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Rates of foliar penetration of Fe(III) chelates of imidodisuccinic acid (IDHA), ligninsulfonic acid (Natrel), and citric acid (ammonium ferric citrate) were studied at 20 degrees C using a leaf disk method. After drying of the donor droplets, the humidity over the donor residues was maintained at 100% because Fe(III) chelates deliquesce only when humidity is higher than 90%. The wetting agent Glucopon 215 CSUP was added at a concentration of 0.2 g L(-1) to all donor solutions. With fully expanded stomatous broad bean leaves, penetration of Fe-IDHA followed first-order kinetics and rate constants of penetration were higher in light (0.073 h(-1)) than in the dark (0.042 h(-1)). Permeability of broad been leaves to CaCl2 was about 8 times higher than to Fe-IDHA. Doubling the Fe-IDHA concentration in the donor from 2.5 to 5 mmol L(-1) decreased rate constants of Fe-IDHA penetration by a factor of 2.2. Adding the silicon surfactant Break Thru S240 at 10 g L(-1) to the donor induced infiltration of open stomata and about 80% of the applied Fe-IDHA penetrated during droplet drying, while with Glucopon 215 CSUP stomatal infiltration was not observed. With broad bean leaves, penetration of Natrel and ammonium ferric citrate also followed first-order kinetics and rate constants were also higher in light than in the dark. Adaxial astomatous surfaces of fully expanded pear, apple, and grapevine leaves were practically impermeable to Fe-IDHA while stomatous abaxial leaf surfaces were permeable, but rate constants of penetration decreased with time and differed greatly among species. Astomatous surfaces of young unfurling grapevine and peach leaves were permeable to Fe-IDHA, but permeability of stomatous surfaces was much higher. The effect of light on permeability of stomatous leaf surfaces is attributed to the presence of aqueous pores in cuticles over guard cells, and it is suggested that permeability of these pores increases as stomata open. Consequences of these results for foliar applications of Fe chelates are discussed.

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Plant cuticles are lipid membranes with separate diffusion paths for lipophilic non-electrolytes and hydrated ionic compounds. Ions are lipid insoluble and require an aqueous pathway across cuticles. Based on experimental data, the aqueous pathway in cuticles has been characterized. Aqueous pores arise by hydration of permanent dipoles and ionic functional groups. They can be localized using ionic fluorescent dyes, silver nitrate, and mercuric chloride. Aqueous pores preferentially occur in cuticular ledges, at the base of trichomes, and in cuticles over anticlinal walls. Average pore radii ranged from 0.45 to 1.18 nm. Penetration of ions was a first order process as the fraction of the salt remaining on the cuticle surface decreased exponentially with time. Permeability of cuticles to ions depended on humidity and was highest at 100% humidity. Wetting agents increased rate constants by factors of up to 12, which indicates that the pore openings are surrounded by waxes. The pores in cuticular ledges of Helxine soleirolii allowed passage of berberine sulphate, which has a molecular weight of 769 g mol(-1). Increasing the molecular weight of solutes from 100 to 500 g mol(-1) decreased the rate constants of penetration by factors of 7 (Vicia faba) and 13 (Populus canescens), respectively. Half-times of penetration of inorganic salts and organic ions across Populus cuticles and Vicia leaf surfaces varied between 1 and 12 h. This shows that penetration of ionic compounds can be fairly rapid, and ions with molecular weights of up to 800 g mol(-1) can penetrate cuticles that possess aqueous pores.

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The postinfection activities of copper hydroxide [Cu(OH)2] and copper sulfate (CuSO4) against apple scab (Venturia inaequalis) were evaluated in an in vitro study. Our intention was to support the aim of reducing copper application rates by appropriate timing of applications. Experiments were conducted at 20 degrees C with leaf disks and isolated cuticular membranes (CM) of Malus x domestica 'Gloster' and 'Elstar'. Conidia of V. inaequalis were used as the inoculum. In untreated controls, 7.9 and 33.2% of germinated conidia formed primary stromata 24 and 48 h after inoculation, respectively. Treatments with copper compounds were applied 24 and 48 h after inoculation, which was 16 and 40 h after infection had occurred. When working with CM and using fluorescein diacetate as a vital stain, vital and dead stromata could be distinguished. Treatment effects were assessed 72 h after inoculation by counting vital (fluorescing) primary stromata. With leaf disks, the number of stromata was counted using KOH-aniline blue fluorescence staining. Cu(OH)2 and CuSO4 showed postinfection activity and killed primary stromata, provided that the surface of the CM was kept wet. Cu(OH)2 was more effective than CuSO4 and was able to kill all primary stromata 24 h after inoculation at concentrations of 116 and 231 mg L(-1). When Cu(OH)2 was applied at 116 mg L(-1) to leaf disks 24 h after inoculation, the number of primary stromata did not significantly differ from the control. Results indicate different modes of action for the highly water soluble CuSO4 and the slightly soluble Cu(OH)2. This supports the hypothesis that spore exudates react with insoluble copper compounds and form highly toxic copper complexes. Application of Cu(OH)2 to dry CM did not kill primary stromata. Hence, for Cu(OH)2 to exert postinfection activity, leaves must be wet. In the field, this cannot be guaranteed and a postinfection application of Cu(OH)2 cannot be recommended.

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Time courses of cuticular penetration of FeCl3 and Fe(III) complexes of citric acid, EDTA, EDDHA (Sequestrene 138Fe), imidodisuccinic acid (IDHA), and ligninsulfonic acid (Natrel) were studied using astomatous cuticular membranes (CMs) isolated from Populus x canescens leaves. At 100% relative humidity, the Fe(III) chelates disappeared exponentially with time from the surface of the CMs; that is, penetration was a first-order process that can be described using rate constants or half-times of penetration (t(1/2)). Half-times ranged from 20 to 30 h. At 90% humidity, penetration rates were insignificant with the exception of Natrel, for which t(1/2) amounted to 58 h. Rate constants were independent of temperature (15, 25, and 35 degrees C). Permeability decreased with increasing Fe chelate concentration (IDHA and EDTA). At 100% humidity, half-times measured with FeIDHA were 11 h (2 mmol L(-1)), 17 h (10 mmol L(-1)) and 36 h (20 mmol L(-1)), respectively. In the presence of FeEDTA, penetration of CaCl2 was slowed greatly. Half-times for penetration of CaCl2, which were 1.9 h in the absence of FeEDTA, rose to 3.12 h in the presence of an equimolar concentration of EDTA and 13.3 h when the FeEDTA concentration was doubled. Hence, Fe chelates reduced permeability of CMs to CaCl2 and to the Fe chelates themselves. It is suggested that Fe chelates reduced the size of aqueous pores. This view is supported by the fact that rate constants for calcium salts were about 5 times higher than for Fe chelates with the same molecular weights. Adding Tween 20 (5 g L(-1)) as a humectant did not increase permeability to FeIDHA at 90% humidity and below, while addition of glycine betaine did. Penetration of FeCl3 applied at 5 g L(-1) (pH 1.5) was not a first order process as rate constants decreased rapidly with time. Only 2% of the dose penetrated during the first 2 h and less than that in the subsequent 8 h. Recovery was only 70%. This was attributed to the formation of insoluble Fe hydroxide precipitates on CMs. These results explain why in the past foliar application of Fe compounds had limited success. Inorganic Fe salts are instable and phytotoxic because of low pH, while Fe chelates penetrate slowly and 100% humidity is required for significant penetration rates. Concentrations as low as reasonably possible should be used. These physical facts are expected to apply to stomatous leaf surfaces as well, but absolute rates probably depend on leaf age and plant species. High humidity in stagnant air layers may favor penetration rates across stomatous leaf surfaces when humidity in bulk air is below 100%.

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Equilibrium sorption of n-alkyl esters (dimethyl suberate, diethyl suberate, diethyl sebacate, dibutyl suberate and dibutyl sebacate) and monodisperse alcohol ethoxylates (diethylene glycol, tetraethylene glycol, pentaethylene glycol, hexaethylene glycol and octaethylene glycol monododecyl ether) between the reconstituted cuticular waxes of Stephanotis floribunda Brongn (Madagascar jasmine) or Hordeum vulgare L (barley) leaves and an external aqueous receptor solution was determined. Logarithms of the wax/receptor partition coefficient (K(wax/rec)) of the n-alkyl esters increased linearly with the number of C-atoms. With alcohol ethoxylates, log K(wax/rec) decreased linearly with the number of ethylene oxide units. For both groups of compounds, K(wax/rec) increased with increasing lipophilicity. The values of K(wax/rec) in Stephanotis wax were between 5 and 16 times higher than in barley wax. It is argued that this difference was due to different chemical composition and crystallinity of the waxes. Mobility of [14C]2,4-dichlorophenoxybutyric acid (2,4-DB) in reconstituted Stephanotis and barley wax was increased by a factor of 2-8 by both n-alkyl esters and alcohol ethoxylates. Effects on the mobility of 2,4-DB were linearly related to the internal concentrations of n-alkyl esters and alcohol ethoxylates in reconstituted Stephanotis or barley wax. At the same internal concentrations the effect of n-alkyl esters on the mobility of 2,4-DB in wax exceeded that of alcohol ethoxylates by between 1 and 2 orders of magnitude. Results are discussed in relation to formulating systemic pesticides.

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Effects of diethyl suberate (DESU), diethyl sebacate (DES), dibutyl suberate (DBSU), dibutyl sebacate (DBS), and tributyl phosphate (TBP) on diffusion of 14C-2,4-dichlorophenoxy butyric acid (2,4-DB) across cuticular membranes (CM) was studied. Astomatous CM were isolated enzymatically from Stephanotis floribunda Brongn. leaves, and diffusion was measured at 20 degrees C. The alkyl-substituted dicarboxylic acids constitute a homologous series with carbon numbers increasing from C12 to C18. Molecular weights increased only moderately from 230.0 (DESU) to 314.5 (DBS), while partition coefficients varied over orders of magnitude from 92 (DESU), to 1213 (DES), to 15,988 (DBSU), to 210,762 (DBS). All the above compounds turned out to be accelerators as they increased 2,4-DB mobility by up to 40-fold with accelerator concentrations in the CM ranging from only 9.2 to 105 g kg(-1). Efficacy (2,4-DB mobility in the presence/mobility in the absence of accelerators) increased with increasing concentrations of accelerators in CM or in reconstituted cuticular waxes. Plotting efficacy vs accelerator concentration in the CM resulted in straight lines, and their slopes increased in the order DBS (0.14), DBSU (0.31), DES (0.51), and DESU (0.85). Hence, DESU was the most powerful accelerator in this series as it increased 2,4-DB mobility in the CM about 6 times more than DBSU. Waxes constitute the major barrier in plant cuticles, and plots of efficacy vs accelerator concentration in Stephanotis wax were also linear, but compared to CM slopes were steeper by factors of 3.20 (DBS), 2.97 (DBSU), 2.70 (DES), and 1.62 (DESU). TBP was similarly effective as DESU, but plots of efficacy vs concentration were not linear, and curves approached a plateau at 60-80 g kg(-1). These data are discussed with regard to suitability of these accelerators for formulating systemic pesticides.

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Effects of diethylsuberate (DESU), tributyl phosphate (TBP), and monodisperse ethoxylated alcohols (EAs) on rate constants of penetration (k) of model solutes across astomatous cuticular membranes isolated from Madagascar ivy (Stephanotis floribunda) and pear (Pyrus communis) leaves were studied. Model solutes (selected on the basis of their octanol/water partition coefficients, K(ow)) were iprovalicarb (log K(ow) = 3.18), metribuzin (log K(ow) = 1.60), and methyl glucose (MG) (log K(ow) = -3.0). K(ow) varied by more than 6 orders of magnitude. Accelerators had wax/water partition coefficients (log K(ww)) ranging from 1.75 (DESU) to 4.32 (C(12)E(2)), and their equilibrium concentrations in Stephanotis wax varied from 0 to about 160 g kg(-)(1). Accelerators increase solute mobility in cuticles by increasing fluidity of cutin and waxes. This effect was quantified by plotting log k versus the accelerator concentration in wax. With the lipophilic solutes metribuzin and iprovalicarb, these plots were linear. Slopes of these plots characterize the intrinsic activities of the accelerators, and they decreased in the order DESU (0.029) > TBP (0.015) > EAs (0.01). Using these intrinsic activities, the effects of accelerators on rate constants of penetration can be calculated for any accelerator concentration in wax. For instance, at 50 g kg(-)(1), rate constants for lipophilic solutes increased by factors of 28 (DESU), 5.6 (TBP), and 3.2 (C(12)E(n)()), respectively. Permeability of cuticles for the hydrophilic MG was not increased by DESU, TBP, C(12)E(2), and C(12)E(4), while C(12)E(6) and C(12)E(8) increased it. Small hydrophilic solutes such as MG can access aqueous pores in cuticles, and this pathway is not affected by changes in fluidity of amorphous waxes. After rate constants of penetration of ionic CaCl(2) were compared with those for nonionic MG, it was concluded that 60% of the MG diffused across aqueous pores, while 40% used an alternative pathway. Because the solubility of MG in wax is extremely low, it is unlikely that MG diffused along the lipophilic pathway used by metribuzin and iprovalicarb. This agrees with the observation that DESU and TBP had no effect on rate constants for MG. An alternative pathway of unknown properties is suggested. It is speculated that C(12)E(6) and C(12)E(8) sorbed in cuticles might have generated a polar pathway for MG.

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Size selectivity of aqueous pores in Vicia leaf cuticles was investigated by measuring the penetration of calcium salts into the abaxial surface of detached leaves. Molecular weights of salts ranged from 111 g mol(-1) to 755 g mol(-1). Penetration in light at 20 degrees C and 100% humidity was a first order process and rate constants of penetration ranged from 0.39 h(-1) (CaCl2) to 0.058 h(-1) (Ca-lactobionate). Penetration was a first order process in the dark as well, but the rate constants were smaller by a factor of 1.82. Plotting logarithmatised rate constants versus anhydrous molecular weights resulted in straight lines both in light and in the dark. The slopes per hour were very similar and the average slope was -1.2 x 10(-3) mol g(-1). Hence, size selectivity was not affected by stomatal opening, and in light or darkness permeability of Vicia cuticles decreased by a factor of 2.9 when molecular weight increased from 100 g mol(-1) to 500 g mol(-1). Silver nitrate was preferentially precipitated as silver chloride in guard cells, glandular trichomes and at the base of trichomes. It was concluded that these precipitates mark the location of aqueous pores in Vicia leaf cuticles. The size selectivity of aqueous pores in Vicia leaf cuticles is small compared to that observed in poplar leaf cuticles, in which permeability decreased by a factor of 7-13 for the same range of molecular weights. It is also much smaller than size selectivity of the lipophilic pathway in cuticles. These findings suggest that active ingredients of pesticides, growth regulators and chemical inducers with high molecular weights penetrate leaves at higher rates when formulated as ions.

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Potassium and calcium salts of glyphosate were obtained by titrating glyphosate acid with the respective bases to pH 4.0, and rates of penetration of these salts across isolated astomatous cuticular membranes (CMs) were measured at 20 degrees C and 70, 80, 90, and 100% humidity. K-glyphosate exhibited first-order penetration kinetics, and rate constants (k) increased with increasing humidity. Ca-glyphosate penetrated only when the humidity above the salt residue was 100%. At 90% humidity and below, Ca-glyphosate formed a solid residue on the CMs and penetration was not measurable. With Ca-glyphosate, the k value at 100% humidity decreased with time and the initial rates were lower than for K-glyphosate by a factor of 3.68. After equimolar concentrations of ammonium oxalate were added to Ca-glyphosate, high penetration rates close to those measured with K-glyphosate were measured at all humidities. Adding ammonium sulfate or potassium carbonate also increased rates between 70 and 100% humidity, but they were not as high as with ammonium oxalate. The data indicate that at pH 4.0 one Ca2+ ion is bound to two glyphosate anions. This salt has its deliquescence point near 100% humidity. Therefore, it is a solid at lower humidity and does not penetrate. Its molecular weight is 1.82 times larger than that of K-glyphosate, and this greatly slows down rates of penetration, even at 100% humidity. The additives tested have low solubility products and form insoluble precipitates with Ca2+ ions, but only ammonium oxalate binds Ca2+ quantitatively. The resulting ammonium salt of glyphosate penetrates at 70-100% humidity and at rates comparable to K-glyphosate. The results contribute to a better understanding of the hard water antagonism observed with glyphosate. It is argued that other pesticides and hormones with carboxyl functions are likely to respond to Ca2+ ions in a similar fashion. In all of these cases, ammonium oxalate is expected to overcome hard water antagonism.

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Little is known about the permeability of plant cuticles to ionic molecules with hydration shells that render them lipid insoluble and limit their diffusion to narrow aqueous pores. Therefore, the permeation of cuticular membranes to ionised calcium salts with anhydrous molecular weights ranging from 111 to 755 g mol(-1) was studied. Penetration was a first-order process and rate constants (k) (proportional to permeability) decreased exponentially with molecular weight. Plots of log k vs. molecular weight had slopes of -2.11x10(-3) and -2.80x10(-3), respectively, depending on the year in which the cuticular membranes were isolated. This corresponds to decreases in permeability by factors of about 7 to 13 when molecular weight increased from 100 to 500 g mol(-1). This size selectivity is small compared to the dependence on molecular weight of solute mobility in Populus cuticles. A decrease in mobility of neutral molecules by more than 3 orders of magnitude has been reported [A. Buchholz et al. (1998) Planta 206:322-328] for the same range of molecular weights. Hence, discrimination of large ionic species diffusing in aqueous pores (polar pathway) is much smaller than that for neutral solutes diffusing in cutin and waxes (lipophilic pathway). This indicates that formulating large solutes as ionic species would be advantageous.

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Penetration of glyphosate salts across isolated poplar (Populus canescens (Aiton) Sm) cuticular membranes (CM) was studied using Na+, K+, NH4+, trimethylsulfonium+ (TMS) and isopropylamine+ (IPA) as cations. After droplet drying, humidity over the salt residues on the outer surfaces of the CM was kept constant, and cuticular penetration was monitored by sampling the receiver solution facing the inner surfaces of the CM. Glyphosate salts disappeared exponentially with time from the surfaces of the CM. This first-order process could be quantitatively described using rate constants (k) or half-times (time for 50% penetration; t1/2). Humidity strongly affected the velocity of penetration, as k increased by factors of 5.3 (K-glyphosate), 6.9 (TMS-glyphosate), 7.1 (NH4-glyphosate), 8.5 (Na-glyphosate) and 10.5 (IPA-glyphosate) when humidity was increased from 70 to 100%. Depending on the type of cation and humidity, t1/2 varied between 4 and 70h, but the humidity effect was statistically significant only at 100% humidity, when half-times were highest with IPA-glyphosate and lowest with TMS-glyphosate. Glyphosate acid penetration was measured only at 90% humidity and found to be extremely slow (t1/2 = 866 h). Adding 0.2 g litre-1 of a wetter (alkylpolyglucoside) to the donor increased IPA-glyphosate rate constants by about four times, but increasing concentration produced no further increase in k. When donors contained 0.2 g litre-1 wetter, further additions of 4 g litre-1 Ethomeen T25 did not change rate constants measured with IPA-glyphosate at 90% humidity, while Genapol C-100 and diethyl suberate increased k by only 35%. Concentration of IPA-glyphosate (1, 2 and 4 g litre-1) did not influence k at 90% humidity, and pH of donor solutions (4.0, 7.7, 9.5) had no effect on k of K-glyphosate at 90% humidity. Temperature (10 to 25 degrees C) had only a small influence on velocity of penetration of IPA-glyphosate and K-glyphosate, as energies of activation amounted to only 4.26 and 2.92 kJ mole-1, respectively. These results are interpreted as evidence for penetration of glyphosate salts in aqueous pores.

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The distribution of pentachlorophenol and 2,4-dichlorophenoxyacetic acid in conifer needles was studied following uptake from aqueous solutions for up to 21 h. Desorption kinetics revealed two compartments (C1 j and C2 ) in which organics were sorbed reversibly and one compartment (Cs ) from which organics could not be desorbed. Reversible sorption proceeded very rapidly (less than 1 h for equilibration) and was made up of sorption to the surfaces of cuticular waxes and sorption in waxes and cutin. C3 appears to be identical with the apoplast and the symplast of needles. Penetration into C3 proceeded slowly and remained steady up to 21 h (longer periods not tried). The distribution of organics between the three compartments varied with time of loading and depended on plant species and properties of solutes. Data are discussed in relation to methods of exposure monitoring.