Gating in grapevine: relationship between application of the fungicide fludioxonil and circadian rhythm on photosynthesis.
ABSTRACT The aim of this study was to determine the impact of the fludioxonil (fdx) fungicide on the diurnal fluctuation in grapevine photosynthesis. Therefore, fdx treatment was performed at the end of flowering, at 8 am, 12 am or 7 pm. The study was performed in experimental field and several photosynthesis parameters were followed one day after treatment. Morning fdx treatment induced (i) a significant and simultaneous drop of both photosynthesis (Pn) and stomatal conductance between 8 am and 4 pm and (ii) an increase of intercellular CO(2) concentration when compared to control plants. On the contrary, evening fdx treatment did not affect Pn whereas midday treatment caused Pn increase after 4 pm. These data suggest that (i) morning fdx treatment results in a non-stomatal limitation of Pn, (ii) midday treatment is more suitable to treat grapevine with fdx and (iii) a phenomenon of gating was noticed.
- SourceAvailable from: Nathalie Vaillant-Gaveau[Show abstract] [Hide abstract]
ABSTRACT: Fungicides are widely used to control pests in crop plants. However, it has been reported that these pesticides may have negative effects on crop physiology, especially on photosynthesis. An alteration in photosynthesis might lead to a reduction in photoassimilate production, resulting in a decrease in both growth and yield of crop plants. For example, a contact fungicide such as copper inhibits photosynthesis by destroying chloroplasts, affecting photosystem II activity and chlorophyll biosynthesis. Systemic fungicides such as benzimidazoles, anilides, and pyrimidine are also phytotoxic, whereas azoles stimulate photosynthesis. This article focuses on the available information about toxic effects of fungicides on photosynthesis in crop plants, highlighting the mechanisms of perturbation, interaction, and the target sites of different classes of fungicides.Photosynthesis Research 02/2012; 111(3):315-26. · 3.15 Impact Factor
Gating in grapevine: Relationship between application of the fungicide
fludioxonil and circadian rhythm on photosynthesis
Anne-Noe ¨lle Petit, Florence Fontaine, Christophe Clement, Nathalie Vaillant-Gaveau*
Laboratoire de Stress, De ´fenses et Reproduction des Plantes, URVVC-SE EA 2069, Universite ´ de Reims Champagne-Ardenne,
UFR Sciences Exactes et Naturelles, Ba ˆtiment 18, Moulin de la Housse, BP 1039, F-51687 REIMS Cedex 2, France
The period of fdx spraying was an important parameter in stress response: the midday fdx treatment is more suitable to treat grapevine with fdx.
a r t i c l e i n f o
Received 26 March 2008
Received in revised form 17 July 2008
Accepted 22 July 2008
a b s t r a c t
The aim of this study was to determine the impact of the fludioxonil (fdx) fungicide on the diurnal
fluctuation in grapevine photosynthesis. Therefore, fdx treatment was performed at the end of flowering,
at 8 am, 12 am or 7 pm. The study was performed in experimental field and several photosynthesis
parameters were followed one day after treatment. Morning fdx treatment induced (i) a significant and
simultaneous drop of both photosynthesis (Pn) and stomatal conductance between 8 am and 4 pm and
(ii) an increase of intercellular CO2concentration when compared to control plants. On the contrary,
evening fdx treatment did not affect Pn whereas midday treatment caused Pn increase after 4 pm. These
data suggest that (i) morning fdx treatment results in a non-stomatal limitation of Pn, (ii) midday
treatment is more suitable to treat grapevine with fdx and (iii) a phenomenon of gating was noticed.
? 2008 Elsevier Ltd. All rights reserved.
Grey mould, caused by the fungus Botrytis cinerea, is a serious
disease in grapevine (Vitis vinifera L.) culture, affecting both the
quantity of harvest and the quality of wine produced from infected
grapes (Bulit and Dubos, 1988). Up to now, this disease is exclu-
sively controlled in vineyards by chemical fungicides. Three
preventive applications are generally recommended: at the end of
flowering (BBCH 69), at the bunch closure (BBCH 77) and at
the beginning of berry ripening (BBCH 81). Fludioxonil (fdx) [4-
a contact fungicide recommended for the control of B. cinerea and is
widely used against this phytopathogen (Rosslenbroich and Stue-
bler, 2000). Fdx is a non-systemic molecule, which inhibits spore
germination, germ-tube elongation and mycelium growth of B.
cinerea (Leroux,1996). Moreover, fdx increases the glycerol content
in the fungus, leading to a perturbation of its osmoregulation
potential (Pillonel and Meyer, 1997).
In grapevine, pesticides significantly affect plant physiology (for
review see Ref. Saladin and Cle ´ment, 2005). Fdx affects several
physiological parameters including leaf water and nitrogenous
contents. The photosynthetic apparatus is also temporarily affected
as revealed by fluctuation of CO2assimilation and photosynthetic
pigment contents (Saladin et al., 2003a,b). This may alter the whole
plant physiology since carbon metabolism is involved in grapevine
yield and vigour. Fdx applied during flowerand berry development,
may thus have significant consequences on berry yield. Indeed,
during berry ripening, there is a strong competition for sugar
nutrients between the reserve restitution in the woody part of the
plant and berries during maturation (Zapata et al., 2004). The use of
reserves to counteract the photosynthesis decrease, fungicide-
induced effects, may alter berry development and thus, affect
grapevine growth and yield.
In plants, there is an important interaction between carbohy-
drate metabolism and circadian rhythm (Bla ¨sing et al., 2005). Plants
have endogenous biological rhythms that enable them to daily
processes such as germination, growth, enzyme activity, flower
opening, fragrance emission (Hotta et al., 2007) and most param-
eters of photosynthesisincluding
exchange and CO2fixation (Yakir et al., 2007). In addition, recent
data reveal that the circadian rhythm modulates the ability to
respond to abiotic stresses such as cold (Fowler et al., 2005),
mechanical stimulation (Anderson-Bernadas et al., 1997) and wind
(Gaal and Erwin, 2005).
The aim of the study was to determine whether circadian
rhythm modulates grapevine ability to respond to fdx chemical
stress. The impact of fdx on the diurnal fluctuation in grapevine
* Corresponding author. Tel.: þ33 3 26 91 85 87; fax: þ33 3 26 91 34 27.
firstname.lastname@example.org (F. Fontaine), email@example.com (C.
Clement), firstname.lastname@example.org (N. Vaillant-Gaveau).
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/envpol
0269-7491/$ – see front matter ? 2008 Elsevier Ltd. All rights reserved.
Environmental Pollution 157 (2009) 130–134
photosynthesis was evaluated by following different aspects of gas
exchange in experimental vineyards (V. vinifera L. cv. Chardonnay)
at the end of flowering (BBCH 69).
2. Materials and methods
2.1. Plant material and treatment
Experiments were performed on Chardonnay grapevines (V. vinifera L.) grafted
on 41B rootstock on the experimental vineyardin Reims (France). Plants were grown
during 3 years in PVC containers with 100 L of compost.
The fdx fungicide (under formulated products Geoxe?) was sprayed using
the recommended concentration (4 mM) at the end of flowering (BBCH 69).
During our experiment the sun rose around 6 am local time. The photoperiod
was 15 h day/9 h night. Eight plants were sprayed in the morning (8 am), 8 in
the midday (12 am) and 8 in the evening (7 pm). Eight control plants in non-
treated areas were chosen as perfectly healthy and received none botryticide
2.2. Gas exchanges
The net photosynthesis (Pn), the stomatal conductance (gs), the intercellular
CO2concentration (Ci), the transpiration rate (T), the incident photon flux density
(PFD) and the air temperature (Tair) were measured simultaneously using a portable
infraredgas analyser (LI-Cor Model 6400, Lincoln, NE, USA).The infrared gas analysis
system was equipped with a clamp-on leaf cuvette that exposed 6 cm2of leaf area.
Humidity was fixed at 30%. CO2concentration was maintained at a constant level of
360 mmol l?1using a LI-6400-01 CO2 injector with a high-pressure liquid CO2
cartridge source. Gas exchanges measurements were performed on mature sun-
exposed leaves throughout the natural photoperiod. Three replicate measurements
per plant were conducted one day after treatment between 8.30 am and 7 pm.
Regression associated to Pn response to incident natural PFD was represented.
The apparent quantumyield of CO2fixation (FCO2) was calculated as theslope of the
linear portion of the response curves between 0 and 300 mmol photonsm?2s?1.
Dark respiration (Rd) was calculated for x¼0 and compensation point (G) for y ¼0.
Using high PFD (>1200 mmol m?2s?1), the slope of curves was calculated (Bigot
et al., 2007). Moreover, response of gs, Ci and T to incident natural PFD was
Fig. 1. Photon flux density, PFD (A) and air temperature, Tair (B) over the photoperiod. The values given are the means ? SD (n ¼ 8).
Fig. 2. Net photosynthesis (A), stomatal conductance (B), transpiration rate (C) and intercellular CO2concentration (D) in leaves after fdx spraying at various moments of
photoperiod (morning, midday, evening). The control represents plants without fdx treatment. The values given are the means ?SD (n ¼8). Means for a considered parameter were
not significantly different when marked by the same letter.
A.-N. Petit et al. / Environmental Pollution 157 (2009) 130–134131
2.3. Statistical analysis
Eight replicate plants per treatment were carried out (n ¼8). All data were
analyzed using the Mann and Whitney test at the 0.05 probability level.
3.1. Gas exchanges
was about 1300 mmolm?2s?1, peaking at 1700 mmolm?2s?1at 1.30
7 pm. Tair increased along with PFD to reach 38?C at 1.30 pm
(Fig.1B). By 4 pm, Tair dropped to 35?C and continued to decrease
down to 32?C at 7 pm.
Between 8.30 am and 11 am, Pn, gs and T were maximal in
control plants (Fig. 2A–C). They decreased until 1.30 pm and
increased again from 1.30 pm to 4 pm. Morning fdx treatments
induced significant drop of Pn between 8 am and 4 pm when
compared to control plants and a significant increase at 7 pm
(Fig. 2A). This Pn reduction was simultaneous to either a Ci increase
(at 8 am, Fig. 2D) or a gs decrease (at 1.30 pm) or both (at 11 am).
These data suggest that morning fdx treatment results in a non-
stomatal limitation of Pn. In opposite, at 7 pm, Pn increase was
associated with Ci decrease and stable gs, suggesting a stomatal
regulation compared to control plants. Midday fdx treatment only
induced Pn and gs increase after 4 pm while Ci remained stable.
Therefore, Pn regulation was stomatal after midday treatment. On
the contrary, fdx treatment applied in the evening did not affect Pn
at none time of the day after treatment.
3.2. Light response curves
Regression associated to responses of Pn, gs, Ci and T to PFD was
represented in Fig. 3. With higher light intensity Pn, gs and T
increased until 900 mmolm?2s?1then declined while Ci increased
whatever the period of fdx treatment. Treated leaves had a lower
photosynthetic capacity than control leaves. Treated plants at the
morning responded in a lower extent to the light than control
plants. In morning treated leaves, Pn max was strongly inhibited
compared to the control (Fig. 3A, Table 1). Pn max of leaves treated
at the midday and the evening has little inhibition. Apparent
quantumyield of CO2fixation (FCO2) showed a significant decrease
with fdx treatment (Table 1). The strongest inhibition was
measured in treated plants in the evening while the lowest
reduction was emphasized following the midday treatment.
Dark respiration (Rd) increased according to the period of
treatment. Higher Rd increase was noted following the morning
treatment. Lowest modifications occurred after the midday treat-
ment. Compensation point declined after fdx treatment whatever
application period, especially after evening treatment. Using the
high PFD (>1200 mmolm?2s?1), all the slopes of curves were
negative (Fig. 3, Table 1), meaning that there was photoinhibition.
However, higher inhibitionwas measured in midday treated plants.
Our results provide new insights into the dynamic approach of
fdx possible effects on grapevines photosynthetic activity and
importance of the time of fungicide application. Fdx induced
Fig. 3. Net photosynthesis (A), stomatal conductance (B), transpiration rate (C) and intercellular CO2concentration (D) in leaves during the day after spraying of fdx at morning,
midday, or evening, exposed under natural PFD. The control represents plants without fdx treatment. The values given are the means ?SD (n ¼8).
nomial regression for the morning values (r2¼0.9078),
values (r2¼ 0.7857).
Indicate a poly-
for the control for the midday values (r2¼0.9221),for the evening values (r2¼0.7449) and
A.-N. Petit et al. / Environmental Pollution 157 (2009) 130–134 132
a strong Pn inhibition accompanied by gs, T, FCO2and Rd declines.
The greater accumulated carbon gain was by the grapevine treated
in the midday. It was þ3%, þ14% and þ14% compared to control
plants and to plants treated at the morning and at the evening,
respectively. These data complement preliminary information on
fdxeffects on grapevine physiology. Saladin et al. (2003a,b) showed
that, in vitro, both water content and osmotic potential decreased
in fdx treated leaves. Carbohydrate accumulated, suggesting that
plantlets could react to the stress through an active osmoregulation
process by uptaking sugars from the medium. Moreover, in vine-
yard, fdx modified leaf water content and carbohydrate levels,
whereas nitrogenous compounds accumulated transiently. Effects
of fungicides on photosynthesis have been already revealed by
modifications of photosynthetic activity, chlorophyll fluorescence,
and pigment contents (Van Iersel and Bugbee, 1996; Benton and
Cobb, 1997; Tort and Tu ¨rkyilmaz, 2003; Untiedt and Blanke, 2004;
Xia et al., 2006). In vitro studies using isolated chloroplasts or
excised leaves have reported a direct effect of copper on the
photosynthetic electron transport chain, lipid peroxidation of
thylakoid membranes, or with the alteration of lipid chloroplast
membrane, affecting the light reaction processes, especially those
associated with PSII (Alaoui-Sosse ´ et al., 2004). Krugh and Miles
(1996) showed that the fungicide tributyltin chloride hinders PSI
electron transport or in some other way inhibits the oxidation of
the PSII electron transport components.
4.1. Stress responses and circadian rhythm
We have measured one variation in grapevine responses to the
same fdx spraying applied at different times of the day. Morning fdx
spraying induced depression of carbon fixation between 8 am and
1.30 pmwhereas midday fdx spraying stimulated Pn after 7 pm. This
phenomenon is so-called gating. Hotta et al. (2007) have reported
that external stimuli of equal strength applied at different times of
thedaycanresultindifferentintensitiesof response. One example of
gating is the diurnal variation in the inhibition of stem elongation by
wind (Gaal and Erwin, 2005). When wind perturbation was given to
Cosmos bipinnatus at different times of the day, the most intense
effect on growth was observed when wind was applied during the
day (Gaal and Erwin, 2005). Similarly, inhibition of stem growth in
the legume Phaseolus vulgaris by mechanical stimulation was also
greatest atthe beginningof the day (Anderson-Bernadas et al.,1997).
4.2. Photosynthesis and circadian rhythm
Between 8 am and 1.30 pm, Pn reduction one day after the
morning fdx spraying was attributed to a non-stomatal limitation.
On the contrary, Pn increases at 7 pm seems to be linked to
a stomatal regulation. Similarly, Maroco et al. (2002) have shown
that grapevine photosynthesis can be regulated via both stomatal
and non-stomatal processes in drought conditions.
In our work, stomatal regulation was observed. Plants, such as
grapevine, promote stomatal opening, allowing CO2uptake and
fixation, as soon as sufficient light is available to drive photosyn-
thesis. It is known that stomata close around midday and start
closing long before dusk (Webb, 1998). These responses were
traditionally considered as a consequence of the water status of the
leaf, but recent works demonstrated that, at least in well-watered
plants, they are due to endogenous circadian control of the guard
cell (Dodd et al., 2005). Regulation of stomatal pore size, during
favourable environmental conditions optimizes CO2uptake against
water loss. However, under stress conditions, stomatal closure
prevents water loss. Guard cells are able to integrate many internal
and external signals and produce an appropriate turgor response
that results in guard cell movements allowing to control stomatal
aperture (Hetherington and Woodward, 2003).
Non-stomatal limitation noticed after fdx treatment can be
explained bystimulation of photorespiration and day-respiration at
higher temperatures, for example, at 1.30 pm (Mohotti and Lawlor,
2002). Short-term temperature increases stimulate photosynthesis
until the temperature optimum is reached (Saxe et al., 2001).
Increasing air temperature decreases Rubisco specificity for and
solubility of CO2more than O2, which stimulates photorespiration
and reduces photosynthesis (Saxe et al., 2001). In addition,
following sub-saturating PFD (100–300 mmolm?2s?1), Pn was
strongly limited by electron transport (FCO2inhibition) for treated
plants in the morning and in the evening. This phenomenon has
been previously reported as a result of a reduced RuBP regenera-
tion, which may be limited when (i) light-harvesting and electron
transport produce ATP and NADPH; (ii) the stromal bisphophatases
regenerate RuBP in the photosynthetic carbon reduction cycle and
(iii) end-product synthesis consumes triose-phosphates and
regenerates inorganic phosphate for photophosphorylation (Allen
and Ort, 2001).
Interpreting the effect of fdx spraying on grapevine photosyn-
thesis, we showed for the first time, a phenomenon of gating. Our
work strongly suggests that the period of fdx spraying was an
important parameter in stress response. These data suggest that
morning fdx treatment results in a non-stomatal limitation of Pn.
Midday treatment is more suitable to treat grapevine with fdx
because it is the treatment which disrupts least the photosynthesis.
Alaoui-Sosse ´, B., Genet, P., Vinit-Dunand, F., Toussaint, M., Epron, D., Badot, P.M.,
2004. Effect of copper on growth in cucumber plants (Cucumis sativus) and its
relationships with carbohydrate accumulation and changes in ion contents.
Plant Science 166, 1213–1218.
Allen, D.J., Ort, D.R., 2001. Impacts of chilling temperature on photosynthesis in
warm-climate plants. Trends in Plant Science 6, 36–42.
Anderson-Bernadas, C., Cornelissen, G., Turne, C.M., Koukkari, W.L., 1997. Rhythmic
nature of thigmomorphogenesis and thermal stress of Phaseolus vulgaris L.
shoots. Journal of Plant Physiology 151, 575–580.
Benton, J.M., Cobb, A.H., 1997. The modification of phytosterol profiles and in vitro
photosynthetic electron transport of Galium aparine L. (cleavers) treated with
the fungicide, epoxiconazole. Plant Growth Regulation 22, 93–100.
Bigot, A., Fontaine, F., Cle ´ment, C., Vaillant-Gaveau, N., 2007. Effect of the herbicide
flumioxazin on photosynthetic performance of grapevine (Vitis vinifera L.).
Chemosphere 67 (6), 1243–1251.
Bla ¨sing, O.E., Gibon, Y., Gu ¨nther, M., Ho ¨hne, M., Morcuende, R., Osuna, D., Thimm, O.,
Usadel, B., Scheible, W.R., Stitt, M., 2005. Sugars and circadian regulation make
Analyses of photosynthetic light response curves: maximal photosynthesis (Pn max), the apparent quantum yield of CO2fixation (FCO2), dark respiration (Rd), compensation
point (G), and slope with PFD >1200 mmolm?2s?1of grapevine one day after treatment
Period of fdx applicationPn max (mmolm?2s?1)
Slope with PFD >1200 mmolm?2s?1
Means for a considered parameter were not significantly different when marked by the same letter (n ¼8).
A.-N. Petit et al. / Environmental Pollution 157 (2009) 130–134133
major contributions to the global regulation of diurnal gene expression in
Arabidopsis. The Plant Cell 17, 3257–3281.
Bulit, J., Dubos, B., 1988. Botrytis Bunch Rot and Blight. In: Pearson, R.C.,
Goheen, A.C. (Eds.), Compendium of Grape Diseases. APS Press, Paul, pp. 13–14.
Dodd, A.N., Salathia, N., Hall, A., Ke ´vei, E., Toth, R., Nagy, F., Hibberd, J.M., Millar, A.J.,
Webb, A.A.R., 2005. Plant circadian clocks increase photosynthesis, growth,
survival, and competitive advantage. Science 309, 630–633.
Fowler, S.G., Cook, D., Thomashow, M.F., 2005. Low temperature induction of Arabi-
Gaal, T.V., Erwin, J.E., 2005. Diurnal variation in thigmotropic inhibition of stem
elongation. HortTechnology 15, 291–294.
Hetherington, A.M., Woodward, F.I., 2003. The role of stomata in sensing and
driving environmental change. Nature 424, 901–908.
Hotta, C.T., Gardner, M.J., Hubbard, K.E., Baek, S.J., Dalchau, N., Suhita, D., Dodd, A.N.,
Webb, A.A.R., 2007. Modulation of environmental responses of plants by
circadian clocks. Plant, Cell and Environment 30, 333–349.
Krugh, B.W., Miles, D.,1996. Monitoring the effects of five ‘‘nonherbicidal’’ pesticide
chemicals of terrestrial plants using chlorophyll fluorescence. Environmental
Toxicology and Chemistry 15, 495–500.
Leroux, P.,1996. Recent developments in the mode of action of fungicides. Pesticide
Science 47, 191–197.
Maroco, J.P., Rodrigues, M.L., Lopes, C., Chaves, M.M., 2002. Limitations to leaf
modelling approaches. Functional Plant Biology 29, 451–459.
Mohotti, A.J., Lawlor, D.W., 2002. Diurnal variation of photosynthesis and photo-
inhibition in tea: effects of irradiance and nitrogen supply during growth in the
field. Journal of Experimental Botany 53 (367), 313–322.
Pillonel, C., Meyer, T., 1997. Effect of phenylpyrroles on glycerol accumulation
and protein kinase activity of Neurospora crassa. Pesticide Science 49,
Rosslenbroich, H.J., Stuebler, D., 2000. Botrytis cinerea – history of chemical control
and novel fungicides for its management. Crop Protection 19, 557–561.
Saladin, G., Magne ´, C., Cle ´ment, C., 2003a. Physiological stress responses of Vitis
vinifera L. to the fungicides fludioxonil and pyrimethanil. Pesticide Biochemistry
and Physiology 77, 125–137.
Saladin, G., Magne ´, C., Cle ´ment, C., 2003b. Effects of fludioxonil and pyrimethanil,
two fungicides used against Botrytis cinerea, on carbohydrate physiology in Vitis
vinifera L. Pest Management Science 59, 1083–1092.
Saladin, G., Cle ´ment, C., 2005. Physiological Effects of Pesticides on Cultivated Crops.
In: Livingston, J.V. (Ed.), Agriculture and Soil Pollution: New Research. Nova
Science Publishers, New York, pp. 53–86.
Saxe, H., Cannell, M.G.R., Johnsen, Ø, Ryan, M.G., Vourlitis, G., 2001. Tree and forest
functioning in response to global warming. New Phytologyst 149, 369–400.
Tort, N., Tu ¨rkyilmaz, B., 2003. Physiological effects of captan fungicide on pepper
(Capsicum annuum L.). Plant Pakistan Journal of Biological Sciences 6, 2026–
Untiedt, R., Blanke, M.M., 2004. Effects of fungicide and insecticide mixtures on
apple tree canopy photosynthesis, dark respiration and carbon economy. Crop
Protection 23, 1001–1006.
Van Iersel, M.W., Bugbee, B.,1996. Phytotoxic effects of benzimidazole fungicides on
bedding plants. Journal of the American Society for Horticultural Science 121,
Webb, A.A.R.,1998. Stomatal Rhythms. In: Lumsden, P.J., Millar, A.J. (Eds.), Biological
Rhythms and Photoperiodism in Plants. Bios Scientific Publications, Oxford, pp.
Xia, X.J., Huang, Y.Y., Wang, L., Huang, L.F., Yu, Y.L., Zhou, Y.H., Yu, J.Q., 2006.
Pesticides-induced depression of photosynthesis was alleviated by 24-epi-
brassinolide pretreatment in Cucumis sativus L. Pesticide Biochemistry and
Physiology 86, 42–48.
Yakir, E., Hilman, D., Harir, Y., Green, R.M., 2007. Regulation of output from the plant
circadian clock. FEBS Journal 274, 335–345.
Zapata, C., Dele ´ens, E., Chaillou, S., Magne ´, C., 2004. Partitioning and mobilization of
starch and N reserves in grapevine (Vitis vinifera L.). Journal of Plant Physiology
A.-N. Petit et al. / Environmental Pollution 157 (2009) 130–134134