Plant and Soil 266: 195–203, 2004.
© 2004 Kluwer Academic Publishers. Printed in the Netherlands.
195
Temporary flooding increases iron phytoavailability in calcareous
Vertisols and Inceptisols
M. Velázquez1 , M.C. del Campillo1 & J. Torrent1,2
1 Departamento
de Ciencias y Recursos Agrı́colas y Forestales, Universidad de Córdoba, Apdo. 3048, 14080
Córdoba, Spain. 2 Corresponding author∗
Received 7 October 2003. Accepted in revised form 9 March 2004
Key words: Fragaria × ananassa, flooding, iron availability, iron chlorosis, iron oxides, Lupinus albus, reduction
Abstract
Temporary soil flooding before cultivation alleviates iron chlorosis in crops grown on some calcareous Mexican
Vertisols. In order to investigate the effectiveness of such practice we carried out experiments with ten calcareous
Vertisols from Mexico and eight calcareous Inceptisols from Spain. In an incubation experiment, we studied the
release of Fe2+ into the solution of soil suspensions in sealed vials with 5 mM CaCl2 . In a pot experiment,
we measured the leaf SPAD value (i.e. an estimate of leaf chlorophyll concentration) of lupin and strawberry
sequentially grown on a soil–sand mixture previously flooded for 30 days (SPADf value) and on a non-flooded
(control) mixture (SPADc value). The amount of Fe2+ released by the soil at day 58 and the increase in oxalateextractable Fe (Feo ) upon incubation in vials were larger on average for the Inceptisols than for the Vertisols. The
SPADc values for lupin and strawberry were (i) larger for the Vertisols than for the Inceptisols (probably because the
Vertisols contain little carbonate and induce less Fe chlorosis than the Inceptisols) and (ii) correlated with Feo , and
with citrate/ascorbate- and DTPA-extractable Fe (Feca , FeDTPA ). The SPADf –SPADc difference was (i) much larger
for the Inceptisols than for the Vertisols and (ii) correlated with the increases in Feo and Feca caused by flooding
and with the amount of Fe2+ released in the incubation experiment. We hypothesize that the weak response of the
Vertisols to flooding was partly a result of their history including flooding episodes in the field, so a steady state
had been reached in which the pool of Fe compounds undergoing reductive dissolution and reprecipitating upon
oxidation as poorly crystalline Fe oxides (the main source of phytoavailable Fe) remained relatively constant and
thus changed little after pot flooding. The Inceptisols, which had never been flooded in the field, were capable
of releasing Fe from sources other than poorly crystalline Fe oxides upon flooding, thus making this treatment
effective against Fe chlorosis. Our results point to the need to further study those soil chemical and mineralogical
properties that are related to increases in Fe phytoavailability upon temporary soil flooding.
Abbreviations: ACCE – active calcium carbonate equivalent; CCE – calcium carbonate equivalent; Feca –
citrate/ascorbate-extractable Fe; FeDTPA – DTPA-extractable Fe; Feo – acid oxalate-extractable Fe.
Introduction
The occurrence of iron (Fe) deficiency chlorosis in
plants grown on calcareous soils is strongly influenced
by the content and solubility of soil Fe oxides (a term
used here to designate Fe oxides, hydroxides, and
oxyhydroxides). Thus, the content in poorly crystal∗ FAX No: +34957218440. E-mail: [email protected]
line, relatively soluble Fe oxides, as measured by acid
oxalate extractable Fe (Feo ), has been found to be
negatively correlated with the degree of Fe chlorosis
in soybean and sorghum (Loeppert and Hallmark,
1985), chickpea and sunflower (del Campillo and Torrent, 1992), and olive trees (Benítez et al., 2002).
These poorly crystalline oxides generally supply most
of the Fe(III) that is chelated by phytosiderophores,
microbial siderophores or any other natural or arti-
196
ficial chelating agents and finally acquired by plant
roots through different mechanisms (Hell and Stephan,
2003).
In aerated calcareous soils, the concentration of
non-complexed Fe3+ in solution is very low because
of the low solubility of Fe oxides at the alkaline pH
of these soils. On waterlogging, however, reductive
dissolution of poorly crystalline Fe oxides results in
a significant release of Fe2+ into the soil solution
(Ponnamperuma, 1972). One would then expect Fe
chlorosis in plants to be alleviated during the wet
periods of the growing season or under excessive irrigation. However, the opposite is generally observed,
likely as a result of the Fe chlorosis-inducing effect
of the bicarbonate ions that accumulate in the solution of wet calcareous soils (Lindsay and Thorne,
1954). These field observations are consistent with
those of pot experiments in which soils were subjected to different moisture regimes (Inskeep and Bloom,
1986).
Little is known on the effects of temporary soil
flooding before cultivation on Fe phytoavailability;
however, one can assume that the corresponding redox
changes influence the nature and amount of the more
soluble Fe oxides and other phytoavailable Fe forms
(de Mello et al., 1998). Longoria (1973) reported
that the development of Fe chlorosis in sugarcane and
sorghum grown on calcareous Vertisols of Mexico was
largely prevented if the soils were flooded and allowed
to dry below field capacity before planting. Such a
practice has been successfully adopted by some farmers in northeastern and central Mexico. In this paper,
we report laboratory and growth chamber experiments
designed to examine the effect of temporary flooding on changes in Fe phytoavailability to lupin and
strawberry in calcareous soils. To this end, a number
of Vertisols from Mexico and Inceptisols from Spain
with a different Fe chlorosis-inducing capacity were
studied.
Materials and methods
Soils and soil analyses
Samples were collected from the surface horizon of
ten calcareous Vertisols from Mexico and eight calcareous Inceptisols from Spain. The Mexican Vertisols
were located in the Zamora Valley (northwestern Mexico; ∼ 20◦ 07 N lat. ∼ 102◦25 W long.), where mean
annual rainfall is 820 mm, the mean annual temperature 21 ◦ C, and the moisture regime ustic (Soil
Survey Staff, 1999). These soils had developed on
Tertiary and Quaternary basalts, lay on low landscape
positions, and were occasionally flooded by nearby
streams in the wet season. The Spanish Inceptisols
were collected in Andalusia (southern Spain), at
∼ 38◦12 N lat. ∼ 3◦ 30 W long., where mean annual
rainfall is about 600 mm, the mean annual temperature 16 ◦ C, and the moisture regime xeric (Soil Survey
Staff, 1999). They had developed mainly on marls and
calcareous marls of Tertiary age.
Soil samples were air-dried and ground to pass
a 2-mm sieve before analysis. The clay content was
determined by the pipette method, pH by potentiometric measurement in a 1:2 soil:water suspension, and organic carbon by dichromate oxidation.
The calcium carbonate equivalent (CCE) was determined from the weight loss upon treatment with
6 M HCl for 20 min, and the active calcium carbonate equivalent (ACCE) or ‘active lime’ according
to Drouineau (1942). Citrate/bicarbonate/dithioniteextractable Fe (Fed ), acid oxalate-extractable Fe (Feo ),
citrate/ascorbate-extractable Fe (Feca ), and DTPAextractable Fe (FeDTPA ) were determined according
to Mehra and Jackson (1960), Schwertmann (1964),
Reyes and Torrent (1997), and Lindsay and Norvell
(1978), respectively. Total Fe (Fet ) was determined
by H2 SO4 –HClO4 digestion. Clay minerals in the
clay fraction were identified in oriented aggregates
by conventional X-ray diffraction methods, using a
Siemens D5000 diffractometer. Visible diffuse reflectance spectra (400–700 nm, 0.5-nm steps) of one finely
ground (< 20 µm) 250–300 mg sample of each of the
intact and incubated soils were acquired using a Varian
Cary 1E spectrophotometer equipped with a BaSO4 –
coated integrating sphere 73 mm in diameter. The
powdered samples were pressed firmly by hand into
the 8 × 17 rectangular holes of white plastic holders
so that the resulting mounts could be placed vertically without the powder falling into the sphere. The
white standard was powder BaSO4 (Merck DIN 5033).
The CIE 1931 color-matching functions weighted by
the relative spectral radiant power distribution of CIE
Standard Illuminant C were used to calculate the tristimulus values, X, Y, Z from the reflectance values.
The data tabulated by Wyszecki and Stiles (1982) at
5-nm intervals in the 380–770 nm range were used in
all these calculations. Then, the chromaticity coordinates, x = X/(X + Y + Z), y = Y/(X + Y + Z),
z = Z/(X + Y + Z) were converted to the CIE
1976 (L∗ a ∗ b∗ ) coordinates with the aid of free software downloaded from Munsell Color’s site (www.
197
Munsell.com). These coordinates provide good information about the content and nature of Fe oxides in
soil samples because L∗ is lightness, positive a ∗ value
is degree of ‘redness’, and positive b∗ value is degree
of ‘yellowness’.
Soil incubation in vials
We added 4-g soil samples to 30-mL vials containing
25 mL of 5 mM CaCl2 . The vials were immersed
in an ultrasonic bath for 20 s to remove gas bubbles
and capped with a rubber septum that was secured
tightly with a metal stopper. This soil–5 mM CaCl2
suspension was then shaken in a reciprocating shaker
at 2.5 Hz for 30 min and finally kept in a dark room
at 25 ◦ C. Samples from the supernatant solution were
taken at 7, 14, 21, 28, 35, 42, 49 and 58 days using
a syringe (to pierce the septum) and centrifuged at
∼ 2400 g for 10 min. A 0.5-mL portion of the clear
supernatant was then taken and immediately acidified
with 1 mL of 0.1 M Na-acetate buffer at pH 3.5 to
prevent oxidation of Fe2+ . After each sampling, an
amount of 5 mM CaCl2 equal to the amount of solution taken was injected into the vial and the vial gently
shaken. Dissolved Fe was always determined by the
o-phenanthroline method (Olson and Ellis, 1982). At
the end of the experiment the water of the suspension was evaporated at a temperature of 25 ◦ C and
the solid phase dried and analyzed for Feo and colour characteristics. These incubation tests were done
in triplicate.
Pot experiments
Mixtures consisting of 30 g of soil and 90 g of 0.2–
0.5 mm silica sand (which was Fe oxide- and clay-free
and had been washed previously with water) were
placed in 250-mL polyethylene pots with a drain at
the bottom. The pots were slowly immersed in a tray
filled with 5 mM CaCl2 so that the final solution level
was 2 cm above the mixture surface. Then, the pots
were covered with filter paper and kept in the dark
at 25 ◦ C for 30 d. The supernatant in each pot was
then removed with a pipette and the pot was allowed
to drain for 2 d before planting two lupin (Lupinus
albus L. cv. Multolupa) seedlings obtained by germinating seeds on moist paper towels for 3 d. After one
week, plants were thinned to one per pot, and pots
were randomly arranged and re-randomized each day
in a growth chamber equipped with fluorescent lamps
(light intensity: ∼ 100 µmol m−2 s−1 ) with a 14/10 h
light/dark photoperiod, and a day/night temperature of
28/10 ◦ C. The pots were watered daily to field capacity
using distilled water, and Hoagland nutrient solution
was added twice a week. The youngest fully expanded
leaves in each plant were selected to estimate the leaf
chlorophyll content, using a Minolta SPAD-502 apparatus (Minolta Co. Ltd., Osaka, Japan) the readings
of which are so-called ‘SPAD units’. SPAD values
(average of five readings per leaf) were measured 10,
14, and 21 days after planting, at which time plants
were harvested and plant height, leaf length, shoot
dry weight, and root dry weight measured. The soil–
sand mixture in each pot was dried, gently ground to
pass through a 2-mm sieve, transferred into a clean
polyethylene pot and watered to field capacity before planting a strawberry plant (Fragaria × ananassa
Guedès cv. Camarosa). The pots were placed in a
growth chamber under light and temperature conditions identical with those used for the lupin plants.
SPAD values were measured on the youngest fully
expanded leaf at 26, 32 and 42 days after planting,
at which time plants were harvested, and leaf length,
shoot, and root dry weight measured. During cropping of both lupin and strawberry, the SPAD value and
leaf chlorophyll content per unit surface were measured on leaves ranging widely in degree of chlorosis.
Chlorophyll was extracted with 96% ethanol and determined spectrophotometrically as described in detail
by Benítez et al. (2002). After harvest, the soil–
sand mixture was dried, ground to pass through a
2-mm sieve and analyzed for Feo , Feca , and FeDTPA
in duplicate.
Statistical analyses
Regression, Student’s t-test, and paired Student’s
t-test analyses were performed using either Statistix 7.0 (Analytical Software, Tallahassee, FL, USA)
or CoStat (CoHort Software, Minneapolis, MN, USA)
programs. Unless otherwise stated, the term ‘significant’ is used here to denote significance at P < 0.05,
and the symbols ∗ , ∗∗ , and ∗∗∗ after a correlation coefficient denote significance at P < 0.05, < 0.01, and
< 0.001, respectively.
Results and discussion
Soil properties
The general properties of the soils are shown in
Table 1. Most soils had an alkaline pH (range: 6.7–
8.4), and organic carbon contents from 9 to 20 g kg−1.
198
Table 1. General properties of the soils
Organic
Clay
Soil C
Clay CCEa ACCEa pH ECa
Feat
Fead
Feao Feo /Fed Fed /Fet mineralsb
———– g kg−1 ———–
dS m−1 —— g kg−1 ——
Mexican Vertisols
101 12
540
102 13
500
103 15
640
104 16
510
105 13
500
106 18
480
107 15
560
108 19
360
109 16
660
110 14
540
80
73
55
52
66
56
62
74
55
54
19
23
16
16
20
15
18
15
16
16
7.5
7.7
6.9
7.3
7.8
7.3
7.6
7.7
6.7
7.0
0.19
0.14
0.09
0.11
0.12
0.18
0.13
0.23
0.08
0.07
35.9
2.15
31.8
1.44
37.0
2.10
28.5
1.44
39.2
1.71
30.7
1.35
35.5
1.62
28.9
0.99
52.1
5.30
58.1 10.60
0.81
0.37
0.98
0.51
0.61
0.53
0.63
0.43
2.40
3.41
0.38
0.26
0.47
0.35
0.36
0.39
0.39
0.43
0.45
0.32
0.06
0.04
0.06
0.05
0.04
0.04
0.04
0.03
0.10
0.18
S, K
S, K
S, K
S, K
S, K
S, K
S, K
S, K
S, K
S, K
Spanish Inceptisols
201 15
260
204 14
350
205 15
250
209 20
540
210 10
200
214 17
190
223 9
370
236 15
140
570
460
555
45
610
685
680
840
260
265
265
20
200
240
285
310
7.6
7.3
7.4
7.7
7.5
7.8
8.4
7.5
0.23
0.26
0.28
0.15
0.25
0.40
0.17
0.36
15.2
2.62
27.0
4.63
15.9
2.37
49.9 15.40
14.4
2.66
11.5
2.66
16.6
2.45
8.1
1.25
0.43
0.55
0.34
1.33
0.35
0.27
0.53
0.22
0.16
0.12
0.14
0.09
0.13
0.10
0.22
0.18
0.17
0.17
0.15
0.31
0.18
0.23
0.15
0.15
S, I, K
S, I, K
S, I, K
I, K
S, I, K
S, I, K
S, I, K
S, I, K
a CCE, calcium carbonate equivalent; ACCE, active calcium carbonate equivalent; EC, electrical conductivity in the 1:5 soil:water extract; Feo , oxalate-extractable Fe; Fed , citrate/bicarbonate/dithionite-extractable
Fe; Fet , total Fe.
b S, smectite; I, illite; K, kaolinite.
CCE and ACCE ranged from 45 to 840 g kg−1 and
from 15 to 310 g kg−1 , respectively, the (Spanish) Inceptisols being much richer in carbonates than
the (Mexican) Vertisols. The Vertisols were generally
richer in clay than the Inceptisols, smectite being the
dominant or most abundant clay mineral in 17 of the
soils. The values for the Fed /Fet ratio (0.03–0.31) suggest that the degree of weathering of the soils was
low to moderate. In 15 soils, the respective Fed and
Feo contents were < 5 g kg−1 and < 1 g kg−1 , which
are typical of soils capable of inducing Fe chlorosis
in sensitive plants (Loeppert and Hallmark, 1985;
Benítez et al., 2002). The Feo /Fed ratio, which measures the ratio between poorly crystalline and total Fe
oxides, was higher for the Vertisols (range: 0.26–0.47)
than for the Inceptisols (range: 0.09–0.22).
Soil incubation experiments
The Inceptisols released more Fe2+ into the solution
on average than did the Vertisols (Figure 1). The
amount of Fe2+ released by the Vertisols was small
until day 28 of incubation (0.35–7.15 mg kg−1 ) because some time was needed for the initial amount
of oxygen in the vial to be used by microorganisms.
Then, the amount of Fe2+ released increased slowly
and reached 7–31 mg kg−1 at day 58 in four soils but
only < 3 mg kg−1 in the other six soils. The amount of
Fe2+ released by the Inceptisols increased with time;
most soils exhibited a maximum at day 28 that was followed by a decrease and a second maximum at day 49.
Generally, changes were not substantial after about
one month of anaerobic incubation, consistent with
a study involving widely contrasting soils of temperate areas (Scalenghe et al., 2002). This suggests that
the onset of relatively stable redox conditions occurs
roughly one month after saturation.
Because the Vertisols and the Inceptisols differed
little in Feo content (which provides a measure of
poorly crystalline, relatively soluble Fe oxides), it is
difficult to explain why the former released so little
Fe2+ relative to the latter. One possible reason is
that Mn oxides, which were more abundant in the
199
Figure 1. Temporal variation of the amount of Fe2+ released into the solution of 1:5 soil −5 mM CaCl2 suspensions incubated in sealed vials
for (a) Mexican Vertisols and (b) Spanish Inceptisols. Bar = standard error.
Vertisols than in the Inceptisols (data not shown), act
as the main electron acceptors (Patrick and Jugsujinda,
1992). One other reason might be the higher clay
content (and higher cation exchange capacity) of the
Vertisols relative to the Inceptisols, which would result in a higher proportion of the Fe2+ released being
retained by the cation exchange complex. In general
terms, all patterns are largely influenced by the precipitation reactions that remove Fe2+ from the solution
and to organic matter mineralization, which releases
organically complexed Fe (Willet et al., 1989; de
Mello et al., 1998).
Table 2 shows the values of the CIE 1976 (L∗ a ∗ b∗ )
coordinates (L∗ = lightness; positive a ∗ value = degree of ‘redness’; positive b∗ value = degree of ‘yellowness’) of the non-incubated (control) soils and their
changes upon incubation in sealed vials. As per Student’s paired t-test changes were significant for some
colour coordinates and soils groups. Thus, lightness
increased significantly upon incubation in most soils,
probably because some organic matter was mineralized during the process. The a ∗ (redness) coordinate
decreased significantly in both soil groups, and the
b∗ (yellowness) coordinate decreased significantly in
the Inceptisols and four Vertisols. The corresponding
changes (a ∗ , b ∗ ; Table 2) for the Inceptisols were
significantly correlated with the amount of Fe2+ released during incubation (r = −0.82∗ and −0.75∗,
respectively). This suggests that (i) reductive dissolu-
tion affected significantly Fe oxides (which typically
have colours ranging from red to yellow), and (ii) the
Fe(III) oxides that precipitated after the soil dried and
Fe2+ was oxidized were less coloured than the original
Fe oxides that underwent dissolution. The a ∗ /b∗ ratio decreased significantly upon incubation (i.e. there
was a decrease in the redness/yelloweness ratio) for
both soil groups. This indicates that the original Fe
oxides (or other soil components) that are affected by
reductive dissolution are redder than the Fe(III) oxides
that precipitate after reoxidation. One can then hypothesize that (i) some of the minerals that dissolved are
essentially reddish brown varieties of ferrihydrite and
small amounts of relatively insoluble hematite, which
is red (Schwertmann, 1991; Scheinost and Schwertmann, 1999), and (ii) the newly formed Fe(III) oxides
are mainly in the form of coatings and small masses
of yellowish brown rather than reddish brown ferrihydrite. It should be noted that most of the soils
studied were clayey and silicate clays (particularly
smectite) hinder the formation of crystalline Fe oxides
by maintaining an ionic environment rich in Al and Si
(Schwertmann and Taylor, 1989).
Incubation of soils with 5 mM CaCl2 in closed
vials resulted in a significant increase in Feo content
(Feo ) in one Vertisol and in all Inceptisols (Feo ,
Table 2). This suggests that the long incubation under anaerobic conditions resulted in the release of Fe
from various sources (e.g., Fe oxides more crystalline
200
Table 2. Changes in oxalate-extractable Fe (Feo ) and in CIE (1976) L∗ a ∗ b∗ coordinates upon incubation of a
soil–5 mM CaCl2 suspension in a sealed vial
Soil
Feao
g kg−1
L∗
L∗
a∗
CIE (1976) L∗ a ∗ b∗ coordinates
a ∗
b∗
b∗
a ∗ /b∗
(a ∗ /b∗ )
Mexican Vertisols
101
–
102
0.17
103
−
104
−
105
−
106
−
107
−
108
−
109
−
110
−
56.6
55.8
52.8
52.8
55.6
52.9
53.6
54.7
52.2
53.9
1.5
2.3
1.6
4.0
3.1
3.7
3.9
3.3
4.3
3.7
1.62
1.64
1.83
1.75
1.66
1.76
1.60
1.54
2.35
3.47
−0.07
−0.11
−0.09
−0.07
−0.19
−0.13
−0.07
−0.07
−0.24
−0.37
7.65
7.62
7.35
7.19
7.47
7.58
6.55
6.74
9.44
13.79
0.45
0.33
−0.13
0.19
0.27
−0.05
0.79
0.05
−0.25
−0.19
0.21
0.22
0.25
0.24
0.22
0.23
0.24
0.23
0.25
0.25
−0.02
−0.03
−0.01
−0.01
−0.03
−0.01
−0.03
−0.01
−0.02
−0.02
Spanish Inceptisols
201
0.92
204
1.34
205
0.94
209
1.75
210
0.43
214
0.84
223
0.44
236
0.41
77.8
70.1
77.5
56.3
78.6
70.0
79.5
76.7
0.5
2.3
−0.1
4.6
0.1
0.1
0.5
0.1
0.55
1.54
0.57
6.78
0.60
1.98
0.00
1.40
−0.23
−0.87
−0.35
−1.90
−0.25
−0.33
−0.15
−0.29
11.92
14.83
12.10
21.46
11.13
11.18
8.57
9.04
−2.79
−2.35
−2.79
−2.63
−2.63
−1.29
−0.44
−0.91
0.05
0.10
0.05
0.32
0.05
0.18
0.00
0.15
−0.02
−0.05
−0.03
−0.06
−0.01
−0.01
−0.02
−0.01
a In this column, where no value is shown, the increment was not significantly different from zero according to
Student’s t test.
than ferrihydrite, organic Fe complexes, and Fe-rich
smectite or illite) that were not oxalate-soluble, and
that the Fe2+ released precipitated upon reoxidation
as oxalate-soluble oxides. For the Inceptisols, Feo
was positively correlated (r = 0.83∗ ) with Fed –Feo ,
which is a measure of the content in crystalline Fe
oxides, and with the clay content (r = 0.75∗); this
suggests that reduction may have caused the release of
some Fe from crystalline Fe oxides and Fe-containing
silicate clays. This was not observed in the Vertisols,
probably because their history included a substantial number of episodes of temporary flooding. Consequently, a steady state had probably been reached in
which the pool of Fe oxides and other oxalate-soluble
Fe compounds that were reductively dissolved during each flooding episode [and later reprecipitated as
poorly crystalline Fe(III) oxides] remained essentially
unchanged.
Pot experiments
Changes in Fe forms induced by flooding
Table 3 shows the values of Feo , Feca , FeDTPA in
the intact (control) soils and their increases (Feo ,
Feca , FeDTPA ) as a result of flooding followed by
successive lupin and strawberry cropping. Significant
increases in Feo were recorded for five Inceptisols. By
contrast, Feca increased significantly in four Vertisols
and three Inceptisols. Therefore, citrate/ascorbate is
a little more sensitive extractant than oxalate to detect the effects caused by flooding on Fe forms, even
though both extractants have been shown to selectively
dissolve the poorly crystalline Fe oxides (Reyes and
Torrent, 1997). Fourteen soils exhibited a significant
increase in FeDTPA , which is often used to predict Fe
phytoavailability (Lindsay and Norvell, 1978). The
Feo , Feca , FeDTPA values were correlated with
the Feo values recorded in the vial experiments
(Table 2), which indirectly suggests that cropping did
not substantially modify the effect of flooding on Feo ,
Feca and FeDTPA .
201
Table 3. Extractable Fe formsa and their increments after soil
flooding in pots and successive lupin and strawberry cropping
Soil
Feo
Febo Feca
Febca FeDTPA FebDTPA
————- g kg−1 ——————– mg kg−1 ——–
Mexican Vertisols
101
0.81
102
0.37
103
0.98
104
0.51
105
0.61
106
0.53
107
0.63
108
0.43
109
2.40
110
3.41
–
–
–
–
–
–
–
–
–
–
0.58
0.35
0.72
0.35
0.29
0.49
0.50
0.51
2.25
3.51
–
0.16
0.30
0.34
0.31
–
–
–
–
–
4.4
2.3
16.4
6.9
4.6
3.4
5.0
3.0
52.0
49.5
9.6
10.3
11.2
10.7
9.8
8.6
13.6
9.4
–
6.5
Spanish Inceptisols
201
0.43
0.46
204
0.55
0.51
205
0.34
0.67
209
1.33
0.44
210
0.35
–
214
0.27
0.23
223
0.53
–
236
0.22
–
0.59
0.62
0.40
2.49
0.59
0.38
0.67
0.19
0.73
0.77
0.89
–
–
–
–
–
6.7
12.0
7.8
19.0
7.1
9.9
7.6
4.6
34.0
37.0
32.8
40.5
–
–
–
18.5
a Fe , oxalate-extractable Fe; Fe , citrate/ascorbate-extractable Fe;
o
ca
FeDTPA , DTPA-extractable Fe.
b In this column, where no value is shown, the increment was not
significantly different from zero according to Student’s t-test.
Plant growth and leaf chlorophyll content
Both lupin and strawberry plants grew vigorous and
exhibited no signs of iron chlorosis until day 10
and 12, respectively, after planting. The chlorotic
symptoms (viz. interveinal yellowing and, eventually,
generalised leaf yellowing and necrosis) developed
more slowly in strawberry than in lupin. Previous experiments had shown that these symptoms dissapear
when pots are fertilized with an effective Fe source
(Fe-EDDHA).
The SPAD value can be used as a convenient measure of the leaf chlorophyll concentration per unit surface; in fact, these variables were found to be highly
correlated (r = 0.95∗∗∗ for lupin, and 0.96∗∗∗ for
strawberry) and no statistically significant curvature
(which would indicate SPAD saturation) was observed
at high chlorophyll concentrations. The effect of previous flooding on chlorosis prevention can thus be easily
envisaged by plotting the SPAD value for plants grown
on previously flooded soil–sand substrates (SPADf ,
y-axis) versus that for plants grown on the control
(non-flooded) substrates (SPADc , x-axis). As shown
in Figure 2, the SPADc values for both crops were,
on average, substantially larger in plants grown on
Vertisols than in plants grown on Inceptisols; only a
few of the former exhibited visual signs of chlorosis.
We hypothesized that such differences were partly due
to the relatively low CCE and ACCE content of the
Vertisols, which resulted in a proportion of only 10–
20 g CCE kg−1 in some of the soil–sand mixtures used
as substrates. This hypothesis was supported (data not
shown) by tests showing that the degree of chlorosis
exhibited by lupin increased substantially when calcareous sand (0.2–0.5 mm in size) was added to the
substrates to match the CCE content of the original
soil.
As can be seen in Figure 2, the points corresponding to most Inceptisols lay well above the 1:1 line.
By contrast, the points for the Vertisols lay generally
above, but close to, or even below the 1:1 line. Paired
t-tests showed that flooding had exerted a significant
effect on the lupin and strawberry SPAD values for the
Inceptisols, but only on the strawberry SPAD value for
the Vertisols. Flooding also had a significant effect on
plant height, leaf length, shoot dry weight, and root
dry weight of lupin grown on the Inceptisols (data not
shown).
Leaf chlorophyll content in relation with extractable
Fe forms
The SPADc values of the plants grown on the Inceptisols were correlated with Feo (r = 0.91∗∗ for
both lupin and strawberry), Feca (r = 0.93∗∗ for
lupin, and r = 0.94∗∗∗ for strawberry), and FeDTPA
(r = 0.87∗∗ for lupin, and r = 0.79∗ for strawberry),
as expected from the known predictive value of these
extractable Fe forms (Benítez et al., 2002; Lindsay
and Norvell, 1978). This was not the case with the
Vertisols, given that, as stated above, most of them
had a limited capacity to induce chlorosis under the
conditions of this study.
As noted earlier, the increment in SPAD due to the
flooding treatment [i.e. SPAD = (SPADf –SPADc )]
was very small or even negative for the Vertisols. This
was probably due to (i) the high SPADc values, and
(ii) the fact that Feo , Feca , and FeDTPA were
either relatively small or not significantly different
from zero (Tables 2 and 3). These small changes are
consistent, as noted when discussing the vial experiments, with the steady state that was probably reached
202
Figure 2. SPAD values for the leaves of pot-grown (a) lupin and (b) strawberry. SPADf = values for plants in previously flooded pots;
SPADc = values for plants in control (not previously flooded) pots.
Figure 3. Relationships between the SPADf /SPADc ratio for
pot-grown lupin and the af∗ /ac∗ and bf∗ /bc∗ ratios recorded in the
vial experiment (subscripts: f, soil incubated for 58 days; c, intact
soil).
by the Vertisols in terms of amounts of oxalatesoluble, reducible Fe oxides and other Fe-containing
minerals. The high effectiveness of flooding for the
Inceptisols (high SPAD values, Figure 1) likely reflects the fact that these soils are never (or at least
rarely) waterlogged in nature. Thus, a long 30-day
flooding treatment resulted in reductive dissolution of
some Fe compounds that had never been dissolved
before. The Fe so released was later reoxidized to
poorly crystalline Fe(III) oxides that contributed significantly to increase the Feo , Feca , and FeDTPA , and,
consequently, helped to remediate Fe chlorosis.
If all soils were considered, then SPAD was significantly correlated with Feo (r = 0.49∗ for lupin,
and 0.51∗∗ for strawberry) and Feca (r = 0.75∗∗∗
for lupin, and 0.71∗∗ for strawberry), but not with
FeDTPA . These results, as well as the former correlations of SPADc with Feo , Feca , and FeDTPA , suggest
that Feo and Feca predict soil chlorosis inducing capacity better than does FeDTPA . It should also be noted
that SPAD was correlated with the amount of Fe2+
released at day 58 (r = 0.70∗∗ for lupin, and 0.60∗
for strawberry), and with Feo in the vial experiments
(r = 0.52∗ for lupin, and 0.54∗ for strawberry). Thus,
the increase in Fe phytoavailability seems to be related
to the amount of Fe that can be mobilized by reductive
dissolution of some non-oxalate-soluble Fe-containing
compounds. The redox changes resulting from the
flooding treatment may increase also the phytoavailability of the oxalate- or citrate/ascorbate-soluble Fe
oxides. The latter hypothesis is supported by the correlations between the SPADf /SPADc ratio for lupin
(the more responsive plant) and the ratios between the
final and initial a ∗ and b∗ coordinates in the vial experiments (i.e. af∗ /ac∗ and bf∗ /bc∗ , data in Table 2). Briefly,
these correlations (Figure 3) indicate that the increase
in Fe phytoavailability is somehow related to changes
affecting the nature of the Fe oxides (i.e. the more coloured and redder oxides become, upon reduction and
reoxidation, paler, yellower Fe oxides). Unfortunately,
the low concentrations of Fe oxides in both untreated
and treated soils preclude identification of the mineral
species involved in these transformations by means of
instrumental techniques such as X-ray diffraction and
Mössbauer spectroscopy.
203
Conclusions
The flooding treatment helped to prevent Fe chlorosis
in lupin and strawberry grown on pots with soil–quartz
sand substrates. The effect was larger in the Spanish
Inceptisols than in the Mexican Vertisols, probably
because (i) the Vertisols were less calcareous and
induced less Fe chlorosis than did the Inceptisols,
and (ii) flooding caused a significant increase in extractable Fe forms (Feo , Feca , and FeDTPA ) in most
Inceptisols, but not in most Vertisols. We hypothesize that periodical flooding episodes in the history of
the Vertisols probably had stabilized the amount of
Fe oxides and other compounds that could be reductively dissolved and reprecipitated as poorly crystalline
Fe(III) oxides. The colour changes observed in the vial
experiments indicate that the Fe oxides precipitating
after reoxidation of dissolved Fe are less coloured and
red than the original Fe oxides.
In summary, the results of this study are consistent
with the Mexican farmers’ observation that temporary
flooding before cultivation can alleviate Fe chlorosis
in some sensitive crops. They also indicate that the
effects of such a practice are likely to be affected by
soil properties –mostly by the content and nature of
reducible Fe-containing compounds– as well as by the
moisture regime history of the soil. Further research
into the soil chemical and mineralogical properties
that cause the increase in Fe phytoavailability following flooding is thus needed. Finally, it must be noted
that the effect of temporary flooding has only been
tested for Strategy I plant species but not for graminaceous species, in which Fe chelation is more relevant
than Fe reduction.
Acknowledgements
M. Velázquez wishes to thank the Mexican Consejo
Nacional de Ciencia y Tecnología for funding her stay
in the Departamento de Ciencias y Recursos Agrícolas
y Forestales, Universidad de Córdoba.
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Section editor: D.E. Crowley