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AUSTRALIAN JOURNAL
OF PLANT PHYSIOLOGY
An International Journal of Plant Function

Volume 28, 2001

CSIRO 2001

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Aust. J. Plant Physiol., 2001, 28, 801806

Response of oxidative metabolism to the application

of carbendazim plus boron in tobacco
Pablo C. Garca, Rosa M. Rivero, Luis R. Lpez-Lefebre, Esteban Snchez, Juan M. Ruiz
and Luis RomeroA
Department of Plant Biology, Faculty of Sciences, University of Granada, 18071-Granada, Spain.
ACorresponding author; email: lromero@ugr.es
Abstract. In view of the essential role of oxidative process in the development of pathogen resistance in plants, the
aim of the present study was to determine the individual effect of a fungicide, as well as to determine the combined
effect of the fungicide and boron (B) on superoxide dismutase (SOD), guaiacol peroxidase (GPX), catalase (CAT),
and ascorbate peroxidase (APX) activities and H2O2 levels in tobacco plants (Nicotiana tabacum L. cv. Tennessee
86). The fungicide applied was carbendazim (carb) at a concentration of 2.6 mM. Boron was applied as H3BO3 at:
1.6 mM (B1), 4 mM (B2), 8 mM (B3), 16 mM (B4), 32 mM (B5), and 64 mM (B6). The results indicated that foliar
application of carbendazim by itself does not increase SOD, GPX, CAT or APX activities or H2O2 foliar
accumulation. The combined application of carbendazim and B increased SOD, GPX, CAT, and APX activities,
especially in carb-B3 and carb-B4. This effect may signify an additional tolerance mechanism to pathogenic
infection, given the participation of these enzymes in the early phases of the plantpathogen interactions.
Key words: Boron, carbendazim, Nicotiana tabacum L., oxidative metabolism.

Introduction
Plants react to pathogen attack through a variety of active
and passive defence mechanisms. At the site of infection, a
hypersensitive response (HR) is often initiated in resistant
plants, which is frequently manifested as necrotic lesions
resulting from host-cell death. In addition, the distal
uninfected parts of the plants usually develop systematic
acquired resistance (SAR) which results in enhanced
long-lasting resistance against the same or even unrelated
pathogens (Wendehenne et al. 1998).
Active oxygen species (AOS), such as superoxide
radicals (O2), hydroxyl (OH), and primarily hydrogen
peroxide (H2O2), play a fundamental role in the defence
processes of the plant, being involved in determining the
result of plantpathogen interactions. The key role of AOS is
based on the fact that H2O2 has been demonstrated to be
toxic to microorganisms, in addition to leading to oxidative
cross-linking of the glycoproteins of the cell wall (Bolwell
and Wojtaszek 1997), reducing the susceptibility to enzymatic degradation (Brisson et al. 1994), inducing SAR
(Chen et al. 1993), and finally orchestrating the hypersensitive cell death response (Tenhacken et al. 1995). The

AOS production in plants is regulated by the enzymatic

action of superoxide dismutase (SOD), guaiacol peroxidase
(GPX), catalase (CAT), and ascorbate peroxidase (APX),
among others, which act jointly to achieve an optimal
balance of AOS.
On the other hand, certain deficiencies in mineral
nutrients, such as zinc (Zn), manganese (Mn), iron (Fe),
copper (Cu), magnesium (Mg), potassium (K), and B, can
modulate these enzymatic activities (Cakmak et al. 1995).
Specifically, the role of B bears mentioning, since this
nutrient acts through such oxidative enzymes as polyphenol
oxidase (PPO), and highly active oxidized compounds such
as quinones, which are also responsible for producing AOS
(Pillinger et al. 1994; Ruiz et al. 1998, 1999).
Similarly, it has been suggested that certain fungicides
used in order to mitigate or prevent pathogen attack, could
be involved in activating certain defensive responses of
plants (Nemestothy and Guest 1990). However, the fact that
such substances could influence the enzymatic activities of
the oxidative process, has scarcely been studied.
Therefore, the present study was designed to ascertain
experimentally, with the foliar application of the micro-

Abbreviations used: AOS, active oxygen species; APX, ascorbate peroxidase; BSA, bovine serum albumin; carb, carbendazim; CAT, catalase;
DTT, 1,4-dithio-DL-threitol; EDTA, ethylenediamine tetraacetic acid; GPX, guaiacol peroxidase; HEPES, 4-(2-hydroxyethyl)-piperazine1-ethanesulfonic acid disodium salt; HR, hypersensitive response; H2O2, hydrogen peroxide; NBT, nitro blue tetrazolium;
PMSF, phenylmethanesulfonyl fluoride; PPO, polyphenol oxidase; PVP, polyvinylpyrrolidone; SAR, systemic acquired resistance;
SOD, superoxide dismutase.
CSIRO 2001

10.1071/PP00098

0310-7841/01/080801

802

nutrient B and the fungicide carbendazim on tobacco plants,

the effects that these two substances can have on the H2O2
levels and enzymatic activities related to SOD, GPX, CAT,
and APX, with the final aim of inducing SAR in
non-infected plants. The plants chosen for this experiment
were tobacco, because prior research in our laboratory has
revealed that, in this plant, B substantially influences of B in
the metabolism of phenolic compounds, which are known to
have an outstanding role in plant defensive responses (Ruiz
et al. 1999).
Carbendazim was used because (1) it is one of the most
widely used fungicides in south-eastern Spain, a zone of
intensive agriculture, and (2) because, being a fungicide of
broad preventive spectrum, it is applied to a large proportion
of the crop that is not even infected by the pathogen (Tomlin
1994).
Materials and methods
Crop design and plant sampling
Seeds of Nicotiana tabacum L. cv. Tennessee 86 were sown in May
(1999) in southern Spain (Granada). The seedlings were grown in trays
of 200-well trays using peat moss as media culture, inside an
experimental greenhouse for 45 d, and then transferred to individual
pots (25 cm upper diameter, 17 cm lower diameter, 25 cm in height)
filled with vermiculite. The plants were grown in a cultivation chamber
under controlled environmental conditions with relative humidity of
6080%, temperature of 30/20C (day/night), and a 16-h photoperiod
at a photosynthetically active photon flux density (PPFD) of
350 mol m2 s1 (measured at the top of the plants with a 190 SB
quantum sensor, Li-Cor Inc., Lincoln, NE, USA). For 1 month (from
day 45 until day 75 after sowing), before the experimental treatments,
the plants received a nutrient solution of: 6 mM KNO3, 2 mM NaH2PO4,
1.5 mM CaCl2, 1.5 mM MgSO4, 5 M Fe-EDTA, 2 M MnSO4,
1 M ZnSO4, 0.25 M CuSO4, 0.1 M (NH4)6Mo7O24 and 2.5 M
H3BO3. The nutrient solution (pH 5.56.0) was renewed every 3 d.
At 75 d after sowing, we performed a foliar spraying of the
treatments. The treatments were applied to plants to runoff as aqueous
foliar sprayings containing the surfactant Tween 20 (0.5% v/v), using a
stainless steel sprayer. The fungicide carbendazim (benzimidazole 2-il
methyl carbamate; C9H9N3O2) was applied at 100% purity and a
concentration of 2.6 mM, as recommended for foliar spraying.
Boron was applied in the form of H3BO3: 1.6 mM (B1), 4 mM (B2),
8 mM (B3), 16 mM (B4), 32 mM (B5), or 64 mM (B6). The B dosage
chosen was within the range normally applied to foliage (Haggag et al.
1995; Bondok 1996; Singh and Riwari 1996). In all, there were eight
treatments: one without carbendazim and without B (control), one with
only carbendazim, and six combinations of carbendazim with each
concentration of B (carb-Bx). The experimental design was a
randomized complete block with eight treatments, arranged in
individual pots with four plants per treatment, each replicated three
times.
Each treatment was applied three times fortnightly. The plants were
sampled beginning approximately at the 14-leaf stage, just before the
onset of flowering. From the same plants, leaves were sampled once, on
day 120 after sowing, from nodes 1013. All the sampled leaves were
in the mature state, with lengths of more than 10 cm. The material was
rinsed three times in distilled H2O after disinfecting with non-ionic
detergent at 1% (v/v) (Decon 90, Merk) (Wolf 1982), then blotted on
filter paper. A subsample of leaves was used fresh for the analysis of
enzymatic activities of SOD, GPX, CAT, APX and H2O2 performing

P. C. Garcia et al.

triplicate assays for each extraction, while the other subsample was
dried in a forced-air oven at 70C for 24 h.
Plant analysis
Extraction and assay of SOD (EC 1.15.1.1).
SOD activity was assayed by monitoring the inhibition of
photochemical reduction of nitro blue tetrazolium (NBT), using the
method of Giannopolitis and Ries (1977) and Beyer and Fridovich
(1987). Frozen leaf samples were weighed and homogenized on ice
with a mortar and pestle for 2 min with 1/4 teaspoon of sand and 10 mL
of homogenizing solution containing 50 m M 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid disodium salt (HEPES) buffer and
0.1 mM Na2 ethylenediamine tetraacetic acid (EDTA) at pH 7.6. The
homogenate was centrifuged at 4C for 15 min at 15 000 g, then filtered
through qualitative filter paper (MN 617, Macherey-Nagel, Germany)
to produce the crude extract.
For total SOD assay, a 5-mL reaction mixture contained
50 mM HEPES (pH 7.6), 0.1 mM EDTA, 50 mM Na2CO3 (pH 10.0),
13 mM methionine, 0.025% (w/v) Triton X-100, 63 M NBT,
1.3 M riboflavin, and an appropriate aliquot of enzyme extract. The
reaction mixtures were illuminated for 15 min; light intensity was
380 mol m2 s1. Identical reaction mixtures that were not illuminated
were used to correct for background absorbance. One unit of SOD
activity was defined as the amount of enzyme required to cause 50%
inhibition of the reduction of NBT as monitored at A560.
Extraction and assay of GPX (EC 1.11.1.7) and CAT (EC 1.11.1.6)
The method used was a modified version of that proposed by Kalir
et al. (1984) and Badiani et al. (1990). Fresh plant material was ground
with 50 mM Trisacetate buffer, pH 7.5, 5 mM 2-mercaptoethanol,
2 mM 1,4-dithio-DL-threitol (DTT), 2 mM EDTA, 0.5 mM phenyl
methanesulfonyl fluoride (PMSF), and 1% (w/v) polyvinylpyrrolidone
(PVP). The homogenate was filtered through two layers of Miracloth
and centrifuged for 30 min at 37 000 g. The pellet was discarded and
the supernatant used for GPX and CAT assays.
The GPX activity was determined by following the change of A485
due to guaiacol oxidation (Kalir et al. 1984; Ruiz et al. 1998). The
reaction mixture contained 100 mM Trisacetate buffer, pH 5.0,
1 mM guaiacol and 0.003 mM H2O2. To test whether the reaction was
due to peroxidase, control assays contained catalase from bovine liver
(EC 1.11.1.6) (Fluka, Madrid, Spain) (420 units in 0.1 mL H2O). To
determine whether the reaction was enzymatic, the sample extract was
boiled and assayed.
The CAT activity was determined by following the consumption of
H2O2 at A240 for 5 min in 3 mL of a reaction mixture (Kalir et al. 1984;
Rao et al. 1997). The reaction mixture contained 25 m M Trisacetate
buffer, pH 7.0, 0.8 mM EDTA-Na and 20 mM H2O2. Both enzyme
assays were performed at 25C with equal amounts of protein (100 g).
Extraction and assay of APX (EC 1.11.1.11)
The method used was a modified version of that proposed by
Nakano and Asada (1981). Frozen leaf samples were weighed and
homogenized on ice with a mortar and pestle for 2 min with
1/4 teaspoon of sand and 5 mL of homogenizing solution containing
100 mM of phosphate-K, pH 7.5. The homogenate was filtered through
two layers of Miracloth and centrifuged for 20 min at 15 000 g. The
pellet was discarded and the supernatant used for APX assays.
The APX activity was determined by following the change of A290
for 5 min due to consumption of ascorbate (Nakano and Asada 1981).
The reaction mixture (2.5 mL), contained 100 m M phosphate-Na
buffer, pH 7.5, 0.2 mM H2O2, 0.5 mM ascorbate-Na.
Protein content was measured following the Bradford method, with
bovine serum albumin (BSA) as a standard (Bradford 1976).

Carbendazim and oxidative metabolism in tobacco

The method used was a modified version of that proposed by

MacNevin and Uron (1953) and Brennan and Frenkel (1977).
Hydroperoxides form a specific complex with titanium (Ti4+), which
can be measured by colorimetry. Peroxides were extracted by
homogenizing 1 g foliar tissue in 2 mL cold acetone. The homogenate
was filtered and the filtrate brought to 3 mL with water. Two mL of a
titanium reagent (20% titanic tetrachloride in concentrated HCl, v/v)
were added to 2-mL samples of the peroxide extract, followed by the
addition of 2 mL concentrated NH 4OH to precipitate the
peroxide-titanium complex (MacNevin and Uron 1953). After
centrifugation (5 min at 10000 g), the supernatant was discarded and
the precipitate solubilized in 7.5 mL of 2 N H2SO4, washed repeatedly
with acetone, and brought to final volume of 10 mL with water. The
absorbancy of the resulting solutions was read at A415 against a water
blank. The concentration of the peroxide in the extracts was
determined by comparing the absorbancy against a standard curve
representing titanium-H2O2 complex from 0.1 to 1 mM. The
hydro-peroxides, as extracted and estimated by the above procedure,
represent the total peroxides.
Estimation of total boron
Total B was analysed after digestion of dry and milled leaf material
with 6 M H2SO4 and H2O2. For the measurement of the B concentration
in leaf tissues, the azomethine-H+ method was followed, and
absorbance was read at A410 (Wolf 1974). The B concentration was
expressed as g (g dry weight)1.
Statistical analysis
The data shown are mean values s.e. Differences between treatment
means were compared using the l.s.d. at the 0.05 probability level.
Also, a correlation analysis was performed between the different
variables. Levels of significance were represented by: * P < 0.05,
** P < 0.01, *** P < 0.001 and n.s., not significant.

Results and discussion

Effect of carbendazim
The oxidative metabolism in plants is mediated by reduced
oxygen species such as the radicals O2 and H2O2, which
are inevitable by-products of cell metabolism. These free
radicals can cause lipid peroxidation, protein denaturing and
DNA mutations (Cadenas 1989). The first step that leads to
oxidative stress may be the formation of O2 radicals
(Cadenas 1989). In response, SOD reacts with O2 radicals,
catalysing their dismutation to H2O2. Therefore, SOD
appears to be central to the defence mechanisms of plants
against oxidative stress (Van Loon et al. 1986).
It is worth emphasizing the effect of certain pesticides
and herbicides, such as Paraquat on SOD activity, since
direct effects on the chloroplast can induce the formation of
harmful O2 species (Rabinowitch et al. 1987). In our
experiment, the foliar application of carbendazim increased
SOD activity by only 8% over control (P > 0.05, Table 1),
which does not imply a positive effect of the fungicide on
the defensive capacity of tobacco plants against the
oxidative stress.
Directly related to SOD activity is H2O2 concentration,
since this compound is formed almost entirely during cell

metabolism, from the dismutation of O2 catalysed by SOD

(Auh and Murphy 1995). In our experiment, the foliar levels
of H2O2 in plants treated with carbendazim were similar to
those of control (P > 0.05, Fig. 1).
On the other hand, H2O2 can be eliminated by many
scavenger enzymes in order to avoid accumulation to toxic
levels. One of these enzymes, CAT, catalyses the dismutation of newly formed H2O2, producing H2O and O2. It has
also been demonstrated that Paraquat induces CAT activity
in many cases (Rabinowitch et al. 1987). In our experiment,
the CAT activity in the plants treated with carbendazim
increased by 29%, although this rise was not significant in
relation to the CAT activity in control (P > 0.05, Table 1).
Another type of enzyme involved in the detoxification of
H2O2 is GPX. This enzyme is often found in the cell wall,
where it utilizes H2O2 to generate phenoxy compounds, such
as lignin, suberin and extensin, and thus can have defensive
functions (Vance et al. 1980). Such GPX activity can also be

Table 1. Response of oxidative metabolism to the application of

carbendazim or carbendazim plus Boron in leaves of tobacco plants
Data are means of three tobacco plants s.e. Units: SOD, g1 fresh wt;
POD, mol guaiacol oxidized mg1 protein min1; CAT, mol H2O2
reduced mg1 protein min1; APX, mol ascorbate reduced mg1
protein min1
Treatments
Control
Carb
Carb-B1
Carb-B2
Carb-B3
Carb-B4
Carb-B5
Carb-B6
l.s.d.

SOD

GPX

CAT

APX

2062.3 28.3
2219.5 31.3
2545.0 38.2
2787.4 36.5
2838.0 47.3
2878.9 45.9
2745.9 33.5
2654.5 35.2
72.97

1.32 0.17
1.47 0.21
1.88 0.25
3.23 0.36
3.85 0.31
3.53 0.35
2.31 0.30
1.91 0.33
0.401

0.26 0.01
0.33 0.02
0.37 0.01
0.54 0.02
0.68 0.04
0.65 0.03
0.35 0.02
0.26 0.02
0.105

0.078 0.01
0.074 0.01
0.129 0.02
0.204 0.02
0.386 0.03
0.392 0.03
0.112 0.01
0.109 0.01
0.061

100

LSD = 5.671
90
80

Boron, g leaf1

Extraction and quantification of H2O2

803

70
60
50
40
30
20
10
0

control

carb

carb-B1 carb-B2 carb-B3 carb-B4 carb-B5 carb-B6

Fig. 1. H2O2 concentration in tobacco leaves in response to the

application of carbendazim or carbendazim plus Boron. Data are
means of three tobacco plants. s.e. is presented as T-bars.

804

P. C. Garcia et al.

induced by other environmental factors, as in the case of

Paraquat (Hughes and Holton 1981). In our experiment, the
application of carbendazim did not significantly increase
GPX activity with respect to peroxidase activity in control
plants (P > 0.05, Table 1).
On the other hand, APX can reduce some organic
hydroperoxides. This enzyme is essential in the ascorbateglutation cycle (Asada and Takahashi 1987). Also, APX
genes are rapidly induced by various stress conditions, such
as Paraquat, ethylene, drought and thermal shock, which
suggests a major role for APX in stress tolerance (Mittler and
Zilinskas 1994). In our experiment, the APX activity in the
plants treated with carbendazim decreased by only 6% in
relation to the APX activity in control (P > 0.05, Table 1).
Finally, the application of carbendazim resulted in a
significant increase in foliar dry weight with respect to
controls (P < 0.001; Fig. 2). Tripathi et al. (1982) observed
that the application of carbendazim at a concentration of
20 g mL1, inhibits the breakdown of chlorophyll as well as
of RNA and proteins by protecting against the efflux of ions
responsible for the cell-membrane degradation. This fact
could explain the beneficial effect of carbendazim on the
foliar biomass production in our experiment.
The explanation of the results of GPX, CAT, and APX
activities (Table 1) could be that the activities of these
antioxidant enzymes are triggered about by the rise in the
H2O2 level, this being minimum among control plants and
those treated with carbendazim. In short, our results show
that carbendazim applied to tobacco plants unafflicted by
biotic or abiotic agents did not augment the activity of
antioxidant enzymes (SOD, GPX, CAT and APX), since it
did not give rise to a significant accumulation of H2O2.
These results appear to confirm the conclusion proposed
previously by Ruiz et al. (1999), indicating that the principal
effect of carbendazim application is increased biosynthesis
and accumulation of phenolic compounds. This may foster

stronger pathogen resistance in plants, given the essential

role of phenolic compounds in the lignification and suberization of the cell wall (Daayf et al. 1997).
Combined effect of carbendazim and boron
There were significant differences in B concentrations in
leaves between treatments (P < 0.001, Fig. 3). In general, all
the plants showed appropriate B levels, except for those that
received B5 and B6, concentrations that can be considered
toxic (Shelp 1993). In fact, these plants were characterized
by toxicity symptoms such as necrotic patches on the
margins and tips of older leaves (Nable et al. 1997). Finally,
it should be emphasized that the plants receiving the
intermediate B rates (B2, B3 and B4) had B concentrations of
this micronutrient lower than in carb-B1 treatments, in which
the B concentrations applied were lower (Fig. 3). Recent
work on B mobility has shown evidence of retranslocation
of this micronutrient, principally in foliar application, B
being translocated primarily through the phloem towards
growing meristem tissues (Brown and Shelp 1997). This
mobility in B could account for the decreased foliar
concentrations of this element in the carb-B2, carb-B3 and
carb-B4 treatments, in addition to the possible avoidance of
the appearance of toxicity symptoms appearing in this plant.
In our experiment, the various B treatments induced
significant differences in SOD activity (P < 0.001, Table. 1).
In this case, the highest enzymatic activities were recorded
in treatment carb-B4, and the lowest in carb at 23% lower
than in carb-B4. That is, the highest enzymatic activities
resulted from intermediate B dosages (B2, B3 and B4), these
plants also presenting lower foliar concentrations of this
micronutrient (Fig. 3). Hence, we found an inverse relationship between the B concentration and SOD activity
(r = 0.85**).
With respect to H2O2 levels (Fig. 1), the highest concentrations were reached in carb-B6, and the lowest in carb-B3
10

1.4

LSD = 0.061

LSD = 1.315

H2O2, mol g1 FW

Leaf dry weight, g DW leaf1

1.2

1.0

0.8

0.6

0.4

7
6
5
4
3
2

0.2

1
0

0.0

control

carb

carb-B1 carb-B2 carb-B3 carb-B4 carb-B5 carb-B6

Fig. 2. Leaf dry weight in tobacco in response to the application of

carbendazim or carbendazim plus Boron. Data are means of three
tobacco plants. s.e. is presented as T-bars.

control

carb

carb-B1 carb-B2 carb-B3 carb-B4 carb-B5 carb-B6

Fig. 3. Boron concentration in tobacco leaves in response to the

application of carbendazim or carbendazim plus Boron. Data are
means of three tobacco plants. s.e. is presented as T-bars.

Carbendazim and oxidative metabolism in tobacco

(P < 0.001). In this case, the trend was the opposite to that of
SOD, since the intermediate treatments (principally carb-B3)
gave the lowest H2O2 concentrations. Thus, the relationship
between these parameters was again inverse and significant
(r = 0.89***).
Meanwhile, GPX, CAT and APX activities significantly
changed after B application (P < 0.001, Table. 1), the three
enzymes behaving with a trend similar to that of SOD, since
the highest values appeared in the intermediate treatments
(carb-B3 for GPX and CAT, and carb-B4 for APX), and the
lowest in the extreme treatments (carb for GPX and APX,
and carb-B6 for CAT). By contrast, the behaviour of the
three enzymes proved entirely contrary to that H2O2 (Fig. 1).
Therefore, the GPX, CAT and APX activities showed an
inverse relationship to the H2O2 levels (H2O2GPX,
r = 0.94***; H2O2CAT, r = 0.95***, H2O2APX,
r = 0.83***). These findings appear to signify that
although the intermediate treatments (carb-B3 and carb-B4)
resulted in high SOD activity, they did not lead to a greater
H2O2 accumulation, due to the high GPX, CAT and APX
activities in these treatments (Table 1).
The dynamic of these parameters of the oxidative metabolism studied reflect an effect or direct involvement of B in this
metabolic process. In our experiment, it is evident that the
treatments with the lowest B concentrations (carb-B2, carb-B3
and carb-B4 registered the highest SOD activities (B concentrationsSOD, r = 0.84 ***), together with the highest GPX
activities (B concentrationsGPX, r = 0.87***) CAT
activities (B concentrationsCAT, r = 0.91***), and APX
activities (B concentrationsAPX, r = 0.81***). On the
other hand, the accumulation and subsequent oxidation of the
phenolics by PPO is characteristic of B-deficient tissues.
These results, as shown in Ruiz et al. (1999), arise on using
the carb-B3 and carb-B4 treatments. Oxidation of phenolic
compounds prompts the production of quinones, which are
known to be strongly toxic and responsible for producing
AOS (Pillinger et al. 1994). Therefore, the possible production of AOS from phenolic oxidation in the carb-B3 and
carb-B4 treatments, together with the highest SOD activities
in these treatments (Table 1), should boost H2O2 accumulation; however, such an increase was not detected (Fig. 1). This
may be because, under the normal conditions of our experiment (utilization of non-infected plants), most of the cells
have acquired protection mechanisms to maintain low levels
of AOS (primarily O2 and H2O2) within the cell. In this case,
the intermediate treatment carb-B3, would be the most
efficient maintaining the appropriate levels of AOS and in
preventing oxidative damage in the cell metabolism.
Nevertheless, under certain stress conditions, as in
pathogenic infection, these mechanisms are redirected by a
rapid and transitory production of large quantities of AOS,
called oxidative burst. This is one of the most peculiar
events in the early phase of plantpathogen interactions
(Low and Mrida 1996). In addition, AOS could also act as a

805

signal to promote the defence reactions (SAR) in the plant

(Wu et al. 1997).
Finally, the combined treatments of carbendazim and B
increased the dry weight per leaf (P < 0.001) over values
registered by plants treated with carbendazim alone, the
carb-B3 treatment giving the maximum dry weight per leaf
(Fig. 2). These results show the positive effect that adequate
application amounts of B can have on plant growth (Shelp,
1993; Ruiz et al. 1998). In contrast, in plants treated with B5
and B6, there was a severely diminished foliar dry weight
(Fig. 2), due to the toxicity of B on plant growth (Nable et al.
1997). In this case, the behaviour was opposite to that found
for the H2O2 concentration, since the greatest foliar dry
weight appeared in the intermediate treatments (carb-B2,
3 and 4), and these treatments showed the least H2O2
concentrations (Fig. 1). This finding may explain the negative and significant correlation between the two parameters
(r = 0.79**). These results are logical, since H202 accumulation, being toxic, normally depresses biomass production
(Willekens et al. 1997). This may be due to the fact that O2
and particularly H2O2, which are inevitable by-products of
cell metabolism, can cause lipid peroxidation, protein denaturing and DNA mutations (Cadenas 1989).
In conclusion, our experiment indicates that the combined application of carbendazim and B, particularly the
treatments carb-B3 and carb-B4, can intensify enzymatic
activities of oxidative metabolism (SOD, GPX, CAT, and
APX) in the leaves of tobacco plants. These enzymes have a
central function during the early stages of plantpathogen
interactions, controlling the levels of O2 and especially
H2O2 (Vance et al. 1980; Wu et al. 1997) and one could
speculate that carbendazim sprayed along in combination
with the correct B concentration (B3 and B4) could influence
the defence capacity against pathogens. Our work confirms
the results proposed by other authors, suggesting that certain
wide-spectrum fungicides, besides their antibiotic action
against pathogens, could be involved in the activation of
some defensive responses of the plants (Nemestothy and
Guest 1990; Molina et al. 1998).
References
Asada K, Takahashi M (1987) Production and scavenging of active
oxygen in photosynthesis. In Photoinhibition. (Eds DJ Kyle,
CB Osmond and CJ Arntzen) pp. 227228. (Elsevier: New York)
Auh C-K, Murphy TM (1995) Plasma membrane redox enzyme is
involved in the synthesis of O2 and H2O2 by Phytophthora
elicitor-stimulated rose cells. Plant Physiology 107, 12411247.
Badiani M, De Biasi MG, Felici M (1990) Soluble peroxidase from
winter wheat seedlings with phenoloxidase-like activity. Plant
Physiology 92, 489494.
Beyer WF, Fridovich I (1987) Assaying for superoxide dismutase
activity: some large consequences of minor changes in conditions.
Analytical Biochemistry 161, 559566.
Bolwell GP, Wojtaszek P (1997) Mechanisms for the generation of
reactive oxygen species in plant defence a broad perspective.
Physiological and Molecular Plant Pathology 51, 347366.

806

P. C. Garcia et al.

Bondok MA (1996) The role of boron in regulating growth, yield and

hormonal balance in sugar beet (Beta vulgaris var. vulgaris).
Annals of Agriculture Science (Cairo) 41, 1533.
Bradford MM (1976) A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of protein
dye-binding. Analytical Biochemistry 72, 248254.
Brennan T, Frenkel C (1977) Involvement of hydrogen peroxide in the
regulation of senescence in pear. Plant Physiology 59, 411416.
Brisson LF, Tenhaken R, Lamb CJ (1994) Function of oxidative
cross-linking of cell wall structural proteins in plant disease
resistance. The Plant Cell 6, 17031712.
Brown PH, Shelp BJ (1997) Boron mobility in plants. Plant and Soil
193, 181198.
Cadenas E (1989) Biochemistry of oxygen toxicity. Annual Review of
Biochemistry 58, 79110.
Cakmak I, Kurz H, Marschner H (1995) Short-term effects of boron
germanium and high light intensity on membrane permeability in
boron deficient leaves of sunflower. Physiologia Plantarum 95,
1118.
Chen Z, Silva H, Klessig DF (1993) Active oxygen species in the
induction of plant systemic acquired. resistance by salicylic acid.
Science 162, 18831886.
Daayf F, Schmitt A, Blanger RR (1997) Evidence of phytoalexins in
cucumber leaves infected with powdery mildew following
treatments with leaf extracts of Reynoturia sachaliensis. Plant
Physiology 113, 719727.
Giannopolitis CN, Ries SK (1977) Superoxide dismutase occurrence
in higher plants. Plant Physiology 59, 309314.
Haggag LF, Maksoud MA, Barkouky FMZ (1995) Effect of boron
sprays on sex ratios and fruit quality of mango (Mangfera ndica
L.) cv. Hindi Be-Sinara. Annals of Agricultural Science (Cairo) 40,
753758.
Hughes KW, Holton RW (1981) Levels of superoxide dismutase,
peroxidase and catalase in a tobacco cell line selected for herbicide
resistance. In Vitro Cellular & Developmental Biology Plant 43,
211.
Kalir A, Omri G, Poljakoff-Mayber A (1984) Peroxidase and catalase
activity in leaves of Halimione portulacoides exposed to salinity.
Physiologia Plantarum 62, 238244.
Low PS, Merida JR (1996) The oxidative burst in plant defence:
function and signal transduction. Physiologia Plantarum 96,
533542.
MacNevin WM, Uron PF (1953) Separation of hydrogen peroxide
from organic hydroperoxides. Analytical Chemistry 25, 17601761.
Mittler R, Zilinskas BA (1994) Regulation of pea cytosolic ascorbate
peroxidase and antioxidant enzymes during the progression of
drought stress and following recovery from drought. The Plant
Journal 5, 397405.
Molina A, Hunt MD, Ryals JA (1998) Impaired fungicide activity in
plants blocked in disease resistance signal transduction. The Plant
Cell 10, 19031914.
Nable RO, Bauelos GS, Paull JG (1997) Boron toxicity. Plant and
Soil 198, 181198.
Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by
ascorbate specific peroxidase in spinach chloroplasts. Plant Cell
Physiology 22, 867888.
Nemestothy GN, Guest DI (1990) Phytoalexin accumulation,
phenylalanine ammonia-lyase activity and ethylene biosynthesis in
fosetylAl treated resistant and susceptible tobacco cultivars
infected with Phytophthora nicotianae var. nicotiane.
Physiological and Molecular Plant Pathology 37, 207219.

Pillinger JM, Coope JA, Ridge I (1994) Role of phenolic compounds in

the antialgal activity of barley straw. Journal of Chemical Ecology
20, 15571569.
Rabinowitch HD, Privalle CT, Fridivich I (1987) Effects of paraquat on
the green alga Dunaliella salina: protection by the mimic of
superoxide dismutase, desferal-Mn (IV). Free Radicals in
Biological Medicine 3, 125131.
Rao MV, Paliyath G, Ormrod DP, Murr DP, Watkins CB (1997)
Influence of salicylic acid on H2O2 production, oxidative stress, and
H2O2-metabolizing enzymes: salicylic acid-mediated oxidative
damage requires H2O2. Plant Physiology 115, 137149.
Ruiz JM, Bretones G, Baghour M; Belakbir A, Romero L (1998)
Relationship between Boron and phenolic metabolism in tobacco
leaves. Phytochemistry 48, 269272.
Ruiz JM, Garcia PC, Rivero RM, Romero L (1999) Response of
phenolic metabolism to the application of carbendazim plus boron
in tobacco. Physiologia Plantarum 106, 151157.
Shelp BJ (1993) Physiology and biochemistry of boron in plants.
In Boron and its role in crop production. (Ed. VC Grupta)
pp. 5385. (CRC Press: Charlottetown, Canada)
Singh DP, Riwari RS (1996) Effects of micronutrients on yield and
quality of onion (Allium cepa L.) variety Pusa Red. Recent
Horticulture 3, 111117.
Tenhaken R, Levine A, Brisson LF, Dixon RA, Lamb C (1995)
Function of the oxidative burst in hypersensitive disease resistance.
Proceedings of the National Academy of Sciences USA 92,
41584163.
Tomlin C (1994) The pesticide manual, 10th edn. (The British Crop
Protection Council and The Royal Society of Chemistry:
Cambridge, UK)
Tripathi RK, Tandon K, Schlsser E, Hess WM (1982) Effect of
fungicides on the physiology of plants. Part IV: protection of
cellular organelles of senescent wheat leaves by carbendazim.
Pesticide Science 13, 395400.
Vance CP, Kirk TK, Sherwood RT (1980) Lignification as a mechanism
of disease resistance. Annual Review of Phytopathology 18,
259288.
Van Loon APGM, Pesold-Hurt B, Schatz G (1986) A yeast mutant
lacking mitochondrial manganese-superoxide dismutase is
hypersensitive to oxygen. Proceedings of the National Academy of
Sciences USA 83, 38203824.
Wendehenne D, Durner J, Chen Z, Klessig DF (1998)
Benzothiadiazole, an inducer of plant defences, inhibits catalase
and ascorbate peroxidase. Phytochemistry 47, 651657.
Willekens H, Chamnongpol S, Davey M, Schraudner M, Langebartels
C, Van Montagu M, Inz D, Van Camp W (1997) Catalase is a sink
for H2O2 and is indispensable for stress defence in C3 plants.
EMBO Journal 16, 48064816.
Wolf B (1974) Improvement in the Azomethine-H method for
determination of boron. Communications in Soil Science and Plant
Analysis 5, 3944.
Wolf B (1982) A comprehensive system of leaf analysis and its use for
diagnosing crop nutrient status. Communications in Soil Science
and Plant Analysis 13, 10351059.
Wu G, Shortt BJ, Lawrence EB, Len J, Fitzsimmons KC, Levine EB,
Raskin I, Shah D (1997) Activation of host defence mechanisms by
elevated production of H2O2 in transgenic plants. Plant Physiology
115, 427435.

Manuscript received 10 July 2000, accepted 4 April 2001

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