RESEARCH ARTICLE

Physiological and biochemical response of potato(Solanum tuberosum L. cv. Kara) to O3 and antioxidantchemicals: possible roles of antioxidant enzymesI.A. Hassan

Department of Botany, Faculty of Science, Alexandria University, El-Shatby, Alexandria, Egypt

Keywords

Chlorothalonil; ethylenediurea; glutathione

reductase; ozone (O3); peroxidase; potato

(Solanum tuberosum L. cv. Kara); superoxide

dismutase; yield.

Correspondence

I.A. Hassan, Department of Botany, Faculty of

Science, Alexandria University, 21526

El-Shatby, Alexandria, Egypt.

Email: i_hassan82@hotmail.com

Received: 26 July 2005; revised version

accepted: 6 March 2006.

doi:10.1111/j.1744-7348.2006.00058.x

Abstract

An Egyptian cultivar of potato (Solanum tuberosum L. cv. Kara) was grown in

the field at two locations in northern Egypt: a ‘rural’ and a ‘suburban’ site,

from October 2000 and November 2002. The antiozonant ethylenediurea

(EDU) and the fungicide chlorothalonil (1,3-benzenedicarbonitrile-2,4,5,6 tetra-

chloroisophthalnitrile) were applied as a foliar spray to plants at both sites. It

was found that foliar injury symptoms were reduced greatly in plants treated

with EDU and/or chlorothalonil, and the yield of treated plants was higher

than that of the untreated ones, with the EDU having a greater protection

than chlorothalonil. Antiozonant (EDU) and fungicide (chlorothalonil) combin-

ation sprays were even more effective in reducing O3 injury. Moreover, the

percentage of protection was higher in the rural area than in the suburban

one, and this was associated with higher levels of O3 recorded in the rural

area. The response to O3, EDU, and chlorothalonil of the leaf antioxidant scav-

enger system was examined. Antiozonant-treated plants had the highest

reduced glutathione/oxidised glutathione ratio. The results suggest that EDU

and chlorothalonil do not act directly as antiozonant to inhibit O3 injury but

act through maintaining some antioxidant enzymes during O3 exposure. To

the best of knowledge, this is the first report demonstrating the marked

enhancement of yield and plant oxidative enzymes by fungicides as a mech-

anism of protecting plants against noxious oxidative stress from the environ-

ment in the developing world.

Introduction

Ozone (O3) is the most common phytotoxic pollutant in

the USA, Europe and other industrialised areas of the

world, causing adverse effects on physiology, growth

and yield of agricultural crops (Tonneijack & Van Dijk,

1997; Hassan et al. 1999; Madkour & Laurence, 2002;

El-Khatib, 2003; Hassan, 2004; Carrasco-Rodriguez et al.,

2005).

Tropospheric O3 is almost entirely a secondary pollut-

ant, generated through complex photochemical reaction

sequences that require reactive hydrocarbons, nitrogen

oxides (NOx) and sunlight. Episodes of elevated O3 are

commonly associated with anticyclonic weather (Pearson &

Mansfield, 1993). The meteorological conditions associ-

ated with anticyclones such as high solar radiation, high

temperature and low wind speed are favourable for tro-

pospheric O3 formation, and these are the conditions

prevalent in Egypt (Nasralla & Shakour, 1981; Hassan,

1999).

Very little is known about the impact of O3 on crops and

vegetation in Egypt (Elkiey & Ormrod, 1987; Ali, 1993;

Hassan et al., 1994, 1995; Hassan, 1998), although con-

centrations of O3 that are potentially high enough to

cause yield reductions of sensitive crops have been re-

corded in rural and urban areas in Egypt (55–200 nL L21)

(e.g. WHO/UNEP, 1992; Hassan, 1999).

The phytotoxicity of O3 is due to its high oxidative

capacity through the induction of reactive oxygen spe-

cies (ROS) in exposed plant tissues, such as hydrogen

Annals of Applied Biology ISSN 0003-4746

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197

peroxide (H2O2), superoxdie (O22) and singlet oxygen

(1O2) (Wu and Tiedemann, 2002).

Ethylenediurea (EDU) is a synthetic chemical that can

prevent O3-induced visible injury and shifts in biomass

partitioning and allocation (Foster et al., 1983; Eckardt &

Pell, 1996; Varshney & Rout, 1998), although it is not

effective with all species. A useful recent review of its

use and that of other antiozonant chemicals is given by

Manning (2000). It has been used extensively in the

USA and Western Europe to examine the effects of O3

on plant performance under either natural or environ-

mentally controlled conditions (e.g. Godzik & Manning,

1998; Kuehler & Flaglar, 1999: Manning et al., 2003). It

has been also used in developing countries in a very lim-

ited number of studies (Bambawale, 1989; Hassan et al.,

1995; Varshney & Rout, 1998). However, there is no

ready access to EDU in Egypt, so it was necessary to find

an alternative(s) to counteract the toxic effects of O3 on

plants. Bisessar (1982) reported that the fungicide chlor-

othalonil could be used to mitigate the toxic effects on

potato plants in the field, while the systemic fungicide

benomyl could also be used to effectively control O3

damage on some plant species (Manning et al., 1974;

Foster et al., 1983; Bambawale, 1989). Nevertheless,

chlorothalonil is available in Egypt and very accessible, so

it was worth examining its effects on growth and yield of

some field crops.

Whitaker et al. (1990) reported that leaf lipid composi-

tion and pigment contents of snap bean plants treated

with EDU showed no response after exposure to acute

dose of O3 for 3 h, while galactolipids and phospholipids

increased by 50%. EDU pretreatment of pea plants did

not inhibit ethylene biosynthesis when exposed to O3

(Zilinskas et al., 1990).

Although there are many studies investigating the

changes in growth and yield caused by EDU (Bennett

et al., 1979; Bisessar et al., 1982; Hassan et al., 1995;

Tonneijack & Van Dijk, 1997), only a few studies

focused on the protective mechanisms of EDU (Whitaker

et al., 1990; Pitcher et al., 1992; Lee et al., 1997; Gatta et al.,

1997). Moreover, nothing is known about the mech-

anism of the protective effects of chlorothalonil.

Results from studies investigatingEDU-induced changes

in the activity of antioxidant enzymes or in the level of

metabolites are contradictory. While EDU-induced O3

tolerance in bean plants was correlated with superoxide

dismutase (SOD) induction (Lee & Bennett, 1982),

Pitcher et al. (1992) and Lee et al. (1997) did not detect

significant changes in SOD activity in bean plants after

EDU application. It has been also reported that ascorbate

peroxidase (APX) and guaiacol peroxidase (GPX) activ-

ities were not affected by EDU (Brunschon-Harti et al.,

1995; Lee et al., 1997).

This study was conducted to investigate the response to

O3 of an important Egyptian crop, potato (Solanum tuber-

osum L. cv. Kara), and to assess the effectiveness and to

understand the mechanisms by which the antiozonant

EDU and chlorothalonil induce O3 tolerance in plants, in

order to further the insights into the basis of naturally

acquired O3 tolerance in plants and the factors affecting

stress-induced senescence of EDU and chlorothalonil in

protection of potato from of O3 injury.

Materials and methods

Experimental area

Two locations were chosen for the fieldwork in northern

Egypt. The first was at Al-Montazah Botanical Garden,

12 km east of Alexandria city centre (’suburban’ site); it

is surrounded by theMediterranean Sea to the north, resi-

dential areas to the west and south and a tourist village

(Al-Maamoura) to the east. A screen of large trees and

palms bound the garden. The other location was at Abbis

village (a ‘rural’ site), located 35 km to the south of

Alexandria city in the Nile Delta. The experimental site

was about 2 km from the nearest traffic road and was

surrounded by Eucalyptus, Casuarina and Salix trees. This

was being the main agricultural area of the country. In

both locations, the soil texture was loamy clay.

Cultural methods

Two field experiments were carried out: (I), 1 October

2000 to 10 February 2001; (II), 18 November 2002 to 29

March 2003. Four 11-m � 3-m blocks were chosen in

each of the two field sites. Each block was divided into

four equal plots (2 � 3 m) each of four rows, with 1-m

distance between subplots. The between-rowdistancewas

0.50 m.

Whole tubers of an Egyptian cultivar of potato

(S. tuberosum L. cv. Kara), obtained from a commercial

source, were sown 20 cm apart at both locations on 1

October 2002 in the first experiment (I) and on 18

November 2003 in the second experiment (II). Potato

plant is a winter crop, so they were sown and harvested

during the winter seasons as normal agricultural practice

in Egypt.

Four treatments were distributed in each plot in

a randomised Latin square design: (a) control (no chem-

ical treatments), (b) EDU as foliage spray, (c) spraying

with chlorothalonil (Bravo) and (d) spraying with amix-

ture of EDU and chlorothalonil. No fertilisers or other

fungicides were applied at either location to avoid inter-

ference with the fungicides. The plants were irrigated

once a week.

Physiological and biochemical response of potato to O3 and antioxidant chemicals I.A. Hassan

198 Ann Appl Biol 148 (2006) 197–206 ª 2006 The Authors

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Ozone monitoring

Ambient O3 was determined daily by continuous moni-

toring during daylight hours (8:00–18:00 h Egyptian

local time) using a UV photometer (DASIBI Model 1003-

AH, Hutchinson Corporation, UK), one at each location.

Sample air from the plots was collected 50 cm above

canopy height.

EDU and chlorothalonil application

EDU was dissolved in tap water to a final concentration of

300 mg L21 and dispensed as a foliage spray, with each

plant receiving 300 ml EDU, to one of the four plots in

each of the four blocks. In each case, another plot, not

being treated with EDU, received the same amount of

tap water (control). The third plot in each block received

chlorothalonil as a foliage spray at a concentration of

0.40%. The remaining plot in each block received both

EDU and chlorothalonil together at the same rates as

when applied singly. When chemical sprays were

applied, the remaining plots were sprayed with tap

water. A preliminary experiment carried out in 2000 to

show the effectiveness of four different concentrations of

EDU (100, 200, 300 and 400 mg L21) and chlorothalonil

(0.10, 0.20, 0.30 and 0.40%) on delaying senescence

and preventing visible injury on potato plants showed

that the EDU at a concentration of 300 mg L21 and

chlorothalonil at concentration of 0.40% were efficient

in increasing the yield and delaying senescence (data not

shown). EDU was applied nine times at 10-day intervals,

beginning 15 November 2000, 11 days before flowering

(28 days after emergence and 48 days after sowing),

while the fungicide (chlorothalonil) was applied five

times at 7-day intervals, beginning 14 November 2002.

The same concentrations of EDU and chlorothalonil

were applied in 2002 with a slight modification in the

timetable as plants were sown on 18 November 2002

and the fungicide and antioxidant application started on

17 December 2002 and at the same intervals as described

in the first experiment.

Visible injury and destructive harvest

Foliar injury symptomswere assessed carefully, in the first

experiment on 10 February 2001 (130 days after sowing)

and in the second experiment on 29 March 2003 (102

days after sowing) by counting the number of injured

leaves and estimating the percentage of each leaf’s area

showing injury (on a score of 0 ‘no injury’ to 5 ‘100%

injury’) (Taylor et al., 1990).

The plants were harvested immediately after injury was

scored. Yield was determined by weighing fresh tubers of

the plants treated with different treatments (four rows per

treatment per block per location). Number of tubers per

plant was also counted.

Measurements of hydrogen peroxide

The H202 assay followed the method of Wu & Tiedemann

(2002). Fifteen leaf discs (10-mm diameter) were sub-

merged in 750 lL reagent mixture containing 0.05%

guaiacol and horseradish peroxidase (350 lL L21,

250 U mL21) in 25 mM sodium phosphate buffer (pH

7.0) and incubated for 2 h at 20�C in the dark. Then, a

volume of 250 lL was transferred into 96-well microtitre

plates and the absorbance was immediately measured at

4450 nm in a plate reader photometer (SLT, Spectra,

Dixons Ltd, Pure Chemicals for Laboratories, Switzerland).

Commercial H2O2, which was used for standard curves,

was calibrated by titration with KMnO4.

Antioxidant enzymes assays

Leaves collected from Abbis Village in experiment II were

subjected to biochemical analyses. Extractions of antioxi-

dant enzymes from the leaves of the four treatments (con-

trol, EDU, chlorothalonil and chlorothalonil + EDU) were

performed according to Lee et al. (1997). Leaves were cut

from each treatment and immersed in liquid nitrogen

and kept in a deep freezer at 80�C until the analyses

were performed at the Department of Ecology and Envi-

ronmental Science, Kuopio University, Finland.

Sampleswereweighed and ground at about 0�C in 25 m

Tris–HCl buffer containing 3 mM MgCl2, then the ho-

mogenates were centrifuged at 20 000 for 15 min (Cen-

trifuge 17 S/RS, Heraeus Sepatech). The supernatants

were used for the enzyme assays and the results were

expressed on protein basis (Bradford, 1976).

All assays were performed using a final volume of 1 mL,

with at least duplicate assays undertaken on each sample.

Moreover, the assays were end-point determinations.

SOD(EC 1.15.1.1) activity was monitored according to

Lee et al. (1997). The extraction mixture contained

50 mM phosphate buffer solution (pH 7.8), 13 mM

L-methionine, 63 lM nitro blue tetrazolium and 2 lMriboflavin. The ability of the extract to inhibit the photo-

chemical reduction of nitro blue tetrazolium was

determined at 560 nm (Schimadzu UV-1201 spectropho-

tometer). The amount of the extract resulting in 50%

inhibition of nitro blue tetrazolium reaction is defined as

one unit of SOD activity.

Catalase (EC, 1.11.1.6) activity was assayed in enzyme

extract reaction mixture containing 50 mM phosphate

buffer (pH 7.4). The reaction was started by adding

I.A. Hassan Physiological and biochemical response of potato to O3 and antioxidant chemicals

Ann Appl Biol 148 (2006) 197–206 ª 2006 The AuthorsJournal compilation ª 2006 Association of Applied Biologists

199

10 mM H2O2, and the reduction in absorbance was

determined at 240 nm (Maehly & Chance, 1954).

GPX (EC, 1.11.1.7) activity was determined by adding

50 mM phosphate buffer (pH 6.1), 1% H2O2 and 1%

guaiacol to the extract, and the absorbance was deter-

mined at 470 nm.

APX (EC, 1.11.1.11) activity was determined according

to Maehly & Chance (1954). The reaction mixture con-

tained 50 mM potassium phosphate, 0.5 mM ascorbate,

0.1 mM ethylenedimethyl tartaric acid (EDTA) and 0.1 mM

H2O2, and the absorbance was determined at 290 nm.

Glutathione reductase (GR; EC, 1.6.4.2) activity was

determined according to Lee et al. (1997). The enzyme

activity was monitored by measuring a decrease in ab-

sorbance at 334 nm resulting from oxidation of reduced

nicotine amide dinucleotide (NADH) (6.2 mM21). The

assay mixture contained 0.1 M Tris–HCl (pH 8.0), 1 mM

EDTA, 0.1 mM NADH and 1 mM oxidised glutathione

(GSSG) and the leaf extract.

Glutathione was analysed with a Schimadzu R.F. 1201

high performance liquid chromatography (HPLC), and

peaks were detected by a fluorescence detector using an

excitation wavelength of 340 nm and an emission wave-

length of 420 nm. Total glutathione (GS) and GSSG were

quantified by comparing peak areas with known stand-

ards. Reduced glutathione (GSH) was calculated by sub-

tracting GSSG from GS (Lee et al., 1997).

Protein concentrations of leaf extracts were determined

as described earlier (Bradford, 1976).

Data analysis

Data were subjected to two-way analysis of variance

(ANOVA), using sites and chemical treatments as factors,

followed by a least significant difference test, and P val-

ues � 0.05 were considered significant (using the STAT-

GRAPHICS statistical package, Package 3, UK) based on

plot means, using EDU and chlorothalonil as factors.

Data of visible injury were log transformed prior to

analysis to ensure that they were normally distributed.

There were no covariates used in the ANOVA.

Results

Ambient O3 concentration

The mean monthly concentrations of ambient O3 meas-

ured at the two locations (8:00–18:00 h) are shown in

Fig. 1. The mean 10-h concentration of O3 over the

experimental periods (October 2000 to February 2001

and November 2002 to March 2003) was 78 nL L21 at

Alexandria (75 and 81 nL L21 in experiments I and II,

respectively) and 95.5 nL L21 at Abbis (93 and 98 nL L21

in both experiments, respectively). The mean midday

temperature over the experimental period was almost

the same (19�C at Alexandria and 21�C at Abbis; data

not shown). It is obvious that O3 levels at the rural site

(Abbis) were higher than that at the suburban site

(Alexandria).

Foliar injury

Visible injury symptoms appeared on the upper surfaces

of leaves as pinpoint brown dots, followed by bronze le-

sions, and by the end of the experiment (130 days after

sowing), necrotic spots appeared on older leaves. There

was better protection against foliar injury symptoms by

EDU + chlorothalonil than either EDU or chlorothalonil

when they were applied individually. There was no vis-

ible injury symptoms on leaves treatedwith EDU + chloro-

thalonil in Alexandria in both experiments (2000 and

2003).

In the 2003 experiment, the number of injured leaves

was decreased at Alexandria by 65 and 43% and at Abbis

by 82 and 53%, after treatment with EDU or chlorothalo-

nil, respectively (Table 1), while the degree of injury was

also reduced by 75 and 50% in Alexandria by EDU and

chlorothalonil, respectively (Table 1), and by 75, 66 and

2000 Experiment

60708090

100

1 Oct

2000

1 Nov

2000

1 Dec

2000

1 Jan

2001

1 Feb

2001

Duration of experiment

O3

levl

el (

nl L

-1)

O3

leve

ls (

nl L

-1)

Abbis Montazah

2003 Experiment

60708090

100110

1 Nov

2002

1 Dec

2002

1 Jan

2003

1 Feb

2003

1 Mar

2003

Duration of experiment

Figure 1 Monthly mean concentration of ambient ozone (nL L21) during 2000 and 2003 experiments in the experimental sites. Error bars represent

1 SE of means.

Physiological and biochemical response of potato to O3 and antioxidant chemicals I.A. Hassan

200 Ann Appl Biol 148 (2006) 197–206 ª 2006 The Authors

Journal compilation ª 2006 Association of Applied Biologists

79% in Abbis by EDU, chlorothalonil and EDU + chloro-

thalonil, respectively (Table 1).

In 2003 experiment, the number of injured leaves was

decreased by 63 and 54% in Alexandria and by 78 and

66% in Abbis as a result of spraying with either EDU or

chlorothalonil, respectively, while degree of injury was

reduced by 78 and 68% in Alexandria and by 87 and

80% in Abbis, as a result of the same treatments, respect-

ively (Table 1).

Number of injured leaves on plants grown at Abbis and

treated with EDU + chlorothalonil was reduced by 80 and

83% in experiments I and II, respectively, while degree of

injury was reduced by 83 and 90% in the same experi-

ments, respectively (Table 1).

Effect on yield

Tuber weights from plants, collected from Alexandria,

treated with EDU or/and chlorothalonil were significantly

higher than control plants (without chemical treatment)

by 31, 12 and 24%, in the 2000 experiment, and by 40, 24

and 36% in the 2003 experiment, respectively (Table 2),

while number of tubers increased by 32, 19 and 27% in

the 2000 experiment and by 47, 40 and 45% in the 2003

experiment by the same treatments, respectively (Table 2).

At Abbis, tuber weights increased by 32, 16 and 26%, in

2000 experiment and by 43, 31 and 46% in 2003 experi-

ment, and the number of tubers was also increased by 35,

22 and 33% in 2000 experiment and by 42, 28 and 40%

in 2003 experiment, as a result of treatments with EDU,

chlorothalonil and EDU + chlorothalonil, respectively

(Table 2). Moreover, there were significant interaction be-

tween sites and chemical treatment on yield components

(P < 0.05) (Table 3).

Effects on H2O2

H2O2 accumulation was enhanced in plants treated by

a combination of EDU and chlorothalonil compared with

control (fungicides � O3, P � 0.01; Fig. 2).

Antioxidant enzymes

Although there was a 20% difference in atmospheric O3

between sites, enzyme activities were similar between

sites for a given treatment.

It was found that O3 inhibited activities of SOD and GR,

while antiozonant chemicals (EDU and chlorothalonil)

stimulated these enzymes. Both peroxidase (PX) and cata-

lase showed no response to EDU, chlorothalonil and/or O3.

Table 2 Effect of EDU and/or chlorothalonil on potato tuber yield

Treatment

Alexandria Abbis

Weight (kg) No. of Tubers (per plant) Weight (kg) No. of Tubers (per plant)

Experiment I (2000)

Control 5.76a 4.72a 5.00a 4.51a

EDU 7.52c 6.21c 6.59c 6.10c

Chlorothalonil 6.93b 5.61b 5.80b 5.49b

Chlorothalonil + EDU 7.12c 6.00c 6.30c 6.00c

Experiment II (2003)

Control 5.45a 4.19a 4.27a 4.05a

EDU 7.36c 6.23c 6.13c 5.76c

Chlorothalonil 6.79b 5.78b 5.60b 5.21b

Chlorothalonil + EDU 7.41c 6.08c 6.23c 5.69c

EDU, ethylenediurea. Means not followed by the same letter are significantly different from each other at P � 0.05. Figures are means of plants

per plot.

Table 1 Effect of EDU and/or chlorothalonil on visible injury symptoms of potato leaves

Parameter

Treatment [Alexandria (‘suburban’ site)] Treatment [Abbis (‘rural’ site)]

Control EDU Chl Chl + EDU Control EDU Chl Chl + EDU

Experiment I (2000)

No. of injured leaves 37d 18b 28c 14a 31c 20a 27b 24b

Degree of injury 0.61d 0.15b 0.30c 0.09a 1.30d 0.32b 0.43c 0.27a

Experiment II (2003)

No. of injured leaves 38d 28b 33c 21a 34c 19a 25b 21a

Degree of injury 0.87d 0.19b 0.28c 0.15a 1.94c 0.25a 0.39b 0.20a

EDU, ethylenediurea; Chl, chlorothalonil. Means not followed by the same letter within each site are significantly different from each other at

P � 0.05. Data presented show the mean values (n = 40) for the proportion of sampled leaves that were infected and the leaf area damaged

assessed on a scale where 1 = no visible injury or damaged area and 5 = complete infection or damaged area.

I.A. Hassan Physiological and biochemical response of potato to O3 and antioxidant chemicals

Ann Appl Biol 148 (2006) 197–206 ª 2006 The AuthorsJournal compilation ª 2006 Association of Applied Biologists

201

PX and catalase enzymes showed nonsignificant

response to spray treatments, while activities of SOD and

GR were increased in Alexandria by 58, 75 and 83% and

by 55, 66 and 55% in response to EDU, chlorothalonil

and EDU + chlorothalonil, respectively (Table 4). SOD

and GR were also increased in Abbis in response to these

spray treatments, where SOD was increased by onefold

as a result of spraying with either EDU or chlorothalonil

and by twofold as a result of spraying with EDU + chloro-

thalonil, while GR was increased by 54, 45 and 73% in

response to spraying with EDU, chlorothalonil and EDU +

chlorothalonil, respectively (Table 4).

GS, GSSG and GSH concentrations are shown in

Table 5.

EDU-treated plants grown in O3 had higher concen-

trations of GSH but lower GSSG compared to plants

grown in O3 but that did not receive EDU. Generally,

plants treated with EDU and/or chlorothalonil had higher

GSH/GSSG ratio (;5) than those of control (;1.4).

Discussion

The measurements of O3 in the present study were car-

ried out during the winter season in Egypt (October–

March); O3 levels are likely to be higher during summer

when daylight periods are longer, with higher temperatures

and irradiance. There is evidence that the levels of ambi-

ent O3 in Egypt are higher during summer seasons than

during winters (e.g. Hassan, 1999). The results of this

study showed that in the winter, the levels of ambient

O3 at the rural area (Abbis) were higher than those at

the suburban area (Alexandria), and this supports the

results of our previous study (Hassan et al., 1995), in

which the ambient levels of oxidants recorded in Alex-

andria were lower (56 nL L21) than those recorded in

Abbis (67 nL L21). Moreover, the results are in agree-

ment with other studies, such as Pearson et al. (1988),

who reported higher O3 levels in rural areas in Ontario

than in urban areas, Anjea et al. (1992), who reported

that O3 levels at rural sites in the south-eastern sites in

the USA are higher than those in urban areas, and

Schenone & Lorenzini (1992), who reported higher

levels of O3 at rural sites in Italy than in urban ones.

Sensitivity of potatoes (S. tuberosum L.) to oxidant air

pollutants, particularly ozone (O3), has been documented

with several combinations of experimental techniques

and evaluation criteria, including (a) foliar symptoms in

field trials (e.g. Bambawale, 1989), (b) correlations of

foliar injury and apparent tuber yield reductions in field

trials (e.g. Mosley et al., 1978; Bisessar, 1982), (c) tuber

yield under controlled conditions (Pell et al., 1980; Pell &

Person, 1984) and (d) tuber yield in the field trials using

antioxidant chemical treatments to reduce O3 damage

(e.g. Clarke et al., 1990).

In agreement with other studies (e.g. Bisessar, 1982),

young leaves were virtually uninjured, with older fully

Table 3 Analysis of variance summary for growth parameters in the two locations

Treatment

Alexandria Abbis

Weight No. of Tubers Weight No. of Tubers

EDU *** ** *** ***

Chlorothalonil * * ** **

EDU � Chlorothalonil *** *** *** ***

Site � EDU ** *** *** ***

Site � chlorothalonil ** ** *** ***

Site � EDU � chlorothalonil *** *** *** ***

EDU, ethylenediurea. *, ** and *** are significant at P � 0.05, 0.01 < P < 0.05 and P � 0.01, respectively.

0102030405060708090

Control EDU Chl Chl +EDU

Control EDU Chl Chl +EDU

H2O

2

(Um

ol H

2O2 h

-1 c

m-2

dis

c)

2000 2003

0102030405060708090

H2O

2

(um

ol H

2O2

h-1 c

m-2

dis

c)

Figure 2 Effects of fungicide treatments on hydrogen peroxide (H2O2) content (lmol H2O2 h21 cm22 disc) in potato leaves. Columns represent

means of 15 replicates ± SE bars.

Physiological and biochemical response of potato to O3 and antioxidant chemicals I.A. Hassan

202 Ann Appl Biol 148 (2006) 197–206 ª 2006 The Authors

Journal compilation ª 2006 Association of Applied Biologists

expanded leaves sustaining more severe O3 injury. This

phenomenon is mainly based on the fact that ageing

leaves contain lower antioxidant levels than younger

leaves (Wu & Tiedemann, 2002).

‘Kara’ potato plants developed more extensive foliar

injury than any other cultivar in the previous studies

conducted in Canada, USA and Europe. Bisessar (1982)

reported that EDU and chlorothalonil reduced O3 injury

on ‘Norchip’ potatoes (a sensitive cultivar) by 50 and

25%, respectively, and the combined effects of both an-

tiozonants were additive in reducing O3 injury on potato

foliage. My results indicated that EDU and chlorothalonil

reduced the degree of injury on potato foliage (averaged

between experiments and locations) by 78 and 65%,

respectively, at O3 concentrations similar to those

reported by Bisessar (1982).

Based on foliar response, Kara plants proved to be more

susceptible to O3 than the cultivar Norchip used in the

developed world (assuming that the prevailing air pol-

lutant is O3).

In this study, there were no early blight symptoms on

both plants in either location. Moreover, there were no

symptoms of other fungal infections.

The higher foliar injury symptoms at Abbis than Alex-

andria reflected the higher levels of ambient O3 recorded

at Abbis (95.5 nL L21) than at Alexandria (78 nL L21).

Moreover, very low level of foliar injury symptoms on

plants treated with both EDU and chlorothalonil in

Alexandria indicated that the combined effect of EDU +

chlorothalonil gave better protection than either EDU or

chlorothalonil when applied individually. Nevertheless,

EDU gives better protection than chlorothalonil, and this

is in agreement with the results of Bisessar (1982), who

reported the same order of protection of this treatment

on potato cv. Norchip. Furthermore, Clarke et al. (1990)

reported a protective effect of EDU against foliar injury of

different cultivars of potatoes and soybeans.

The EDU approach has been frequently used as feasible

and inexpensive tool to assess crop yield losses caused by

oxidant air pollution without toxic effects per se (e.g.

Brennan et al., 1987; Gatta et al., 1997; Tonneijack & Van

Dijk, 1997). In a previous study on Egyptian cultivars of

radish (Raphanus sativus L. cv. Baladey) and turnip (Bras-

sica rapa L. cv. Sultani) 25 and 20% losses in the yield of

both cultivars, respectively, were recorded, using EDU

under Egyptian field conditions (Hassan et al., 1995) at

Abbis site.

The yield response observed in the present study sug-

gests that ambient air pollution (viz. O3) reduces tuber

weight and tuber number, with a greater effect on tuber

number at both locations. Plants treated chemically with

EDU and/or chlorothalonil had significantly higher yield

than nontreated plants (control), and this is in agree-

ment with the results of Bisessar (1982), Foster et al.

(1983), Pell & Pearson (1984) and Clarke et al. (1990).

In her through review, Polle (1998) stated that there

was interaction between O3 exposure nutrient supply on

growth and physiology on birch plants. She reported a

reduction in activities of antioxidants enzymes in leaves

of birch plants that had been sufficiently supplied with

nutrients, but not in starved plants. These findings were

supported with the results of Pfirmann et al. (1993) who

found very little interaction between O3 exposures and

nutrient treatments in Norway spruce. However,

Table 4 Effects of O3 and EDU on concentrations of antioxidant enzymes (lmol min21 mg21 protein)

Treatment

Alexandria Abbis

SOD Catalase GPX APX GR SOD Catalase GPX APX GR

Control 0.12a 30.3a 0.18a 0.64a 0.09a 0.09a 31.2a 0.15a 0.71a 0.11a

EDU 0.19b 31.2a 0.17a 0.69a 0.14b 0.18b 31.7a 0.16a 0.76a 0.17b

Chl 0.21b 29.4a 0.19a 0.72a 0.15a 0.19b 31.0a 0.14a 0.79a 0.16b

Chl + EDU 0.22b 30.1a 0.17a 0.73a 0.14b 0.25b 32.1a 0.17a 0.80a 0.19b

EDU, ethylenediurea; Chl, chlorothalonil; SOD, superoxide dismutase (units mg protein21); GPX, guaiacol peroxidase; APX, ascorbate peroxidase;

GR, glutathione reductase. Means not followed by the same letter are significant different from each other at P � 0.01.

Table 5 Effects of O3 and EDU on GSSG, GSH and GS (lg g21 fresh weight)

Treatment

Alexandria Abbis

GSSG GSH GS GSH/GSSG GSSG GSH GS GSH/GSSG

Control 37c 51a 88a 1.38 39c 54a 90a 1.38

EDU 15a 71b 86b 4.73 18a 75b 88b 4.17

Chl 18b 80b 88b 4.44 20b 82b 88b 4.10

Chl + EDU 13a 91c 104b 7.00 11a 88c 105b 8.00

EDU, ethylenediurea; Chl, chlorothalonil; GSSG, oxidised glutathione; GSH, reduced glutathione; GS, total glutathione.

I.A. Hassan Physiological and biochemical response of potato to O3 and antioxidant chemicals

Ann Appl Biol 148 (2006) 197–206 ª 2006 The AuthorsJournal compilation ª 2006 Association of Applied Biologists

203

although there is an evidence of possible interaction

between O3 and fertilisers, there is no evidence about

interaction between antiozonant chemicals and nutrient

supply that could affect the response of plants to O3.

The results of yield should be treated with caution espe-

cially as no fertilisers were added in the experiments

described here. In Egypt, as a common agricultural prac-

tice, NPK is always added to potato fields to get a reason-

able number of tubers. However, in the present study, no

fertilisers were added to prevent interference with the

antiozonant fungicides. It is worth to investigate the inter-

action between fertilisers, antiozonant fungicides and

ozone in the future.

The biochemical mechanisms by which EDU protects

plants against O3 are hard to identify (Brunschon-Harti

et al., 1995; Eckardt & Pell, 1996; Lee et al., 1997). There

are many mechanisms that have been suggested, but all

are contradictory (Lee et al., 1981; Bennett et al., 1984;

Stevens et al., 1988; Whitaker et al., 1990). SOD in leaves

is the primary scavenger for free radicals generated both

from normal physiological activities such as photosyn-

thesis and respiration and from exposure to oxidative

stress factors like O3. SOD is likely to be central in the

defence against toxic ROS accumulation.

H2O2 is decomposed by catalase and PX very effi-

ciently (Wu & Tiedemann, 2002). Much research has

been recently focused on the potential importance of

antioxidant enzymes of the Halliwell–Asada pathway,

such as APX and GR, in removing H2O2 formed under

oxidative stress such as O3 (Hernandez et al., 1999; Wu &

Tiedemann, 2002).

Higher activities of certain scavenger enzymes along

with several antioxidants could be the agents that

protect plants against O3 (Larson, 1995; Wellburn &

Wellburn, 1997). However, this was not the case in this

investigation as the activities of catalase and PX showed

no significant change after EDU application, which

would indicate a protective action against O3 phytotoxic-

ity. This agrees with the results of Brunschon-Harti et al.

(1995) and Lee et al. (1997), who reported that there

were no differences in the activities of PX and catalase

between control and EDU-treated bean plants. However,

Bennett et al. (1984) reported that catalase and PX could

act to regulate injurious oxyradical and peroxyl con-

centrations in cells to determine equilibrium rates. The

differences in these findings may be because of method-

ology and experimentation.

In the present investigation, both EDU and chlorotha-

lonil increased the activities of GR and SOD. The induction

of SOD coincided with an activity increase in GR, an

enzyme involved in scavenging H2O2. This is of some

importance because the increase in H2O2 resulting from

higher SOD activity required an increased capacity of

enzymatic H2O2 decomposition. Such cooperation may

play a crucial role in preventing plants from O3 injury

(Carrasco-Rodriguiz et al., 2005). The results of the pres-

ent work support previous views that cooperation

between H2O2-scavenging enzymes and SOD plays an

important role in the resistance of plants to environmen-

tal stress (Wu & Tiedemann, 2002).

The effect of O3 on the various antioxidant enzymes

was inconsistent in both induction and degradation.

While fungicide-treated plants showed an increase in the

activities of SOD and GR in presence of O3, other en-

zymes (catalase and APX) showed no induction. It is

possible that the induction had occurred earlier and was

missed by sampling time.

Regarding glutathione, EDU treatment resulted in

higher GSH/GSSG ratios than for control plants, and this

is in agreement with the results of Lee et al. (1997), who

stated that EDU-treated bean tissues previously exposed

to O3 maintained high levels of GS and had higher GSH/

GSSG than ozonated leaves. Therefore, it is expected

that GSH/GSSG be high in EDU-treated leaves after

fumigation with O3 especially as the GR activity of EDU-

treated leaves was high under O3 stress (Lee et al., 1997).

The increase in GSH/GSSG ratio as a result of an increase

inGSH in sprayed leaves versus controlwas associatedwith

the decline in GSH content. So it is clear from the results of

the present study that EDU can maintain glutathione and

SOD under O3 stress or may even synthesize more mole-

cules (Lee et al., 1997; Tonneijack & Van Dijk, 1997).

Glutathione functions in the stabilisation of antioxidant

enzymes and detoxification of ROS (Rennenberg, 1995).

In conclusion, the results of the present investigation,

clearly, indicate that this Egyptian variety of potato is sen-

sitive to ambient O3 and showed that the protective

effects of EDU and chlorothalonil are not because of

a positive effect on these chemicals themselves but are

because of negative effects of ambient pollution on con-

trol plants. Moreover, they maintained SOD, GR and GS

levels, and this may indicate that EDU and chloro-

thalonil do not act directly as antiozonant to inhibit O3

but act through maintaining some antioxidant enzymes

during O3 exposure (Lee et al., 1997; Tonneijack & Van

Dijk, 1997). It is, therefore, important to note that the

use of fungicides for controlling fungal diseases in plants

grown for O3 pollution research will generate misleading

results because some of the fungicides will strongly

diminish the response of plants to O3 and reduce O3

injury (Wu & Tiedemann, 2002).

Acknowledgements

I am indebted to the continuous financial support by

the British Council. I also thank Prof. A.W. Davison and

Physiological and biochemical response of potato to O3 and antioxidant chemicals I.A. Hassan

204 Ann Appl Biol 148 (2006) 197–206 ª 2006 The Authors

Journal compilation ª 2006 Association of Applied Biologists

Dr J. Barnes from University of Newcastle (UK) for

the generous supply of EDU; Prof. Edward Lee, USA, for

his interest in this work and his help in methodologies of

antioxidant enzymes; Prof. S. Barakat, Prof. S. Khalil and

Prof. A.A. Aal, Alexandria University, for their corrections

and guidance and Eng. Ahmed Ismaeil for the access to

Al-Montazah Botanical Garden in Alexandria. Last but

not the least, I thank from the bottom of my heart Prof.

J.N.B. Bell of Imperial College for revising this paper and

his valuable comments and corrections. My deepest

thanks to anonymous reviewers for their invaluable sug-

gestions and encouraging comments.

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