Improving soil oxygenation with hydrogen peroxide injection into heavy clay loam soil: Effect on plant water status, CO 2 assimilation and biomass of avocado trees

Abstract

Commercial avocado production in Chile has expanded to areas with poorly drained soils presenting low oxygenation over significant periods of time throughout the year. In many of these areas, irrigation management is difficult because plantations are often placed on slopes of hills. Poorly aerated soils combined with irrigation design and management problems can limit avocado fruit production and quality, particularly if hypoxia stress occurs between spring and the beginning of summer. It is well known that avocado trees are very sensitive to waterlogging and the relatively low productivity of this species may be related to root asphyxiation. Therefore, in order to get adequate yield and fruit quality, proper irrigation management and better soil oxygen conditions in avocado orchards are necessary. The objective of this study was to evaluate the effect of the hydrogen peroxide (H 2O 2) injection into the soil as a source of molecular oxygen, on plant water status, net CO 2 assimilation and biomass of avocado trees established in clay loam soil with water content at field capacity. Three-year-old 'Hass' avocado trees were planted outdoors in containers filled with heavy loam clay soil with moisture content kept at field capacity. Plants where divided into 2 treatments, those with H 2O 2 injected into the soil through subsurface drip irrigation and plants in soil with no H 2O 2 added (control). In addition to determining physical soil characteristics, net CO 2 assimilation (A), transpiration (T), stomatal conductance (g s) and shoot and root biomass were determined for plants in each treatment. Injecting H 2O 2 into the soil significantly increased the biomass of the aerial portions of the plant, but had no significant effect on measured A, T or g s. The increased biomass of the aerial portions of plants in treated soil indicates that H 2O 2 injection into heavy loam clay soils may be a useful management tool in poorly aerated soil.

Full text

97
1 Instituto de Investigaciones Agropecuarias, Centro Regional de
Investigación La Cruz, Chorrillos 86, La Cruz, Chile.
* Corresponding author (pgil@inia.cl ).
2 Centro Regional de Estudios en Alimentos Saludables. Blanco
1623, Of. 1402, Valparaíso, Chile.
3 Ponticia Universidad Católica de Chile, Facultad de Agronomía e
Ingeniería Forestal, Casilla 306-22, Santiago, Chile.
Received: 03 January 2008.
Accepted: 28 August 2008.
ABSTRACT
In Chile, avocado (Persea americana Mill.) orchards are often located in poorly drained, low-oxygen soils, situation
which limits fruit production and quality. The objective of this study was to evaluate the effect of injecting soil with
hydrogen peroxide (H2O2) as a source of molecular oxygen, on plant water status, net CO2 assimilation, biomass and
anatomy of avocado trees set in clay loam soil with water content maintained at eld capacity. Three-year-old ‘Hass’
avocado trees were planted outdoors in containers lled with heavy loam clay soil with moisture content sustained at
eld capacity. Plants were divided into two treatments, (a) H2O2 injected into the soil through subsurface drip irrigation
and (b) soil with no H2O2 added (control). Stem and root vascular anatomical characteristics were determined for
plants in each treatment in addition to physical soil characteristics, net CO2 assimilation (A), transpiration (T),
stomatal conductance (gs), stem water potential (SWP), shoot and root biomass, water use efciency (plant biomass
per water applied [WUEb]). Injecting H2O2 into the soil signicantly increased the biomass of the aerial portions
of the plant and WUEb, but had no signicant effect on measured A, T, gs, or SWP. Xylem vessel diameter and
xylem/phloem ratio tended to be greater for trees in soil injected with H2O2 than for controls. The increased biomass
of the aerial portions of plants in treated soil indicates that injecting H2O2 into heavy loam clay soils may be a useful
management tool in poorly aerated soil.
Key words: stomatal closure, net photosynthesis, root histology, oxygen injection, root hypoxia, subsurface drip
irrigation.
INTRODUCTION
Avocado trees are very sensitive to waterlogging
(Schaffer et al., 1992; Whiley and Schaffer 1994;
Schaffer and Whiley, 2002). An excess or lack of water
during growth limits avocado fruit production and
quality, particularly if stress occurs between spring and
the beginning of summer (Whiley et al., 1988a; 1988b).
Therefore, proper irrigation management in avocado
orchards is necessary to insure adequate yield and fruit
quality (Lahav and Whiley, 2002). In Chile, commercial
avocado production has expanded to areas with poorly
drained low-oxygen soils. Thus, root asphyxiation is
a growing concern for avocado growers when trees are
planted on these marginal soils.
In heavy clay, compacted, saturated soils, or when
subsurface drainage is impeded, an inadequate oxygen
concentration in the root zone can negatively affect
the biological functioning of plants (Letey, 1961). For
avocado trees, root hypoxia or anoxia usually results in
reduced stomatal conductance (gs), transpiration (T), net
CO2 assimilation (A), root and shoot growth, as well as
inhibited leaf expansion, moderate to severe stem and leaf
wilting, leaf abscission, and root necrosis (Schaffer et al.,
1992; Schaffer, 1998; Schaffer and Whiley, 2002). Stolzy
et al. (1967) reported that when the oxygen diffusion
rate (ODR) in the soil was lower than 0.17 µg cm-2 min-1,
there occurred 44 to 100% damage to roots of ‘Mexicola’
avocado trees. Ploetz and Schaffer (1987) observed a
synergistic relationship between Phytophthora root rot
and avocado root hypoxia, resulting in considerably more
root damage than that caused by either stress alone.
Root anoxia or hypoxia often results in increased
concentrations of 1-aminocyclopropane-1-carboxylic
EFFECT OF INJECTING HYDROGEN PEROXIDE INTO HEAVY CLAY
LOAM SOIL ON PLANT WATER STATUS, NET CO2 ASSIMILATION,
BIOMASS, AND VASCULAR ANATOMY OF AVOCADO TREES
Pilar M. Gil M.1, 2*, Raúl Ferreyra E.1, 2, Cristián Barrera M.1, Carlos Zúñiga E.1, and Luis Gurovich R.3
CHILEAN JOURNAL OF AGRICULTURAL RESEARCH 69 (1): 97-106 (JANUARY-MARCH 2009)
SCIENTIFIC NOTE

98 CHILEAN J. AGRIC. RES. - VOL. 69 - Nº 1 - 2009
acid (ACC), ethylene and abscisic acid (ABA) in leaves
(Bradford and Yang, 1980; Kozlowski, 1997). Elevated
concentrations of ACC and ABA in leaves of ooded
plants can accelerate abscission (Kozlowski, 1997).
Additionally, an increase in leaf ABA concentration
has been involved as a stimulus for stomatal closure in
ooded plants (Kramer and Boyer, 1995; Else et al., 1995;
Kozlowski, 1997). Finally, low soil oxygen content can
result in root tissue damage, inhibition of the vegetative
and reproductive growth, changes in plant anatomy and
morphology (i.e., development of hypertrophic stem
lenticels, adventitious roots or root and stem aerenchyma,
and alterations in the relationship between xylem
andphloem), premature senescence, and plant mortality
(Schaffer et al., 1992; Drew, 1997; Kozlowski, 1997).
At the present time, few methods exist to alleviate
growth and production problems due to lack of oxygen
in the root zone in avocado orchards. For other fruit tree
species, the use of ood-tolerant rootstocks (Schaffer and
Moon, 1991; Striegler et al., 1993; Gettys and Sutton,
2001) has been tested, but there are currently no ood-
tolerant rootstocks available for avocado trees. In Israel,
a few clonal rootstocks have been selected for soils with
poor aeration (Ben-Ya´acov and Zilberstaine, 1999),
but they have not been massively used in commercial
orchards. In Chile, to reduce problems caused by root
asphyxia in avocado orchards, trees are often planted on
raised beds to increase the volume of soil occupied by
roots thus improving drainage of irrigation and rain water.
However, the use of raised beds can result in signicant
soil erosion of steep hillside orchards and can cause water
obstruction problems when heavy rains wash soil from
raised beds into canals and streams.
Atmospheric enrichment of air surrounding the canopy
with CO2 or O2 has been used to enhance photosynthesis
of several crop species, thereby stimulating plant growth
and/or yield (Cave et al., 1981; Mortensen, 1984; Heij
and van Uffelen, 1984). However, increasing the oxygen
content of the rhizosphere to improve root metabolism
is a less-studied technique. In pepper plant production
(Capsicum annuum L. var. annuum), injecting air into the
water through subsurface drip irrigation (SDI) increased
the oxygen concentration and partial pressure in the root
zone, as well as N absorption xation, and reduced plant
stress leading to a 39% increase in fruit weight (Goorahoo
et al., 2001). Injecting air into the root zone also increased
biomass and fruit production, as well as water use
efciency of zucchini (Cucurbita pepo L. subsp. pepo),
soybean (Glycine max (L.) Merr.), and cotton (Gossypium
hirsutum L.) in heavy clay soils (Bhattarai et al., 2004).
An alternative technique to injecting oxygen into the
soil is the use of hydrogen peroxide (H2O2). Hydrogen
peroxide has been successfully utilized as an oxygen
source for in situ remediation in a saturated aquifer (Zappi
et al., 2000). The natural decomposition of H2O2 provides
molecular oxygen needed for aerobic metabolism of
microorganisms and roots. H2O2 typically dissociates in
the soil to produce one-half mole of dissolved oxygen per
mole of H2O2 as shown by the equation: H2O2 + H2O →
0.5 O2 + 2 H2O. This reaction can be catalyzed by iron
or by catalase enzyme, which is ubiquitous in aerobic
organisms (Petigara et al., 2002). Bhattarai et al. (2004)
found that injecting H2O2 through the irrigation system into
a heavy clay soil, which was saturated or at eld capacity,
increased biomass and yield of zucchini, soybean, and
cotton after treatments of 1, 3, and 4 months, respectively.
Injecting H2O2 into the soil through the irrigation system
may also be an effective method to alleviate potential root
asphyxia of avocado trees in ood-prone or slow-draining
soils. If this method is successful for avocado, adequate
productivity of this very ood-sensitive fruit crop may
be sustained in areas where soils are at risk of becoming
decient in oxygen.
The objective of this study was to evaluate the
effect of injecting H2O2 into a low air content soil
through subsurface drip irrigation on plant water status,
photosynthesis, biomass, and anatomy of avocado trees.
MATERIALS AND METHODS
Plant material
The experiment was conducted with 3-year-old
‘Hass’ avocado trees (Persea americana Mill.) grafted
onto ‘Mexícola’ avocado rootstock seedlings that were
planted in a heavy clay loam soil in 200-L containers.
These were constructed by placing a white plastic mesh
sustained by a structure of metal wire around a mass of
soil. The soil was obtained from a fallow hillside with
typical soil characteristics of avocado orchards planted
on hillsides. Physical characteristics of the soil are shown
in Table 1. The soil was steam-sterilized and periodically
treated with metalaxil and fosetyl-Al fungicides to avoid
Phytophthora root damage. Plants were irrigated with a
localized irrigation system consisting of 16 drippers (0.5
L h-1) per plant. The irrigation frequency varied from 2 to
4 times per day (according to daily evapotranspiration)
in order to maintain a relatively constant water content
near eld capacity (-0.33 kPa). Irrigation water and soil
analyses indicated no salt or carbonate problems. Each
tree was fertilized once a week from October to March
with 145 g N, 10 g P, 63 g K, and 14 g Mg.
Climatic conditions
The outdoor study site was located at the INIA
Regional Research Center in La Cruz (32º49’ S; 71º13’ W),
Valparaíso Region, Chile. The region has a humid marine

99
P. GIL et al. – EFFECT OF INJECTING HYDROGEN PEROXIDE…
Mediterranean climate with a mean annual temperature of
14.5 ºC, a minimum mean temperature of 5.2 ºC (July),
and a maximum mean temperature of 29.3 ºC (January).
The nine-month period from September to May is frost-
free. The mean total annual precipitation in the region is
328.5 mm with over 80% of it occurring from May to
August.
Experimental design
The experiment was carried out from November 2006
to March 2007 (spring-summer season). Plants were
divided into two treatments, a control treatment (T0) and an
H2O2 injection treatment (T1). The T0 treatment consisted
of frequently irrigated trees to maintain soil water content
near eld capacity and a seasonal mean of total soil
aeration at 17%. The T1 treatment consisted of frequently
irrigated trees to maintain soil water content near eld
capacity and a seasonal mean of total soil aeration at
16%, plus a 1 mg kg-1 of H2O2 (50%) solution injected
into the soil through the irrigation system at the end of
the irrigation period. The injection time corresponded to
10% of the total irrigation period. The H2O2 concentration
was chosen following the methodology described by
Bhattarai et al. (2004). The total amount of H2O2 applied
from November to March of the 2006-2007 summer
season was approximately 700 mL per tree; the water
volume applied each day was calculated from estimated
crop evapotranspiration, using a Class A evaporation pan
with a coefcient (Kb) of 0.8 and crop coefcient (Kc) of
0.72 (Gardiazábal et al., 2003). The drippers were buried
in the soil at a depth of 3 cm. The diluted H2O2 solution
was injected from a 200-L container. The experimental
design was a completely randomized design with ve
replications per treatment.
Data collection
Soil physical properties. Soil bulk density (BD) was
determined by using the cylinder method (Blake and
Hartage, 1986). Final BD values were the mean of three in
situ measurements and one laboratory measurement. Total
soil porosity was calculated as described by Danielson and
Sutherland (1986) using a real density soil value of 2.64 g
cm-1, which is a typical value in most mineral-originated
soils (Blake and Hartage, 1986). Soil macroporosity (air
capacity) in situ was calculated as explained by Ball and
Smith (1991). The in situ value was compared with a
laboratory air capacity measurement obtained using the
methodology illustrated by Carrasco (1997). The soil
water content at eld capacity (FC) was determined six
times in situ during the experimental period by means
of the Cassel and Nielsen (1986) method; the six in situ
laboratory measurements were pooled to obtain a mean
FC value. The FC was calculated by subtracting the
percentage of macropores from the percentage of total
porosity; the remaining percentage of pores corresponded
to the total microporosity, which when lled with water
equals the water content at eld capacity (Danielson and
Sutherland, 1986). The soil water content at eld capacity
was calculated by multiplying the gravimetric water
content (ω) by the BD value as expressed by Cassel and
Nielsen (1986).
Soil moisture. Soil water content was measured daily by
means of frequency domain reectometry (FDR) at a soil
depth of 30 cm using a Diviner probe (Diviner 2000,
Sentek Sensor Technologies, Stepney, Australia). The
soil water content was also determined gravimetrically
(ω) and volumetrically (θ) at a soil depth of 30 cm. The
ω water content was used to calibrate the FDR probe
soil moisture data (mm) and FC determination in each
soil replication. Calibration equation examples were ω
= 1.2089 x mm - 14.795 (R2 = 0.86) and ω = 0.9336 x
mm - 7.6278 (R2 = 0.86), for T1 and T0 soils respectively.
The ω of the soil was determined by the formula: ω
= [(wet soil weight - dry soil weight)/dry soil weight] ∗
100. The soil θ was computed by multiplying ω with the
BD value.
Soil air content. Volumetric air content of the soil was
calculated as described by Benavides (1994). In brief,
soil air content was calculated as the result of total
porosity minus the mean of soil water content during the
experimental period (% Soil air content = % Porosity - θ).
Soil oxygen diffusion rate (ODR), CO2 and O2 content.
The soil oxygen diffusion rate (ODR) was measured
Table 1. Physical characteristics of heavy loam clay soil.
T0 Loam clay 1.4 20.0 46.0 28.6 17.5 17.4
T1 Loam clay 1.5 18.3 43.8 27.3 16.5 15.6
Values are means of in situ and laboratory measurements; FC: eld capacity; BD: bulk density.
Tmt
Soil
texture
%
MicroporosityPorosity
Air
capacity
Air
content
FC
ω
g cm3
BD

100 CHILEAN J. AGRIC. RES. - VOL. 69 - Nº 1 - 2009
on three dates throughout the experimental period
(12/27/07, 01/24/07, and 03/15/07) with a Pt-electrode
(Oxygen Diffusion Meter, Eijkelkamp, Netherlands) as
illustrated by Letey and Stolzy (1964). Measurements
were made during the morning hours with two irrigation
pulses applied during that period; the Pt-electrode
was inserted at a depth of 15 cm into the soil. Soil air
was sampled at a depth of 30-cm by using a “point-
source soil atmospheric sampler” as found in Staley
(1980). Samples were collected on two dates (22/02/07
and 14/03/07) during the morning before starting to
irrigate. Samples were analyzed by injecting a 1 mL
headspace sample into a gas chromatograph (Perkin-
Elmer AutoSystem XL, Waltham, Massachusetts,
USA) equipped with a thermal conductivity detector
(TCD) and a CTR-1 column.
Plant water relations. Stomatal conductance (gs) and
transpiration (T) were measured with a Li-1600 steady-
state porometer (LI-COR, Lincoln, Nebraska, USA) as
demonstrated in Prive and Janes (2003). Both gs and
T were measured at 2-wk intervals during the morning
(09:00-11:00) and in the afternoon (13:00-16:00).
Three mature, sun-exposed leaves from each plant were
measured. Stem (xylem) water potential (SWP) was
calculated at the same frequency as for gs and T, whereas
for SWP determinations, three sun-exposed leaves per tree
were covered with plastic and aluminum foil and excised
30 min after covering (Meyer and Reicosky, 1985). The
SWP of the excised leaves was immediately measured
with a pressure chamber in accordance with Scholander et
al. (1965). Leaves were excised and SWP was measured
during the morning (09:00-11:00) and in the afternoon
(13:00-16:00).
Net CO2 assimilation. Net CO2 assimilation (A) was
measured once a month with an open system Li-6400
portable gas analyzer (LI-COR, Lincoln, Nebraska,
USA). Measurements were made from 10:00 to 13:00 by
taking three mature leaves from each plant, all of similar
size and light exposure, located in the middle of a spring
shoot. Measurements were made with a photosynthetic
photon ux (PPF) ranging from 1300 to 1900 µmol m-2 s-1,
which is above the light saturation point for maximum net
CO2 assimilation of avocado leaves (Whiley and Schaffer,
1994). Reference CO2 concentration in the leaf cuvette
ranged from 375 to 400 ppm, and the airow rate into the
cuvette was set at 200 µmol s-1.
Plant water use efciency (WUEb). Water use efciency
expressed as total plant dry matter produced in relation to
the amount of water applied (WUEb) was calculated by
dividing the nal total plant dry weight by the volume of
water supplied to the plants from the time of planting to
harvest (Bhattarai et al., 2004).
Biomass and leaf area. Plants were harvested at the end
of the study period when aerial parts were separated from
the roots and the fresh weights of leaves, shoots, and wood
were determined using a digital scale (Shanghai SP-300,
Shanghai Huade Weighing Apparatus, Shanghai, China).
Shoot refers to the current season’s branches whereas
wood refers to the older trunk and branches. Tissues were
then oven-dried at 70 ºC for 3 days when leaf shoot and
wood dry weights were calculated with an electronic scale
(Transcell ESW-5M, Transcell Technology, Buffalo Grove,
Illinois, USA). Root density was found by subsampling
roots with a 4.6-cm diameter, 1-m long tube sampler
(Split tube sampler, Eijkelkamp, Netherlands) inserted
into the soil (Ferreyra et al., 1984; 1989). Depending on
the depth of the soil in the sampled pot, the depth of the
soil sampled for root density ranged between 35 and 45
cm. Root samples were washed and separated from the
soil in order to determine fresh weights. Roots were then
oven-dried at 70 ºC for 3 days and root dry weight and
density (g cm-3) were measured for each plant. Total root
dry weight was estimated by multiplying the root density
by the total soil volume in each pot.
After detaching and weighing all the leaves of each
tree, 300 leaves from each tree were randomly sampled
and leaf area was measured with a portable leaf area
meter (model LI-3000C, LI-COR, Lincoln, Nebraska,
USA). The sample was weighed with an electronic scale
(Transcell ESW-5M, Transcell Technology, Buffalo
Grove, Illinois, USA) and the total leaf area per plant was
then estimated by multiplying the area/weight ratio of the
300 sub-sampled leaves per plant by the total leaf weight
per plant.
Vascular anatomy of active roots and spring shoots.
Three 2-mm diameter pieces of active roots and three
2-mm diameter pieces of spring shoots were sampled
from three plants (replications) in each treatment at the
end of the experiment. Finer roots were selected for
histological examination because it has been suggested
that these are the most active in direct uptake of water and
minerals (Zilberstaine et al., 1992). Samples were xed
in a formalin-acetic acid-alcohol solution (10 formalin: 5
acetic acid: 50 ethanol, by volume) (Ruzin, 1999). The
tissue was embedded in water-soluble wax. Wax blocks
with a thickness of 6-18 µm were cut from the embedded
shoot and root tissues and 5-µm-thick sections were cut
from the tissue and wax blocks using a rotary microtome
(Spencer 820 Microtome, American Optical Co., Buffalo,
New York, USA). Sections were stained with safranin and
fast green.

101
Histological sections were observed at 100X for
roots and 40X for shoots using a Leitz orthoplan optical
microscope with an incorporated semiautomatic camera
(Leitz, Wetzlar, Germany). Images were analyzed for mean
vessel area and total xylem area using Sigma Scan Pro 5.0
software (Systat Software, Richmond, California, USA).
Scion Image for Windows Beta 4.02 (Scion Corporation,
Frederick, Massachusetts, USA) was used to determine
the mean number of vessels per root in xylem tissue. To
work out the xylem/phloem ratio in shoots and roots, both
areas were measured in each photomicrograph using the
Sigma Scan Pro 5.0 software and the ratio was obtained
by dividing the xylem area by the phloem area.
Climatic variables. Temperature and relative humidity
were continuously monitored throughout the experiment
with a Hobo datalogger (Onset Computer Corporation,
Pocasset, Massachusetts, USA) and vapor pressure decit
was calculated using these variables.
Data Analysis
Data was expressed as means ± standard error (SE). Effects
of treatment on ODR, CO2 and O2 soil concentrations,
gs, T, SWP, A, leaf area, biomass, xylem/phloem ratio,
and root xylem vessel diameter were analyzed utilizing
ANOVA and the Bonferroni test using the SAS statistical
software (SAS Institute, 1989).
RESULTS AND DISCUSSION
Water content and physical soil properties such as
texture and structure are the factors that most affect soil
aeration. The higher the soil water content, the lower the
air volume, and greater are the limitations to the aerobic
metabolism of the roots (Letey, 1961; Blokhina et al.,
2003). Fine textured soils have a greater water retention
capacity than coarser textured soils. Therefore, a slight error
in the irrigation rate or frequency may lead to continuous
anaerobic conditions in the root zone (Letey, 1961;
Blokhina et al., 2003). The physical soil characteristics
measured in this experiment are summarized in Table 1.
The mean air content in the soil was determined from this
data as well as the mean gravimetric soil water content
(θ) during the season (Table 1). Both volumetric soil
water content (ω) and θ (data not shown) were similar
for each treatment throughout the experimental period.
Furthermore, laboratory analysis of Phytophthora in soil
samples taken at the end of the experimental period did
not indicate the presence of this soil pathogen.
Although plants in the present study were set in a
loam clay-textured soil maintained near eld capacity
with average soil air content lower than 17% (Table 1),
ODR never reached limit values for avocado (0.17 µg cm-2
min-1, Stolzy et al., 1967). The mean ODR throughout the
experimental period is shown in Table 2. While ODR was
higher in the T1 treatment, differences between treatments
were not statistically signicant (P > 0.1). Moreover, no
signicant differences were found between treatments in
CO2 or O2 concentrations in the soil (Table 2), although
the O2 soil concentration tended to be higher in soil treated
with H2O2.
P. GIL et al. – EFFECT OF INJECTING HYDROGEN PEROXIDE…
Table 2. Oxygen diffusion rate (ODR) and soil gas content.
Control 0.34 ± 0.03 4.97 ± 0.23 0.39 ± 0.09
H2O2 injection 0.39 ± 0.04 5.16 ± 0.12 0.40 ± 0.05
Signicance NS NS NS
Values represent means ± statistical error. ODR was the mean of three
measurement dates, soil gas concentration was the mean of two measurement
dates. NS: no signicant difference (Bonferroni Test, P > 0.1).
Treatment
µg cm-2 m-1
ODR %O2%CO2
Injecting H2O2 into heavy loam clay soil managed at
near eld capacity water content during 4 months resulted
in an increase in biomass of the aerial portion of avocado
trees as well as higher WUEb. Plants in the H2O2 injection
treatment had signicantly higher wood (shoots plus old
wood) and leaf dry weights than plants in the control
treatment (P ≤ 0.05); the wood and leaf dry weights were
27% and 28% higher, respectively for the T1 over the
controls (Table 3). The T1 treatment was also signicantly
higher in total plant dry weight than the controls (P ≤ 0.1).
However, no signicant difference in root density (data
not shown) or total root dry weight between treatments
was noted (Table 3). Leaf area was signicantly (43.1%)
greater (P ≤ 0.05) for plants in the T1 treatment than in
the controls (Table 3). WUEb calculated from the total
biomass divided by the total water supply showed
statistical differences between treatments (P ≤ 0.05)
(Table 3). Similar results have been reported for zucchini,
soybean, and cotton (Bhattarai et al., 2004). An increase
in growth has also been observed in tomato (Lycopersicon
esculentum Mill.) plants cultivated in ooded conditions
when H2O2 was added to the ood solution (Bryce et
al., 1982). Similar results were also identied for maize
(Zea mays L.), where a signicant gain in biomass was
due to the application of H2O2 to soil exhibiting excellent
structure and adequate irrigation rates (Melsted et al.,
1949).
Although injecting H2O2 into the soil signicantly
increased the biomass of avocado trees, no signicant
effect of H2O2 injection on gs, T, A or SWP was noted.
No signicant differences between treatments for gs T, A,
or SWP (P > 0.1) were observed (Table 4). Although it is
well-known that prolonged root hypoxia causes stomatal
closure and lowers transpiration in avocado trees (Schaffer

102 CHILEAN J. AGRIC. RES. - VOL. 69 - Nº 1 - 2009
et al., 1992; Schaffer, 1998; Schaffer and Whiley, 2002), it
was not possible to observe any signicant improvement
of gs and T as a result of adding H2O2 to the soil oxygen
supply in this study. The same phenomenon was found by
Bhattarai et al. (2004) for zucchini, soybean, and cotton.
The greater biomass of plants in the H2O2 soil
injection treatment as opposed to the plants in the
control treatment should have resulted in a greater net
CO2 assimilation by plants in the treated soil. However,
no signicant difference was perceived in the measured
net CO2 assimilation rate between the H2O2 soil injection
and control treatments (Table 4). Nevertheless, it must
be pointed out that net CO2 assimilation was measured
on a leaf area basis in this study. Trees in the H2O2 soil
injection treatment had a greater total leaf area than those
in the control treatment (Table 3). Therefore, on a whole-
plant basis, net CO2 assimilation was signicantly higher
for plants in the H2O2 injection treatment than in the
control treatment, presumably accounting for the greater
biomass of plants in soil injected with H2O2. Moreover, an
extra oxygen supply due injecting H2O2 to the soil might
enhance the ATP production in roots resulting in increased
energy for plant metabolic processes, including growth.
Although soil measurements of O2, CO2, and ODR did not
show a signicant effect on the H2O2 treatment, it must be
pointed out that measurements were not continuous and
were performed only three times during the measurement
season. As a consequence, it was not possible to see
changes in either O2 and CO2 composition or ODR, during
a day with several irrigation pulses. It may therefore be
presumed that changes would be momentary and that
those specic changes in the concentration of O2 in the
soil could generate a better root oxygenation and thus ATP
production.
The xylem/phloem ratio in roots and shoots, the
number and mean area of root xylem vessels, and the
total root xylem areas are shown in Table 5. An example
of the anatomical features of xylem vessels in roots and
spring shoots is shown in Figure 1. Although for almost
all histological variables measured the roots and shoots of
treated plants had a larger xylem system, the differences
were only statistically signicant for the spring shoot
xylem/phloem ratio (P ≤ 0.15). Histological examination
of avocado roots revealed larger xylem mean vessel
diameters for plants in the H2O2 injection treatment than
those in the control treatment, indicating that H2O2 soil
injection leads to root anatomy improvement in avocado
more than growth of the root system. However, the
difference in xylem anatomy between treatments was not
statistically signicant, probably due to the high variability
in the size of xylem vessels in individual plants. Larger
xylem vessels would increase the water conduction
capacity allowing for better development of the aerial
portion of the plant. According to Poiseuille’s law, the
water ow in a vascular conduit is related to its radius by
a factor to the fourth power, meaning that a slight increase
Table 3. Final biomass, leaf area, and water use efciency (WUEb) of avocado trees.
T0 2 706.6 ± 149.8 877.32 ± 26 833.26 ± 75 996 ± 158 66 524 ± 8.1 2.41 ± 0.1
T1 3 181.9 ± 147.1 1 111.50 ± 24 1 067.48 ± 13 1 003 ± 171 95 185 ± 11.8 2.83 ± 0.1
Sig. * ** ** NS ** **
Values represent treatment means ± statistical error. * P ≤ 0.1; ** P ≤ 0.05.
NS indicates no signicant difference between treatments according to Bonferroni test (P > 0.1); T0: control treatment; T1: H2O2 injection treatment.
Tmt
Total
biomass
cm2
Wood
biomass
g dry weight
Leaf
biomass
Root
biomass Leaf area WUEb
g L-1
Table 4. Effect of injecting H2O2 into heavy clay loam soil maintained at eld capacity on water relations and physiological
variables of avocado plants.
gs, cm s-1 0.49 ± 0.03 0.51 ± 0.03 NS 0.29 ± 0.02 0.28 ± 0.02 NS
T, µg cm-2 s-1 4.50 ± 0.18 4.80 ± 0.25 NS 5.30 ± 0.24 4.80 ± 0.22 NS
SWP, kPa -0.60 ± -0.05 -0.63 ± -0.04 NS -0.92 ± -0.04 -0.88 ± -0.04 NS
A, µmol s-1 m-2 4.86 ± 0.48 5.38 ± 0.58 NS
Values are means ± statistical error. T0: control treatment; T1: H2O2 injection soil treatment; gs: stomatal conductance; T: transpiration; SWP: soil water
potential; A: net CO2 assimilation; AM: 09:30 to 11:00; PM: 13:00 to 3:00. NS indicates no signicant difference between treatments according to
Bonferroni test (P > 0.1).
AM
T0
PM
T1Signicance T0T1Signicance

103
in xylem vessel diameter could result in a signicant
increase in water conductivity through it. Further indirect
evidence of increased water conductivity in avocado trees
in the H2O2 injection treatment was also observed in the
histological sections of the spring shoots where a higher
xylem/phloem ratio was observed. According to several
researchers (Kozlowski, 1997; Hsu et al., 1999; Liao and
Lin, 2001), plants in low-oxygen soils exhibit a reduction
in the xylem/phloem ratio. Therefore, in the present study
of avocado trees, the increased xylem/phloem ratio in
plants in the H2O2 soil injection treatment might be an
indirect indication that H2O2 increased the oxygen content
of the root zone.
CONCLUSIONS
Injecting H2O2 through the irrigation system into
a soil with low air content signicantly increased the
biomass of the aerial portions of the plant and water use
efciency (plant biomass per water applied [WUEb]), but
had no signicant effect on measured CO2 assimilation,
transpiration, stomatal conductance, or steam water
potential. Furthermore, xylem vessel diameter and the
xylem/phloem ratio tended to be greater for trees in soil
injected with H2O2 than in the controls.
The increased biomass of the aerial portions of plants
in treated soil indicates that H202 soil injection might
have potential as a method for improving soil oxygen
content in a heavy clay loam soil. However, the present
study was conducted with trees in containers, and before
this method can be used to mitigate damage caused by
low soil aeration in avocado orchards, further studies are
needed to evaluate the practical and economic feasibility
of using hydrogen peroxide on a larger scale in orchards.
ACKNOWLEDGEMENTS
This study was nanced by INNOVA-CORFO
(Chile). We thank Dr. Jaime Salvo from INIA La Cruz
for providing equipment and assistance for photosynthetic
measurements, Dr. Bruno Delippi and Dr. Reynaldo
Campos from INIA La Platina for the use of equipment
and assistance with gas analysis methodology, Mrs.
Marisol Pérez from INIA La Platina for assistance with
laboratory measurements, Mr. Raúl Eguiluz from INIA
La Platina for soil laboratory analysis, Dr. Bruce Schaffer
from the Tropical Research and Educational Center,
University of Florida, for his helpful editorial comments,
and Ms. Claudia Fassio from the Ponticia Universidad
Católica of Valparaíso for her assistance with histological
P. GIL et al. – EFFECT OF INJECTING HYDROGEN PEROXIDE…
Table 5. Effect of injecting H2O2 to heavy clay loam soil maintained at eld capacity on root and shoot vascular
anatomy.
Spring shoot xylem/phloem relationship 1.5 2.0 *
Root xylem/phloem relationship 1.5 1.4 NS
Number of root xylem vessels 59.8 63.6 NS
Mean xylem vessel area, µm2 2 392.5 2 418.4 NS
Xylem total area, µm2 146 630 154 202 NS
T0: control treatment; T1: soil H2O2 injection treatment. Values are means; NS: no signicant difference according to Bonferroni test (P > 0.15).
* (P > 0.15).
T0T1Signicance
Figure 1. Anatomical features in avocado root and shoot vascular tissue. A) Root section from a plant in the H2O2 soil
injection treatment (100X). B) Spring shoot section from a plant in the H2O2 soil injection treatment (40X). “X”
indicates xylem tissue; “P” indicates phloem tissue.
A B

104 CHILEAN J. AGRIC. RES. - VOL. 69 - Nº 1 - 2009
analysis. We also thank Mr. Cristóbal Gentina from the
Universidad del Mar and Ms. María José Pino from the
Instituto de Educación Rural for their cooperation during
several outdoor measurements. This work is part of a Ph.D.
thesis in Agricultural Science (PM Gil) at the Ponticia
Universidad Católica de Chile, Facultad de Agronomía e
Ingeniería Forestal.
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RESUMEN
Efecto de la inyección de peróxido de hidrógeno en
suelo franco arcilloso pesado, sobre el estado hídrico,
asimilación neta de CO2, biomasa y anatomía vascular
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drenados con bajo contenido de oxígeno, lo que limita
producción y calidad de fruta. El objetivo de este estudio
fue evaluar el efecto de la inyección de peróxido de
hidrógeno (H2O2) al suelo como fuente de O2, sobre el
estado hídrico, asimilación de CO2, biomasa y anatomía
de paltos en suelo franco arcilloso con contenido de
humedad cercano a capacidad de campo. Paltos cv. Hass
de 3 años fueron establecidos en condiciones ambientales
no forzadas, plantados en contenedores con suelo franco
arcilloso pesado cuya humedad fue mantenida cercana
a capacidad de campo. Las plantas se dividieron en
dos tratamientos: inyección de H2O2 al suelo mediante
riego por goteo subsupercial y plantas sin inyección de
H2O2 (control). Además de determinar las características
físicas del suelo, se determinó la asimilación neta de
CO2 (A), transpiración (T), conductancia estomática
(gs), potencial hídrico xilemático (SWP), biomasa aérea
y radical, eciencia de uso del agua (materia seca total
producida en relación a la cantidad de agua aplicada
[WUEb]) y características anatómicas vasculares en
brotes y raíces. La inyección de H2O2 al suelo aumentó
signicativamente la biomasa aérea de las plantas y
también el WUEb, pero no tuvo efectos signicativos en
A, T, gs, o SWP. El diámetro de los vasos xilemáticos y la
relación xilema/oema tendieron a ser mayores en árboles
situados en suelos tratados con H2O2 comparados con los
controles. El aumento en la biomasa aérea de paltos en
suelo tratado indica que la inyección de H2O2 en suelos
franco arcillosos pesados puede ser una útil herramienta
de manejo en suelos pobremente aireados.
Palabras clave: conductancia estomática, fotosíntesis
neta, histología radical, inyección de oxígeno, hipoxia
radical, riego por goteo subsupercial.

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