Environmental Pollution 197 (2015) 247e255
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Screening agrochemicals as potential protectants of plants against ozone phytotoxicity Costas J. Saitanis a, *, Dimitrios V. Lekkas a, Evgenios Agathokleous a, 1, Fotini Flouri b a b
Laboratory of Ecology and Environmental Sciences, Agricultural University of Athens, Iera Odos 75, Votanikos 11855, Athens, Greece Laboratory of Pesticide Science, Agricultural University of Athens, Iera Odos 75, Votanikos 11855, Athens, Greece
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 September 2014 Received in revised form 3 November 2014 Accepted 6 November 2014 Available online 26 November 2014
We tested seven contemporary agrochemicals as potential plant protectants against ozone phytotoxicity. In nine experiments, Bel-W3 tobacco plants were experienced weekly exposures to a) 80 nmol mol1 of ozone-enriched or ozone-free air in controlled environment chambers, b) an urban air polluted area, and c) an agricultural-remote area. Ozone caused severe leaf injury, reduced chlorophylls' and total carotenoids' content, and negatively affected photosynthesis and stomatal conductance. Penconazole, (35% ± 8) hexaconazole (28% ± 5) and kresoxim-methyl (28% ± 15) showed higher plants’ protection (expressed as percentage; mean ± s.e.) against ozone, although the latter exhibited a high variability. Azoxystrobin (21% ± 15) showed lower protection efficacy and Benomyl (15% ± 9) even lower. Trifloxystrobin (7% ± 11) did not protect the plants at all. Acibenzolar-S-methyl þ metalaxyl-M (Bion MX) (6% ± 17) exhibited the higher variability and contrasting results: in some experiments it showed some protection while in others it intensified the ozone injury by causing phytotoxic symptoms on leaves, even in control plants. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Fungicides Bion MX Biomonitoring Tobacco Azoxystrobin Benomyl Penconazole Greece
1. Introduction Ambient ozone level has risen over the last four decades, it is still being gradually increasing, and it is expected to be a major menace for cultivated plants and natural ecosystems in the near future (Fishman et al., 2010). The nowadays occurring ambient ozone levels are high enough to negatively affect wild plant species (Bermejo et al., 2003; Feng et al., 2014; Agathokleous et al., 2014a) and field-growing cultivated plants (Booker et al., 2009; Avnery et al., 2011), especially in the Mediterranean countries (Saitanis and Karandinos, 2001a; Saitanis, 2003). Retardation of plants' growth and severe yields’ losses in crop plants, due to increased ozone levels, have been reported (Booker et al., 2009). Fishman et al. (2010) estimated the global economic loss to the farming community to exceed $10 billion annually.
* Corresponding author. E-mail addresses: [email protected] (C.J. Saitanis), [email protected] (E. Agathokleous), [email protected] (F. Flouri). 1 Present address: Silviculture and Forest Ecological Studies, Hokkaido University, Sapporo, Hokkaido 060-8589, Japan. http://dx.doi.org/10.1016/j.envpol.2014.11.013 0269-7491/© 2014 Elsevier Ltd. All rights reserved.
The main goal, nowadays, is the reduction of ambient ozone levels through the reductions of its precursors (NOx, VOCs). This, however, is considered a long-term goal. Thus, methods for protection of plants against ozone should be urgently developed. Many substances have been tested as potential protectants of plants against ozone phytotoxicity, among which agrochemicals, such as fungicides, herbicides, insecticides, and plant growth regulators (Manning, 2000). Substances that cause stomatal closure, such as phenylmercuric acetate and monoethyl esters of decenylsuccinic acid have been tested many years ago (Rich, 1964; Seidman et al., 1965), and they have been found to protect plants. Similar protective efficacy has also been obtained by abscisic acid (ABA), which also induces stomata closure (Lin et al., 2001). In a recent study, Francini et al. (2011) found that the antitranspirant di-1-p-menthene significantly protected Pinto bean plants from acute ozone injury, but this substance did not work on tobacco plants (Agathokleous et al., 2014b). However, the stomatal closure induced by such substances does not only impede ozone entry to leaf tissues, but simultaneously impedes CO2 uptake, which, in turn, may lead to undesirable yield loss due to reduced CO2 assimilation.
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A well-known promising antioxidant is EDU (N-[2-(2-oxo-1imidazolidinyl)ethyl]-N'-phenylurea; abbreviated EDU for ethylenediurea) (Carnahan et al., 1978; Feng et al., 2010; Manning et al., 2011). However, the major disadvantage of EDU is that it is not commercially available (only very few laboratories and only for research purpose can prepare it). A short review of the available publications shows that many biocides or some antioxidant substances, available in commerce, may offer protection of plants against ozone. Since 1972, benomyl (a fungicide now being banned) was tested as plant-protectant against ozone (Reinert and Spurr, 1972; Manning et al., 1973a, b, c) with positive results. When benomyl was applied as a foliar spray to field growing bean plants resulted to a 70e80% suppression of oxidant injury (Manning et al., 1973b), while when applied in Bel-W3 plants mitigated leaf injury up to ~60% (Reinert and Spurr, 1972). Azoxystrobin and epoxiconazole fungicides have also been reported possessing antiozonate properties (Wu and Von Tiedemann, 2002). Although numerous substances have been tested and proposed as potential antiozonants over the years, there is a lack of understanding of their antiozonate mechanisms for all of them. Even for EDU, which is the most studied antiozonate substance, the actual underline mechanism has not been uncovered yet (Agathokleous et al., 2014c). In the framework of this investigation, we tested seven contemporary agrochemicals as potential plant-protectants against ozone phytotoxicity. We focused on agrochemicals because they are commercially available and they are extensively used in intensive agriculture. The knowledge that some agrochemicals exhibit e as a side effect e antiozonate property, gives to them an advantage, when compared with other homologous agrochemicals, so they would be preferable in agricultural practice. Besides, such agrochemicals would also be used by researchers in setting up control group plants in studies aiming to assess crop yield loss by ozone under field conditions. 2. Material and methods 2.1. Plant materials As experimental plant, the Bel W3 tobacco (Nicotiana tabacum L.) variety was used. This variety is known to be hypersensitive to ozone (Saitanis and Karandinos, 2001b) with a threshold of sensitivity of about 40 nmol mol1 of ozone for few hours. Thus it is not cultivated and often the results coming from experiments based on this variety e especially those dealing with yield e are not representative to what really happens in cultivations. However, because of its high sensitivity to ozone, it is widely used as a useful tool in laboratory and field experiments to reveal the mechanisms of plants’ response to ozone. Seeds of Bel-W3 variety were planted in peat. After germination, seedlings were transferred to 12 cm d plastic pots (one seedling per pot) filled with commercial soil (Floran, STEDIP corp.). When plants reached the fifth leaf stage of growth, they were selected for uniformity and divided equally between treatments’ groups. The number of plants used per agrochemical treatment (subgroup) differed among experiments (8-20 plants per agrochemical per ozone treatment per experiment), with a total of about 800 plants. Within each chamber the positions of the plants were randomly rotated, at least once daily, in order to minimize any chamber edge effects.
2.2.1. Laboratory experiments In seven experiments, sets of Bel-W3 tobacco potted plants were transferred to two identical walk-in chambers (230 190 170 cm Model 60R - CDR corp.) under the same conditions: 14:10 (L:D) h photoperiod, 65 ± 3% relative humidity and 28 ± 0.5 C temperature. Both chambers were supplied with purified air, by passing it through dry purafil (KMnO4) and activated charcoal filters, in order to minimize contamination by the ambient air pollutants. In one of the chambers the filtered air was enriched with ozone. Ozone was produced by an electric generator (Air-Zone® XT-6000) that uses a new patented technology producing no NOx byproducts. Teflon lines led sample air from the chamber to a UV ozone analyser (Environnement S.A O3 42M). The ozone concentration within the chamber was stabilized via a feedback controller (CDR corp.). The wind speed within the chambers, above the plants’ canopy, was about 2 m s1. In each experiment, the plants of the “ozone chamber” were exposed to 80 nmol mol1 of ozone for 8 h per day for 7 days (AOT40: 2240 nmol mol1 h); this level is very close to the ambient ozone levels occurring in rural - agricultural areas nowadays (Saitanis, 2003). Those plants constituted the “ozone exposed” main group (hereafter: OZþ) while the plants of the chamber provided with filtered air constituted the “ozone-free” e control main group (hereafter: OZ). In each experiment, the plants of each chamber were further subdivided to eight subgroups. The plants of each of the seven subgroups were sprayed with one of the agrochemicals, as described below, while those of the eighth subgroup were sprayed with distilled water and constituted the “control subgroup”. 2.2.2. Field experiments Two field experiments were additionally conducted to test the potential protective role of the used agrochemicals under ambient conditions. One ambient experiment was conducted in the urban (polluted) campus of Agricultural University of Athens (AUA). In this experiment 9e12 plants per treatment were used. The second ambient experiment (six plants per treatment) was conducted at the agricultural area of Aliartos, located about 70 km away (NW) from Athens. In the overall data analysis, the plants exposed to ambient urban (AUA) or rural (Aliartos) environment were considered exposed to ozone enriched air (OZþ). 2.3. Agrochemicals tested In each experiment, different subgroups of plants were sprayed with different water solutions of the following seven agrochemicals (in brackets their abbreviation): azoxystrobin [AZOX], benomyl [BENML], hexaconazole [HEX], kresoxim-methyl [KRM], penconazole [PENC], trifloxystrobin [TRIFL] and the mixture acibenzolar-Smethyl þ metalaxyl-M [BION]. The active constituents, the trade names and the used dose rates of the used agrochemicals are shown in Table 1. The spray solution of the applied dose of each agrochemical was prepared according to the instructions indicated on packaging. One more subgroup of plants, per experiment, was sprayed only with water [WATER] and served as control. All the leaves of the treated plants were sprayed on both surfaces until the applied agrochemical run off. 2.4. Parameters measured
2.2. Experiments A total of nine (laboratory and field) experiments were conducted.
After the end of each experiment, the leaf visible injury was estimated and the following parameters were measured in the three middle (fully expanded) leaves of each plant.
C.J. Saitanis et al. / Environmental Pollution 197 (2015) 247e255 Table 1 The fungicides tested for their probable antiozonate traits along with the applied dose rates. Agrochemical Active constituent 1) 2) 3) 4) 5) 6) 7)
Used dosage rate Commercial names
Acibenzolar-S-methyl þ Metalaxyl-M Bion MX 44 WG Azoxystrobin Quadris 25%, SC Benomyl Benlate 50%, WP Hexaconazole Anvil 5%, SC Kresoxim-methyl Stroby 50%, WG Penconazole Topas 10%, EC Trifloxystrobin Flint 50%, WG
45 g/100 lit 150 cc/100 lit 75 g/100 lit 45 cc/100 lit 30 g/100 lit 75 cc/100 lit 22.5 g/100 lit
2.4.1. Visible injury For the visible injury estimation, the Visible Injury Index (V.I.I.) of each leaf of each plant was scored, as the percentage of leaf area showing necrosis or chlorosis, 24 h after the last fumigation. 2.4.2. Pigments’ content and pigments ratios The pigments’ contents [chlorophylls a and b and total carotenoids] were measured in acetone extracts of leaf tissues, of fixed size, using a double beam UV/VIS (Model Lambda 20, Perking Elmer) spectrophotometer, and calculated according to Lichtenthaler (1987). The chlorophyll content was calculated on a “per leaf area” base (mg cm2) (Saitanis et al., 2001). 2.4.3. Stomatal conductance The gs (cm s1) of the abaxial surface of leaves was measured in all plants using a porometer (AP4, Delta-T Devices Ltd). 2.4.4. Quantum yield of photosynthesis The FPSII of leaves was measured using a portable Plant Photosynthesis Meter instrument (PPM of EARS corp.). It was determined from two chlorophyll fluorescence measurements: once under ambient light conditions (Fs) and once at light saturation (F0 m). Then, the FPSII was calculated using the formula FPSII ¼ (F0 m Fs)/ F0 m, as suggested by Genty et al. (1989). This is a non-destructive method which can be easily used for in vivo stress detection in plants. 2.4.5. Superoxide dismutase For the SOD activity measurement, three leaf disks (d ¼ 0.7 cm) per plant were taken and homogenized to constitute a composite sample. The activity was measured according to the method of Beauchamp and Fridovich (1971). The assay mixture was exposed uniformly under a light provided by a 15 W fluorescent lamp for 10 min. Under these light conditions riboflavin oxidizes methionine, which is the electron donor. By donating an electron methionine generates superoxide anions (O: 2 ) that reduce the NBT, giving an insoluble purple coloured formazan. This colour change was measured by spectrophotometer at A560 nm. The presence of SOD inhibits the formazan production. One unit of SOD activity is defined as the amount of enzyme required to produce a 50% inhibition of NBT reduction. 2.5. Statistical analysis Because of the high multicollinearity between the measured parameters, the data of each parameter of each experiment was firstly separately submitted to ANOVA. Prior to this, data were tested for ANOVA assumptions and transformed, if needed, according to the BoxeCox method (Box and Cox, 1964). In these analyses the plants actually constituted the pseudo-replicates. In order to utilize the overall information and to generalize the results,
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the data from all the experiments were pooled and analysed together. To this purpose, and in order to remove the variability between experiments, the data of each parameter and each experiment, were transformed to z scores (standardized), in order to have a mean equals to zero and standard deviation equal to one. Thus, the average z-score of each agrochemical, in each experiment, constituted the data (real-replicate) of the overall analysis. This process allows a fair intercomparison of the measured variables as estimators/indicators of ozone-induced stress and removes any heterogeneity among experiments (Saitanis et al., 2014a). This process has not been applied to Chl a/Chl b and Car T/Chl T ratio parameters. Furthermore, in order a) to group the agrochemical with similar performance and b) to order the agrochemical according to their performance as potential protectants against ozone, Cluster and PCA analyses were applied, based on the effect size observed in the parameters measured in the ozone-treated plants. The effect size, quantifying the size of the difference between the group of ozoneexposed plants treated by an agrochemical (A) and those treated with water (W), was calculated, using the formula (Cohen, 1988):
½Ai W ffi ESi ¼ rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi h i. 2 ðni 1Þ*ðSDi Þ þ nw 1 *ðSDw Þ2 ðni þ nw 2Þ where Ai, SDi and ni stand for the average score, the standard deviation and the number of observations, respectively, in the ozoneexposed plants treated by the agrochemical i and W, SDw and nw stand for the average score, the standard deviation and the number of observations, respectively, in ozone-exposed plants treated only with water (reference group) (see Saitanis et al., 2014a). Data processing and statistical analyses were conducted using STATISTICA v.10 (© StatSoft Inc. 1984e2001) and the XLSTAT (© Addinsoft 1995e2013) software, along with the MS EXCEL 2010 (© Microsoft). 3. Results The mean and the range of values of each parameter measured over all of the nine experiments are synoptically presented in Table 2 while all the results of the two-way ANOVA tests are shown in Table 3. For the visible injury, only one-way ANOVA was conducted because all the plants exposed in the ozone-free air did not exhibit symptoms. No significant interaction effect between ozone and agrochemicals was observed for any of the measured parameters (Table 3). 3.1. Ozone exposure levels The ozone levels during the ambient experiments were not so different from those applied in the controlled environment Table 2 The mean, minimum and maximum values of each parameter measured over all of the nine experiments. Parameter
OZ
OZþ
Mean Min Max Foliar visible injury (%) e 4.74 Total Chlorophylls (mg cm2) Chl-a/b (unitless) 2.96 Carotenoids (mg cm2) 1.74 Car-T/Chl T (unitless) 0.27 Stomatal conductance gs (cm/s) 0.13 Quantum yield (unitless) 0.48 Superoxide dismutase (arbitrary units) 17.53
Mean Min
Max
e e 32.34 10.26 75.51 3.79 5.97 3.55 1.52 6.10 2.71 3.34 2.58 1.05 3.58 1.35 2.21 1.57 0.84 3.22 0.27 0.28 0.30 0.27 0.40 0.07 0.20 0.13 0.06 0.28 0.20 0.81 0.48 0.13 0.74 7.10 32.78 14.67 5.81 35.99
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Table 3 Analysis of variance results of the effects of ozone, agrochemical and Ozone Agrochemical interactions on several parameters measured in Bel-W3 plants. Given that control plants exhibited no injury at all, one way ANOVA was conducted e only for plants exposed to ozone e to test for the effects of agrochemicals on foliar visible injury. Numbers shown in boldface indicate statistically significant effects. Parameter
Foliar visible injury Total Chlorophylls Chl-a/b Carotenoids Car-T/Chl T Stomatal conductance Quantum yield Superoxide dismutase
Ozone (df ¼ 1)
Agrochemical (df ¼ 7)
Ozone agrochemical (df ¼ 7)
F
p
F
p
F
p
e 31.509 4.631 30.382 21.229 26.368 40.013 1.569
e 0.000 0.036 0.000 0.000 0.000 0.000 0.217
3.220 2.314 0.141 2.358 0.227 1.964 1.493 2.545
0.007 0.041 0.994 0.037 0.977 0.046 0.181 0.029
e 0.603 0.089 0.548 0.148 0.415 0.377 0.652
e 0.749 0.998 0.793 0.993 0.888 0.912 0.999
chambers at the laboratory experiments (the AOT40, over the seven exposure days, were 2240 nmol mol1 h at the laboratory exposures, 1642 nmol mol1 h at the urban ambient exposure and 1844 nmol mol1 h at the rural ambient exposure), allowing a comparability of the results (Fig. 1). 3.2. Visible injury All the exposed plants exhibited ozone-induced symptoms; none of the applied agrochemicals offered 100% protection. There was also variability in the overall ozone injury among agrochemicals (Fig. 2; Tables 2 and 3). Although there was a high variability within and between groups, over all, the fungicides of the group of triazoles (PENC and HEX) showed higher average protection efficacy (38.2 ± 8.6 and 30.6 ± 5.5%, respectively). In one of the experiments the protection offered by PENC reached the level of ~80%. KRSM showed about equal protection efficacy with HEX, but with higher variability (28.4 ± 15.3%). AZOX showed lower, but with adequate average protection efficacy (21.2 ± 15.1%). BENML showed even lower (20.1 ± 9%) protection efficacy while TRIFL did not essentially protect the plants at all (10.8 ± 11.3%). BION exhibited the highest variability and contrasting results (2 ± 17.3%). 3.3. Pigments' content and pigments’ ratios
Fig. 2. The average visible injury (in terms of z-scores) observed over all of the nine experiments. The vertical lines represent the standard errors. Columns marked by the same letter do not differ statistically significantly at level of significance a ¼ 0.05, after Bonferroni post hoc test.
plants treated by BENML (~17%). The lowest average chlorophyll content was observed in plants treated by BION, in both OZþ and OZ groups. The chl a/b ratio (Fig. 4) was, over all, reduced by ozone, although neither the effect of the agrochemical nor the interaction effects
Although within each agrochemical the levels of chlorophylls a and b separately or in total, did not differ between ozonized and control plants, overall they were reduced by ozone (by an average of ~25%) and they were affected by the agrochemicals (Fig. 3; Table 3). The highest average reduction was observed in plants treated with water only (~34%), while the lowest was observed in
Fig. 1. The time course of AOT40 in each of the seven experiments (weekly exposures) conducted in controlled environment chambers and in the two experiments conducted under ambient environmental conditions: a) in Athens (urban environment) and b) Aliartos (rural environment).
Fig. 3. The average total chlorophyll content (in z-scores) observed over all of the nine experiments. The measurements were conducted in Bel-W3 plants exposed either to ozone enriched (OZþ: red squared markers interconnected with red dashed line) or to ozone free (OZ: blue filled circles, interconnected with blue dashed line) air and treated either by agrochemical or with water (control group). The vertical lines stand for the standard errors. Means marked by the same letter do not differ statistically significantly at level of significance a ¼ 0.05, after Bonferroni post hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. The ratio of Chl a to Chl b measured in Bel-W3 plants exposed either to ozone enriched (OZþ; red squared markers) or to ozone free (OZ; blue filled circles) air and treated either by agrochemical or by water. The vertical lines stand for the s.e. Means marked by the same letter do not differ statistically significantly at level of significance a ¼ 0.05 after Bonferroni post hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 6. The ratio of total carotenoids to total chlorophyll measured in Bel-W3 plants exposed either to ozone enriched (OZþ; red squared markers) or to ozone free (OZ; blue filled circles) air and treated either by agrochemical or by water. The vertical lines stand for the standard errors. Means marked by the same letter do not differ statistically significantly at level of significance a ¼ 0.05 after Bonferroni post hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
were statistically significant (Table 3); the reduction ranged from 8% in plants treated by TRIFL to 20% in plants treated only with water. In the same way, the carotenoids content was affected by both ozone and agrochemicals (Fig. 5). Overall the ozone-exposed plants exhibited lower levels (~10%) of total carotenoids. In Car-T/Chl-T ratio a significant effect of ozone treatment was found; the ratio was higher (by average ~11%) in the ozone-treated plants (see Fig. 6). However, the substance treatment and the interactions were statistically insignificant (Table 3).
3.5. Quantum yield of photosynthesis Quantum yield of photosynthesis (FPSII) was significantly reduced by ozone (average reduction attributed to ozone ¼ ~15%), but it was not affected by the agrochemicals (Fig. 8). Their interaction effect was also insignificant (Table 3). The highest reduction (~26%) was observed in plants treated by KRM.
3.6. Stomatal conductance 3.4. Superoxide dismutase The SOD activity was significantly different among the groups treated by different agrochemicals (Fig. 7), but it was unaffected by ozone; the interaction effect was not statistically significant (Table 3). Plants treated by AZOX (10.7 units) or KRM (11.5 units) showed the lowest levels in SOD activity, although this was not significantly lower in comparison with SOD activity of control plants sprayed with water. The highest levels were observed in plants treated by BENML (18.8 units) and PENC (18.5 units).
Stomatal conductance was found to be affected by ozone, but not by the agrochemicals; the interaction effect was not statistically significant (Table 3). All the groups exposed to ozone exhibited lower gs (by average ~17%) in comparison with controls (Fig. 9). Although, within each agrochemical group none of the pairwise difference between ozone and control groups was significant, the overall effect of ozone was clear. The reduction ranged from ~6% in the group of HEX to ~30% in the group of PENC. The lower levels of gs were observed in the plants treated by BION (0.092 and 0.077 cm s1, in the OZ and OZþ groups, respectively).
Fig. 5. The average total carotenoids content (in z-scores) observed over all the nine experiments. The measures were conducted in Bel-W3 plants exposed either to ozone enriched (OZþ: red squared markers interconnected with red dashed line) or to ozone free (OZ: blue filled circles, interconnected with blue dashed line) air and treated either by agrochemical or by water (control group). The vertical lines stand for the standard errors. Means marked by the same letter do not differ statistically significantly at level of significance a ¼ 0.05 after Bonferroni post hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 7. The average SOD activity (in z-scores) observed over all the nine experiments. The measures were conducted in Bel-W3 plants exposed either to ozone enriched (OZþ: red squared markers interconnected with red dashed line) or to ozone free (OZ: blue filled circles, interconnected with blue dashed line) air and treated either by agrochemical or by water (control group). The vertical lines stand for the standard errors. Means marked by the same letter do not differ statistically significantly at level of significance a ¼ 0.05 after Bonferroni post hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 8. The average FPSII (in z-scores) observed over all the nine experiments. The measures were conducted in Bel-W3 plants exposed either to ozone enriched (OZþ: red squared markers interconnected with red dashed line) or to ozone free (OZ: blue filled circles, interconnected with blue dashed line) air and treated either by agrochemical or by water (control group). The vertical lines stand for the standard errors. Means marked by the same letter do not differ statistically significantly at level of significance a ¼ 0.05 after Bonferroni post hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.7. Multivariate analysis Based on the effect size of the agrochemicals on the measured parameters, a cluster analysis was performed and the relevant dendrogram has been created (Fig. 10). Four main groups were revealed: the 1st and the 2nd group had as solo members the BIOM and PENC, respectively; the 3rd and 4th group were consisted of three and two agrochemicals, respectively (HEX, BENML & TRIFL and AZOX & KRM respectively) (Fig. 10). All of these parameters are, more or less, inter-correlated and, thus, there is a redundancy in information (Saitanis et al., 2014a). So, Principal Components Analysis, based on the effect size (ESi), was conducted to show the overall performance of the agrochemical in plants’ response to ozone. The resulting biplot is shown in Fig. 11. The first two principal axes explained 75.2% of the total variance, with the PC1 explaining 57.8%. Thus, PC1 would be considered as the major axis for ordinations of the cultivars. By projecting the agrochemicals on the PC1, they can be ordered, in
Fig. 9. The stomatal conductance (z-scores) measured in Bel-W3 plants treated by seven agrochemical or water (control) and either exposed to ozone (OZþ: red squared markers interconnected with red dashed line) or to ozone free (OZ: blue filled circles, interconnected with blue dashed line) air and treated either by agrochemical or by water (control group). The vertical lines stand for the standard error; Means marked by the same letter do not differ statistically significantly at level of significance a ¼ 0.05 after Bonferroni post hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 10. Dendrogram grouping the seven agrochemicals, based on effect size observed in the parameters. Based on their effect size (ESi) of the ozone-induced changes on each of the measured parameters: visible injury (Inj), total chlorophylls (Chl T), total carotenoids (Car), quantum yield of photosynthesis (FPSII), superoxide dismutase (SOD), and stomatal conductance (gs), measured in the ozone treated plants. Dissimilarity measure: Squared Euclidean distance; Agglomeration method: Complete linkage.
terms of their overall performance, as PECN KRM AZOX > BENML HEX > TRIFL >> BION. 4. Discussion Ozone affected significantly most of the measured parameters. It caused visible foliar injury, reduced chlorophylls' and total carotenoids’ content and negatively affected photosynthesis, stomatal conductance and SOD activity in the exposed plants. By the exception of BION, none of the other agrochemicals affected any of the above-mentioned parameters at the plants grown in the chamber supplied with ozone-free air (OZ).
Fig. 11. Biplot of the PCA ordination (with varimax rotation) of the seven agrochemicals, based on their effect size (ESi) of the ozone-induced changes on each of the measured parameters: visible injury (Inj), total chlorophylls (Chl T), total carotenoids (Car), quantum yield of photosynthesis (FPSII), superoxide dismutase (SOD), and stomatal conductance (gs).
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4.1. Ozone exposure levels The applied ozone levels in the chamber experiments were similar to those occurred in the field experiments (Fig. 1) and close enough to those which have been previously recorded in several rural areas of Greece, where ozone e during the summer months and midday-afternoon hours e often reaches or exceeds the level of 70 nmol mol1 (i.e. Saitanis, 2003; Saitanis et al., 2003, 2004, 2014b). Even higher ozone levels have been reported for rural areas in other nchez et al., 2005). Thus, the experiments concountries (e.g. Sa ducted here e in terms of ozone levels e can be considered realistic and the outcome applicable to the agricultural practice. 4.2. Visible injury In some experiments, the plants treated with some agrochemicals exhibited higher degree of symptoms than the reference plants sprayed only with water. As a result, the standard deviation and the standards error lines include negative values. The worst case was that of BION where the overall average was negative. This is not only due to lack of protection against ozone, but also because BION enhanced the leaf visible injury by causing phytotoxic symptoms. Such symptoms were developed also in the group of plants sprayed with BION and grown in the ozone-free chamber. The phytotoxic symptoms caused by BION were similar to those caused by ozone, making the discrimination impossible at the plants treated by both of them. 4.3. Pigments' content and pigments’ ratios Ozone affected the pigments content negatively. Plenty of researches have shown that chlorophyll content is reduced by ozone in several plant species, e.g. in wheat (Pleijel et al., 2006), tobacco (Saitanis and Karandinos, 2001b), and other plant species (Madkour and Laurence, 2002). Our results are in agreement with those of Calatayud and Barreno (2004). Nonetheless, there are many publications reporting no reduction of chlorophyll for some plant species exposed to ozone, e.g. for Cucumis sativus (Agrawal et al., 1993) and Oryza sativa (Welfare et al., 1996). Although ozone is improbable to penetrate cell plasmalemma, secondary byproducts (e.g. free radicals) of ozone oxidations can affect pigment concentrations (Gill and Tuteja, 2010) into plants’ cells. In our study only BION reduced chlorophylls' content while the other agrochemicals had no effects. There are, however, reports supporting that strobilurins increase chlorophylls' content (Grossmann and Retzlaff, 1997; Mercer and Ruddock, 1998). Gopi et al. (2007) found that HEX and PBZ increased the total chlorophylls' content by ~39.1 and 35.9%, respectively, in carrot (Daucus carota L.) plants. Such increase has been attributed to the increase of the cytokinins' level and the stimulation of chlorophylls’ biosynthesis (Fletcher et al., 2000). The two chlorophyll forms (Chl a and Chl b) seem to differ slightly in their sensitivity to the ozone oxidative effect but an effect of agrochemicals was not observed. The latter corresponds to the findings of Khalili et al. (1990) who reported no effect of triazole fungicides on Chl a/b ratio, but disagrees with those of Gopi et al. (2007) who found that hexaconazole and paclobutrazol increased chl b more than chl a. The Chl a/b ratio is considered as an indication of the light-harvesting complex size of photosystem II (PSII) (Ferraro et al., 2003; Pellegrini, 2014). The slight reduction in Chl a/ b ratio, observed in our present study, suggests a slight higher sensitivity to ozone of Chl a in comparison with Chl b. These results are consistent with those of Saitanis et al. (2001) where Chl a was shown to be more vulnerable to ozone oxidative effect in comparison with Chl b, but only by about 10%, which is quite low and
253
hardly detectable. This probably explains the fact that other researchers have reported no effects of ozone to Chl a/b ratio (e.g. Mikkelsen et al., 1995). The total carotenoids were overall reduced by ozone. This is consistent with the results of Calatayud and Barreno (2004) who also reported a reduction in carotenoids in two lettuce varieties exposed to ozone. Similar reduction in carotenoids has been also reported by Madkour and Laurence (2002) who observed that O3 affected the carotenoids' concentration of several Egyptian species and cultivars, but the degree of carotenoids' loss did not always reflect the level of sensitivity to O3. Concerning the effects of agrochemicals on total carotenoids, although the overall effect was significant, no any significant difference was confirmed within agrochemicals between OZþ and OZ groups. This is in contrast with the results of Khalili et al. (1990) where carotenoids' content per unit of area was increased in maize seedlings treated with triazole fungicides. The carotenoids' reduction is considered of high significance for plants’ functions, because carotenoids are lightharvesting pigments contributing to photosynthesis and they seem to protect chlorophylls against oxidative destruction by O2, under high light intensities (Gill and Tuteja, 2010). In our experiments, ozone reduced total chlorophyll more than total carotenoids. Similar higher vulnerability of chlorophylls in comparison to carotenoids has been observed also in Tilia americana plants treated by increased ozone levels (Pellegrini, 2014). Since carotenoids are yellowing pigments, in conjunction with the Chl a/b ratio, they have been suggested as good markers of chlolez-Rodríguez et al., 2001). No effect of roplasts’ adaptation (Gonza the agrochemicals on Car/Chl ratio was observed in our study. In accordance, Khalili et al. (1990) reported no effect of triazole fungicides on Chl/Car ratio. 4.4. Superoxide dismutase Chlorophylls' loss is the major symptom of leaf senescence and prematurity caused by ozone. Senescence is driven by active oxygen species (AOS), the most common of which are superoxide _ radicals (O: 2 ), hydroxyl radicals (OH), hydrogen peroxide (H2O2) and singlet oxygen (1O2), all of which impair metabolic processes (Zhang et al., 2010). Superoxide dismutase (SOD), catalase (CAT), ascorbate-peroxidase (APX), peroxidase (POX) and glutathione reductase (GR) are key enzymes inhibiting the accumulation of active oxygen species in plants' tissues. Several studies have reported that SOD, and other antioxidant enzymes, regulate responses and acclimation of plants to stresses (Wu and VonTiedemann, 2001, 2002; Pandey et al., 2014). There are, however, contradictory reports on the effects of ozone on the antioxidant enzymes activity. Ozone has been reported to induce significant increases in the activity of antioxidant enzymes SOD, POX and CAT after two days of fumigation of spring barley (Hordeum vulgare L.) plants, while, after four days of fumigation, these enzymes declined to a level lower than in non-fumigated plants, due to the oxidative degradation of leaf proteins (Wu and Vo Tiedemann, 2002). Calatayud et al. (2003), reported stimulation of SOD and APX in spinach plants (Spinacia oleracea L.) as a response to ozone fumigation. Significant increase in SOD activity has also been reported by Calatayud et al. (2010) in evergreen plants (Pistacia terebinthus, Viburnum lantana) and deciduous plants (Pistacia lentiscus and Viburnum tinus) exposed to ozone. However, this is not always the case: some studies reported no change, or even reduction, in SOD activity, after exposure to ozone. For example, Kubo et al. (1995) found no difference in total SOD activity levels in Arabidopsis thaliana plants treated with 150 nmol mol1 O3 for 4 days. Esposito et al. (2009) reported, on the one hand, increase of SOD activity in Bel-W3 tobacco plants exposed to ozone, but, on the other hand,
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reduction of SOD in tomato plants. Other studies (Wohlgemuth et al., 2002) have shown that generation and accumulation of superoxide and/or peroxide may differ among species and that SOD activity oscillates in plants growing under stressing environment (Iriti and Faoro, 2008; Esposito et al., 2009). This may depend on the plant species, the stage of growth (Kuk et al., 2003), etc. The forms of the SOD (Fe-SOD, Mn-SOD or Cu/Zn-SOD iso-enzymes) may also differ in response to oxidative stress; the presence of these isoenzymes complicates further the elucidation of the SOD role in plants’ response to ozone (Calatayud and Barreno, 2001; De Sousa et al., 2013). There are studies supporting that strobilurins increase dry matter, gain yield, protein and chlorophyll content and delay senescence (Grossmann and Retzlaff, 1997; Mercer and Ruddock, 1998; Zhang et al., 2010). Several other agrochemicals, including fungicides and herbicides, have been reported possessing antioxidative properties, preventing, thus, ozone induced injury (Agathokleous et al., 2014b). However, little is known about the exact mechanism of their antiozonate action. De Sousa et al. (2013) found that metalaxyl induced oxidative stress to cell suspension cultures of Solanum nigrum L. This is in agreement with our findings. BION (that is a mixture of acibenzolar-S-methyl 4% w/w and metalaxyl-M 37.52% w/w) enhanced the oxidative effects of ozone by causing phytotoxic symptoms. It seems that strobilurins fungicides are effective in delaying senescence in spring wheat and spring burley by inducing activation of antioxidant enzymes (Wu et al., 2001; Zhang et al., 2010). Zhang et al. (2010) found that, in comparison to the untreated plants, the fungicides JS399-19, azoxystrobin, tebuconazole and carbendazim enhanced the SOD activity in the flag leaves of wheat plants. However, Calatayud and Barreno (2001) have reported significant decrease in SOD activity of ozone-exposed tomato (Lycopersicon esculentum Mill.) plants that had not been treated with benomyl, in comparison to the treated ones. Benomyl ameliorated the ozone induced oxidative stress by preventing the peroxidation of membrane lipids and, thus, protecting PSII from ozone oxidative effect.
5. Conclusions After testing the potential of seven agrochemicals to protect plants against ozone, we found that the triazoles fungicides penconazole and hexaconazole offered higher protection to plants against ozone than the other agrochemicals. Kresoxim-methyl showed similar protection efficacy with hexaconazole, but with higher variability. Azoxystrobin showed lower protection, while benomyl showed even lower. Trifloxystrobin did not protect the plants at all. The worst agrochemical was BION, which also caused necrotic spots to the plants not exposed to ozone. Briefly, based on their ozone-protection efficacy, the tested agrochemicals can be ordered as follow: Penconazole Kresoxim-methyl Azoxystrobin > Benomyl Hexaconazole > Trifloxystrobin >> Acibenzolar-Smethyl þ metalaxyl-M. However, the high variability in the antiozonate performance of the tested agrochemicals suggests that the research should be continued until some commercially available and economically affordable substances with even higher antiozonate performance will be identified, for the benefit of the applied intensive agriculture. Besides, the results presented here come from both laboratory and field experiments, using the Bel-W3 tobacco variety, which is hypersensitive to ozone. The effectiveness of the agrochemicals may be species-specific and it may be influenced by environmental factors (e.g. edaphic parameters, etc.) affecting the response of plants to ozone and probably to agrochemicals. Acknowledgement This investigation was partly funded by the EPEAEK “Pythagoras I” project, (European Union, 75%, and the Greek Ministry of National Education and Religious Affairs, 25%; AUA-ELKE Code: 73.00.00.24.0001). We thank the Assistant Professor Georgios Papadopoulos (Division of Informatics, Mathematics and Statistics – Agricultural University of Athens) for the usefull dicussion about the statistical analysis of the data.
4.5. Quantum yield
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Environmental Pollution journal homepage: www.elsevier.com/locate/envpol
Screening agrochemicals as potential protectants of plants against ozone phytotoxicity Costas J. Saitanis a, *, Dimitrios V. Lekkas a, Evgenios Agathokleous a, 1, Fotini Flouri b a b
Laboratory of Ecology and Environmental Sciences, Agricultural University of Athens, Iera Odos 75, Votanikos 11855, Athens, Greece Laboratory of Pesticide Science, Agricultural University of Athens, Iera Odos 75, Votanikos 11855, Athens, Greece
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 September 2014 Received in revised form 3 November 2014 Accepted 6 November 2014 Available online 26 November 2014
We tested seven contemporary agrochemicals as potential plant protectants against ozone phytotoxicity. In nine experiments, Bel-W3 tobacco plants were experienced weekly exposures to a) 80 nmol mol1 of ozone-enriched or ozone-free air in controlled environment chambers, b) an urban air polluted area, and c) an agricultural-remote area. Ozone caused severe leaf injury, reduced chlorophylls' and total carotenoids' content, and negatively affected photosynthesis and stomatal conductance. Penconazole, (35% ± 8) hexaconazole (28% ± 5) and kresoxim-methyl (28% ± 15) showed higher plants’ protection (expressed as percentage; mean ± s.e.) against ozone, although the latter exhibited a high variability. Azoxystrobin (21% ± 15) showed lower protection efficacy and Benomyl (15% ± 9) even lower. Trifloxystrobin (7% ± 11) did not protect the plants at all. Acibenzolar-S-methyl þ metalaxyl-M (Bion MX) (6% ± 17) exhibited the higher variability and contrasting results: in some experiments it showed some protection while in others it intensified the ozone injury by causing phytotoxic symptoms on leaves, even in control plants. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Fungicides Bion MX Biomonitoring Tobacco Azoxystrobin Benomyl Penconazole Greece
1. Introduction Ambient ozone level has risen over the last four decades, it is still being gradually increasing, and it is expected to be a major menace for cultivated plants and natural ecosystems in the near future (Fishman et al., 2010). The nowadays occurring ambient ozone levels are high enough to negatively affect wild plant species (Bermejo et al., 2003; Feng et al., 2014; Agathokleous et al., 2014a) and field-growing cultivated plants (Booker et al., 2009; Avnery et al., 2011), especially in the Mediterranean countries (Saitanis and Karandinos, 2001a; Saitanis, 2003). Retardation of plants' growth and severe yields’ losses in crop plants, due to increased ozone levels, have been reported (Booker et al., 2009). Fishman et al. (2010) estimated the global economic loss to the farming community to exceed $10 billion annually.
* Corresponding author. E-mail addresses: [email protected] (C.J. Saitanis), [email protected] (E. Agathokleous), [email protected] (F. Flouri). 1 Present address: Silviculture and Forest Ecological Studies, Hokkaido University, Sapporo, Hokkaido 060-8589, Japan. http://dx.doi.org/10.1016/j.envpol.2014.11.013 0269-7491/© 2014 Elsevier Ltd. All rights reserved.
The main goal, nowadays, is the reduction of ambient ozone levels through the reductions of its precursors (NOx, VOCs). This, however, is considered a long-term goal. Thus, methods for protection of plants against ozone should be urgently developed. Many substances have been tested as potential protectants of plants against ozone phytotoxicity, among which agrochemicals, such as fungicides, herbicides, insecticides, and plant growth regulators (Manning, 2000). Substances that cause stomatal closure, such as phenylmercuric acetate and monoethyl esters of decenylsuccinic acid have been tested many years ago (Rich, 1964; Seidman et al., 1965), and they have been found to protect plants. Similar protective efficacy has also been obtained by abscisic acid (ABA), which also induces stomata closure (Lin et al., 2001). In a recent study, Francini et al. (2011) found that the antitranspirant di-1-p-menthene significantly protected Pinto bean plants from acute ozone injury, but this substance did not work on tobacco plants (Agathokleous et al., 2014b). However, the stomatal closure induced by such substances does not only impede ozone entry to leaf tissues, but simultaneously impedes CO2 uptake, which, in turn, may lead to undesirable yield loss due to reduced CO2 assimilation.
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A well-known promising antioxidant is EDU (N-[2-(2-oxo-1imidazolidinyl)ethyl]-N'-phenylurea; abbreviated EDU for ethylenediurea) (Carnahan et al., 1978; Feng et al., 2010; Manning et al., 2011). However, the major disadvantage of EDU is that it is not commercially available (only very few laboratories and only for research purpose can prepare it). A short review of the available publications shows that many biocides or some antioxidant substances, available in commerce, may offer protection of plants against ozone. Since 1972, benomyl (a fungicide now being banned) was tested as plant-protectant against ozone (Reinert and Spurr, 1972; Manning et al., 1973a, b, c) with positive results. When benomyl was applied as a foliar spray to field growing bean plants resulted to a 70e80% suppression of oxidant injury (Manning et al., 1973b), while when applied in Bel-W3 plants mitigated leaf injury up to ~60% (Reinert and Spurr, 1972). Azoxystrobin and epoxiconazole fungicides have also been reported possessing antiozonate properties (Wu and Von Tiedemann, 2002). Although numerous substances have been tested and proposed as potential antiozonants over the years, there is a lack of understanding of their antiozonate mechanisms for all of them. Even for EDU, which is the most studied antiozonate substance, the actual underline mechanism has not been uncovered yet (Agathokleous et al., 2014c). In the framework of this investigation, we tested seven contemporary agrochemicals as potential plant-protectants against ozone phytotoxicity. We focused on agrochemicals because they are commercially available and they are extensively used in intensive agriculture. The knowledge that some agrochemicals exhibit e as a side effect e antiozonate property, gives to them an advantage, when compared with other homologous agrochemicals, so they would be preferable in agricultural practice. Besides, such agrochemicals would also be used by researchers in setting up control group plants in studies aiming to assess crop yield loss by ozone under field conditions. 2. Material and methods 2.1. Plant materials As experimental plant, the Bel W3 tobacco (Nicotiana tabacum L.) variety was used. This variety is known to be hypersensitive to ozone (Saitanis and Karandinos, 2001b) with a threshold of sensitivity of about 40 nmol mol1 of ozone for few hours. Thus it is not cultivated and often the results coming from experiments based on this variety e especially those dealing with yield e are not representative to what really happens in cultivations. However, because of its high sensitivity to ozone, it is widely used as a useful tool in laboratory and field experiments to reveal the mechanisms of plants’ response to ozone. Seeds of Bel-W3 variety were planted in peat. After germination, seedlings were transferred to 12 cm d plastic pots (one seedling per pot) filled with commercial soil (Floran, STEDIP corp.). When plants reached the fifth leaf stage of growth, they were selected for uniformity and divided equally between treatments’ groups. The number of plants used per agrochemical treatment (subgroup) differed among experiments (8-20 plants per agrochemical per ozone treatment per experiment), with a total of about 800 plants. Within each chamber the positions of the plants were randomly rotated, at least once daily, in order to minimize any chamber edge effects.
2.2.1. Laboratory experiments In seven experiments, sets of Bel-W3 tobacco potted plants were transferred to two identical walk-in chambers (230 190 170 cm Model 60R - CDR corp.) under the same conditions: 14:10 (L:D) h photoperiod, 65 ± 3% relative humidity and 28 ± 0.5 C temperature. Both chambers were supplied with purified air, by passing it through dry purafil (KMnO4) and activated charcoal filters, in order to minimize contamination by the ambient air pollutants. In one of the chambers the filtered air was enriched with ozone. Ozone was produced by an electric generator (Air-Zone® XT-6000) that uses a new patented technology producing no NOx byproducts. Teflon lines led sample air from the chamber to a UV ozone analyser (Environnement S.A O3 42M). The ozone concentration within the chamber was stabilized via a feedback controller (CDR corp.). The wind speed within the chambers, above the plants’ canopy, was about 2 m s1. In each experiment, the plants of the “ozone chamber” were exposed to 80 nmol mol1 of ozone for 8 h per day for 7 days (AOT40: 2240 nmol mol1 h); this level is very close to the ambient ozone levels occurring in rural - agricultural areas nowadays (Saitanis, 2003). Those plants constituted the “ozone exposed” main group (hereafter: OZþ) while the plants of the chamber provided with filtered air constituted the “ozone-free” e control main group (hereafter: OZ). In each experiment, the plants of each chamber were further subdivided to eight subgroups. The plants of each of the seven subgroups were sprayed with one of the agrochemicals, as described below, while those of the eighth subgroup were sprayed with distilled water and constituted the “control subgroup”. 2.2.2. Field experiments Two field experiments were additionally conducted to test the potential protective role of the used agrochemicals under ambient conditions. One ambient experiment was conducted in the urban (polluted) campus of Agricultural University of Athens (AUA). In this experiment 9e12 plants per treatment were used. The second ambient experiment (six plants per treatment) was conducted at the agricultural area of Aliartos, located about 70 km away (NW) from Athens. In the overall data analysis, the plants exposed to ambient urban (AUA) or rural (Aliartos) environment were considered exposed to ozone enriched air (OZþ). 2.3. Agrochemicals tested In each experiment, different subgroups of plants were sprayed with different water solutions of the following seven agrochemicals (in brackets their abbreviation): azoxystrobin [AZOX], benomyl [BENML], hexaconazole [HEX], kresoxim-methyl [KRM], penconazole [PENC], trifloxystrobin [TRIFL] and the mixture acibenzolar-Smethyl þ metalaxyl-M [BION]. The active constituents, the trade names and the used dose rates of the used agrochemicals are shown in Table 1. The spray solution of the applied dose of each agrochemical was prepared according to the instructions indicated on packaging. One more subgroup of plants, per experiment, was sprayed only with water [WATER] and served as control. All the leaves of the treated plants were sprayed on both surfaces until the applied agrochemical run off. 2.4. Parameters measured
2.2. Experiments A total of nine (laboratory and field) experiments were conducted.
After the end of each experiment, the leaf visible injury was estimated and the following parameters were measured in the three middle (fully expanded) leaves of each plant.
C.J. Saitanis et al. / Environmental Pollution 197 (2015) 247e255 Table 1 The fungicides tested for their probable antiozonate traits along with the applied dose rates. Agrochemical Active constituent 1) 2) 3) 4) 5) 6) 7)
Used dosage rate Commercial names
Acibenzolar-S-methyl þ Metalaxyl-M Bion MX 44 WG Azoxystrobin Quadris 25%, SC Benomyl Benlate 50%, WP Hexaconazole Anvil 5%, SC Kresoxim-methyl Stroby 50%, WG Penconazole Topas 10%, EC Trifloxystrobin Flint 50%, WG
45 g/100 lit 150 cc/100 lit 75 g/100 lit 45 cc/100 lit 30 g/100 lit 75 cc/100 lit 22.5 g/100 lit
2.4.1. Visible injury For the visible injury estimation, the Visible Injury Index (V.I.I.) of each leaf of each plant was scored, as the percentage of leaf area showing necrosis or chlorosis, 24 h after the last fumigation. 2.4.2. Pigments’ content and pigments ratios The pigments’ contents [chlorophylls a and b and total carotenoids] were measured in acetone extracts of leaf tissues, of fixed size, using a double beam UV/VIS (Model Lambda 20, Perking Elmer) spectrophotometer, and calculated according to Lichtenthaler (1987). The chlorophyll content was calculated on a “per leaf area” base (mg cm2) (Saitanis et al., 2001). 2.4.3. Stomatal conductance The gs (cm s1) of the abaxial surface of leaves was measured in all plants using a porometer (AP4, Delta-T Devices Ltd). 2.4.4. Quantum yield of photosynthesis The FPSII of leaves was measured using a portable Plant Photosynthesis Meter instrument (PPM of EARS corp.). It was determined from two chlorophyll fluorescence measurements: once under ambient light conditions (Fs) and once at light saturation (F0 m). Then, the FPSII was calculated using the formula FPSII ¼ (F0 m Fs)/ F0 m, as suggested by Genty et al. (1989). This is a non-destructive method which can be easily used for in vivo stress detection in plants. 2.4.5. Superoxide dismutase For the SOD activity measurement, three leaf disks (d ¼ 0.7 cm) per plant were taken and homogenized to constitute a composite sample. The activity was measured according to the method of Beauchamp and Fridovich (1971). The assay mixture was exposed uniformly under a light provided by a 15 W fluorescent lamp for 10 min. Under these light conditions riboflavin oxidizes methionine, which is the electron donor. By donating an electron methionine generates superoxide anions (O: 2 ) that reduce the NBT, giving an insoluble purple coloured formazan. This colour change was measured by spectrophotometer at A560 nm. The presence of SOD inhibits the formazan production. One unit of SOD activity is defined as the amount of enzyme required to produce a 50% inhibition of NBT reduction. 2.5. Statistical analysis Because of the high multicollinearity between the measured parameters, the data of each parameter of each experiment was firstly separately submitted to ANOVA. Prior to this, data were tested for ANOVA assumptions and transformed, if needed, according to the BoxeCox method (Box and Cox, 1964). In these analyses the plants actually constituted the pseudo-replicates. In order to utilize the overall information and to generalize the results,
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the data from all the experiments were pooled and analysed together. To this purpose, and in order to remove the variability between experiments, the data of each parameter and each experiment, were transformed to z scores (standardized), in order to have a mean equals to zero and standard deviation equal to one. Thus, the average z-score of each agrochemical, in each experiment, constituted the data (real-replicate) of the overall analysis. This process allows a fair intercomparison of the measured variables as estimators/indicators of ozone-induced stress and removes any heterogeneity among experiments (Saitanis et al., 2014a). This process has not been applied to Chl a/Chl b and Car T/Chl T ratio parameters. Furthermore, in order a) to group the agrochemical with similar performance and b) to order the agrochemical according to their performance as potential protectants against ozone, Cluster and PCA analyses were applied, based on the effect size observed in the parameters measured in the ozone-treated plants. The effect size, quantifying the size of the difference between the group of ozoneexposed plants treated by an agrochemical (A) and those treated with water (W), was calculated, using the formula (Cohen, 1988):
½Ai W ffi ESi ¼ rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi h i. 2 ðni 1Þ*ðSDi Þ þ nw 1 *ðSDw Þ2 ðni þ nw 2Þ where Ai, SDi and ni stand for the average score, the standard deviation and the number of observations, respectively, in the ozoneexposed plants treated by the agrochemical i and W, SDw and nw stand for the average score, the standard deviation and the number of observations, respectively, in ozone-exposed plants treated only with water (reference group) (see Saitanis et al., 2014a). Data processing and statistical analyses were conducted using STATISTICA v.10 (© StatSoft Inc. 1984e2001) and the XLSTAT (© Addinsoft 1995e2013) software, along with the MS EXCEL 2010 (© Microsoft). 3. Results The mean and the range of values of each parameter measured over all of the nine experiments are synoptically presented in Table 2 while all the results of the two-way ANOVA tests are shown in Table 3. For the visible injury, only one-way ANOVA was conducted because all the plants exposed in the ozone-free air did not exhibit symptoms. No significant interaction effect between ozone and agrochemicals was observed for any of the measured parameters (Table 3). 3.1. Ozone exposure levels The ozone levels during the ambient experiments were not so different from those applied in the controlled environment Table 2 The mean, minimum and maximum values of each parameter measured over all of the nine experiments. Parameter
OZ
OZþ
Mean Min Max Foliar visible injury (%) e 4.74 Total Chlorophylls (mg cm2) Chl-a/b (unitless) 2.96 Carotenoids (mg cm2) 1.74 Car-T/Chl T (unitless) 0.27 Stomatal conductance gs (cm/s) 0.13 Quantum yield (unitless) 0.48 Superoxide dismutase (arbitrary units) 17.53
Mean Min
Max
e e 32.34 10.26 75.51 3.79 5.97 3.55 1.52 6.10 2.71 3.34 2.58 1.05 3.58 1.35 2.21 1.57 0.84 3.22 0.27 0.28 0.30 0.27 0.40 0.07 0.20 0.13 0.06 0.28 0.20 0.81 0.48 0.13 0.74 7.10 32.78 14.67 5.81 35.99
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Table 3 Analysis of variance results of the effects of ozone, agrochemical and Ozone Agrochemical interactions on several parameters measured in Bel-W3 plants. Given that control plants exhibited no injury at all, one way ANOVA was conducted e only for plants exposed to ozone e to test for the effects of agrochemicals on foliar visible injury. Numbers shown in boldface indicate statistically significant effects. Parameter
Foliar visible injury Total Chlorophylls Chl-a/b Carotenoids Car-T/Chl T Stomatal conductance Quantum yield Superoxide dismutase
Ozone (df ¼ 1)
Agrochemical (df ¼ 7)
Ozone agrochemical (df ¼ 7)
F
p
F
p
F
p
e 31.509 4.631 30.382 21.229 26.368 40.013 1.569
e 0.000 0.036 0.000 0.000 0.000 0.000 0.217
3.220 2.314 0.141 2.358 0.227 1.964 1.493 2.545
0.007 0.041 0.994 0.037 0.977 0.046 0.181 0.029
e 0.603 0.089 0.548 0.148 0.415 0.377 0.652
e 0.749 0.998 0.793 0.993 0.888 0.912 0.999
chambers at the laboratory experiments (the AOT40, over the seven exposure days, were 2240 nmol mol1 h at the laboratory exposures, 1642 nmol mol1 h at the urban ambient exposure and 1844 nmol mol1 h at the rural ambient exposure), allowing a comparability of the results (Fig. 1). 3.2. Visible injury All the exposed plants exhibited ozone-induced symptoms; none of the applied agrochemicals offered 100% protection. There was also variability in the overall ozone injury among agrochemicals (Fig. 2; Tables 2 and 3). Although there was a high variability within and between groups, over all, the fungicides of the group of triazoles (PENC and HEX) showed higher average protection efficacy (38.2 ± 8.6 and 30.6 ± 5.5%, respectively). In one of the experiments the protection offered by PENC reached the level of ~80%. KRSM showed about equal protection efficacy with HEX, but with higher variability (28.4 ± 15.3%). AZOX showed lower, but with adequate average protection efficacy (21.2 ± 15.1%). BENML showed even lower (20.1 ± 9%) protection efficacy while TRIFL did not essentially protect the plants at all (10.8 ± 11.3%). BION exhibited the highest variability and contrasting results (2 ± 17.3%). 3.3. Pigments' content and pigments’ ratios
Fig. 2. The average visible injury (in terms of z-scores) observed over all of the nine experiments. The vertical lines represent the standard errors. Columns marked by the same letter do not differ statistically significantly at level of significance a ¼ 0.05, after Bonferroni post hoc test.
plants treated by BENML (~17%). The lowest average chlorophyll content was observed in plants treated by BION, in both OZþ and OZ groups. The chl a/b ratio (Fig. 4) was, over all, reduced by ozone, although neither the effect of the agrochemical nor the interaction effects
Although within each agrochemical the levels of chlorophylls a and b separately or in total, did not differ between ozonized and control plants, overall they were reduced by ozone (by an average of ~25%) and they were affected by the agrochemicals (Fig. 3; Table 3). The highest average reduction was observed in plants treated with water only (~34%), while the lowest was observed in
Fig. 1. The time course of AOT40 in each of the seven experiments (weekly exposures) conducted in controlled environment chambers and in the two experiments conducted under ambient environmental conditions: a) in Athens (urban environment) and b) Aliartos (rural environment).
Fig. 3. The average total chlorophyll content (in z-scores) observed over all of the nine experiments. The measurements were conducted in Bel-W3 plants exposed either to ozone enriched (OZþ: red squared markers interconnected with red dashed line) or to ozone free (OZ: blue filled circles, interconnected with blue dashed line) air and treated either by agrochemical or with water (control group). The vertical lines stand for the standard errors. Means marked by the same letter do not differ statistically significantly at level of significance a ¼ 0.05, after Bonferroni post hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. The ratio of Chl a to Chl b measured in Bel-W3 plants exposed either to ozone enriched (OZþ; red squared markers) or to ozone free (OZ; blue filled circles) air and treated either by agrochemical or by water. The vertical lines stand for the s.e. Means marked by the same letter do not differ statistically significantly at level of significance a ¼ 0.05 after Bonferroni post hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 6. The ratio of total carotenoids to total chlorophyll measured in Bel-W3 plants exposed either to ozone enriched (OZþ; red squared markers) or to ozone free (OZ; blue filled circles) air and treated either by agrochemical or by water. The vertical lines stand for the standard errors. Means marked by the same letter do not differ statistically significantly at level of significance a ¼ 0.05 after Bonferroni post hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
were statistically significant (Table 3); the reduction ranged from 8% in plants treated by TRIFL to 20% in plants treated only with water. In the same way, the carotenoids content was affected by both ozone and agrochemicals (Fig. 5). Overall the ozone-exposed plants exhibited lower levels (~10%) of total carotenoids. In Car-T/Chl-T ratio a significant effect of ozone treatment was found; the ratio was higher (by average ~11%) in the ozone-treated plants (see Fig. 6). However, the substance treatment and the interactions were statistically insignificant (Table 3).
3.5. Quantum yield of photosynthesis Quantum yield of photosynthesis (FPSII) was significantly reduced by ozone (average reduction attributed to ozone ¼ ~15%), but it was not affected by the agrochemicals (Fig. 8). Their interaction effect was also insignificant (Table 3). The highest reduction (~26%) was observed in plants treated by KRM.
3.6. Stomatal conductance 3.4. Superoxide dismutase The SOD activity was significantly different among the groups treated by different agrochemicals (Fig. 7), but it was unaffected by ozone; the interaction effect was not statistically significant (Table 3). Plants treated by AZOX (10.7 units) or KRM (11.5 units) showed the lowest levels in SOD activity, although this was not significantly lower in comparison with SOD activity of control plants sprayed with water. The highest levels were observed in plants treated by BENML (18.8 units) and PENC (18.5 units).
Stomatal conductance was found to be affected by ozone, but not by the agrochemicals; the interaction effect was not statistically significant (Table 3). All the groups exposed to ozone exhibited lower gs (by average ~17%) in comparison with controls (Fig. 9). Although, within each agrochemical group none of the pairwise difference between ozone and control groups was significant, the overall effect of ozone was clear. The reduction ranged from ~6% in the group of HEX to ~30% in the group of PENC. The lower levels of gs were observed in the plants treated by BION (0.092 and 0.077 cm s1, in the OZ and OZþ groups, respectively).
Fig. 5. The average total carotenoids content (in z-scores) observed over all the nine experiments. The measures were conducted in Bel-W3 plants exposed either to ozone enriched (OZþ: red squared markers interconnected with red dashed line) or to ozone free (OZ: blue filled circles, interconnected with blue dashed line) air and treated either by agrochemical or by water (control group). The vertical lines stand for the standard errors. Means marked by the same letter do not differ statistically significantly at level of significance a ¼ 0.05 after Bonferroni post hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 7. The average SOD activity (in z-scores) observed over all the nine experiments. The measures were conducted in Bel-W3 plants exposed either to ozone enriched (OZþ: red squared markers interconnected with red dashed line) or to ozone free (OZ: blue filled circles, interconnected with blue dashed line) air and treated either by agrochemical or by water (control group). The vertical lines stand for the standard errors. Means marked by the same letter do not differ statistically significantly at level of significance a ¼ 0.05 after Bonferroni post hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 8. The average FPSII (in z-scores) observed over all the nine experiments. The measures were conducted in Bel-W3 plants exposed either to ozone enriched (OZþ: red squared markers interconnected with red dashed line) or to ozone free (OZ: blue filled circles, interconnected with blue dashed line) air and treated either by agrochemical or by water (control group). The vertical lines stand for the standard errors. Means marked by the same letter do not differ statistically significantly at level of significance a ¼ 0.05 after Bonferroni post hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.7. Multivariate analysis Based on the effect size of the agrochemicals on the measured parameters, a cluster analysis was performed and the relevant dendrogram has been created (Fig. 10). Four main groups were revealed: the 1st and the 2nd group had as solo members the BIOM and PENC, respectively; the 3rd and 4th group were consisted of three and two agrochemicals, respectively (HEX, BENML & TRIFL and AZOX & KRM respectively) (Fig. 10). All of these parameters are, more or less, inter-correlated and, thus, there is a redundancy in information (Saitanis et al., 2014a). So, Principal Components Analysis, based on the effect size (ESi), was conducted to show the overall performance of the agrochemical in plants’ response to ozone. The resulting biplot is shown in Fig. 11. The first two principal axes explained 75.2% of the total variance, with the PC1 explaining 57.8%. Thus, PC1 would be considered as the major axis for ordinations of the cultivars. By projecting the agrochemicals on the PC1, they can be ordered, in
Fig. 9. The stomatal conductance (z-scores) measured in Bel-W3 plants treated by seven agrochemical or water (control) and either exposed to ozone (OZþ: red squared markers interconnected with red dashed line) or to ozone free (OZ: blue filled circles, interconnected with blue dashed line) air and treated either by agrochemical or by water (control group). The vertical lines stand for the standard error; Means marked by the same letter do not differ statistically significantly at level of significance a ¼ 0.05 after Bonferroni post hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 10. Dendrogram grouping the seven agrochemicals, based on effect size observed in the parameters. Based on their effect size (ESi) of the ozone-induced changes on each of the measured parameters: visible injury (Inj), total chlorophylls (Chl T), total carotenoids (Car), quantum yield of photosynthesis (FPSII), superoxide dismutase (SOD), and stomatal conductance (gs), measured in the ozone treated plants. Dissimilarity measure: Squared Euclidean distance; Agglomeration method: Complete linkage.
terms of their overall performance, as PECN KRM AZOX > BENML HEX > TRIFL >> BION. 4. Discussion Ozone affected significantly most of the measured parameters. It caused visible foliar injury, reduced chlorophylls' and total carotenoids’ content and negatively affected photosynthesis, stomatal conductance and SOD activity in the exposed plants. By the exception of BION, none of the other agrochemicals affected any of the above-mentioned parameters at the plants grown in the chamber supplied with ozone-free air (OZ).
Fig. 11. Biplot of the PCA ordination (with varimax rotation) of the seven agrochemicals, based on their effect size (ESi) of the ozone-induced changes on each of the measured parameters: visible injury (Inj), total chlorophylls (Chl T), total carotenoids (Car), quantum yield of photosynthesis (FPSII), superoxide dismutase (SOD), and stomatal conductance (gs).
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4.1. Ozone exposure levels The applied ozone levels in the chamber experiments were similar to those occurred in the field experiments (Fig. 1) and close enough to those which have been previously recorded in several rural areas of Greece, where ozone e during the summer months and midday-afternoon hours e often reaches or exceeds the level of 70 nmol mol1 (i.e. Saitanis, 2003; Saitanis et al., 2003, 2004, 2014b). Even higher ozone levels have been reported for rural areas in other nchez et al., 2005). Thus, the experiments concountries (e.g. Sa ducted here e in terms of ozone levels e can be considered realistic and the outcome applicable to the agricultural practice. 4.2. Visible injury In some experiments, the plants treated with some agrochemicals exhibited higher degree of symptoms than the reference plants sprayed only with water. As a result, the standard deviation and the standards error lines include negative values. The worst case was that of BION where the overall average was negative. This is not only due to lack of protection against ozone, but also because BION enhanced the leaf visible injury by causing phytotoxic symptoms. Such symptoms were developed also in the group of plants sprayed with BION and grown in the ozone-free chamber. The phytotoxic symptoms caused by BION were similar to those caused by ozone, making the discrimination impossible at the plants treated by both of them. 4.3. Pigments' content and pigments’ ratios Ozone affected the pigments content negatively. Plenty of researches have shown that chlorophyll content is reduced by ozone in several plant species, e.g. in wheat (Pleijel et al., 2006), tobacco (Saitanis and Karandinos, 2001b), and other plant species (Madkour and Laurence, 2002). Our results are in agreement with those of Calatayud and Barreno (2004). Nonetheless, there are many publications reporting no reduction of chlorophyll for some plant species exposed to ozone, e.g. for Cucumis sativus (Agrawal et al., 1993) and Oryza sativa (Welfare et al., 1996). Although ozone is improbable to penetrate cell plasmalemma, secondary byproducts (e.g. free radicals) of ozone oxidations can affect pigment concentrations (Gill and Tuteja, 2010) into plants’ cells. In our study only BION reduced chlorophylls' content while the other agrochemicals had no effects. There are, however, reports supporting that strobilurins increase chlorophylls' content (Grossmann and Retzlaff, 1997; Mercer and Ruddock, 1998). Gopi et al. (2007) found that HEX and PBZ increased the total chlorophylls' content by ~39.1 and 35.9%, respectively, in carrot (Daucus carota L.) plants. Such increase has been attributed to the increase of the cytokinins' level and the stimulation of chlorophylls’ biosynthesis (Fletcher et al., 2000). The two chlorophyll forms (Chl a and Chl b) seem to differ slightly in their sensitivity to the ozone oxidative effect but an effect of agrochemicals was not observed. The latter corresponds to the findings of Khalili et al. (1990) who reported no effect of triazole fungicides on Chl a/b ratio, but disagrees with those of Gopi et al. (2007) who found that hexaconazole and paclobutrazol increased chl b more than chl a. The Chl a/b ratio is considered as an indication of the light-harvesting complex size of photosystem II (PSII) (Ferraro et al., 2003; Pellegrini, 2014). The slight reduction in Chl a/ b ratio, observed in our present study, suggests a slight higher sensitivity to ozone of Chl a in comparison with Chl b. These results are consistent with those of Saitanis et al. (2001) where Chl a was shown to be more vulnerable to ozone oxidative effect in comparison with Chl b, but only by about 10%, which is quite low and
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hardly detectable. This probably explains the fact that other researchers have reported no effects of ozone to Chl a/b ratio (e.g. Mikkelsen et al., 1995). The total carotenoids were overall reduced by ozone. This is consistent with the results of Calatayud and Barreno (2004) who also reported a reduction in carotenoids in two lettuce varieties exposed to ozone. Similar reduction in carotenoids has been also reported by Madkour and Laurence (2002) who observed that O3 affected the carotenoids' concentration of several Egyptian species and cultivars, but the degree of carotenoids' loss did not always reflect the level of sensitivity to O3. Concerning the effects of agrochemicals on total carotenoids, although the overall effect was significant, no any significant difference was confirmed within agrochemicals between OZþ and OZ groups. This is in contrast with the results of Khalili et al. (1990) where carotenoids' content per unit of area was increased in maize seedlings treated with triazole fungicides. The carotenoids' reduction is considered of high significance for plants’ functions, because carotenoids are lightharvesting pigments contributing to photosynthesis and they seem to protect chlorophylls against oxidative destruction by O2, under high light intensities (Gill and Tuteja, 2010). In our experiments, ozone reduced total chlorophyll more than total carotenoids. Similar higher vulnerability of chlorophylls in comparison to carotenoids has been observed also in Tilia americana plants treated by increased ozone levels (Pellegrini, 2014). Since carotenoids are yellowing pigments, in conjunction with the Chl a/b ratio, they have been suggested as good markers of chlolez-Rodríguez et al., 2001). No effect of roplasts’ adaptation (Gonza the agrochemicals on Car/Chl ratio was observed in our study. In accordance, Khalili et al. (1990) reported no effect of triazole fungicides on Chl/Car ratio. 4.4. Superoxide dismutase Chlorophylls' loss is the major symptom of leaf senescence and prematurity caused by ozone. Senescence is driven by active oxygen species (AOS), the most common of which are superoxide _ radicals (O: 2 ), hydroxyl radicals (OH), hydrogen peroxide (H2O2) and singlet oxygen (1O2), all of which impair metabolic processes (Zhang et al., 2010). Superoxide dismutase (SOD), catalase (CAT), ascorbate-peroxidase (APX), peroxidase (POX) and glutathione reductase (GR) are key enzymes inhibiting the accumulation of active oxygen species in plants' tissues. Several studies have reported that SOD, and other antioxidant enzymes, regulate responses and acclimation of plants to stresses (Wu and VonTiedemann, 2001, 2002; Pandey et al., 2014). There are, however, contradictory reports on the effects of ozone on the antioxidant enzymes activity. Ozone has been reported to induce significant increases in the activity of antioxidant enzymes SOD, POX and CAT after two days of fumigation of spring barley (Hordeum vulgare L.) plants, while, after four days of fumigation, these enzymes declined to a level lower than in non-fumigated plants, due to the oxidative degradation of leaf proteins (Wu and Vo Tiedemann, 2002). Calatayud et al. (2003), reported stimulation of SOD and APX in spinach plants (Spinacia oleracea L.) as a response to ozone fumigation. Significant increase in SOD activity has also been reported by Calatayud et al. (2010) in evergreen plants (Pistacia terebinthus, Viburnum lantana) and deciduous plants (Pistacia lentiscus and Viburnum tinus) exposed to ozone. However, this is not always the case: some studies reported no change, or even reduction, in SOD activity, after exposure to ozone. For example, Kubo et al. (1995) found no difference in total SOD activity levels in Arabidopsis thaliana plants treated with 150 nmol mol1 O3 for 4 days. Esposito et al. (2009) reported, on the one hand, increase of SOD activity in Bel-W3 tobacco plants exposed to ozone, but, on the other hand,
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reduction of SOD in tomato plants. Other studies (Wohlgemuth et al., 2002) have shown that generation and accumulation of superoxide and/or peroxide may differ among species and that SOD activity oscillates in plants growing under stressing environment (Iriti and Faoro, 2008; Esposito et al., 2009). This may depend on the plant species, the stage of growth (Kuk et al., 2003), etc. The forms of the SOD (Fe-SOD, Mn-SOD or Cu/Zn-SOD iso-enzymes) may also differ in response to oxidative stress; the presence of these isoenzymes complicates further the elucidation of the SOD role in plants’ response to ozone (Calatayud and Barreno, 2001; De Sousa et al., 2013). There are studies supporting that strobilurins increase dry matter, gain yield, protein and chlorophyll content and delay senescence (Grossmann and Retzlaff, 1997; Mercer and Ruddock, 1998; Zhang et al., 2010). Several other agrochemicals, including fungicides and herbicides, have been reported possessing antioxidative properties, preventing, thus, ozone induced injury (Agathokleous et al., 2014b). However, little is known about the exact mechanism of their antiozonate action. De Sousa et al. (2013) found that metalaxyl induced oxidative stress to cell suspension cultures of Solanum nigrum L. This is in agreement with our findings. BION (that is a mixture of acibenzolar-S-methyl 4% w/w and metalaxyl-M 37.52% w/w) enhanced the oxidative effects of ozone by causing phytotoxic symptoms. It seems that strobilurins fungicides are effective in delaying senescence in spring wheat and spring burley by inducing activation of antioxidant enzymes (Wu et al., 2001; Zhang et al., 2010). Zhang et al. (2010) found that, in comparison to the untreated plants, the fungicides JS399-19, azoxystrobin, tebuconazole and carbendazim enhanced the SOD activity in the flag leaves of wheat plants. However, Calatayud and Barreno (2001) have reported significant decrease in SOD activity of ozone-exposed tomato (Lycopersicon esculentum Mill.) plants that had not been treated with benomyl, in comparison to the treated ones. Benomyl ameliorated the ozone induced oxidative stress by preventing the peroxidation of membrane lipids and, thus, protecting PSII from ozone oxidative effect.
5. Conclusions After testing the potential of seven agrochemicals to protect plants against ozone, we found that the triazoles fungicides penconazole and hexaconazole offered higher protection to plants against ozone than the other agrochemicals. Kresoxim-methyl showed similar protection efficacy with hexaconazole, but with higher variability. Azoxystrobin showed lower protection, while benomyl showed even lower. Trifloxystrobin did not protect the plants at all. The worst agrochemical was BION, which also caused necrotic spots to the plants not exposed to ozone. Briefly, based on their ozone-protection efficacy, the tested agrochemicals can be ordered as follow: Penconazole Kresoxim-methyl Azoxystrobin > Benomyl Hexaconazole > Trifloxystrobin >> Acibenzolar-Smethyl þ metalaxyl-M. However, the high variability in the antiozonate performance of the tested agrochemicals suggests that the research should be continued until some commercially available and economically affordable substances with even higher antiozonate performance will be identified, for the benefit of the applied intensive agriculture. Besides, the results presented here come from both laboratory and field experiments, using the Bel-W3 tobacco variety, which is hypersensitive to ozone. The effectiveness of the agrochemicals may be species-specific and it may be influenced by environmental factors (e.g. edaphic parameters, etc.) affecting the response of plants to ozone and probably to agrochemicals. Acknowledgement This investigation was partly funded by the EPEAEK “Pythagoras I” project, (European Union, 75%, and the Greek Ministry of National Education and Religious Affairs, 25%; AUA-ELKE Code: 73.00.00.24.0001). We thank the Assistant Professor Georgios Papadopoulos (Division of Informatics, Mathematics and Statistics – Agricultural University of Athens) for the usefull dicussion about the statistical analysis of the data.
4.5. Quantum yield
References
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