Agricultural and Forest Meteorology    Reduction of transpiration through foliar application of chitosan Marco Bittelli  Markus Flury  Gaylon S

Agricultural and Forest Meteorology Reduction of transpiration through foliar application of chitosan Marco Bittelli Markus Flury Gaylon S - Description

Campbell Everett J Nichols Department of Crop Soil Sciences Washington State University Pullman WA 99164 USA Decagon Devices Inc Pullman WA 99164 USA Vanson Inc Redmond WA 98052 USA Received 27 June 2000 received in revised form 1 December 2000 acc ID: 28363 Download Pdf

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Agricultural and Forest Meteorology Reduction of transpiration through foliar application of chitosan Marco Bittelli Markus Flury Gaylon S

Campbell Everett J Nichols Department of Crop Soil Sciences Washington State University Pullman WA 99164 USA Decagon Devices Inc Pullman WA 99164 USA Vanson Inc Redmond WA 98052 USA Received 27 June 2000 received in revised form 1 December 2000 acc

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Agricultural and Forest Meteorology Reduction of transpiration through foliar application of chitosan Marco Bittelli Markus Flury Gaylon S

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Agricultural and Forest Meteorology 107 (2001) 167–175 Reduction of transpiration through foliar application of chitosan Marco Bittelli , Markus Flury , Gaylon S. Campbell , Everett J. Nichols Department of Crop Soil Sciences, Washington State University, Pullman, WA 99164, USA Decagon Devices, Inc., Pullman, WA 99164, USA Vanson, Inc., Redmond, WA 98052, USA Received 27 June 2000; received in revised form 1 December 2000; accepted 8 December 2000 Abstract In this study, we investigate the potential of chitosan, a natural beta-1-4-linked glucosamine polymer, to reduce plant

transpiration. Chitosan was applied foliarly to pepper plants and water use was monitored. Peppers were grown in pots in growth-chambers, where transpiration was measured by weighing pots. In an accompanying field study, water use was determined by monitoring soil moisture depletion with time domain reflectometry. An automated irrigation system replenished the water used each day. Plant biomass and yield were determined to calculate biomass-to-water ratios. Differences in canopy resistance between control and chitosan treated plants were analyzed with the aid of the Penman–Monteith

equation. Scanning electron microscopy (SEM) and histochemical analyses demonstrated that chitosan induced closure of the plant’s stomata, resulting in decreased transpiration. Foliar application of chitosan reduced water use of pepper plants by 26–43% while maintaining biomass production and yield. We suggest that chitosan might be an effective antitranspirant to conserve water use in agriculture. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Evapotranspiration; Water balance; Soil water; Irrigation 1. Introduction Chitosan is a natural, biodegradable polysaccharide polymer

which serves as major structural component of the exoskeleton of crustaceans and insects (Muz- zarelli, 1977). Chitosan, together with other polysac- charides and proteins, is also found in the cell walls of a variety of fungi. Chitosan is commercially derived from shells of crabs, shrimp, and lobsters. These shells accumulate as waste byproducts of shellfish process- ing. Thus the raw product for chitosan is abundant. The chemical exists as a linear polysaccharide poly- mer composed of repeating monomeric units of the Corresponding author. Tel.: 1-509-335-1719; fax: 1-509-335-8674.

E-mail address: (M. Flury). amino sugars, glucosamine and -acetyl- -glucos- amine. Chitosan induces the expression of a variety of genes involved in plant defense responses, that, in some cases, result in increased synthesis of sec- ondary plant metabolites (Loschke et al., 1983; Walker-Simmons et al., 1983). Transcriptional activa- tion, induced by both chitosan and jasmonic acid, of genes encoding phenylalanine ammonia lyase and pro- tease inhibitors, suggests that chitosan may influence pathways involving jasmonic acid (Walker-Simmons et al., 1983; Farmer and

Ryan, 1990; Doares et al., 1995). Jasmonates exhibit some activities similar to the plant hormone abscisic acid (ABA), which plays a key role in the regulation of water use by plants (Sembdner and Parthier, 1993). Increased levels of ABA result in closure of stomata and reduced tran- spiration (Willmer and Fricker, 1996; Leung and 0168-1923/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S0168-1923(00)00242-2
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168 M. Bittelli et al. / Agricultural and Forest Meteorology 107 (2001) 167–175 Giraudat, 1998). Thus, manipulating the ABA signa- ling

pathway offers the possibility to reduce water consumption by plants (Grill and Ziegler, 1998). Re- cently, it was found that the enzymatic addition of the 1-carbon group farnesyl to a protein resulted in stom- atal closure of Arabidopsis (Pei et al., 1998). Direct evidence that foliar chitosan applications also reduce stomatal apertures has been reported by (Lee et al., 1999). These authors demonstrated that chitosan in- hibited light-induced opening of stomata in tomato and Commelina communis via inducing H produc- tion in the guard cells. The reported effects of chito- san on stomatal

aperture suggest the possibility that chitosan might be a valuable antitranspirant with useful agricultural applications. The purpose of this study was to determine the extent to which chitosan affects plant transpiration and biomass production under controlled growth-chamber and field conditions, and to further investigate direct effects of chitosan on stomatal opening and closing. The ultimate purpose of the study is to determine whether water can be conserved or plant water re- lations favorably altered in water-limited agricultural applications by using chitosan. 2. Materials and

methods 2.1. Plant species, chemicals, and chemical application Pepper ( Capsicum sp.) was chosen as model plant. Plants were grown from seed in small seedling pots, and then transplanted to growth-chamber pots or to the field. Chitosan (obtained from Vanson, Inc., Redmond, WA) at a concentration of 1 g L was dissolved in a 0.1% (w/w) lactic acid solution. This solution was sprayed onto the plant leaves with a commercial hand-held sprayer. Control plants were treated with the lactic acid solution only, without chitosan. The solutions were sprayed on abaxial and adaxial sur- faces of the

leaves, in such a way to obtain complete coverage of the plant. The number of spray-shots per plant was kept constant, with each plant receiving ap- proximately 20 ml solution per treatment. The degree of polymerization of the chitosan used was 130 and the degree of -acetylation was 24%. A preliminary test with different chitosan concen- trations showed that 1 g L was optimal to reduce evapotranspiration of the pepper plants. Smaller con- centrations (0.1 g L ) showed less effects; larger concentrations (10 g L ) did not show enhanced effects. 2.2. Growth-chamber study Plants were grown in 8 L

pots containing a green- house mix peat soil enriched with a slow release ferti- lizer. The soil was covered with aluminum foil to minimize evaporation. Average air temperature was 20 C, relative humidity 61%, and illumination was provided by metal–halide lamps for 13 h per day. Water use of pepper plants was measured daily by weighing the pots. The amount of transpired water was then replaced to keep the soil water content roughly constant during the course of the experiment. Six plants each were used for treatment and control. 2.3. Field study An experimental plot was set up in Pullman,

Wash- ington. The soil at this site was a Palouse silt loam (Pachic Ultic Haploxeroll) with 14% clay, 74% silt, and 12% sand. Particle size distribution was mea- sured with the pipette method after organic matter was removed with H (Gee and Bauder, 1986). The water-holding capacity of the soil at a matric potential of 33Jkg (field capacity) was about 0.25 m based on the soil moisture characteristic. This was also the measured water content at the beginning of the experiment. Pepper plants were grown in rows of five plants each. The rows were 2.5 m in length with the in- dividual

plants spaced 50 cm apart. Three rows for treatment and control were arranged in a randomized block design. The center-to-center distance between the rows was 100 cm. Before plants were transplanted, the plot was tilled to a depth of 30 cm with a Multiple Tyne Rototiller and a 16% N, 16% P, 16% K fertilizer was applied at a rate of 1523 kg ha during tillage. The peppers were transplanted from the greenhouse on 20 June 1998 (day of year, DOY, 171). Weeds were controlled manually during the entire course of the experiment. Chitosan and control solutions were applied foliarly to the pepper plants

once a week with
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M. Bittelli et al. / Agricultural and Forest Meteorology 107 (2001) 167–175 169 a hand-held sprayer. The application was on abaxial and adaxial sides of the pepper leaves. A drip irrigation system was installed with one dripper for each plant. When activated, the drippers supplied water at a constant rate of 7.56 L h . Irri- gation lines for each row were equipped with electric valves. A 30 cm long transmission line oscillator (TLO) (CS-615, Campbell Scientific, Logan, UT) was installed at the center of each row to measure the soil water content. The

TLOs were installed ver- tically with the upper end of the rods 1 cm below the soil surface. Electric valves and TLO sensors were connected to a data logger (CR23X, Campbell Scientific, Logan, UT). The soil water content was measured on an hourly basis. The field capacity of 0.25 m was used as the threshold value to ini- tiate irrigation. If the water content measured for a given TLO sensor at midnight was smaller than the field capacity, the soil was irrigated with enough wa- ter to restore it to 0.25 m . We calculated the amount of water needed to bring the soil water con-

tent back to field capacity based on an estimated vol- ume of soil (50 cm between plants 100 cm between rows 30 cm deep TLOs) and the measured volu- metric water content. The datalogger automatically supplied the required amount of water by controlling the duration of the irrigation for each row. No surface ponding or runoff occurred at the applied irrigation rates. The amount of water applied each day was, therefore, equal to the evapotranspiration plus deep percolation. The irrigation control assured that the soil water contents of all plots were similar. The evaporation and deep

percolation losses from all plots were therefore similar, since these processes are controlled by the soil water content. Any differences in water use among plots consequently represented actual differences in transpiration, even though the amount of water applied overestimated water use by transpiration. Meteorological parameters were recorded with a weather station. Air temperature, relative humidity, solar radiation, and wind speed were measured at 2 m above ground, precipitation was measured at 50 cm above ground. The air vapor pressure deficit was calculated from relative humidity

and air tempera- ture. Meteorological sensors were recorded with a CR10X datalogger (Campbell Scientific, Inc., Logan, UT). 2.4. Biomass determination and statistical data analysis To compare biomass production of control and treated plants, pepper plant biomass was measured. Pepper fruits were harvested during the course of the experiment when the peppers exceeded 7 cm in length. At the end of the experiment, stems, leaves and fruits were harvested and separated using a razor blade. Total dry biomass for the individual plant parts was determined by drying at 60 Cinanovenfor 72 h. For

fruits, fresh biomass is reported. Differences between the two treatments were ana- lyzed as paired comparisons by an analysis of variance (ANOVA). Statistical tests were conducted for water use, total dry matter, and fresh biomass. 2.5. Stomatal conductance Stomatal conductance was measured at the abaxial side of the pepper leaves with a steady state porome- ter (LI-1600, Li-Cor, Inc., Lincoln, NE) (Weyers and Meider, 1990). Stomatal conductance was recorded on two leaves for each plant. Four pepper plants for control and four pepper plants for treatment were measured. Measurements were made

on pepper plants grown in the growth chamber on an hourly basis from 6.00 am to 6.00 pm. To assure precise measure- ments, relative humidity, temperature and flow rate in the cuvette were recorded at the beginning of ev- ery measurement, and the instrument was allowed to equilibrate for 30 min before readings were taken. In the field plot, no reproducible measurements could be made because of interferences due to wind. 2.6. Scanning electron microscopy Chitosan was applied to pepper plants in the same manner as described in the growth-chamber and field studies. Chitosan was

applied to the leaves 24 h before the leaves were cut from the plant and prepared for SEM. SEM-preparation was done between 2.00 and 3.00 pm, to avoid possible mid-day closure of stom- ata. Capsicum sp. leaves were cut into small pieces (2 cm 2 cm) and were prepared for SEM according to standard procedures where the epidermal strips is fixed in ethanol (Weyers and Meider, 1990) followed by a critical point drying technique (Cohen, 1979).
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170 M. Bittelli et al. / Agricultural and Forest Meteorology 107 (2001) 167–175 Samples were gold coated for 6 min with a Hummer V

Technics sputter coater. Stomatal apertures may be affected by the preparation procedures, but compar- ative interpretations of stomatal openings should be informative (Weyers and Meider, 1990). A Hitachi S570 SEM was used for observations. Stomatal apertures were measured with a micrometer. An area of 0.5 cm was selected for aperture mea- surements and 40 stomata were selected randomly for aperture determination. 2.7. Histochemical analysis Stomatal opening is associated with accumula- tion of K in guard cells. The accumulation of K can be demonstrated by a histochemical analysis. The

preparation of the leaves with sodium hexanitrocobal- tate(III) [Na Co NO ] results in the formation of a black precipitate of cobalt sulphide if high con- centrations of K are present. Preparation procedures followed the standard protocol as described in the literature (Willmer and Fricker, 1996; Fischer, 1971; Green et al., 1990). All staining procedures were made in a tray of crushed ice. Epidermal strips were analyzed and photographed using confocal optical microscopy. 2.8. Canopy resistance Since no direct measurements of stomatal resistance were obtained from the field experiments,

the meteoro- logical measurements were used along with the water use estimates to compute canopy resistance of treated and control plots. The Penman–Monteith equation was used for these calculations, generally following the method given by Allen et al. (1998). Since the water use values included evaporation and deep percolation losses, the estimates of resistance obtained are not ex- act, but they are nevertheless useful for comparisons. The potential reference evapotranspiration ET (kg m day ) was calculated as (Monteith and Unsworth, 1990) ET .R G/ . D/=r γ. =r (1) where is the

slope of the saturation vapor pressure temperature relationship (Pa ), the daily net radiation flux density (MJ m day ), the soil heat flux density (MJ m day ), the latent heat of vaporization for water ( 45 MJ kg ), the vapor pressure deficit (kPa), the mean air density at constant pressure (kg m ), the specific heat of air at constant pressure (MJ kg ), the psychrometer constant (Pa ), is the resistance (s m ) of vapor flow from the evaporating surface into the air above the canopy, called the aerodynamic resistance, and the resistance (s m ) of vapor flow

due to transpiration and soil evaporation, called the surface or canopy resistance. For a grass reference surface, the resistance is calculated according to Allen et al. (1998) as 208 (2) where is the wind speed (m s )at2mabove ground. The surface resistance includes the stom- atal conductance and is as such the parameter in the Penman–Monteith equation that is affected by chitosan application. The parameters and in Eq. (1) and in Eq. (2) were directly measured during the field experiment. The soil heat flux was assumed to be 10% of the net radiation (Clothier et al., 1986). The

crop evapotranspiration for pepper plants ET was then calculated by multiplying the reference ET by a crop coefficient ET ET (3) where the values for K were taken as 0.6, 1.05, and 0.9 for initial, mid-season, and end-of-later-season stages (Allen et al., 1998). In our experiment, the ini- tial stage ran from DOY 171 to 220, the mid-season stage from DOY 220 to 260 and the later stage from DOY 260 to 271. All parameters, except , in Eq. (1) are known or have been measured, so that can be used as an adjustable parameter to match modeled and measured evapotranspiration. By varying the re-

sistance , we can account for the chitosan-induced stomatal closure. 3. Results and discussion Over the 47 days of the growth-chamber study, the chitosan-treated plants used 26% less water than the
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M. Bittelli et al. / Agricultural and Forest Meteorology 107 (2001) 167–175 171 Fig. 1. Daily water use of pepper plants under well-watered con- ditions in the growth chamber. Data represent averages of six control and six treated plants. control plants, a difference significant at 05 6) (Fig. 1). The meteorological conditions at the field site are characterized by few

intense rainfall events, amount- ing cumulatively to 116.4 mm during the 168-day period (Fig. 2). The weather was always windy, with wind speeds ranging from 0.5 to 5.5 m s . The av- erage vapor pressure deficit ranged from 0.1 to 5 kPa, indicating a high atmospheric evaporative demand. From the onset of the measurements in the field, the control plants used considerably more water per day than the chitosan-treated plants (Fig. 3). During the 84-day study, the chitosan-treated plants used 43% less water than the control plants, a difference signif- icant at a level of 05. The

downward peaks in Fig. 3 correspond to precipitation events when the soil moisture was replenished by natural rainfall, thus reducing the water used as measured by the TLO. In both the growth-chamber and the field study, the reduced water use of chitosan-treated plants did Table 1 Averaged biomass, total yield, water use, and biomass-to-water ratio for single pepper plants in the field study ( 3) Treatment Biomass (g) Yield (g) Cumulative water use (L) Biomass-to-water ratio (g L Mean S.D. Mean S.D. Mean S.D. Mean S.D. Control 133.7 24.2 634 95 81.9 15.8 7.8 0.6 Chitosan 115.3 17.2

607 25 46.9 11.7 13.7 4.3 Biomass refers to the dry matter of leaves and stems harvested at the end of the experiment. Yield refers to the fresh matter of pepper fruits, harvested continuously during the course of the experiment when their size exceeded 7 cm length. Biomass-to-water ratio is defined here as yield of pepper per unit volume of water consumed by the plants. Different at a significance level of 05 (ANOVA). Different at a significance level of 1 (ANOVA). Fig. 2. Meteorological parameters during the field experiment: (A) daily rainfall; (B) mean, maximum and

minimum daily air temperature; (C) daily averaged water vapor pressure deficit; and (D) daily averaged wind speed. not adversely affect biomass production. Dry mat- ter of leaves and stems, and fresh matter of yield of treated plants were not statistically different from control plants (Table 1). Since water consump- tion was less for the chitosan-treated plants, the
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172 M. Bittelli et al. / Agricultural and Forest Meteorology 107 (2001) 167–175 Fig. 3. Water use of pepper plants under field conditions: (A) daily precipitation; (B) daily water use estimated by soil

moisture measurements in the top 30 cm of the soil. Data represent water use of single plants, averaged over all treated and all control plants, respectively. biomass-to-water ratio was significantly better in the treated plants compared to the control plants. On an average, the biomass-to-water ratio in the field study was 7.8gkg for the control plants compared to 13.7 g kg for the treated plants. Similar differences were found in the growth-chamber study. Foliar application of chitosan resulted in a reduction of stomatal conductance compared to control plants in the

growth-chamber study (Fig. 4). The stomatal conductance shows a pronounced increase during the day with a maximum around noon. The initial drop of stomatal conductance for the control leaves observed at 1.00 pm might indicate a mid-day partial closure of stomata, as has been reported for some plant species (Willmer and Fricker, 1996). This initial drop of con- ductance was not observed for chitosan treated leaves. The systematic difference between control and treated leaves supports the results from the water-use mea- surements, and indicates that chitosan causes partial stomatal closure. The

canopy resistance required to match the control plot evapotranspiration, using the Penman Monteith equation, was 70 s m (Fig. 5), about the same as the resistance assumed for reference crop Fig. 4. Stomatal conductance of control and chitosan-treated leaves during daytime. Data represent hourly measurements made on two leaves of four pepper plants for control and treatment each in the growth chamber ( 8 for each curve). Vertical bars denote 1 S.D. evapotranspiration (Allen et al., 1998). To match cal- culations with measurements for the chitosan-treated plants, we increased the surface

resistance to 210sm (Fig. 5). This threefold increase in surface resistance for chitosan-treated plants is substantially greater than shown in Fig. 4 for growth chamber plants. This is consistent with the differences in tran- spiration observed for growth chamber (Fig. 1) and field grown pepper (Fig. 3). These differences could be related to the higher evaporative demand in the field or the higher photosynthetically active radiation levels. Scanning electron microscopy and a histochemical analysis in conjunction with confocal optical micro- scopy confirmed that foliar

application of chitosan Fig. 5. Measured and simulated water use for a single pepper plant during the field experiment: (A) control measured; (B) control simulated ( 70 s m ); (C) treatment measured; (D) treatment simulated ( 210 s m ).
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M. Bittelli et al. / Agricultural and Forest Meteorology 107 (2001) 167–175 173 Fig. 6. Representative SEM images of abaxial side of pepper leaves: (A) control leaf, stomata open; (B) chitosan-treated leaf, stomata mostly closed. The images shown are each representative of five specimens of 2 cm 2 cm from different parts of a pepper

leaf. For each specimen, 20 fields of view were examined. Bar is 50 m. induced closure of the stomata. Fig. 6 shows represen- tative SEM images of a control and chitosan-treated leaf, clearly indicating the difference in stomatal opening. Control leaves had mostly open stom- ata, chitosan-treated leaves had mostly closed or partially-closed stomata. Stomatal apertures were 82 30 m for the control leaf and 0 92 85 for the chitosan-treated leaf, a difference statisti- cally significant at a level of 05 ( 40). Lee et al. (1999) reported that stomatal apertures for chitosan-treated

tomato leaves were reduced as com- pared to control plants, an observation consistent with our findings for pepper. Potassium is a main regulator of the osmotic po- tential in the guard cells, thereby controlling opening and closing of the stomata (Willmer and Fricker, 1996; Fig. 7. Confocal optical microscopy images of abaxial side of pepper leaves after histochemical treatment: (A) control leaf; (B) chitosan-treated leaf. Dark areas indicate accumulation of K. El- lipsoidal cells are stomata. The images shown are each represen- tative of five specimens of 3 cm 3 cm from different

parts of a pepper leaf. For each specimen, 20 fields of view were examined. Bar is 50 m. Fischer, 1971). Low K concentrations result in stom- atal closure, and vice versa. Fig. 7 shows the results of the histochemical analysis. The dark colored areas in Fig. 7A are guard cells, indicating K accumula- tion and thus open stomata. No accumulation of K in Fig. 7B indicates that stomata are closed. Some of the dark areas are not associated with stomatal guard cells, and are likely K contaminations caused by breaking stomatal guard cells during the preparation procedure. Chitosan induced a

decrease of K in the guard cells, indicating that foliar application of chitosan induces a biochemical process leading to stomatal closure. 4. Conclusions Water use, stomatal conductance, electron micro- scopy, and histochemical analyses demonstrate that
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174 M. Bittelli et al. / Agricultural and Forest Meteorology 107 (2001) 167–175 foliar application of chitosan reduces transpiration of pepper plants through partial or full closure of stomata. Reduced water use was found under growth-chamber as well as field conditions. Compared to the opti- mal environmental conditions

in the growth-chamber study, the pepper plants in the field experiment were exposed to more variable, and more realistic, meteo- rological and soil conditions. The consistent findings with respect to reduced water use and increased biomass-to-water ratio through foliar chitosan appli- cation are encouraging. The water conservation for pepper plants was considerable, and of the same order of magnitude reported for wheat, barley, and tomato treated with ABA analogs (Rademacher et al., 1987; Rademacher et al., 1989). Reduction in yield and biomass from chitosan-induced closure of

stomata were not statistically significant. Evapotranspiration modeled using the Penman- Monteith equation showed good agreement between measured and calculated evapotranspiration for con- trol plants. For chitosan-treated plants, simulation results indicate a three fold increase of stomatal resis- tance as compared to control plants ( 210s m versus 70 s m ). A stomatal resistance of 210 s m found by the evapotranspiration simulations corrobo- rates the interpretation of partial closure of stomata; for completely closed stomata the resistance would be about 840 s m (Allen et al., 1998).

Chitosan is commercially prepared as a waste byproduct from the shells of shrimp, crab, and lobster (Muzzarelli, 1977), and as such, its use as an anti- transpirant in crop production seems economically feasible. In view of the increasing demand for limited resource water, the possibility to reduce crop transpi- ration, particularly in irrigated agriculture, would be advantageous. Acknowledgements We thank J.B. Mathison, O. Badini, V.R. Franceschi, and V.J. Lynch-Holm for help with the experiments. We also thank P. Chevalier, J.P. Reganold, and V.R. Franceschi for reviewing the manuscript, and

M. Evans for discussing the statistical analysis. This work was funded by the Washington Technology Center and Vanson, Inc. References Allen, R.G., Pereira, L.S., Raes, D.R., Smith, M., 1998. Crop evapotranspiration: guidelines for computing crop water requi- rements. FAO Irrigation and Drainage Paper 56, Food and Agricultural Organization of the United Nations, Rome. Clothier, B.E., Clawson, K.L., Printer, P.J., Moran, M.S., Reginato, R.J., Jackson, R.D., 1986. Estimation of soil heat flux from net radiation during the growth of alfalfa. Agric. For. Meteorol. 37, 319–329. Cohen, A.L.,

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Physics, 2nd Edition. Butterworths, London. Muzzarelli, R.A.A., 1977. Chitin. Pergamon Press, Oxford. Pei, Z.M., Ghassemian, M., Kwak, C.M., McCourt, P., Schroeder, J.I., 1998. Role of farnesyltransferase in ABA regulation of guard cell anion channels and plant water loss. Science 282, 287–290. Rademacher, W., Maisch, R., Liessegang, J., Jung, J., 1987. Water consumption and yield formation in crop plants under the influence of synthetic analogues of abscisic acid. In: Hawkins, A.F., Stead, A.D., Pinfield, N.J. (Eds.), Plant Growth Regulators for Agricultural and Amenity Use. BCPC

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