ± 0. 02 a
FeIDHA+GLY 8.9 ± 0.6 ab 0.57 ± 0. 02 74.2 ± 7. 9 a 18.6 ± 28. 3 ab 0.36 ± 0. 07 a .20 ± 0. 02 a
FeIDHA+PS 9.6 ± 0.7 ab 0.62 ± 0. 13 50.6 ± 10.7 b 186. 5 ± 6.7 ab 0.28 ± 0. 07 ab 0.15 ± 0. 04 ab
FeEDTA+APG 11.6 ± 1. 3 a 0.69 ± 0. 12 50.2 ± 0. 5 b 235. 7 ± 21. 4 ab 0.21 ± 0.01 ab 0.13 ± 0. 01 ab
Control -Fe 7.3 ± 1.1 b 0.43 ± 0. 12 41.5 ± 3. 3 b 306. 3 ± 73. 1 a 0.15 ± 0. 03 b 0.09 ± 0. 01 b
Data are means ± standard error (SE) of three independent replicates, except forSPAD values (average of 18 measurements). Different letters
in the same column denote significant differences among the treatments (P ? 0. 05).
DW: Dry weight basis
SPAD was measured periodically in all the levels of leaves. Two-way ANOVA
analysis showed that SPAD values measured for each treatment in the third level of
leaves were strongly influenced (at P ≤ 0.001) by the treatment applied and by the age of
the plants (days since the first application of the treatments), but interaction between
both factors did not occur. Average SPAD values are reported in Table 3, and indicate
that for all the treatments average SPAD was higher than for control –Fe. The best
results corresponded to plants treated with Fe-compounds combined with APG as
adjuvant (FeIDHA+APG and FeEDTA+APG), while the other treatments were not
statistically different to control –Fe.
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CAPÍTULO IV
separately for each factor using one-way ANOVA. The treatments strongly affected Fe
and Mn concentration and Fe/Mn and Fe/(Mn+Cu+Zn) ratio in roots (at P ≤ 0.001 in
all the cases).
The data corresponding to dry weight, Fe and Mn concentration and Fe/Mn and
Fe/(Mn+Zn+Cu) ratio in non treated leaves and roots at the end of the experiment are
reported in Table 4. In non treated leaves, statistical differences due to the treatments
were not observed for any of the parameters studied. However, when micronutrient
concentrations and ratios were analyzed in roots, IDHA treatments including supply of
Fe to roots (FeIDHA+APG II and FeIDHA+U) were the most effective and presented
statistical differences with control –Fe. Nevertheless in most of cases these treatments
were not statistically different to control FeEDTA (where foliar applications were not
performed). The only treatment in which Fe was not added to the nutrient solution
(FeIDHA+APG I), did not show statistical differences with control –Fe. With regard to
roots dry weight, the highest values were found for FeIDHA+U and FeEDTA+APG,
while statistical differences were not observed in roots dry weight between
FeIDHA+APG I (without Fe supply through the nutrient solution) and control –Fe.
SPAD was measured periodically in all the levels of leaves. Two-way ANOVA
analysis showed that SPAD values for each treatment in the third level of leaves were
strongly influenced (at P ≤ 0.001) by the treatment applied and by the age of the plants
(days since the first application of the treatments), but interaction between both factors
did not occur. Average SPAD values are reported in Table 4, and indicate that all the
treatments except FeIDHA+APG I (without Fe added to the nutrient solution) where
statistically different to control -Fe. On the other hand, very low SPAD measurements
were recorded when foliar sprays were not performed (control FeEDTA). The best
treatments improving SPAD values were FeIDHA+U and FeEDTA+APG.
88
Table 4: SPAD values, non treated leaves and roots dry weight, non treated leaves and roots Fe and Mn concentrations, and non treated leaves and roots Fe/Mn and Fe/(Mn+Zn+Cu) ratios
in soybean plants sprayed with the Fe chelates tested, at the end of the Experiment II.
Biomass Fe Concentration Mn Concentration Fe/
SPAD Fe/Mn
(g plant-1 DW) (µg g- 1 DW) (µg g -1 DW) (Mn+Cu+Zn)
Non treated Non treated Non treated Non treated Non treated
leaf Root leaf Root leaf Root leaf Root leaf Root
FeIDHA+APG I 4.4 ± 0.7 d 0.6 ± 0.1 ab 47.9 ± 1.9 c 215.9 ± 36.6 a 0.2 ± 0.1 c 0.20± 0.02 b
FeIDHA+APG II 10.1± 1.4 bc 1.2 ± 0.1ab 0.9 ± 0.3 ab 46.5 ± 0.5 ns 135.3 ± 21. 7 a 70.8 ± 1.8 ns 23.3 ± 2.1 b 0.7 ± 0.01 ns 5.9 ± 1.1 b 0.4 ± 0.1 ns 2.3 ± 0.3 a
FeIDHA+U 14.8± 1.4 a 1.5 ± 0.4 a 1.6 ± 0.5 a 45.1 ± 8.9 105.3 ± 9.1 ab 63.8 ± 12.1 12.5 ± 1.9 b 0.8 ± 0.3 8.7 ± 0.9 a 0.5 ± 0.2 2.3 ± 0.1 a
FeEDTA+APG 14.1± 1.6 ab 0.5 ± 0.2 b 1.6 ± 0.6 ab 40.7 ± 3.3 89.4 ± 10.4 b 70.4 ± 20.6 8.6 ± 0.2 b 0.7 ± 0.2 10.4 ± 1.2 a 0.4 ± 0.1 2.0 ± 0.1 a
Control -Fe 3.2± 0.4 d 0.5 ± 0.1 b 33.1 ± 1.8 c 194.8 ± 26.9 a 0.2 ± 0.1 c 0.10± 0.04 b
Control FeEDTA 6.5± 0.7 cd 0.6 ± 0.1 ab 1.1 ± 0.2 ab 31.4 ± 4.2 140.4 ± 17.9 a 97.6 ± 25.5 15.8 ± 0.4 b 0.4 ± 0.1 8.9 ± 1.1 a 0.2 ± 0.1 2.5 ± 0.2 a
Data are means ± standard error (SE) of three independent replicates or 24 SPAD measurements. Different letters in the same column denote significant differences among the treatments (P < 0.05,
n = 3).
DW: Dry weight basis.
FeIDHA + APG I and Control –Fe did not developlevels of non treated leaves.
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CAPÍTULO IV
4. DISCUSSION
The IDHA is a biodegradable chelating agent recently proposed for its agricultural
use (Mitschker et al., 2004). Consequently, at present few works evaluating the
effectiveness of IDHA/Fe3+ to overcome Fe chlorosis are available. The ability of this
chelate to solve Fe deficiencies when applied to roots under controlled (Villén et al.,
2007; Lucena et al., 2008) or field conditions (Lucena et al., 2008) has been tested
successfully, but due to its relatively low stability its application through foliar sprays is
also under consideration. In this sense, several field trials to asses its performance have
been carried out in the last years (Fernández et al., 2006, 2008), although to our
knowledge the foliar application of IDHA/Fe3+ chelates under controlled conditions to
correct Fe deficiencies in plants has not been studied yet. One of the objectives of this
work was to evaluate the behavior of this chelate under controlled conditions. So, all the
possible sources of Fe would be fixed in the experimental design and any improvement
in plant Fe nutrition could be attributed to the Fe supplied through the treatments.
In the studies assessing the effectiveness of IDHA/Fe3+ foliar sprays when applied
in field to peach trees (Fernández et al., 2006, 2008), the IDHA/Fe3+ behaved as
efficiently as EDTA/Fe3+ if the appropriate adjuvant was employed, although the results
were always very variable. Since in soil experiments a fraction of soluble Fe is always
available for plants, even under calcareous soils conditions, foliar sprays are not the only
source of Fe. Thus, the results will be influenced by soil conditions and Fe availability in
it.
In the first experiment described in this work, foliar sprays were the only source of
Fe for plants. In general, the IDHA/Fe3+ behaved as efficiently as the EDTA/Fe3+. Root
Fe concentration and the other nutritional parameters reported in Table 3 suggest that
redistribution of Fe from leaves to roots occurred. Evidence of translocation from leaves
has been observed in other experiments working with soybean, cucumber and tomato
57 59
and Fe chelates labeled with Fe (Rodríguez-Lucena et al., submitted), and Fe
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CAPÍTULO IV
Three biodegradable adjuvants were tested in this first experiment: i) two non-ionic
products (polyoxyethylene (20) sorbitan monooleate (PS) and an oligomeric
alkylpolyglucoside (APG) product) and ii) an anionic glycine based (GLY) solution
(Table 1). Non-ionic adjuvants are the most commonly used in foliar applications, since
the interaction with the active ingredient are minimized, although not completely
avoided (Fernández et al., 2009), in this type of compounds. At the same time, they
increase the cuticular penetration of the active ingredient through complex interactions
between the active ingredient, the adjuvants and leaf surfaces (Stock and Holloway,
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CAPÍTULO IV
1993). PS adjuvants have not been commonly applied in foliar formulations. In the case
of APG, these adjuvants favor cuticular penetration of solutions in general (Schönherr et
al., 2001) and Fe solutions in particular (Fernández et al., 2006). For anionic adjuvants, it
is important to keep in mind that these molecules may interact with the electrolyzed
cations of the Fe-compound solutions, forming large molecules that may block cuticular
pores and interfere with the process of leaf Fe penetration (Fernández and Ebert, 2005).
However when applied through foliar sprays, GLY could penetrate leaf surfaces and was
translocated to other plant organs (Espasa-Manresa, 1983), so solutions based in this
amino acid may improve Fe penetration.
Independently of the Fe compound and the adjuvant employed, leaf burn was
always observed in treated leaves, even when the non-phytotoxic APG surface active
agent was used. This observation is in accordance with the results obtained by Fernández
et al. (2009), who detected the ionization of a non-ionic adjuvant in the present of
EDTA. However, these symptoms were similar to the necrotic spots observed for
control –Fe, so it was not possible to confirm if they were due to the foliar sprays (the
damage observed on leaves might be related to the Fe-compound applied, to the
dissolution of the cuticle as a negative consequence of the application of adjuvants, to a
detrimental interaction between the adjuvant and the Fe-compound) or to the severe
chlorosis of the plants.
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CAPÍTULO IV
The results of the Experiment I indicated that foliar applied Fe can re-green leaves
and be distributed to other plant organs, although due to the limited absorption of Fe
through leaf surfaces (Fernández et al., 2009) and to the low mobility of Fe in the
phloem (Briat et al., 2007; Kim y Guerinot, 2007) this type of applications can not fully
satisfy plant Fe demand. Since Fe fertilization through foliar sprays can only be used as a
complementary technique to direct application to soil or to nutrient solution of Fe
chelates (Pestana et al., 2001; Álvarez-Fernández et al., 2004), in the second experiment
presented in this work foliar sprays were applied as a complement to the application of
Fe chelates to the nutrient solution of soybean plants grown in hydroponics under
calcareous soil conditions.
The results of this assay confirm that even if Fe foliar sprays can not fully
substitute conventional supply of Fe chelates to roots, they contribute to overcome Fe
chlorosis when applied as a complementary source of Fe. Under these conditions, re-
greening of leaves and redistribution of Fe from treated leaves to other organs occurred.
Foliar application of Fe chelates when EDTA/Fe3+ was added to the nutrient solution
(FeIDHA+APG II, FeIDHA+U and FeEDTA+APG) promoted increments in root Fe
concentration. This suggests that Fe was translocated to roots, as already observed when
labeled Fe was used (Hüve et al., 2003; Nikolic et al., 2003; Rodríguez-Lucena et al.,
2009); and/or that uptake of Fe from the nutrient solution was enhanced due to the
transmission of signals from the shoot (Romera et al., 2006) that activate Fe uptake
mechanism by roots. None of the treatments tested was clearly more efficient than the
rest favoring redistribution of Fe to non treated organs, but the highest SPAD values
corresponded to FeIDHA+U.
Though the APG adjuvant was the most efficient product used in combination
with IDHA/Fe3+, the interaction between both compounds has been described in
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CAPÍTULO IV
previous works. Thus, the effectiveness of the APG was compared with urea (U) when
added in IDHA/Fe3+ formulations.
The use of U in foliar sprays favors Fe assimilation by leaves (Swietlik and Faust,
1984), due to its capacity to increase the permeability of leaf membranes and to its
surfactant properties. At the same time, these applications constitute an additional source
of nitrogen for the plant, although urea must be supplied at low rates to avoid the
inhibition of photosynthesis (Orbovic et al., 2001). The extra supply of nitrogen related
to the application of urea may explain the high SPAD values for the FeIDHA+U
treatment.
Regarding burn signs on leaf surfaces, they were less abundant in the mildly
chlorotic plants used in this experiment. However, except for FeIDHA+U, treated
leaves always presented dark spots that could be associated to leaf damage. This
confirms that the use of APG affected negatively the leaf surface even if this type of
adjuvants have been described as non-phytotoxic. For U negative interactions among
sprays components and leaf surfaces did not seem to occur. Nevertheless, it should be
kept in mind that the additional supply of nitrogen might have helped to improve leaves
status.
5. CONCLUSIONS
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ACKNOWLEDGEMENTS
This work was partly supported by ADOB, and by the Spanish Ministry of Science
and Education Project AGL2007—63756, co-financed with FEDER and the
Commission of European Communities. P. Rodríguez-Lucena was on a Spanish Ministry
of Science and Education “FPI” pre-doctoral contract co-financed by the European
Social Fund.
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REFERENCES
Briat J F, Curie C and Gaymard F. 2007. Iron utilization and metabolism in plants.
Plant Biol. 10:276-282.
Fernández V and Ebert G. 2005. Foliar Iron Fertilization: A Critical Review. J. Plant
Nutr. 28:2113-2124.
Fernández V, Río V, Abadía J and Abadía A. 2006. Foliar Iron Fertilization of Peach
(Prunus persica (L.) Batsch): Effects of Iron Compounds, Surfactants and Other
Adjuvants. Plant Soil. 289:239-252.
Kim A S and Guerinot M L. 2007. Mining iron: iron uptake and transport in plants.
FEBS Letters 581:2273-2280.
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Lindsay W L. 1979. Chemical Equilibria in Soils. John Wiley and sons. N.Y., USA.
Lindsay W L and Schwab A P. 1982. The chemistry of iron soils ans its availability to
plants. J. Plant Nutr. 5:821-840.
Lindsay W L. 1991. Iron oxide solubilization by organic matter and its effect on iron
availability. In: Iron nutrition and Interaction in Plants. Eds. Chen Y and Hadar Y.
Kluwer Academic Pub., The Netherlands.
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Schönherr J and Baur P. 1996. Effects of temperature, surfactants and other adjuvants
on rates of uptake of organic compounds, p. 135-155. In: G. Kerstiens (ed.). Plant
cuticles-An integrated functional approach. Bios Sci. Publisher Ltd.., Oxford,
United Kingdom.
Swietlik D and Faust M. 1984. Foliar nutrition of fruit crops. Hort. Rev. 6:287–356.
98
Capítulo V:
RESUMEN
101
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102
CAPÍTULO V.1
ABSTRACT
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CAPÍTULO V.1
1. INTRODUCTION
Most olive oil production is located in countries within the Mediterranean basin.
Spain is the world’s major producer, with a mean output of 1,100,000 tons per year for
the 2001-2007 period (International Olive Council database, 2008). Technical
improvements in oil extraction systems in recent decades have favored the increase in
production. Today, around 90 % of olive mills in Spain use the so-called “continuous
two-phase system”, whose main disadvantage is the production of two-phase olive mill
wastes (OMW), commonly known as “alperujos”. OMW are solid and very wet by-
products, with acidic pH and low consistency, rich in organic matter, potassium and
nitrogen, also containing fats, water-soluble carbohydrates and phenols. It is estimated
that 4,000,000 tons of OMW are generated yearly in Spain, and their disposal constitutes
a technical, financial and environmental concern (Alburquerque et al., 2004). The
application of OMW to soils as amendments or plant nutrient fertilizers (in most of
cases after composting of the raw material) is one of their possible uses (Alburquerque et
al., 2006; Plaza et al., 2008).
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CAPÍTULO V.1
Although their composition means that OMW can complex micronutrients, several
constraints impair their direct use as micronutrient fertilizers. The two-phase system
OMW have an important solid fraction formed by the remaining parts of the olive
(stone, pulp, etc.), with fats being a significant part of this organic matter (around 13 %
according to Alburquerque et al., 2004). Accordingly, OMW have to be processed before
they can be used to complex micronutrients. One such modification is that proposed by
Canet-Benavent (2006), which suggests a series of steps in which the liquid fraction of
the original OMW was modified (by centrifugation, biological treatment, alcoholic
fermentation, filtration and concentration) to produce a valued and multi-purpose
concentrate (OMWm).
This work assesses the ability of the OMWm extract to overcome micronutrient
deficiencies. Firstly, a study was made of the structural characteristics of the OMWm and
its ability to complex Fe, Zn, Mn and Cu. The stability of the complexes at different
pHs, their reactivity with soils and their perdurability under agronomic conditions were
also tested. Two plant experiments were then conducted with soybean (Glycine max. cv
Stine 0480) chlorotic plants grown in hydroponics to study the efficacy of
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CAPÍTULO V.1
The OMWm was kindly supplied by Ges-Biolives, and derived from fresh OMW
subjected to a series of steps to obtain a valued by-product (Canet-Benavent, 2006). The
valorized extract has an important fulvic fraction (270 g kg-1), acidic pH (5.5) and lower
total organic matter content than raw OMW (330 g kg-1). With regard to the nutrient
content, nitrogen (3.5 g kg-1), phosphorous (10 g kg-1) and potassium (55 g kg-1) are
the most significant. The mass spectra of the resulting OMWm consist largely of low
molecular weight fractions, with the most abundant appearing in the range from M/z
160 to 460 g mol-1.
The OMWm was freeze-dried and analyzed by Fourier Transform Infrared (FTIR)
spectroscopy. FTIR spectra were obtained on a FTIR Bruker IFS60v spectrophotometer
with a MTC detector and diffuse reflectance (DRIFT) accessory. Spectra of OMWm
samples were recorded in the 4000-400 cm-1 region at a resolution of 4 cm-1 in the
diffuse reflectance mode.
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CAPÍTULO V.1
After determining the highest complexing ability of the OMWm for each metal,
OMWm-metal complexes were formed by adding the appropriate amount of metal
solution (200 gL-1 FeSO47H2O, FeCl36H2O, CuSO45H2O, and 100 gL-1 ZnSO4H2O
and MnSO4H2O) to the OMWm. To ensure that the whole amount of metal is
complexed by the OMWm, an excess of complexing agent was used in the formation of
the complexes, with the metal:OMWm ratio being 1:1.1. Soluble and complexed
elements were determined. The precipitation method of Villén et al. (2007) was used for
the complexed elements. The soluble element content was measured according to
official EC methods 9.2 and 9.4 (Regulation (EC) Nº 2003/2003 of the European
Parliament and of the Council, relating to fertilizers. Regulation (EC) Nº 2003/2003 of
the European Parliament and of the Council, relating to fertilizers.).
Interaction of OMWm complexes with soils (Soil Experiment I): The interaction
experiments were performed according to Álvarez-Fernández et al. (2002). In brief, 2 g
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CAPÍTULO V.1
of two calcareous soils (S1 and S2) collected from different Spanish regions (S1 from
Valencia and S2 from Lérida) were allowed to interact with 5 mL of 4.010-4 M metal-
OMWm solution, 5 mL of a solution containing 2.010-2 M CaCl2 and 2.010-3 M
HEPES (pH 8) in 60 mL sterile polyethylene flasks. The soils had already been
characterized elsewhere (Álvarez-Fernández et al., 2002) and their main characteristics
are reported in Table 1. In addition, blanks for both the complexes and the soils were
provided, prepared in a similar manner, albeit without the addition of the soil or the
complexes, respectively. The flasks were shaken for 1 h at 25 ºC and 56 min-1 and then
allowed to stand for 3 days in a thermostatized incubator at 25 ºC.
Table 1. Chemical and Physical Characteristics of the Soils Used in the Interaction Assays
S1 S2
Sand (gkg-1) 650 460
Silt (gkg-1) 120 280
Clay (gkg-1) 230 260
Texture Sandy loam Sandy clay loam
pH (H2O) 7.69 7.82
pH (KCl) 7.15 7.23
EC, extract, 1:5 (dSm-1) 0.235 0.188
OM oxidizable (gkg-1) 8.0 24.0
[N], Kjeldahl (gkg-1) 0.79 1.40
C/N 10.5 9.9
CaCO3 (gkg-1) 150 179
Active lime (gkg-1) 40 52
Macronutrient (Soltanpour, cmolckg-1)
Ca 1.76 1.55
Mg 1.72 0.66
K 1.26 1.02
Na 0.150 0.047
Micronutrient (Soltanpour, mgkg-1)
Fe 14.3 27.0
Mn 13.0 5.4
Cu 1.46 47.1
Zn 1.08 4.3
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CAPÍTULO V.1
Its effectiveness was compared with two commercial products used to correct
micronutrient deficiencies: a synthetic chelate commonly used to treat Fe chlorosis
(Fe(III)EDTA), and a commercial lignosulfonate (pulp paper natural polymer obtained
during the sulfite pulping process when original lignin is broken down, Fe(III)LS)
(Rodríguez-Lucena et al., in press). In both cases, the soluble element was measured
according to official EC methods 9.2 and 9.4 (Regulation (EC) Nº 2003/2003 of the
European Parliament and of the Council, relating to fertilizers.). The percentage of Fe
complexed by the LS (6.9 %) was determined following the precipitation methodology
of Villén et al. (2007), while the method proposed by Lucena et al. (1996) was used for
Fe chelated by EDTA (12.9 %). For both products, the complexed vs. soluble Fe was
100 %. Furthermore, one control treatment without Fe addition was fixed (Control –Fe).
Soybean plants (Glycine max L. cv Stine 0480) were used in the biological
experiments. Soybean seeds were kindly supplied by Prof. R. J. Goos (North Dakota
State University, Fargo, USA). The seeds were washed with distilled water for 30 minutes
and then placed in trays, on filter paper moistened with distilled water and with another
filter paper placed over them. Thirty mL of distilled water and 20 mL of CaSO4 1.010-3
M were added. The trays were placed in a thermostatized stove, in darkness, at 28 ºC, for
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CAPÍTULO V.1
three days. After this time, seedlings were transferred to a growth chamber, where they
were grown under controlled climatic conditions: day/night photoperiod, 16/8 h;
temperature (day/night) 30/25 ºC; R.H. (day/night) 50/70 %. The seedlings of similar
development were placed on a perforated plate, floating over containers with
continuously aerated 1/5 diluted nutrient (with 5 µM Fe(III)EDTA) solution for five
days.
The diluted nutrient solution was then replaced by a full-strength solution, with the
following composition (macronutrients in M): 1.010-3 Ca(NO3)2, 9.010-4 KNO3, 3.010-
4
MgSO4, 1.010-4 KH2PO4; (cationic micronutrients in M, as EDTA buffered
micronutrient solution) 2.510-6 MnSO4, 1.010-6 CuSO4, 10.010-6 ZnSO4, 1.010-6 NiCl2,
1.010-6 CoCl2, 115.510-6 EDTANa2, 2.3110-4 M KOH; (anionic micronutrients, in M)
35.010-6 NaCl, 10.010-6 H3BO3, 5.010-8 Na2MoO4. The pH was adjusted to 7.5 with
KOH 1.0 M, and buffered with HEPES 1.0 x 10-4 M. The seedlings were kept in this
solution for six days, until chlorotic symptoms were observed.
After this time, the plants were transferred to 2 L polyethylene pots containing 2 L
of full-strength nutrient solution. The stems of two plants were wrapped together with
foam and placed in the pots (three holes in the lid, three pairs of plants per pot). This
nutrient solution had the same composition as the initial one, except in the cationic
micronutrients content (not buffered micronutrient solution, in M): 1.010-6 MnSO4,
5.010-7 CuSO4, 5.010-7 ZnSO4, 1.010-7 NiCl2, 1.010-7 CoCl2. The pH was adjusted to
7.5 with KOH 1.0 M, and buffered with HEPES 1.010-4 M, and 0.2 gL-1 of solid
CaCO3 per pot were added. Water was added every two days, and the solution was
renewed every week. At this point, Fe treatments were applied, being repeated 8 and 15
days after transplanting in both experiments.
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CAPÍTULO V.1
Table 2. OMWm complexing ability and formation of complexes with Fe, Zn, Mn and Cu
OMWm complexing ability Metal-OMWm complexes
relating to fertilizers. .
FW: fresh weight
In both assays, three replicates per treatment were performed. During the
experiments, SPAD readings were regularly taken with a chlorophyll meter (Minolta
SPAD-502) for all the leaf stages (average of three readings per leaf). Whole plants were
sampled after transplanting at 8 days (two pairs of plants) and 22 days (one pair of
plants). The sampled roots, stems and leaves were separated and washed (Álvarez-
Fernández et al., 2001), weighed and dried at 65 ºC for three days. Micronutrients in the
leaves were determined by AAS after dry digestion procedure.
Data were statistically evaluated using Analysis of Variance (ANOVA) with SPSS
15.0 software to assess the significance of the main factors and interactions. Means were
also compared using Duncan’s test at P ≤ 0.05 in order to find significant differences
between treatments.
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CAPÍTULO V.1
The FTIR spectrum for the OMWm is presented in Figure 1. Infrared bands were
interpreted according to Plaza et al. (2008) and Ait Baddi et al. (2004), and its main
features were:
660
930
2970 2935
2885
1260
1045
1400
3201
3409
1595
1720
1120
(1) a broad band at 3409 cm-1 due to O-H stretching of phenol, alcohol or carboxyl
OH and also to amide and amine N-H stretching; (2) bands at 2970-2935 cm-1 and at
2885 cm-1 due to aliphatic C-H stretching; (3) a band at 1720 cm-1 due to C=O stretching
of COOH, and also ascribed to other carbonyl groups such as ketones and aldehydes; (4)
a band at 1595 cm-1 that could be considered a group of unresolved absorptions mainly
due to aromatic C=C, C=O stretching of amide groups, quinonic C=O and/or C=O of
H-bonded conjugated ketones; (5) a band at 1400 cm-1 attributed to stretching of anti-
symmetric COO-, aliphatic C-H bending, O-H bending and C=O stretching of phenolic
groups; (6) a band at 1260 cm-1 due to C-O stretching of aryl ethers and phenols and C-
O stretching and O-H deformation of carboxylic groups; (7) a band at 1120 cm-1 due to
alcoholic group vibrations; (8) a band at 1045 cm-1 due to C-O stretching of
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CAPÍTULO V.1
carbohydrates and polysaccharides; and (9) peaks at 930-660 cm-1 attributed to C-H
bending of substituted aromatic groups.
The FTIR study indicates that the complexing groups able to complex
micronutrients, mostly phenolic and carboxylic, were abundant. These structures would
be included in the low molecular fractions shown by the mass spectra, and could be
attributed to organic acids and natural chelating agents, such as siderophores and
phytosiderophores. On the other hand, the major bands observed in the spectra are the
same as those observed in the fulvic fraction acids extracted from OMW in the first
stage of the composting process (Ait Baddi et al., 2004), suggesting that the fulvic
fraction is very important in the OMWm studied here, and supports its potential ability
to form metal complexes (Stevenson, 1994). The abundance of carboxylic and phenolic
groups capable of complexing metals also supports this hypothesis.
The ability of OMWm to complex Fe, Zn, Cu and Mn was evaluated. In the case
of Fe complexes, they are usually prepared with Fe(II) (from FeSO4) when commercial
complexes are manufactured and its natural oxidation to Fe(III) is expected to occur. In
this experiment, the complexing ability of OMWm with both forms of Fe and the
stability of the Fe complexes were studied in order to evaluate the influence of the initial
status of Fe in the behavior of the complex.
Figure 2 shows the measured vs. added element when the complexing ability was
studied. The curves obtained are consistent with those described by Villén et al. (2007),
having a rising segment that corresponds with the complexing process, followed by
another decreasing one that implies the coagulation of the material through the excess of
metal. In addition, for each metal Table 2 presents the percentage of element that
remains in solution with regard to the added element (obtained from the intersection
point of the two lines in figure 2). As elucidated from the FTIR spectra, all the elements
studied could be complexed by the OMWm, especially in the case of Fe(III) and Cu,
although a fraction of the added element flocculated and/or precipitated as from the
beginning of the addition of the metal solutions to the OMWm (the measured element
was always lower than the added element), especially in the case of Zn and Mn. This
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CAPÍTULO V.1
indicates that both the strong and the weak complexing sites produce coagulation of the
complex or precipitation of the metal. In addition, different complexation abilities were
measured for Fe(II) and Fe(III).
The different behavior for both Fe forms suggests that the expected Fe(II)
oxidation did not occur in the experimental period and that less stable Fe complexes
were formed with divalent than with trivalent Fe. Hence, the formation of Fe-OMWm
complexes with Fe(III) would be recommended.
Once the complexing ability was known, the metal-OMWm complexes were
formed and characterized. The highest percentage of complexed element with regard to
soluble element corresponded to Cu, Zn and Fe(III). In any case, for all the elements the
amount of metal complexed and the degree of complexation exceeded the requirements
of Spanish Regulation Order APA/863/2008, so they can be commercialized.
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CAPÍTULO V.1
Figure 2. Complexing ability for OMWm with Fe (divalent and trivalent), Zn, Cu and Mn.
115
CAPÍTULO V.1
The effect of pH on the amount of element remaining in solution after three days
is reported in Figure 3. For Fe(III), the recovery was negligible in the alkaline pH range.
In the case of Cu, the solubility of the complex was high in the entire pH interval
studied, decreasing only between pH 6 to 8.
This behavior was ascribed to the solubility of inorganic Cu species below pH 6 and to
the strong complexation processes occurring over pH 8. Mn and Zn complexes readily
maintained a high amount of the element in the pH range corresponding to alkaline
soils, with the complexed element decreasing sharply when the pH was above 9. Figure 4
reports the amount on Fe, Zn, Cu or Mn remaining in solution with regard to the total
amount of element added in the interaction of the complexes with both calcareous soils
(Soil Experiment I). Soil blanks showed that the contribution of the soils to the amount
of metal found in solution was negligible (data not shown). After three days of
interaction, the percentage of metal recovered was very low, especially for
Zn(II)OMWm. The highest values corresponded to the complexes formed with trivalent
Fe and Mn, although in both cases the metal remaining in solution was below 8 %.
Two-way ANOVA indicated that there were no statistical differences in the percentage
of recoverable metal according to the type of soil used or to the metal interacting with
the soil (data not shown).
S1 S2
n 12
o
it
u
lo10
s
e
h
t 8
n
i
g
n
i 6
n
ia
er 4
m
la
te 2
m
%0
Fe (II) Fe (III) Zn Cu Mn
Figure 4. Percentage of metal remaining in a 2.010-2 10-3
M CaCl2 and 2.0 M HEPES (pH 8)
solution with regard to the total element added (410-4 M) for the different OMWm complexes
after three days of interaction with the two soils studied (Soil Experiment I).
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CAPÍTULO V.1
n 100
o
ti
lu
o
s 80
e
h
t
n
i 60
g
n
i
n
ia 40
m
er
la 20
te
m
% 0
5 6 7 8 9 10 11
pH
Figure 3. Percentage of Fe(II), Fe(III), Zn, Cu and Mn remaining in a 1.010-1 M CaCl2 solution with
regard to the total element added (1.010-2 M) for the different OMWm complexes at different pHs
after three days of interaction.
data. The study of complexing ability indicated that OMWm formed stable complexes
with Fe, especially with Fe(III), under the conditions described in the methodology used
(Villén et al., 2007), after 24 hours of interaction at pH 9. Since the experiment testing
the stability of the complexes at different pHs showed that soluble Fe did not remain in
the solution in the alkaline pH range, it can be concluded that Fe-OMWm complexes
were not stable after three days in the presence of Ca2+. However, soluble Fe was
recovered after three days of interaction for both experiments with calcareous soils (Soil
Experiments I and II); the percentage of recoverable Fe being higher when the pH of
the initial metal-OMWm solution was not fixed and Ca2+ was not added to the solution.
The different concentration of Fe-OMWm used in the pH assay and in both soil
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CAPÍTULO V.1
experiments could explain these differences. For LS polymers, their structural swelling at
high alkaline pHs has been described (Pang et al., 2008) and this phenomenon has been
related to the ionic groups inside the molecule and its branched structure in aqueous
solution. This would favor Ca2+ complexation because the polymer “opens” and more
complexing sites can be used to form complexes. Although a similar study has not been
performed for OMWm, the similarities between both complexing agents with regard to
% metal remaining in the solution
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CAPÍTULO V.1
the presence of functional groups able to complex metals (mostly carboxylic and
phenolic hydroxyl radicals) suggest that swelling and expansion could also occur in the
case of OMWm. The lower concentrations used in both soil experiments would have
favored swelling to a higher extent, promoting the contact between the Fe and the
complexing sites and, consequently, the complexation process. Regarding the differences
between both soil experiments, it is important to bear in mind that the pH and Ca2+
concentrations were fixed in the assay Soil Experiment I (pH 8 and 2.010-2 M CaCl2)
whereas they were not in the second one. Theoretical modeling using Visual Minteq
ver.2.52 confirmed this hypothesis, since the initial pH and Ca2+ concentrations
decreased when they were not fixed, favoring the presence of soluble Fe.
For Zn, Mn and Cu complexes, more than 60 % of the initial amount of metal
remained in the solution at alkaline pH when the complexes did not interact with soils
(Figure 3). The low concentration of element remaining after interaction with soils
(Figure 4 and 5) suggests that in this case the sorption of the complex or the
displacement of the metal occurred. This supposition is confirmed by the results of the
experiment of perdurability under agronomic conditions.
In general, all the treatments recorded a low ability to recover plant Fe deficiencies.
Since Fe(III)OMWm complexes performed better than Fe(II)OMWm with regard to
their complexing ability and in the stability experiments, trivalent Fe was used to form
the Fe-containing compounds used in the biological experiments. The trend in the
SPAD Index from the beginning of the experiment showed that the highest re-greening
values corresponded to the Fe(III)EDTA treatment, followed by Fe(III)OMWm
(without statistical differences with Fe(III)EDTA), Fe(III)LS and Control –Fe. The
average values in the second level of leaves were 12.9 ± 1.1(a), 10.0 ± 0.6(a), 5.4 ± 0.7(b)
and 5.7 ± 1.1(b), respectively, with different letters denoting statistical differences
between treatments.
Two-way ANOVA of plant dry weight, leaf Fe concentration and Fe/Mn ratio in
leaves was performed, using the treatments and sampling time as factors (Table 3). The
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CAPÍTULO V.1
analysis revealed that statistical differences due to the interaction between both factors
occurred for plant dry weight and leaf Fe concentration.
Table 4. Effect of the different Fe-containing compounds applied to the nutrient solution on the dry
weight and leaf Fe concentration and Fe/Mn ratio in soybean plants grown in hydroponics after 8 and 22
days.
Dry weight Fe Concentration
Fe/Mn
(g plant-1 DW) (µg g-1 DW)
8 days after the 1st application of the treatments
Fe(III)OMWm 0.94 ± 0.02 ns 48.5 ± 5.8 a 0.49 ± 0.14 ns
Fe(III)LS 0.79 ± 0.09 40.1 ± 5.7 ab 0.29 ± 0.06
Fe(III)EDTA 1.01 ± 0.15 44.5 ± 3.9 ab 0.38 ± 0.05
Control -Fe 0.72 ± 0.05 31.5 ± 0.4 b 0.22 ± 0.01
22 days after the 1st application of the treatments
Fe(III)OMWm 1.46 ± 0.12 b 37.4 ± 0.3 b 0.19 ± 0.03 b
Fe(III)LS 1.17 ± 0.09 b 31.7 ± 2.5 bc 0.11 ± 0.01 c
Fe(III)EDTA 2.61 ± 0.31 a 65.9 ± 2.2 a 0.38 ± 0.02 a
Control -Fe 0.96 ± 0.06 b 26.9 ± 1.3 c 0.16 ± 0.01 bc
Data are means ± SE. Different letters within the same column denote significant differences between the
treatments (P < 0.05). ns: non-significant. DW: dry weight basis.
Table 4 reports biometric data for all treatments at both sampling times. For the
first sampling time there were no statistical differences between the treatments tested
with regard to plant dry weight, but at the end of the assay Fe(III)EDTA treated plants
recorded higher growth than the rest. The same behavior was observed for Fe
concentration and Fe/Mn ratio in leaves. Statistical differences between treatments were
observed only at the end of the assay, with Fe(III)EDTA treated plants recording the
highest values. After this chelate, Fe(III)OMWm was the most efficient compound,
while Fe(III)LS was not statistically different to the Control –Fe.
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CAPÍTULO V.1
most efficient treatment, which may be explained by the percentage of chelated Fe,
which was higher than the amount of element complexed by Fe(III)OMWm and
Fe(III)LS. In other experiments, we have shown that synthetic chelates were more
efficient than complexes at recovering Fe chlorotic plants (Rodríguez-Lucena et al., in
press). Regarding the effectiveness of Fe complexes, Fe(III)LS was less efficient than
Fe(III)OMWm in spite of the higher complexing capacity of the amount of Fe
complexed (6.9 % and 3.0 %, respectively) and although the stability of Fe(III)OMWm
at alkaline pHs was lower than that described for Fe(III)LS in a previous paper (Lucena
et al., 2009). In this case, complexation and plant development were not correlated.
Stability constants may explain the lower effectiveness of Fe(III)LS. As could be
elucidated from the FTIR spectra (Figure 1), the OMWm studied was rich in functional
groups able to complex metals, and the results reported here suggest that the OMWm
molecule could release Fe to the plant more easily that the LS. Stability constants are
usually used to explain this behavior. However, stability constants in this type of
polymers correspond to a range of values that are highly dependent on several factors,
such as pH, pe + pH and others, so comparison is difficult. The Fe/ligand ratio also has
to be considered to explain the effectiveness of complexes, since low stability ones have
a high ratio (Stevenson, 1994). When weakly bound Fe is released, the remaining Fe
would be strongly bonded and may not be available for use by the plants. Thus, higher
concentrations should be tested in order to improve the effectiveness of complexes with
regard to synthetic chelates.
Foliar Application
The same Fe compounds applied to the nutrient solution were tested when applied
through foliar sprays. None of the treatments was particularly effective at recovering
plants suffering from severe chlorosis. One-way ANOVA revealed that the SPAD Index
recorded statistical differences between treatments. The trend in the SPAD Index during
the assay showed that Fe(III)EDTA favored re-greening of leaves to a higher extent than
the other treatments. Fe(III)OMWm was also effective at increasing SPAD values, while
Fe(III)LS was not statistically different to the Control –Fe. The average values in the
second level of leaves were 20.3 ± 3.0(a), 15.9 ± 3.1(ab), 10.5 ± 1.4(b) and 5.2 ± 1.0(c),
respectively, with different letters denoting statistical differences among treatments.
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CAPÍTULO V.1
Two samplings were made in order to determine plant dry weight. For this
parameter, statistical differences between treatments and for sampling time were studied
using two-way ANOVA, as well as for the interaction between both factors, which
revealed no interaction between them. The dry weight of the plants was influenced solely
by the time elapsed after the application of the foliar sprays (at P ≤ 0.001), while no
statistical differences between treatments were observed. Since contamination of the
treated leaves due to the foliar sprays has been observed previously (Rodríguez-Lucena
et al., in press), Fe concentration and Fe/Mn ratio were measured only in roots at the end
of the experiment. Statistical differences between treatments were observed for both
parameters (at P ≤ 0.01 and P ≤ 0.05 for leaf Fe concentration and Fe/Mn ratio,
respectively).
Table 5. Effect of the different Fe-containing compounds applied through foliar sprays on the dry
weight and root Fe concentration and Fe/Mn ratio in soybean plants grown in hydroponics after 22
days.
Dry weight Fe Concentration
Fe/Mn
(g plant-1, DW) (µg g-1 DW)
Fe(III)OMWm 0.74 ± 0.06 ns 65.1 ± 9.1 b 0.18 ± 0.04 b
Fe(III)LS 0.97 ± 0.15 129.6 ± 16.5 a 0.38 ± 0.06 a
Fe(III)EDTA 0.96 ± 0.23 65.7 ± 6.5 b 0.15 ± 0.02 b
Control -Fe 0.82 ± 0.19 54.1 ± 1.7 b 0.17 ± 0.02 b
Data are means ± SE. Different letters within the same column denote significant differences
between the treatments (P < 0.05); ns: non-significant; DW: dry weight basis.
Fe concentration and Fe/Mn ratio in roots are reported in Table 5 and indicate
that a significant redistribution of Fe foliarly applied from leaves to roots occurred only
for Fe(III)LS, but this did not improve the general nutritional status of the plants.
The redistribution to roots of Fe applied through foliar sprays has been observed
previously working with labeled Fe and mildly chlorotic plants complexed by humic
substances and LS (Nikolic et al., 2003; Rodríguez-Lucena et al., 2009). The foliar
application of Fe is usually performed in plants with an additional supply of Fe through
the roots (Álvarez-Fernández et al., 2004; Rodríguez-Lucena et al., 2009). Thus, given
the low mobility of Fe through the phloem, the performance of Fe(III)OMWm should
be considered when applied to mildly chlorotic plants receiving an additional supply of
Fe through the roots.
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CAPÍTULO V.1
4. CONCLUSIONS
The OMWm extracts tested in the work are valorized by-products capable of
complexing Fe, Zn, Cu and Mn. The OMWm formed micronutrient complexes suitable
for commercialization according to Spanish legislation, although given their low stability
under alkaline soil conditions their use is recommended solely for application to the
nutrient solution in hydroponic systems or when applied through foliar sprays. Due to
the low concentrations of complexes applied in micronutrient fertilization, the
composting of the extract can be omitted and raw materials could be used. Further
research is necessary to elucidate the structural characteristics that may improve the
stability of the complexes, and FTIR analysis may be a useful tool in the structural
analysis of modified OMWm. Hence, Fe-OMWm complexes constitute a promising eco-
compatible and cheap alternative to synthetic chelates for dealing with micronutrient
deficiencies when applied foliarly or to the nutrient solution of hydroponically grown
plants.
ACKNOWLEDGMENTS
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CAPÍTULO V.1
REFERENCES
Ait Baddi G A, Hadifi M, Cegarra J, Albuquerque J A, Gonzálvez J, Gilard V and
Revel J. 2004. Characterization of fulvic acids by elemental and spectroscopic
(FTIR and 13C-NMR) analyses during composting of olive mill wastes plus straw.
Bioresour. Technol. 93:285-290.
Canet-Benavent, M. 2006. Method for the industrialised production of olive mill by-
products and product thus obtained. Patent WO/2006/ 058938 A3,
Fernández V and Ebert G. 2005. Foliar Iron Fertilization: A Critical Review. J. Plant
Nutr. 28:2113-2124.
Fernández V, Río V, Abadía J and Abadía A. 2006. Foliar iron fertilization of peach
(Prunus persica (L.) Batsch): effects of iron compounds, surfactants and other
adjuvants. Plant Soil 289:239-252.
Lindsay W L and Schwab A P. 1982. The chemistry of iron in soils and its availability
to plants. J. Plant Nutr. 5:821-840.
Lindsay W L. 1972. Zinc in soils and plant nutrition. Adv. Agron. 24: 147-186.
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Lindsay W L. 1979. Iron. In: Chemical Equilibria in Soils; Lindsay W L. John Wiley &
Sons: New York; pp. 129-149.
Lucena J J. 2006. Synthetic iron chelates to correct iron deficiency in plants. In: Iron
Nutrition in Plants and Rhizospheric Microorganisms; Barton L L and Abadía J.,
Eds. Springer-Verlag Academic Publishers, Dordrecht. pp 103-127.
Papadopoulos P and Rowell D L. 1989. The reactions of copper and zinc with
calcium carbonate surfaces. J. Soil Sci. 40: 39-48.
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Uygur V and Rimmer D L. 2000. Reactions of zinc with iron coated calcite surfaces at
alkaline pH. Eur. J. Soil Sci. 51:511-516.
126
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RESUMEN
59
Los LS mostraron escasa capacidad para solubilizar Fe de hidróxido férrico
amorfo, y la movilidad de los complejos 59Fe(III)-LS en una columna se suelo (pH 7,5)
fue muy inferior a la del 59Fe(III)-EDDHA. Sin embargo, su eficacia fue similar a la del
59
Fe(III)-EDDHA al ser aplicados a plantas de tomate en hidroponía pura, indicando
que la reducida efectividad de estos complejos en aplicaciones a suelo estaría relacionada
con su retención en el mismo.
Por tanto, puede concluirse que los complejos Fe-LS constituyen un alternativa
eficaz, barata y respetuosa con el ambiente para corregir la clorosis férrica al ser
aplicados por vía foliar o a la disolución nutritiva de plantas desarrolladas en hidroponía
pura.
127
CAPÍTULO V.2
a
Agricultural Chemistry Department, Universidad Autónoma de Madrid, 28049 Madrid,
Spain
b
Dipartimento di Scienze Agrarie e Ambientali, Università degli Studi di Udine, Via delle
Scienze 208, 33100, Udine, Italia.
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CAPÍTULO V.2
ABSTRACT
Results show that LS had a low capability to solubilize 59Fe-hydroxide and that
preformed 59Fe(III)-LS complexes had poor mobility through a soil column (pH 7.5) and
scarce stability when interacting with soils compared to 59Fe(III)-EDDHA. However
when 59Fe(III)-LS were supplied to roots in a hydroponic system, they demonstrated an
even higher capability to fed Fe-deficient tomato plants than 59Fe(III)-EDDHA. Hence,
data here presented indicate that the low Fe use efficiency from Fe-LS observed in soil-
applications is due to interactions of these Fe-sources with soil colloids rather than to
the low capability of roots to use them.
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CAPÍTULO V.2
1. INTRODUCTION
Lignosulfonates (LS) are pulp paper polymers obtained during the sulfite pulping
process, when original lignin is broken down and fragments are made water-soluble by
the introduction of sulfonic acid groups. In the lignin molecule, -OH radicals can be
bonded to carbon and behave as carboxylic groups like in organic acids. On the other
hand, in soils the degradation of lignin can work as humic substances precursor. These
characteristics are consistent with the capability of LS to complex Fe and other metals,
and are at the basis of their possible use as Fe chlorosis correctors.
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CAPÍTULO V.2
applied to calcareous soils or to the nutrient solution (Rico et al., 1996; Martín-Ortiz et
al., 2009). Comparison of LS and HEDTA (N-(hydroxyethyl)-ethylenediaminetriacetic
acid) as Fe suppliers when applied to butterhead lettuce grown in hydroponics revealed
that LS were almost as efficient as HEDTA in keeping Fe in solution and providing Fe
for plant uptake (Demeyer et al., 2001).
Fe-LS have shown low stability in interaction experiments under calcareous soil
conditions (Goos et al., 2001). The authors found that negligible Fe remained in the
solution after one day of incubation with this type of soil. On the other hand, we have
observed (data unpublished) that from 20 to 30 % of initially complexed Fe was found in
the solution after three days of interaction of different commercial Fe-LS with different
soil materials (peat, illite and ferrihydrite).
Given the low stability shown by Fe-LS when applied to soils or to nutrient
solution, foliar application of Fe-LS complexes can be used as an alternative way to
overcome Fe chlorosis. This type of applications have been tested by several authors
with synthetic chelates, natural complexes and inorganic salts (Pérez-Sanz et al., 1996;
Pestana et al., 2001; Nikolic et al., 2003; Álvarez-Fernández et al., 2004; Fernández et al.,
2006) with variable results, due to the diverse factors controlling foliar absorption
(characteristics concerning the treated leaf, the type of molecule applied, surfactant, etc.)
and difficulties in achieving an optimum formulation of foliar fertilizer.
Although LS have a good ability to complex Fe, to our knowledge there are few
studies evaluating the effectiveness of these complexes to cure Fe chlorosis, and in most
of cases are referred to commercial complexes. Thus, the influence of the characteristic
of the LS in the use efficiency at the root and leaf levels of the Fe-LS, has not been
evaluated. In addition no information concerning the mechanisms of Fe acquisition from
Fe-LS sources at the root and leaf levels are available to date. In the present work four
LS, differing in their physical and chemical characteristics, were compared with regard to
59
their ability to solubilize Fe from sparingly soluble Fe-hydroxide and from soils.
Mobility of 59Fe-LS complexes through soil columns was also evaluated. Furthermore,
the different efficiency of 59Fe-LS complexes to supply Fe to cucumber and tomato
plants through root (nutrient solution) or foliar applications was studied, also evaluating
the long- and short-distance transport of foliarly applied 59Fe.
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CAPÍTULO V.2
59
The freshly precipitated amorphous Fe-hydroxide was obtained insolubilizing
59
FeCl3 at alkaline pH (Guzmán et al., 1994); the specific activity was 23 KBq µmol-1 Fe.
59
Fe-EDDHA or 59Fe-EDTA complex (specific activity of 11 and 65 KBq µmol-1
Fe, respectively) were prepared according to Römheld and Marschner (1986) by mixing
59
FeCl3 with o,o-EDDHA or EDTA with a molar ratio of 1:1.1.
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CAPÍTULO V.2
solution containing 59Fe-LS and that of 59Fe-EDTA were adjusted to pH 5.0 to avoid
altering the ion exchange properties of the cuticle (Fernández et al., 2005) and added
with Tween 80 (non ionic surfactant; PROBUS, Barcelona, Spain) (0.1 % v/v).
2.2 Solubilization of Fe by LS
The capacity of LS to solubilize Fe from insoluble forms was evaluated with the
experimental approach described by Cesco et al. (2000). The freshly precipitated
59
amorphous Fe-hydroxide (2.5 mL of suspension containing 10 µmol Fe) were
transferred into a dialysis tube (ZelluTrans/Roth 6.0, Ø 16mm, exclusion limit of 10-12
kDa, Karlsruhe, Germany) and mixed with 7.5 mL of nutrient solution (Solution A) with
the following composition: 0.7 mM K2SO4, 0.1 M KCl , 2 mM Ca(NO3)2, 0.5 mM
MgSO4, 0.1 mM KH2PO4 (10 mM Hepes-KOH, pH 7.5). Thereafter, the dialysis tube
was transferred into a 250 mL glass containing 230 mL of the same nutrient solution A.
To maintain homogeneity throughout the time-course of the experiment in both
solutions, continuous aeration was provided inside and outside the dialysis tube. Both
solutions, inside and outside the dialysis tube, were continuously aerated in order to
maintain them homogeneous during the experiment. The experiment was started by
adding LS inside the dialysis tube (final LS concentration 5 µM). As controls, EDDHA
(final concentration 5 µM) and distilled water were used. For each LS or control, four
replicates were performed. At timed intervals samples from the outside solution were
collected, and the amount of 59Fe was measured by liquid scintillation counting. In this
experiment only the behavior of the three fractions with the lower molecular weight
(LS1, LS2 and LS3) was evaluated.
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The mobility in the soils of the 59Fe complexed by LS was evaluated as described
by Cesco et al. (2000) loading 5 nmol of 59Fe-LS on the top of a column (Ø 16 mm,
height 6.5 mm) containing 7.5 g of soil (Eutric cambisol, pH 6, bulk density 1.1 g cm-3).
The elution of 59Fe through the column was performed loading carefully the solution A,
the flow rate, due to the gravity, being about 2 mL min-1. The first 20 mL of the solution
eluted through the column were collected and the amount of 59Fe was determined by
liquid scintillation counting. For comparison, elution of 59Fe chelated by EDDHA was
tested. Four replicates were established for each of the LS fractions and EDDHA.
Tomato seeds (Lycopersicum esculentum L., cv. Marmande) were germinated for 6 days
on filter paper moistened with 5 mM CaSO4. Then, the seedlings were transferred to 2.2
L plastic vessels with continuously aerated nutrient solution having the following
composition: (macronutrients) 0.7 mM K2SO4 , 0.1 mM KCl , 2 mM Ca(NO3)2, 0.5 mM
MgSO4, 0.1 mM KH2PO4; (micronutrients) 10 µM H3BO3, (pH 7.5); 0.5 µM MnSO4, 0.5
µM ZnSO4, 0.2 µM CuSO4, 10 nM (NH4)6Mo7O24 (adjusted at pH 6.0 with 0.1M KOH).
Nutrient solution was renewed every 3 days. The plants were kept in a growth chamber
under the following controlled climatic conditions: day/night photoperiod, 16/8;
temperature (day/night) 25/20 ºC; RH 65/75 %, light intensity 350 µE m-2s-1. During
the first 21 days of hydroponic culture, the plants were supplied with Fe which was
added to the solution as Fe-EDTA at the final Fe concentration of 5µM. In the
following 7 days, Fe was completely omitted from the nutrient solution.
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CAPÍTULO V.2
Cucumber seeds (Cucumis sativus L., cv. Lungo della Cina) were germinated for 5
days on filter paper moistened with 5 mM CaSO4. Then, the seedlings were transferred
to the nutrient solution described previously, and were kept for 13 days in a growth
chamber with controlled conditions as described for tomato plants. Plants were grown
without any exogenous addition of Fe.
59 59
Uptake of Fe from Fe-LS complexes was evaluated using the procedure
described by Cesco et al. (2002). Briefly, roots of intact Fe-deficient 34-day-old tomato
plants were washed with micronutrient-free nutrient solution for 30 min and then
transferred to beakers containing 250 mL of a freshly prepared micronutrient-free
59
nutrient solution, where Fe-LS complexes was added in order to give a final Fe
concentration of 1 µM and 1.1 µM LS; as control, 1 µM 59Fe-EDDHA has been used.
Four replicates per treatment (one plant per replicate) were performed. Uptake solution
was buffered at pH 7.5 with 10 mM HEPES-KOH and the uptake period lasted 24
hours. In order to limit photo-chemical reduction phenomena of Fe in the nutrient
solution (Zancan et al., 2006) added by the Fe-sources, during the entire experiment,
beakers have been covered. After the uptake period, plants were transferred to a freshly
59 59
prepared Fe-free nutrient solution for 10 min, root extraplasmatic Fe pool was
removed by 1.2 g L-1 sodium dithionite and 1.5 mM 2,2’-bipyridyl in 1 mM Ca(NO3)2
under N2 bubbling according to the method described by Bienfait et al. (1985) and then
roots and aerial part were harvested separately. Roots and shoots were oven-dried at
80°C, weighed, ashed at 550°C, and suspended in 1 M HCl for 59Fe determination by
liquid scintillation counting. The 59Fe uptake rate, measured as nmol 59Fe, is referred to
the whole plant (root + shoot) and is presented per g dry weight of roots in 24 hours.
The 59Fe translocation rate is presented as nmol 59Fe measured in shoot per g of root dry
weight in 24 hours.
Uptake and translocation of foliar applied 59Fe by intact cucumber and tomato
plants were evaluated using the procedure described by Nikolic et al. (2003). Briefly, the
fully-expanded young leaf of cucumber and the apical portion of fully-expanded young
leaves of tomato plants were immersed for 1 minute in 40 mL 59Fe-labeled solution
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136
CAPÍTULO V.2
described by Nikolic and Römheld (1999). Leaf discs were incubated for 40-60 min in
continuous-orbital shaking solution containing 0.5 mM CaSO4, 250 mM Sorbitol, 100
µM BPDS, 10 mM MES-KOH (pH 6.0) in the dark at 25°C; Fe-sources (Fe-LS
complexes or Fe-EDTA, three replicates per Fe source) were added at a final Fe
concentration of 10 µM. Ferric reduction was determined as formation of the Fe(II)-
BPDS complex at 535 nm after the leaf discs incubation.
3. RESULTS
Table 2: 59Fe solubilization from 59Fe-hydroxide by LS after 22 h of treatment; the LS were added at a
final concentration of 5 µM to a dialysis tube containing 10 µmol 59Fe-hydroxide.
The addition to the solution containing amorphous 59Fe-hydroxide of EDDHA at a final concentration
of 5µM solubilized 163.3 ± 2. 7 nmol 59Fe.
Solubilized 59Fe (nmol 59Fe)
LS1 24.0 ± 1.4 a
LS2 22.9 ± 1.1 ab
LS3 21.6 ± 0.9 b
Control 21.2 ± 1.1 b
Data are means ± SD of four replicates, and refer to the amount of 59Fe recovered in the outer solution.
Different letters in the same column denote significant differences among the treatments (P < 0.05).
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CAPÍTULO V.2
The study of 59Fe-LS complexe stability in soil (Eutric cambisol) showed that the
amount of Fe recoverable after the interaction of the complexes with a soil was
negligible (0.4 % and 0.2 % after 1 h and 24 h from the beginning of the interaction,
respectively, data not shown). The mobility in the soil of Fe complexed by LS was tested
by measuring the 59Fe content in the solutions eluted from soil columns previously
59 59
loaded with different Fe-LS complexes. Figure 1 shows that Fe-LS could move
through the soil column, the highest elution levels being observed for 59Fe-LS2 and 59Fe-
LS4 (Fig. 1). In any case for all the LS tested, less than 5 % of the total 59Fe loaded onto
the soil column could be recovered in the 20 mL of eluted solution (Table 3). In the case
40
Fe-LS1
30
20
10
0
40 0 5 10 15 20
30
Fe-LS2 2/59Fe3+
20
Eluted Fe (pmol)
10
40
30 Fe-LS3
20
10
0
40
30 Fe-LS4 4/59Fe3+
20
10
0
1000 0 5 10 15 20
800
Fe-EDDHA EDDHA/59Fe3+
600
400
200
0
0 5 10 15 20
Solution eluted (mL)
Figure 1: Elution patterns of 59Fe, pre-loaded as 59Fe-LSs or 59Fe-EDDHA, through a soil (Eutric
cambisol) column. The eluent is a nutrient solution (A, buffered with 10 mM HEPES-KOH pH 7.5)
containing no Fe. Data are means ± SD of two independent experiments with three replicates.
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CAPÍTULO V.2
of Fe-EDDHA the recovery of 59Fe accounted to 63% of the chelate loaded on to the
column.
The ability of LS to solubilize native Fe from Eutric cambisol soil samples was
evaluated loading different amounts (50, 500 or 5000 nmol) of LS2 (LS able to form Fe-
LS complexes with the highest mobility through the soil column) on the top of soil
columns and measuring the elution of Fe. The amount of Fe collected outside of the
column was slightly increased (+3%) with respect to the control (water) only when
5µmol of LS2 were applied (data not shown). For comparison, the loading of 50nmol of
EDDHA caused an increase of about 42 % of Fe recovered outside the column (data
not shown).
pmol %
Fe-LS1 87 ± 11 1.7 ± 0.2 c
Fe-LS2 230 ± 36 4.6 ± 0.7 a
Fe-LS3 107 ± 13 2.1 ± 0.3 c
Fe-LS4 163 ± 26 3.3 ± 0.5 b
Data are means ± SD of four replicates. Different letters in the same
column denote significant differences among the treatments (P < 0.05).
3.2 Utilization of 59Fe from 59Fe-LS by roots of intact Fe-deficient tomato plants
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CAPÍTULO V.2
EDDHA treated plants. Roots of intact Fe-deficient tomato plants were also able to
reduce Fe(III) complexed by LS.
Table 4: Uptake and translocation of 59Fe by Fe-deficient tomato plants treated in nutrient solution (pH 7.5) with 59Fe-LS or
59Fe-EDDHA (1µM Fe).
Values of Fe(III)-LS or Fe(III)-EDDHA reduction (10 µM Fe) by roots of intact plants, are also reported.
Uptake Translocation Reduction of FeIII-sources
(nmol 59Fe g-1 root DW in 24h) (nmol 59Fe g-1 root DW in 24h) (nmol Fe2+ g-1 root FW h-1)
Fe-LS1 2044 ± 385 a 263 ± 97 a 150 ± 52 b
Fe-LS2 1720 ± 218 a 194 ± 65 ab 231 ± 37 b
Fe-LS3 1992 ± 140 a 299 ± 122 a 163 ± 48 b
Fe-LS4 1241 ± 101 ab 55 ± 14 c 239 ± 63 b
Fe-EDDHA 1120 ± 147 b 147 ± 59 b 634 ± 107 a
Data are means ± SD (four replicates for the uptake and translocation experiment, three replicates in the case of the Fe(III)-
reduction assay). Different letters within the same line denote significant differences among the treatments (P<0.05).
3.3 Uptake and translocation of foliar applied 59Fe-LS complexes in intact Fe-
deficient tomato and cucumber plants
140
Table 5: Uptake and translocation of foliarly applied 59Fe-LS or 59Fe-EDTA in Fe-deficient tomato plants measured 7-days after leaf application.
Values in parentheses represent percentage of 59Fe translocated to the untreated part of the plants. Values of 59Fe distributed into untreated different
plant tissues (expressed as percentage 59Fe translocation) are also reported.
Distribution of 59Fe in different untreated plant tissues (%)
Uptake Translocation
the Fe-source.
Untreated leaf
(nmol 59Fe g-1 treated-leaf DW ) Old leaves New leaves Stem Root
section
Fe-LS1 35 ± 6 c 6 ± 1 c (18) 9.4 ± 1.4 b 3.9 ± 0.6 a 55.6 ± 8.9 a 16.1 ± 2.8 a 15.0 ± 2.4 ab
Fe-LS2 53 ± 20 bc 11 ± 2 b (21) 9.5 ± 1.7 b 3.3 ± 0.4 a 67.7 ± 11.2 a 8.1 ± 1.2 b 11.4 ± 2.0 b
Fe-LS3 106 ± 49 ab 14 ± 2 b (13) 15.4 ± 2.2 a 3.8 ± 0.5 a 58.5 ± 8.2 a 9.2 ± 1.3 b 13.1 ±1.8 ab
Fe-LS4 83 ± 26 b 10 ± 2 b (12) 20.8 ± 4.1 a 3.3 ± 0.5 a 48.4 ± 10.1 a 10.0 ± 1.9 b 17.5 ± 3.5 a
Fe-EDTA 254 ± 90 a 59 ± 7 a (25) 16.0 ± 1.7 a 4.0 ± 0.4 a 54.4 ± 5.9 a 10.0 ± 0.9 b 15.6 ± 1.7 a
Data are means ± SD of three replicates. Different letters within the same line denote significant differences among the treatments (P < 0.05).
Table 6: Uptake and translocation of foliar applied 59Fe-LSs or 59Fe-EDTA in Fe-deficient cucumber plants measured 7-days
after leaf application. Values in parentheses represent percentage of 59Fe translocated to the untreated part of the plants. Values
of 59Fe distributed into untreated different plant tissues (expressed as percentage 59Fe translocation) are also reported.
Uptake Translocation Distribution of 59Fe in different untreated plant tissues (%)
(nmol 59Fe g-1 treated-leaf DW ) Old leaves New leaves Stem Root
Fe-LS1 151 ± 68 ab 9 ± 2 b (6) 5.0 ± 0.7 b 30.0 ± 4.1 c 46.6 ± 7.9 a 18.4 ± 2.2 c
Fe-LS2 214 ± 26 a 9 ± 1 b (4) 7.6 ± 0.8 a 54.9 ± 5.9 a 10.1 ± 0.9 c 27.4 ± 3.0 b
Fe-LS3 146 ± 30 ab 6 ± 2 bc (4) 4.9 ± 0.9 b 40.1 ± 7.8 bc 17.4 ± 2.9 b 37.6 ± 5.4 ab
Fe-LS4 105 ± 15 b 4 ± 1 c (4) 5.1 ± 1.1 b 37.4 ± 7.4 bc 22.6 ± 4.1 b 34.9 ± 4.9 b
Fe-EDTA 232 ± 61 a 21 ± 4 a (9) 3.3 ± 0.6 c 41.1 ± 6.8 b 5.4 ± 1.0 d 50.2 ± 9.5 a
Data are means ± SD of three replicates. Different letters within the same line denote significant differences among the
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the untreated plant tissues were recorded, particularly when Fe-LS were used. With the
exception of the oldest leaves, Table 6 shows that the micronutrient was distributed
essentially in similar amount inside the different untreated plant tissues, irrespective of
Table 7 shows that Fe(III)-LS complexes could be reduced by tomato leaf discs at
levels comparable or even higher than those observed using Fe(III)-EDTA. Leaf discs of
cucumber plants also exhibited Fe(III)-reducing capacity, but at very low levels especially
when Fe-LS complexes were used.
4. DISCUSSION
In the present work we tested lignosulfonates (LS) of different origins with respect
to their capacity to provide complexed Fe to plant roots or leaves. In particular, two LS
were obtained through sulfite treatment of hardwood (eucalyptus; LS1) and softwood
(spruce; LS4) sources. By means of industrial transformations of LS1, new
lignosulfonates enriched in functional groups able to complex Fe were obtained (LS2
and LS3).
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CAPÍTULO V.2
Data presented here show that LS possess a limited capacity to mobilize Fe from
barely soluble Fe-hydroxide (Table 2). Since this experiment was run at pH 7.5, which is
closed to that encountered in calcareous soils, it might be concluded that LS are not
particularly efficient in complexing endogenous Fe when added to this kind of soils. This
idea is further supported by the low mobility of the pre-formed Fe-LS complexes along a
soil column (Table 3 and Figure 1), as well as by the low solubilization by LS of
endogenous Fe from Eutric cambisol soil samples (data not shown). These data are
consistent with known low stability of Fe-LS complexes in alkaline solution (Goos et al.
2001). Furthermore, in a separate experiment (unpublished data) we could demonstrate
that after adding Fe-LS to a 1 mM CaCl2 solution buffered at pH 6.5, about 80-85 % of
Fe remained in solution while the percentage of soluble Fe strongly decreased when the
pH of the solution increased above 6.5. This behavior appears to be different from that
of other natural Fe-sources like e.g. Fe complexed to a water-extractable humic fraction
(Cesco et al., 2000). The low mobility of the Fe-LS complexes could be also ascribed to
factors like the binding of the complexes to clay and organic particles of the soil as
observed for the siderophores ferrioxamine B (Powel et al., 1980). Moreover, in a
separate experiment (unpublished results) we also observed that Fe-LS complexes could
interact with solid soil components like ferrihydrite and peat. Notwithstanding the
general low capacity to mobilize Fe and limited mobility of the Fe-LS complexes,
differences among the LS fractions tested were observed with LS2 being the most
efficient. Moreover, our data show that, under the experimental conditions used in the
present work, the modification of the original LS1, performed in order to increase the
acidity of the products, only ameliorates the performance of the original product in the
case of LS2.
Although data reported above indicate that LS and their Fe complexes might play a
minor role in providing Fe to plants in the soil, when the roots of intact Fe-deficient
tomato plants were put in contact with the different 59Fe-LS complexes in a hydroponic
system buffered at pH 7.5, they could absorb and translocate 59Fe at levels even higher
59
than those observed supplying synthetic Fe-EDDHA (Table 4). The Fe(III)-LS
complexes could be reduced by roots of intact plants indicating that the utilization of
these Fe-sources occurs, at least in part, via a reduction-based mechanism. However,
rates of reduction were significantly lower than those measured with Fe-EDDHA
possibly suggesting that use of Fe complexed to LS might also occur via an indirect
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CAPÍTULO V.2
mechanism involving ligand exchange with plant-borne chelating agents (Yehuda et al.,
1996; Cesco et al., 2002). No significant differences in plant use (uptake and
translocation of complexed Fe) were observed among LS1, LS2 and LS3, while LS4 was
used at the lowest level. These results indicate that the low Fe use efficiency from Fe-LS
observed in soil-applications (Sadiq and Hussain, 1993) is due to interactions of these
Fe-sources with soil colloids rather than to the low capability of roots to use them.
Cucumber leaves were also able to absorb 59Fe from 59Fe-LS at levels similar to
those obtained with 59Fe-EDTA (Table 6). However, 59Fe translocation to untreated
plant tissues was very poor and not clearly directed toward a specific sink. Fe(III)-
reduction by leaf tissues showed lower values when Fe(III)-LS were used as substrate as
compared to Fe(III)-EDTA.
In conclusion, results of the present work clearly show that Fe applied to leaves as
Fe-LS complexes can be absorbed by tomato and cucumber leaf cells via Fe(III)-
reduction-based mechanism and therefore translocated to untreated plant tissues; these
observations suggest that the practice of foliar application with Fe-LS complexes might
be an eco-compatible and cost-friendly Fe source alternative to synthetic chelates,
although showing a lower efficiency and different use among plant species. Fe-LS
complexes might be used also by roots, and the possible use of these natural Fe-
complexes in hydroponic growth system might be taken into consideration. In
calcareous soil conditions it is unlikely that suitable amounts of Fe would reach the
144
CAPÍTULO V.2
roots. Data here presented also show that treatments to increase the amount of
functional groups did not lead to any clear ameliorative effect on the capability of the
products to increase Fe availability for plants. Nevertheless, indicate that in general
eucalyptus LS presented higher efficiency than those obtained from spruce.
ACKNOWLEDGEMENTS
Work funded by the Italian Ministry for University Education and Research (MIUR), the
Spanish Ministry of Education and Science (Projects AGL2004-07849-C02-01/AGR and
AGL2007—63756) and by the DGUI of the Comunidad Autónoma de Madrid and the
Autónoma University of Madrid (Project CCG07-UAM/AMB-1567/07). P. Rodríguez-
Lucena was supported by a Spanish Ministry of Science and Education “FPI” pre-
doctoral contract co-financed by the European Social Fund.
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CAPÍTULO V.2
REFERENCES
Abadia A, Sanz M, de las Rivas J and Abadia J. 2002. Correction of iron chlorosis by
foliar sprays. Acta Hortic 594:115-121.
Aly S S M and Soliman S M. 1998. Impact of some organic acids on correcting iron
chlorosis in two soybean genotypes grown in calcareous soil. Nutr Cycl Agroecosys
51:185-191.
Bienfait H F, Van den Briel W and Mesland-Mul N T. 1985. Free space iron pools
in roots: generation and mobilization. Plant Physiol 78:596-600.
Chen Y. 1996. Organic matter reactions involving micronutrients in soils and their effect
on plants. In Humic substances in Terrestrial Ecosystems. Piccolo A (Ed), 13:507-
529.
Fernández V and Ebert G. 2005. Foliar Iron Fertilization: A Critical Review. J Plant
Nutr. 28:2113-2124.
Fernández V, Río V, Abadía J and Abadía A. 2006. Foliar iron fertilization of peach
(Prunus persica (L.) Batsch): effects of iron compounds, surfactants and other
adjuvants. Plant Soil 289:239-252.
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Goos R J and Germain S. 2001. Solubility of twelve iron fertilizer products in alkaline
soils. Commun Soil Sci Plant Anal. 32:2317-2323.
Jaeger B, Goldbach H and Sommer K. 2000. Release from lime induced iron
chlorosis by CULTAN in fruit trees and its characterization by analysis. Acta Hort.
531:107-113.
Lindsay W L and Schwab A P. 1982. The chemistry of iron in soils and its availability
to plants. J Plant Nutr. 5:821-840.
Lindsay W L. 1991. Iron oxide solubilisation by organic matter and its effect on iron
availability. In: Iron Nutrition and Interaction in Plants. Eds. Y Chen and Y Hadar.
Kluwer Academic Publishers, Dordrecht. pp. 29-36.
Lucena J J. 2006. Synthetic iron chelates to correct iron deficiency in plants. In: Iron
Nutrition in Plants and Rhizospheric Microorganisms. Eds. Barton L.L., Abadía J.
Springer-Verlag Academic Publishers, Dordrecht. pp 103-127.
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Tagliavini M and Rombola A D. 2001. Iron deficiency and chlorosis in orchard and
vineyard ecosystems—review. Eur J Agronomy 15:71–92.
Zancan S, Cesco S and Ghisi R. 2006. Effect of UV-B radiation on iron content and
distribution in maize plants. Environm Exp Bot. 55:266-272.
148
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RESUMEN
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CAPÍTULO V.3
a
Agricultural Chemistry Department, Universidad Autónoma de Madrid, Francisco Tomás y Valiente
Nº 7, 28049 Madrid, Spain
b
Physical and Analytical Chemistry Department, University of Oviedo, Julián Clavería Nº 8, 33006
Oviedo, Spain.
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ABSTRACT
Four lignosulfonates (LS) were tested with regard to their ability to complex Fe. The
main structural characteristics were elucidated by FTIR spectroscopy and their stability at
different pH was studied. Leaf plasma membrane ferric reductase was tested to evaluate
the capability of the lignosulfonates to supply Fe to the plants by using leaf discs of
cucumber (Cucumis sativus L., cv Ashley) chlorotic plants. The ability of Fe-LS to supply
Fe to cucumber chlorotic plants through foliar sprays was evaluated in two biological
experiments using LS/57Fe3+ in comparison with EDTA/57Fe3+. The first one was
shorter and with one foliar application and in the second two foliar applications were
done. Lignosulfonates were effective to complex Fe and to keep it in solution until pH
7.5. Fe3+ reduction rates by the ferric reductase enzime were higher for LS/Fe3+ than for
EDTA/Fe3+. However, plant experiments indicated that LS complexes were less
efficient than EDTA/57Fe3+, but could provide Fe to cucumber plants though foliar
sprays, and their effectiveness rose when more than one application was performed.
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CAPÍTULO V.3
1. INTRODUCTION
Iron (Fe) chlorosis is a nutritional disorder that induces intervenial leaf yellowing
which can be overcome by application of certain Fe compounds (Carpena, 1966). It is a
widespread agricultural problem, especially in crops grown on calcareous soils, where
calcium carbonate buffers soil solution pH in the range of 7.5-8.5 (Lindsay and Schwab,
1982) and high bicarbonate concentration is present (Lucena, 2000).
Fe-efficient plants have developed two different strategies to assimilate Fe when its
availability is low. Strategy I plants (dicots and nongrass monocots) may increase a
plasma membrane-bound Fe3+ reductase (FC-R) that reduces extracellular chelates/ Fe3+
to Fe2+ (Chaney et al., 1972; Bienfait, 1985). The Fe2+ can be transported into the roots
by a plasma membrane transporter (Fox et al., 1996). Inside the root cells, Fe2+ is
oxidized to Fe3+ and transported as citrate/Fe3+, via xylem, to the leaves (López-Millán et
al., 2009). Subsequently, it a second Fe3+ reduction step is needed for the uptake of
apoplastic Fe2+ by leaf mesophyll cells, that it is mediated by a leaf plasma membrane
ferric reductase (Kim and Guerinot, 2007).
Lignosulfonates (LS) are pulp and paper industrial by-products obtained during the
sulfite pulping process, when original lignin is broken down and fragments are made
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CAPÍTULO V.3
For years the absorption and translocation of Fe in the plant has been studied
using different approaches. Many authors suggest the use of radioactive isotopes as 59Fe
(Cesco et al., 2002). However, the use of this isotope requires specific laboratory
facilities, trained personal and does not allow long-term trials because the isotope activity
drops over time. Mössbauer spectroscopy using 57Fe has also been used in the study of
the Fe chemistry in plants (Kóvacs et al., 2005), but the concentration of Fe in plant
tissues is usually too low to be detected by this technique. As an alternative, ICP-MS
(Inductively Coupled Plasma Mass Spectrometry) can be used for studying the 57Fe in
plant tissues using isotope pattern deconvolution (Rodríguez-Castrillón et al., 2008). The
main advantage of this technique lies in the high precision and the low detection limits.
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CAPÍTULO V.3
Moreover, since 57Fe is a stable isotope, no special requirements are necessary to handle
with it and long-term experiments can be performed.
Although LS have a good ability to complex Fe, to our knowledge most of the
studies evaluating their ability to solve Fe chlorosis are referred to commercial
complexes and do not establish any relationship between the characteristics of the LS
and their effectiveness. In the present work four LS, differing in their physical and
chemical characteristics, were compared with regard to their ability to complex Fe. The
chemical and structural characteristics of the LS and Fe-LS were determined, and the
stability of the Fe-LS at different pH and in the presence of Ca2+ was also evaluated. In
order to test if Fe-LS complexes can be used by leaves to overcome Fe chlorosis, the
activity of leaf plasma membrane ferric reductase was studied in cucumber leaf discs.
Then two biological experiments were performed to test the ability of LS/57Fe3+ to
provide Fe to mildly Fe deficient cucumber plants grown in hydroponics when applied
through foliar sprays. In the first assay, only one application was performed to evaluate
the uptake and redistribution of 57Fe occurred as well as the efficiency of the treatments.
The effect of the number of foliar applications in the effectiveness of the LS/57Fe3+ was
studied in the second experiment.
2.1 Reagents
All reagents used to study Fe complexing ability and determine soluble and
complexed Fe [H2O2, HCl and NaOH (PA, Panreac)], to form the Fe complexes
[FeSO47H2O and FeCl36H2O (PA, Panreac)], to study the effect of pH and Ca2+ on
soluble Fe [CaCl2 (PA, Panreac), and HEPES, MES, CAPS and AMPSO (Merck)], to
measure the leaf plasma membrane reductase activity [CaSO4, KCl (PA, Panreac),
Na2BPDS (Fluka) and Na2EDTA (Tritiplex III, Merck)], to grow plants in the biological
experiments [Ca(NO3)2, KNO3, MgSO4, KH2PO4, MnSO4, CuSO4, ZnSO4, CoSO4,
NiCl2, NaCl, H3BO3, Na2MoO4, CaCO3 (PA, Panreac)] and in the digestion of plant
material [(HNO3, H2O2 and HF (Suprapur, Merck)] were of recognized analytical grade.
The water used for the preparation of reagents or standards conforms to EN ISO
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CAPÍTULO V.3
3696:1987, grade I, free of organic contaminants (Millipore, Milford, USA). 57Fe was
obtained from Isoflex (San Francisco, CA, USA) as elemental powder (metal form, 95.38
% isotopic enrichment). EDDHA/Fe3+ (Torneo ®) used to grow Fe sufficient plants in
the leaf plasma membrane reductase activity was obtained from Bayer Hispania, S.A.
al. (2007), fEuropean Official method for Fertilizers (methods 9.2 and 9.4 EC 2003/2003
regulation).
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CAPÍTULO V.3
2.3 Fe Complexing ability of the LS and formation of the Fe-LS complexes and
Fe-chelates
The LS and Fe-LS samples were analyzed by Fourier Transform Infrared (FTIR)
spectroscopy in order to determine the main structural characteristics of the complexing
agents and complexes. Moreover, the influence of the source of Fe (Fe2+, as FeSO4, or
Fe3+, as FeCl3) in the structural characteristics of the compounds was also evaluated.
After freeze-drying of the LS and the Fe-LS complexes, FTIR analyses were performed.
FTIR spectra were obtained on a FTIR Bruker IFS60v spectrophotometer with a MTC
detector and diffuse reflectance (DRIFT) accessory. Spectra of the samples were
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CAPÍTULO V.3
recorded in the region 4000-400 cm-1 at a resolution of 4 cm-1 using the potassium
bromide pellet method, in the transmittance mode. Fe-LS complexes were prepared by
mixing LS with FeSO4 or FeCl3, the LS:Fe ratio being 1.1:1.0.
LS/Fe2+ were allowed to react in a Ca2+ solution at various pH to study the stability
of the complexes. Five milliliters of LS/Fe2+ solutions were added to 15 mL of 10-2 M
buffer solution (HEPES, MES, CAPS, AMPSO) at different pH (4.0, 5.0, 6.0, 7.0, 7.5,
8.0, 8.5, 9.0, 9.5, 10.0 and 11.0) and 5 ml of 5.010-2 M CaCl2 solution. Samples were
shaken for three days at 25ºC and 56 min-1. Then, the solutions were filtered through
0.45 µm Millipore membranes and final pH was measured with an Orion Research ion
analyzer (EA920). Fe concentration in the filtrate was determined by Atomic Absorption
Spectrometry (AAS, Perkin-Elmer Aanalyst 800) after removal of the organic
compounds (EU Directive 2003/2003, methods 9.3, Official Journal of the European
Union, 21-11-2003, L 304/1). Three replicates per pH were performed.
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CAPÍTULO V.3
solution in a Dycometal type CCK growth chamber provided with fluorescent and
sodium vapour lamps with a 16 h, 30 °C and 50% humidity day and 8 h, 25 °C and 70%
humidity night regime. Water was added every 2 days, and the nutrient solution was
renewed every 7 days. The four LS/Fe3+ and the EDTA/Fe3+ were tested as substrates
for leaf plasma membrane reductase in Fe deficient plants (grown with 5.010-6 M
EDTA/Fe3+). In order to compare the influence of the nutritional status of plants in the
reduction of Fe3+, an additional treatment with EDTA/Fe3+ as substrate and sufficient
Fe supply (5.010-5 M EDDHA/Fe3+) through roots was tested. Blanks without discs but
with the complex or the chelate were also studied. Leaf discs (5 mm diameter) were
punched by a cork borer from the leaf area without main veins of the third and fourth
levels of young leaves of Fe-deficient 25-d-old cucumber plants. Samples containing 40
leaf discs were washed twice for 10 min in 5 mL of washing solution (De la Guardia and
Alcántara, 1996; Nikolic and Römheld, 1999) (5.010-4 M CaSO4, 1.010-2 M MES-KOH
(pH 6.0) and 1.010-3 M KCl). Then the solution was replaced by the 5 mL of the assay
solution (5.010-4 M CaSO4, 1.010-2 M MES-KOH (pH 6.0), 1.010-3 M KCl, 3.010-4 M
BPDS, and 1.010-4 M LS/Fe3+ or EDTA/Fe3+). After vacuum infiltration (32 kPa)
during 20 minutes (2 periods of 10 minutes, after each period the solution was replaced)
leaf discs were incubated for 60 min in the assay solution in continuous-orbital shaking
(125 rpm) in light at 25°C. Reduction rates were determined as formed BPDS3/Fe2+ by
measuring the absorbance at 535 nm against blanks (without leaf discs) with an UV-
Visible Recording Spectrophotometer (UV-160A, Shimadzu) and using extinction
coefficient of 22.14 mM-1 cm-1 (Fox et al., 1996).
Two experiments were carried out to test the ability of the LS/57Fe3+ complexes to
provide Fe to cucumber, which is a Fe-efficient strategy I model plant, through foliar
sprays.
In both experiments the seeds were germinated as described in chapter 6.1. After
germination uniform seedlings were selected and the stem of two plants were wrapped
together with foam and placed in 2 L polyethylene vessels (three holes in the lid, six
plants per pot). The vessels contained 2 L of continuously aerated EDTA buffered
nutrient solution with the same composition than that previously mentioned in chapter
6.1. The pH was buffered at 7.5 with 1.010-4 M HEPES and 0.2 g L-1 of CaCO3 per pot
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CAPÍTULO V.3
were added to simulate the conditions of calcareous soils. Plants were grown in a growth
chamber with controlled climatic conditions: photoperiod (day/night) 16/8 h;
temperature (day/night) 30/25 ºC; R.H. (day/night) 50/70 %. Water was added every 2
days and the nutrient solution was renewed every 7 days.
After 16 days of growth under these conditions chlorotic symptoms appeared and
treatments were applied.
2.6.2.1 Effectiveness of LS/57Fe3+ as foliar source of Fe for cucumber stressed plants. The aim of this
experiment was to evaluate Fe uptake when LS/57Fe3+ complexes were applied through
foliar sprays. One control with EDTA/57Fe3+ was fixed. All the plants (three replicates
per treatment, one pair of plants per replicate) were sprayed with the treatments at the
beginning of the assay and whole plants were sampled after eleven days. Each pair of
plants was sprayed at the beginning of the assay with 2 mL of the products at 5.010-3 M
Fe concentration, by using a nebulizer system. The dose of each treatment was calculated
according to the soluble Fe content of the LS (Table 1). For the EDTA/57Fe3+ plants
were sprayed with 2 mL of 5.010-3 M EDTA/57Fe3+. In order to evaluate the
redistribution of 57Fe to non treated organs only the first four levels of leaves were
treated. Contamination of non treated leaves was avoided by covering them with a
plastic sheet during the application of the foliar sprays. Leaf sprays were applied both on
the adaxial and abaxial leaf surface. All the solutions were adjusted to pH 5.0 to avoid
altering the ion exchange properties of the cuticle. Since LS behave as surface active
agents, surfactants were not included in the foliar solutions. For the treatment with
EDTA/57Fe3+ the non-ionic surfactant Tween 80 (PROBUS) was used at 0.1 % (v/v)
rate. During the experiments, SPAD readings were taken with a chlorophyll meter
(Minolta SPAD-502) for all the leaf stages (average of three readings per leaf). After
sampling plant nutritional status and 57Fe redistribution were studied. The sampled roots,
stems, treated leaves and non treated leaves were separated, weighted and washed with
0.1 % HCl and 0.01 % non-ionic detergent (Tween 80) solution, and rinsed twice with
ultrapure water. Then, samples were dried in a forced air oven at 65 ºC for three days.
Micronutrients were determined in roots, stems, treated leaves and non treated leaves
after microwave (CEM Corporation MARS 240/50) digestion with HNO3 65 %, H2O2
30 % and HF 40 % by AAS. 57Fe quantification was carried out by isotope pattern
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CAPÍTULO V.3
2.6.2.2 Effect of the number of applications. In this assay, only the four LS complexes were
studied (LS/57Fe3+). To evaluate the influence of the number of applications performed
in the effectiveness of the products, all the plants (three replicates per treatment, one
pair of plants per replicate) were sprayed with the LS/57Fe3+ complexes at the beginning
of the experiment while only half of them received a second application (11 days after
since the first application). One control without foliar application of Fe (control –Fe)
was fixed. Whole plants were sampled 21 days after the first application of the
treatments. The same procedures described in chapter 6.2.1. for the preparation and
application of the treatments, SPAD measurements, sampling of plant material (roots,
stems, treated leaves, non treated leaves and flowers), preparation and digestion of
samples, and measurement of micronutrients and 57Fe were followed in this experiment.
Data were statistically evaluated using Analysis of Variance (ANOVA) with the
program SPSS 15.0 to asses the significance of the main factors and interactions. Means
were also compared using Duncan’s test at P ≤ 0.05 in order to find significant
differences between treatments.
Figure 1 presents the measured versus the added element when the complexing
ability was studied. The curves obtained agree with those described by Villén et al.
(2007), having a rising segment that corresponds with the complexing process, followed
by another decreasing segment that implies the coagulation of the material and/or the
precipitation of the excess of Fe. In Table 1 the amount of Fe complexed by each LS,
obtained from the intersection point of the two lines, is showed. Once the complexing
ability was elucidated, FeLS complexes were formed. The percentage of complexed Fe
with regard to soluble Fe was always above 90% (Table 1). For all the LS the amount of
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CAPÍTULO V.3
100
LS1
80
20
0
0 20 40 60 80 100
100
LS2
80
20
0
0 20 40 60 80 100
100
LS3
80
y = 0,98x + 0,15 y = -2,30x + 232,05
60 2 2
R = 1,00 R = 0,97
40
20
0
0 20 40 60 80 100
100
LS4
80 y = 0,94x - 0,82
2
R = 0,99
60
40 y = -2,31x + 298,65
2
R = 0,94
20
0
0 20 40 60 80 100
-1
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CAPÍTULO V.3
metal complexed and the percentage of complexed Fe with regard to soluble Fe were in
the range established by the Spanish Regulations (REAL DECRETO 824/2005), so they
can be commercialized as complexes.
Between the non modified LS tested (LS1 and LS4, derived from eucalyptus and
spruce respectively) the best complexing ability corresponded to LS4. A similar behavior
has been observed by Martín-Ortiz et al. (2009) when the Zn complexing ability of an
eucalyptus and a spruce LS was compared. The modifications performed on LS1
(incorporation of carboxylic (LS2) and sulfonic (LS3) groups to its structure) increased
the complexing ability of LS2 and LS3 until levels above to those recorded LS4. A
similar behavior was observed previously by other authors (Gonçalves and Benar, 2001;
Pang et al., 2008).
The main FTIR bands observed are in agreement with those reported in other
works for lignins, technical lignins and LS (Boeriu et al., 2004; El Mansouri and Salvadó,
2007). The FTIR spectra are shown in Figure 2. All the LS follow a common pattern,
with a broad band 3600-3300 cm-1 corresponding hydroxyl groups in phenolic and
carboxylic acids, less intense bands in the 3000-2800 cm-1 region of the CH stretching in
methyl and methylene, attributed to methoxyl groups, and several bands with variable
intensity in the fingerprint region (1900 to 800 cm-1). In this region the main features
appear at 1770 cm-1 (aromatic acetoxy groups), 1715 and 1630 cm-1 (unconjugated
carbonyl-carboxyl stretching), 1600 to 1500 cm-1 (C=C skeletal vibrations), 1470-1460
cm-1 (C-H deformation combined with aromatic ring vibrations), 1260 cm-1 (C=O
stretch), 1230-1215 cm-1 (C-C, C-O and C=O stretching), 1140 cm-1 (C-H in plane
deformation), 1050 cm-1 (complex vibration associated with the C-O, C-C stretching and
C-OH bending in polysaccharides) and 840 and 810 cm-1 (C-H out-of-plane
deformations). The band appearing at 620-660 cm-1 is assigned to the sulfonic groups (S-
O stretching vibration) formed from the reaction of sodium sulfite with the secondary
OH of the aliphatic side chain of lignins.
The intensity of several bands varies after the formation of the Fe complexes,
indicating that structural changes occur in the molecule due to the addition of the metal..
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CAPÍTULO V.3
A
LS1
B
C
B LS2
C
A
LS3
B
C
A
B LS4
Wavenumber (cm-
Figure 2: FTIR spectra of the lignosulfonates and Fe-LS complexes (A-LS, B-LS/Fe2+
complex, C-LS/Fe3+ complex).
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CAPÍTULO V.3
The bands at 2940, 2830, 1715, 1470, 1500 and 1050 cm-1 were always less intense after
the complexation of Fe (as Fe2+ or Fe3+). The lower intensity of these bands, associated
with CH, carbonyl and carboxyl stretching, CH deformation, aromatic ring vibrations,
C=C aromatic skeletal vibrations and the vibrations of C-O, C-C and C-OH in
polysaccharides, may be related to the formation of Fe phenolates and carboxylates. The
vibration associated to the S-O stretching is also less intense after the addition of Fe,
especially for Fe3+. Some bands exhibit a higher intensity after complexation, though in
this case this behavior is very dependent on the source of Fe added to the LS. An
important increment is observed at 1630 cm-1 when Fe3+ is added that would be ascribed
to a major intensity of the unconjugated carbonyl-carboxyl stretching, maybe due to the
inclusion of the metal in these structures. This new feature might indicate a higher
affinity of these groups for Fe3+ instead of Fe2+. In the case of Fe2+, relevant increases in
intensity appeared at 1150-1140 and 810 cm-1, associated to CH deformations (in plane
and out of the plane, respectively) that would be favored on the presence of divalent Fe.
In general, hardwood (LS1, LS2 and LS3) and softwood (LS4) LS present a similar
pattern, irrespectively of their different origins and characteristics. Various bands have a
higher intensity in the case of LS4 (3600-3300, 1500 and 1140 cm-1), while some bands
are much less intense (2940 and 2830 cm-1). Other features barely detected in the
eucalyptus LS are more pronounced in this LS (the presence of aromatic acetoxy groups
at 1770 cm-1 and C=O stretch at 1260 cm-1),
The modifications on LS1 to form LS2 and LS3 are reflected in the spectra at
specific bands. The intensity of the bands at 2940, 1630 and 1230-1215 cm-1 is higher for
LS2 and LS3 with regard to LS1, indicating major stretching of CH, carbonyl, carboxyl,
C-C,C-O and C=O groups in the modified LS. This confirms the enrichment of the
molecule with groups capable to complex Fe and is in accordance with the complexing
ability obtained for LS2 and LS3 (Table 1). The highest presence of sulfonic groups in
LS3 is not reflected in the spectra neither of the LS nor of the complexes, supporting
their negligible influence on the process of Fe complexation as observed when the
complexing ability was studied.
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CAPÍTULO V.3
% Fe remaining in solution LS 1 LS 2 LS 3 LS 4
100
80
60
40
20
0
3 4 5 6 7 8 9 10 11
pH
pH. For all the complexes nearly 100 % of Fe remains in solution until pH 7.5. In the
case of hardwood complexes (those formed with LS1, LS2 and LS3) precipitation of Fe
begins around pH 8, being the percentage of soluble Fe negligible at pH above 9. LS4
(spruce LS) was the only compound capable to maintain significant amounts of soluble
Fe above pH 8. The best behavior of the LS4 complexes suggests that the number of
strong complexing sites is higher than in the other LS. Since LS4 has the lowest
percentage of carboxylic groups and an intermediate percentage of sulfonic groups, this
may indicate that although these groups promote Fe complexation (Table 1), they
behave like weak complexing sites. Studying Ca2+ complexation by different modified
LS, Pang et al. (2008) concluded that sulfonic groups had little influence on the
complexing process, while the complexing ability was lower when the polymer was
enriched with carboxylic groups. Our results indicated that both groups promoted
complexation (LS2 and LS3), though the complexes formed were less stable than those
formed by LS4 under calcareous soils conditions. Thus, other functional groups capable
to complex metals must be implicated in the formation of stable complexes by LS4.
According to Pang et al. (2008), this better behavior can be ascribed to the presence of
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CAPÍTULO V.3
Although the LS tested showed a good ability to maintain Fe in solution in the range
of alkaline pH and in the presence of Ca2+, it has been demonstrated that the feasibility
of these products to maintain Fe in a soluble form when interacting with soils is scarce
(Goos and Germain, 2001; Rodríguez-Lucena et al., 2009). However, a good
performance of the Fe-LS complexes is expected when applied in hydroponics systems
or as foliar sprays, as it has already been observed in previous works with LS/59Fe3+
complexes (Rodríguez-Lucena et al., 2009).
The ability of leaf discs of cucumber Fe deficient plants to reduce LS/Fe3+ was
studied (Figure 4). The highest reduction rate corresponded to LS3 complexes, followed
by LS1 and LS4. When the non modified eucalyptus LS (LS1) and the spruce one (LS4)
100 A
-1
g FW h
75
B
-1
2+
50 B
nmol Fe
25
C
C
C
0
LS1/Fe3+ LS2/Fe3+ LS3/Fe3+ LS4/Fe3+ EDTA/Fe3+ EDTA/Fe3+
I II
Figure 4: Reduction of 110-4 mol FeL-1 as LS/Fe3+ and EDTA/Fe3+ in light by leaf discs of Fe
deficient cucumber plants. For reduction of EDTA/Fe3+ Fe deficient (EDTA/Fe3+ I) and Fe
sufficient (EDTA/Fe3+ II) plants were compared.
Data are means ± SE of five independent replicates. Different letters among bars denote statistical
differences among the treatments at P ≤ 0.05.
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CAPÍTULO V.3
were compared, statistical differences were not found. Since the main difference between
them is their molecular weight (Table 1), so it can be concluded that the molecular size
of the complexing agent is not related with the ability of plants to reduce the Fe
complexed by it. Modifications on LS1 to form LS2 and LS3 offered variable results, as
LS3 and LS2 present the highest and the lowest reduction rates respectively. The
relationship between these modifications and the reduction rate is not clear. Both
modifications gave higher Fe complexing capacity than the LS1, but the three eucalyptus
LS (LS1, LS2 and LS3) had similar stability at different pH, possibly due to the weak
bonds between the metal and the carboxylic and sulfonic groups. This Fe weakly bound
was expected to be a good substrate for the enzyme, but no correlation was found
between them. LS2 had higher pH (pH 6.8) than the others (around 4.0), this fact might
have a great influence on its chemical and physiological behavior, and may explain the
reduction rates found. The swelling of the polymer may have occurred for LS1 and LS3
when their pH was increased until the one used in the leaf Fe reduction experiment (pH
6), so Fe chelating groups would be more external and more available to be reduced by
the enzyme (Pang et al., 2008).
In the case of EDTA/Fe3+, the high photoreduction (Norvell, 1991)) of the blanks
of this chelate are the reason for the negligible (in chlorotic plants) or very low (in Fe
sufficient plants) reduction rates found once the blanks were subtracted, although non
statistical differences were found. The lower reduction in Fe deficient plants with regard
to Fe sufficient ones is associated to the inhibition of leaf Fe3+ reductase activity due to
The effect of the foliar application of LS/57Fe3+ to chlorotic cucumber plants was
evaluated 11 days after the application of the treatments. One-way ANOVA analysis was
used to elucidate if statistical differences due to the treatments appeared in plants dry
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CAPÍTULO V.3
weight, SPAD Index, total Fe and concentration of 57Fe provided by the complex. This
analysis indicated that 57Fe concentration in roots and non treated leaves was strongly
influenced by the application of the foliar sprays (at P ≤ 0.001 0.01 respectively). The
application of the treatments also influenced significantly total Fe concentration in non
treated leaves (at P ≤ 0.05). For the dry weight, SPAD Index and root total Fe
concentration in roots, statistical differences were not observed.
In Table 2 roots and non treated leaves dry weight, SPAD Index, total Fe
concentration and concentration of 57Fe provided by the complex are reported. Fe
concentration (as total Fe and enriched 57Fe) in treated leaves and stems were also
studied, but since it was not possible to ensure that the spray remaining on their surfaces
was completely removed after washing, these data were not taken into consideration in
the interpretation of the results. Statistical differences among the treatments were only
observed for Fe concentration (as total Fe and enriched 57Fe) in roots and non treated
leaves, although a clear relationship between both Fe concentrations could not be
elucidated with the data here reported. 57Fe measurements indicated that redistribution
of foliar applied Fe occurred, being mostly directed to growing organs (mostly non
treated leaves), in agreement with the results reported in previous works (Hüve et al.,
57
2003; Rodríguez-Lucena et al. , 2009). The highest redistribution of enriched Fe
occurred for EDTA/57Fe3+. Among LS, translocation of 57Fe was only directed towards
non treated leaves, and non statistical differences among them, were detected. Although
none of the LS was clearly more efficient than the rest, 57Fe measurements indicate that
the Fe applied through foliar sprays could be redistributed to non treated organs, so
further research optimizing the experimental conditions is necessary. On the other hand,
phytotoxicity commonly associated to leaf damage due the use of surfactants (Fernández
and Eichert, 2009), was not observed for LS, while burn signs ascribed to the surface
active agent could be appreciated in the plants treated with EDTA/57Fe3+.
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Table 2: Effect of the foliar application of the LS/57Fe3+ and EDTA/57Fe3+ on cucumber plants dry weight (DW), SPAD Index, total Fe and 57Fe concentration supplied by
the complex when one application of LS/Fe3+ complexes was performed (plants were sampled 11 days after the application of the treatments).
Dry weight [Fe] [57Fe] supplied as LS/57Fe3+
SPAD
(g plant-1) (µg g-1 DW) (µg g-1 DW)
Non treated Non treated Non treated Non treated
Root Root Root
leaves leaves leaves leaves
LS1/57Fe3+ 0.31 ± 0.05 ns 0.47 ± 0.15 ns 29.2 ± 2.3 ns 57.9 ± 3.4 ab 81.9 ± 5.8 a 0.5 ± 0.2 b
LS2/57Fe3+ 0.45 ± 0.06 0.52 ± 0.09 27.7 ± 0.2 47.0 ± 2.6 b 40.0 ± 5.4 b 0.2 ± 0.1 b
0.0 b
LS3/57Fe3+ 0.37 ± 0.07 0.55 ± 0.19 30.0 ± 2.3 56.3 ± 3.7 ab 40.6 ± 3.5 b 0.4 ± 0.1 b
LS4/57Fe3+ 0.33 ± 0.02 0.53 ± 0.14 30.4 ± 1.8 68.9 ± 7.4 a 42.1 ± 3.9 b 0.2 ± 0.1 b
EDTA/57Fe3+ 0.36 ± 0.09 0.55 ± 0.15 30.5 ± 0.8 59.7 ± 2.0 ab 40.9 ± 8.7 b 1.7 ± 0.1 a 8.8 ± 2.7 a
Data are means ± SE of three replicates and were analyzed using one-way ANOVA and means were compared with a post-hoc Duncan’s contrast. Different letters within
the same column denote significant differences among the treatments (P ≤ 0.05).
ns: non significant.
CAPÍTULO V.3
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CAPÍTULO V.3
57
Dry weight, total Fe concentration and concentration of Fe supplied by the
complex in roots, flowers and non treated leaves, and SPAD values are reported in Table
3. Redistribution of 57Fe was directed to growing organs (flowers and non treated leaves).
57
Fe measurements corresponding to the plants sprayed twice indicate that two
57
applications favored Fe uptake and redistribution. When only one application was
performed, 57Fe was directed to non treated leaves and flowers, while if the application
57 57
was repeated Fe was also detected in roots. These results indicate that Fe was
translocated to the organs with the highest demand of Fe (flowers at the phenologic
stage of the plants used), but if the application was repeated and more Fe was available,
this translocation was also focused in other organs demanding Fe, and agree with those
obtained in the assay described above (chapter 4.2.1) and with those described in
previous works (Hüve, 2003; Rodríguez-Lucena et al, 2009) for Fe redistribution. On the
other hand, the highest effectiveness of the repeated sprays indicated that saturation of
the routes of penetration of Fe due to the possible precipitation of sprayed Fe did not
occur, even when the number of applications was increased. SPAD values indicate that
the application of the treatments promoted leaf re-greening, which was more favored
when the foliar sprays were applied twice.
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Table 3: Effect of the foliar application of the LS/57Fe3+ on cucumber plants dry weight (DW), SPAD Index, total Fe concentration and 57Fe concentration from 57Fe supplied by the
complex in the experiment comparing the number of applications (plants were sampled 21 days after the first application of the treatments).
Dry weight [57Fe] supplied as LS/57Fe3+
SPAD [Fe] (µg g-1 DW)
(g plant-1) (µg • g-1 DW)
Non treated Non treated Non treated Non treated
Root Root leaves Flowers Root Flowers
leaves leaves leaves
1 application
LS1/57Fe3+ 0.67 ± 0.08 ns 1.43 ± 0.26 ns 24.5 ± 3.0 ab 68.2 ± 0.3 abcd 33.1 ± 0.6 a 53.2 ±2.4 c 0.18 ± 0.01 abc 0.13 ± 0.01 b
LS2/57Fe3+ 0.73 ± 0.17 1.09 ± 0.16 21.0 ± 1.9 ab 62.9 ± 6.1 bcd 28.9 ± 2.3 ab 84.2 ±0.5 a 0.09 ± 0.04 bc 0.15 ± 0.01 b
0.00 c
LS3/57Fe3+ 0.75 ± 0.10 1.22 ± 0.44 21.3 ± 1.5 ab 83.3 ±15.6 ab 26.6 ± 1.99 ab 74.7 ± 6.9 ab 0.13 ± 0.03 abc 0.24 ± 0.02 b
LS4/57Fe3+ 0.52 ± 0.08 1.13 ± 0.21 19.5 ± 0.8 bc 61.9 ± 2.9 bcd 34.2 ± 3.1 a 79.8±6.5 ab 0.07 ± 0.02 c 0.16 ± 0.03 b
2 applications
LS1/57Fe3+ 0.62 ± 0.12 1.14 ± 0.29 19.3 ± 5.0 bc 71.2 ± 8.5 abc 32.4 ± 4.8 a 53.2± 3.8 c 0.02 ± 0.01 bc 0.21 ± 0.05 a 0.21 ± 0.03 b
LS2/57Fe3+ 0.82 ± 0.02 1.35 ± 0.19 21.6 ± 2.2 ab 53.8 ± 6.1 cd 22.5 ± 2.9 ab 63.0 ± 3.1 bc 0.04 ± 0.01 ab 0.18 ± 0.01 abc 0.26 ± 0.01 b
LS3/57Fe3+ 0.73 ± 0.15 1.27 ± 0.23 26.2 ± 0.8 a 45.6 ± 6.1 d 30.7 ± 6.9 ab 49.3 ± 1.7 c 0.06 ± 0.01 a 0. 16± 0.01 abc 0.20 ± 0.01 b
LS4/57Fe3+ 0.75 ± 0.19 1.63 ± 0.36 24.9 ± 0.9 ab 89.7 ± 2.4 a 23.5 ± 5 ab 51.3± 3.2 c 0.05 ± 0.03 ab 0.20 ± 0.04 ab 0.49 ± 0.10 a
Control -Fe 0.65 ± 0.21 1.02 ± 0.35 15.3 ± 0.9 c 60.3 ± 3.9 bcd 20.2 ± 0.7 b 77.6 ± 4.9 ab
Data are means ± SE of three replicates and were analyzed using one-way ANOVA and means were compared with a post-hoc Duncan’s contrast. Different letters within the same column
denote significant differences among the treatments (P ≤ 0.05).
ns: non significant.
CAPÍTULO V.3
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CAPÍTULO V.3
Regarding the differences among LS, none of the complexes was clearly more
efficient than the rest. According to SPAD values and enriched 57Fe concentration in
flowers, LS3/57Fe3+ and LS4/57Fe3+ were slightly more effective than the rest, but solely
when two applications were performed. On the other hand, leaves did not exhibit
toxicity signs related to the damage of leaf surface (Fernández and Eichert, 2009), even if
the number of applications was increased. Taken together, the results of the biological
experiments confirm that LS/Fe3+ were complexes capable to provide Fe to cucumber
deficient plants through foliar sprays, especially when the applications were repeated
periodically. The modifications in the initial eucalyptus LS (LS1) improved the
performance of this type of LS when enriched with carboxylic and sulfonic groups (LS).
In any case, this efficiency was similar to that observed for the spruce non modified LS
(LS4). Moreover, since LS exhibit surfactant properties toxicity signs commonly
associated to the use of surface (Fernández and Eichert, 2009) active agents are not
observed.
All the LS tested in this work present a good ability to complex Fe and high
stability in solution under calcareous soil conditions. Since in previous works it was
observed that these complexes were scarcely stable in interaction with soils irrigated with
a 7.5 pH buffered nutrient solution (Rodríguez-Lucena et al, 2009) their use in
hydroponic systems should be considered. Chemical modifications on original LS1
increasing carboxylic and sulfonic group concentration were useful to increase Fe
complexing ability of LS2 and LS3 until the levels obtained for LS4. When applied
through foliar sprays they are capable to provide Fe to cucumber mildly chlorotic plants,
although in a lesser extent than EDTA/Fe3+, redistribution of Fe being favored when
foliar applications were repeated. Eucalyptus modified LS3 (enriched with carboxylic and
sulfonic groups) behaved similarly to the spruce one (LS4), while LS1 and LS2 were less
efficient. Moreover, given that LS exhibit surfactant properties the use of surface active
agents which can damage leaf surface and interact with the Fe solutions (Fernández and
Eichert, 2009) would not be necessary. Leaf plasma membrane ferric reductase activity
indicated that Fe supplied by LS/Fe3+ complexes could be incorporated by leaf cells.
FTIR analysis revealed that structural changes due to the source of Fe used occurred in
the LS molecule after Fe complexation and might influence their effectiveness, so more
research to elucidate the structural characteristics of LS/Fe2+ and LS/Fe3+ complexes is
advisable.
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CAPÍTULO V.3
ACKNOWLEDGMENTS
We thank Lignotech Ibérica S.A. for providing the LS used in this work. This work was
funded by the Spanish Ministry of Science and Innovation (Project AGL2007-63756)
and by the DGUI of the Comunidad Autónoma de Madrid and the Autónoma
University of Madrid (Project CCG07-UAM/AMB-1567 /07). P. Rodríguez-Lucena was
supported by a Spanish Ministry of Science and Innovation “FPI” pre-doctoral contract
co-financed by the European Social Fund.
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CAPÍTULO V.3
REFERENCES
Aly S S M and Soliman S M. 1998. Impact of some organic acids on correcting iron
chlorosis in two soybean genotypes grown in calcareous soil. Nutr Cycl Agroecosys
51:185-191.
Bienfait H F. 1985. Regulated redox processes at the plasmalemma of plant root cells
and their function in iron uptake. J Bioenerg Biomembr. 17:73-83.
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CAPÍTULO V.3
Fernández V and Eichert T. 2009. Uptake of hydrophilic solutes through plant leaves:
current state of knowledge and perspectives of foliar fertilization. Crit Rev Plant
Sci. 28:36-68.
Fernández V, Río V, Abadía J and Abadía A. 2006. Foliar iron fertilization of peach
(Prunus persica (L.) Batsch): effects of iron compounds, surfactants and other
adjuvants. Plant Soil 289:239-252.
Goos R J and Germain S. 2001. Solubility of twelve iron fertilizer products in alkaline
soils. Commun Soil Sci Plant Anal. 32:2317-2323.
ISO 3696:1987. Water for analytical laboratory use. Specification and test methods.
Kim A S and Guerinot M L. 2007. Mining iron: iron uptake and transport in plants.
FEBS Letters. 581:2273-2280.
Lindsay W L and Schwab A P. 1982. The chemistry of iron in soils and its availability
to plants. J Plant Nutr. 5:821-840.
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Lucena J J. 2006. Synthetic iron chelates to correct iron deficiency in plants. In: Iron
Nutrition in Plants and Rhizospheric Microorganisms. Eds. Barton L.L., Abadía J.
Springer-Verlag Academic Publishers, Dordrecht. pp 103-127.
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CAPÍTULO V.3
- 177 -
Capítulo VI:
Discusión General
CAPÍTULO VI Discusión General
DISCUSIÓN GENERAL
En este Capítulo se discuten los resultados más relevantes de los trabajos incluidos
en esta memoria en relación con los objetivos específicos planteados en el Capítulo II, y
que ya fueron discutidos individualmente en los Capítulos III, IV y V. Se incluye, por
último, una discusión de la posible capacidad de los lignosulfonatos para favorecer la
reducción del hierro en los complejos LS/Fe3+. Este fenómeno podría estar relacionado,
al menos parcialmente, con la limitada eficacia de estos complejos como correctores de
la clorosis férrica.
Aunque los quelatos férricos constituyen un medio eficaz para corregir la clorosis
férrica, su elevado coste y escasa degradabilidad ha llevado a buscar productos
alternativos que aplicados al suelo, a la disolución nutritiva o en rociados foliares
permitan corregir este desorden nutricional.
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CAPÍTULO VI Discusión General
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CAPÍTULO VI Discusión General
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CAPÍTULO VI Discusión General
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CAPÍTULO VI Discusión General
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CAPÍTULO VI Discusión General
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CAPÍTULO VI Discusión General
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CAPÍTULO VI Discusión General
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CAPÍTULO VI Discusión General
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CAPÍTULO VI Discusión General
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CAPÍTULO VI Discusión General
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CAPÍTULO VI Discusión General
Los complejos con Cu, Mn y Zn presentaban una buena capacidad para mantener en
metal en una forma disponible, incluso al pH de suelos calizos, pero eran poco estables
en interacción con este tipo de suelos, indicando que el metal podía ser desplazado del
complejo y precipitar o quedar adsorbido en el suelo. En todo caso, a partir de los
resultados obtenidos en estos experimentos no es posible dilucidar que factores han
favorecido la estabilidad de los complejos, ni los fenómenos que han tenido lugar
(desplazamiento del metal en el complejo y precipitación, coagulación del complejo,
adsorción del complejo y/o del metal). Por ello, para determinar los procesos que han
llevado a obtener estos resultados es necesario emplear técnicas como la Fluorescencia o
la Espectroscopía Mössbauer, que permitan dilucidar las reacciones que ocurren en el
complejo, así como entre el complejo y el suelo.
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CAPÍTULO VI Discusión General
Una vez conocida la capacidad potencial de este tipo de complejos para aportar
hierro a las plantas en condiciones de baja reactividad, se evaluó la capacidad de los
mismos para servir como sustrato para la enzima FC-R, tanto en raíz como en hoja.
La reducción del Fe3+ es un requisito necesario para que el hierro pueda ser
tomado por la raíz (Chaney et al., 1972, Maschner y Römheld, 1995), y se midió en
plantas de tomate desarrolladas en condiciones de deficiencia de hierro (Capítulo V.2,
Tabla 4). Todos los complejos LS/Fe3+ sirvieron como sustrato para la FC-R, si bien la
reducción fue mucho menor que al usar o,o-EDDHA/Fe3+ como sustrato.
Un sistema de reducción similar al observado en raíz tiene lugar en hoja para que el
hierro pueda ser asimilado por la célula vegetal (de la Guardia y Alcántara, 1996;
Kosegarten et al., 1999; Nikolic y Römheld, 1999; Larbi et al., 2001). Este mecanismo
aparece directamente asociado a la fotosíntesis, estando favorecido en presencia de luz,
así como en condiciones de suficiencia de hierro (por la mayor producción de NAD(P)H
y por tanto la mayor actividad fotosintética en plantas sin deficiencias).
La reducción en hoja se ensayó tanto en oscuridad como con luz. Puesto que la
aplicación de rociados foliares tiene lugar habitualmente en plantas con deficiencias de
hierro, los ensayos se realizaron con plantas de tomate y de pepino cloróticas. A
diferencia de lo observado al medir la reducción en raíz, en hoja los quelatos sintéticos
no mostraron un comportamiento claramente más eficaz que los complejos. En
oscuridad y a concentraciones de sustrato bajas (10 µM, próximas a las de Fe-citrato en
el apoplasto de la hoja) se observó un comportamiento distinto para el tomate y el
pepino. La reducción fue mayor en el caso del tomate, lo cual podría asociarse con la
distinta eficacia entre ambas especies frente a la deficiencia de Fe. El tomate es menos
eficiente que el pepino, y la reducción en hoja podría estar favorecida para contrarrestar
la menor adquisición de Fe a través de la raíz. El Lignosulfonato 4 fue el lignosulfonato
más eficaz.
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CAPÍTULO VI Discusión General
lignosulfonatos pueden servían como sustrato para la FC-R en raíz y hoja, si bien
ninguno de los cuatro productos evaluados sobresalía claramente entre los demás, y no
era posible determinar cuáles de sus características habían favorecido o dificultado la
reducción.
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CAPÍTULO VI Discusión General
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CAPÍTULO VI Discusión General
Por otra parte, la translocación desde la hoja tratada a otros órganos vegetales
ocurre vía floema, mediante la formación de complejos entre el hierro y distintas
proteínas. Si bien gran parte de los complejos, mecanismos y transportadores implicados
en la redistribución a través del floema se desconocen, se sabe que este transporte ocurre
principalmente en forma de complejos de Fe2+ con nicotiamina (NA/Fe2+) y de Fe3+
otras proteínas transportadoras de hierro (ITP/Fe3+) (Kim y Guerinot, 2007; Briat et al.,
2007; López-Millán et al., 2009). La escasa redistribución del hierro a través del floema, a
pesar de la existencia de mecanismos específicos para su complejación y transporte, se
asocia a la limitada síntesis de NA, ya que la concentración de hierro en la hoja suele ser
suficiente (Hell et al., 2003). La limitada producción de NA, independiente de la
concentración de hierro en la hoja, ayudaría a explicar la menor redistribución de hierro
en las plantas de pepino, que sin embargo absorbieron más 59Fe.
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CAPÍTULO VI Discusión General
asimilado por la célula vegetal directamente. El peso molecular es otro de los factores
que se ha considerado habitualmente para explicar la eficacia de los productos aplicados
por vía foliar, pero trabajos recientes indican que no existe correlación entre el peso
molecular y la tasa de penetración (Schönherr et al., 2005; Fernández et al., 2006;
Fernández et al., 2008a).
59
Si bien el empleo de Fe nos permitió dilucidar el comportamiento de los
lignosulfonatos que estaban siendo evaluados, la utilización de un isótopo radiactivo
impedía evaluar ciertos factores de importancia en este trabajo, como el estudio de la
redistribución en períodos de tiempo más prolongados, el uso de concentraciones más
elevadas o la aplicación de los productos mediante rociados foliares (en lugar sumergir la
hoja el la disolución del complejo). Por ello, en dos nuevos ensayos (Capítulo V.3) se
evaluó el comportamiento de los cuatro complejos LS/57Fe3+ (concentración de Fe 5
mM) al ser aplicados mediante rociados foliares a plantas de pepino desarrolladas en
hidroponía pura con baja concentración de hierro (5 µM). Además, puesto que los
lignosulfonatos presentan propiedades tensoactivas no se añadió ningún tipo de
surfactante, algo que sí se hizo en el caso del 59Fe.
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CAPÍTULO VI Discusión General
Aunque los experimentos con 57Fe y 59Fe indicaron que los complejos Fe-LS eran
capaces de aportar hierro por vía foliar a las plantas tratadas con rociados foliares, no se
encontraron diferencias en la eficacia de los distintos lignosulfonatos. Las modificaciones
del Lignosulfonato 1 (obteniéndose los Lignosulfonatos 2 y 3) incrementaron su
capacidad complejante de hierro, pero ésto no derivó en una mayor eficacia de los
nuevos agentes complejantes. Estos resultados, junto con los de la actividad FC-R en
hoja, indican que no existe una relación directa entre el buen comportamiento de estos
complejos como sustratos para la FC-R en hoja y su eficacia como fertilizantes foliares.
Lo mismo se ha observado previamente al estudiar un humato férrico con EDTA/Fe3+
(Nikolic et al., 2003). Si bien la fotorreducción del EDTA/Fe3+ hizo que no fuese un
buen sustrato para la FC-R en hoja, este hecho pareció facilitar su absorción a través de
la superficie de la hoja y su entrada en la célula vegetal (sin necesidad de ser reducido
por la misma).
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CAPÍTULO VI Discusión General
este modo sin embargo el uso de complejos deja de ser una alternativa más económica
que los quelatos sintéticos, ya que su precio medio es de 2-4 €kg-1 y 6-12 €kg-1
respectivamente. No obstante, de este modo es posible aprovechar subproductos
derivados de diversas actividades industriales.
Los resultados presentados en esta memoria y los expuestos previamente por otros
autores al estudiar la fertilización foliar con quelatos y complejos de Fe sugieren que la
mayor eficacia mostrada por quelatos como el EDTA/Fe3+ se relaciona con la
fotorreducción de este quelato, que aportaría así el Fe en forma divalente a la planta, que
de este modo lo asimilaría con mayor facilidad.
Por otra parte, se ha señalado anteriormente que una fracción del Fe3+ aportado
para formar los complejos LS/Fe3+ era reducida al añadirla al LS, obteniéndose por tanto
complejos sólo parcialmente oxidados. Un comportamiento parecido ha sido descrito
para la materia orgánica (Skogerboe y Wilson, 1981; Struyk y Sposito, 2001) que en su
proceso espontáneo de oxidación utiliza el Fe3+ y el Mn4+ complejados como aceptores
de electrones. Este fenómeno está favorecido en presencia de ciertos microorganismos.
Se ha observado además su dependencia de la composición del material original
(contenido en carbohidratos, fenoles, etc.) y del pH al cual se encuentra, estando
especialmente favorecida a pH ácido (Chen et al., 2003).
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CAPÍTULO VI Discusión General
Fe más disponible para la planta, que al estar ya reducida debería ser además más eficaz
aportando Fe a la célula vegetal. Por tanto, es importante considerar cuidadosamente la
fuente de Fe empleada al formar los complejos Fe-LS, estando justificado el empleo de
Fe2+ en dicho proceso.
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Capítulo VII:
CAPÍTULO VII Conclusiones
CONCLUSIONES
1. En general, los quelatos mostraron mayor eficacia que los complejos para
mejorar el estado nutricional de plantas de soja cloróticas desarrolladas en
hidroponía pura, ya fuese al ser aplicados a la disolución nutritiva o mediante
rociados foliares.
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CAPÍTULO VII Conclusions
CONCLUSIONS
The results reported in this dissertation confirm that the use of biodegradable Fe
chelates and complexes can be an alternative to the application of the scarcely
biodegradable chelates commonly used in agriculture. The main conclusions obtained
are:
4. The use of Fe isotopes indicated that, although at rates lower than those
observed for EDTA/Fe3+, Fe supplied by LS/Fe3+ complexes was absorbed by
leaves and redistributed from them to growing non treated organs, both
processes being favored when the number of foliar applications performed was
increased. In any case, uptake and redistribution rates were low for all the
LS/Fe3+ and for EDTA/Fe3+.
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Universidad Autónoma de Madrid
Departamento de Química Agrícola