Influence of epiphytic micro-organisms on leaf wettability: wetting of the upper leaf surface of Juglans regia and of model surfaces in relation to colonization by micro-organisms

Wetting of the upper leaf surface of Juglans regia L. and of model surfaces colonized by epiphytic micro-organisms was investigated by measuring contact angles of aqueous solutions buffered at different pH values. During June to October 1995, contact angles of aqueous solutions on the leaf surface of J. regia decreased by angles ranging from 12° (low pH values) to 25° at high pH values. At the end of this vegetation period, wetting was strongly dependent on pH showing significantly lower contact angles with alkaline solutions (pH 9·0) than with acidic solutions (pH 3·0). Contact angle titration measured angles on the leaf surface as a function of the pH of buffered aqueous solutions, covering a pH range from 3·0 to 11·0. Titration curves revealed inflection points around 7·5, indicating the existence of ionizable carboxylic groups at the interface of the phylloplane. Altered leaf-surface wetting properties observed on the intact leaf surface could be simulated in model experiments by measuring contact angles on artificial surfaces colonized by Pseudomonas fluorescens and by epiphytic micro-organisms isolated from the phylloplane of J. regia . Strong evidence is provided that interfacial carboxylic groups derive from epiphytic micro-organisms present on the phylloplane. Results suggest that the age-dependent increase in, and pH dependence of, wetting as leaves mature are related to the presence of epiphytic micro-organisms on the phylloplane. Ecological consequences of increased leaf-surface wetting, concerning the structure of the leaf surface as a microhabitat for epiphytic micro-organisms, are discussed.     

Plant–Microbe Interactions: Wetting of Ivy (Hedera helix L.) Leaf Surfaces in Relation to Colonization by Epiphytic Microorganisms

Abstract Leaf wettability, cuticular wax composition, and microbial colonization of upper and lower leaf surfaces of ivy (Hedera helix L.) was investigated for young and old leaves sampled in June and September. Contact angles of aqueous buffered solutions measured on young leaf surfaces ranged between 76° and 86° and were not dependent on the pH value of the applied droplets. Contact angles measured on old leaf surfaces were up to 32°, significantly lower than on young leaf surfaces. Furthermore, contact angles were significantly lower using aqueous solutions of pH 9.0 compared to pH 3.0, indicating the influence of ionizable functional groups on leaf surface wetting properties. Observed changes in leaf wetting properties did not correlate with different levels of alkanoic acids in cuticular waxes. However, microscopic examination of the leaf surfaces indicated the influence of epiphytic microorganisms on wetting properties of old leaves, since their surfaces were always colonized by epiphytic microorganisms (filamentous fungi, yeasts, and bacteria), whereas surfaces of young leaves were basically clean. In order to analyze the effect of epiphytic microorganisms on leaf surface wetting, surfaces of young and clean ivy leaves were artificially colonized with Pseudomonas fluorescens. This resulted in a significant increase and a pH dependence of leaf surface wetting in the same way as it was observed on old ivy leaf surfaces. From these results it can be deduced that the native wetting properties of leaf surfaces can be significantly masked by the presence of epiphytic microorganisms. The ecological implications of altered wetting properties for microorganisms using the leaf/atmosphere interface as habitat are discussed. Received: 20 March 1999; Accepted: 5 July 1999; Online Publication: 18 July 2000     

The effects of superficial wax on leaf wettability

Experiments are described which provide more information on the role played by superficial waxes in the natural water-repellency of leaf surfaces. Contact angles of water were measured on a variety of leaf surfaces, before and after removal of wax, and on smooth films of the isolated superficial waxes. The differences in wettability of leaf surfaces are not wholly accounted for by differences which occur in the chemical and hydrophobic properties of their superficial waxes. Waxes isolated from leaves exhibiting contact angles less than 90° are usually more hydrophobic than the leaf surface itself. On most leaves exhibiting angles greater than 90° wax is the dominant factor governing water-repellency, the isolated wax normally making at least a 60 % contribution to the contact angle measured on the leaf surface. Additional factors, such as roughness, responsible for the occurrence of contact angles greater than 110° on certain leaf surfaces, reside in the wax layer. The hydrophobic properties of some leaves are unaffected by chloroform washing, revealing that superficial waxes play little part in their wettability.     

Wetting control through topography and surface hydrophilic/hydrophobic property changes by coarse grained simulation

Abstract

The changes of wetting state of water droplet on the solid surface featuring pillared structures are quantitatively studied by Coarse Grained simulation. Our results demonstrate that wetting state changes with the different topography (surface roughness), and it depends on the intrinsic hydrophilic/hydrophobic property of surface as well. Only if the contact angle of water droplet on the smooth surface is larger than 93.13°, the wetting state translates from the Wenzel state to the Cassie state on the rough surface with certain pillar height and width, and the contact angle climb up to the highest point and then remain almost unchanged with the increasing of pillar height and the same pillar distance. However, the wetting state does not change if the contact angle on the smooth surface is 85.1° or less, no matter what pillar structure the surface has. Additionally, the contact angles will remain almost unchanged if the pillar height is higher than a certain value. Our simulation results provide a quantitative understanding about the wetting state of water droplet on solid rough surfaces, and the results show the wetting state can be controlled by combining rough structure design and hydrophilic/hydrophobic property change of surfaces.     

Contact Areas between Waxy Leaf Surfaces and Aqueous Solutions: Quantitative Determination of Specific Leaf Surface Contact Areas

The contact areas (on a molecular scale) between aqueous solutionsand leaf surfaces have been measured in order to obtain informationon the actual areas effective in cuticular transport. For thispurpose, monolayer adsorption of pentachlorophenol (PCP) wasused on leaf surfaces and on artificial surfaces. The adsorptionisotherms were analysed according to the Brunauer-Emmett andTeller (BET) formalism leading to BET constants and actual contactareas between the aqueous PCP solution and the surfaces. BETconstants ranged from 4 to 11, with an arithmetic mean of 6·5.No relationship was found between the chemical composition ofthe surface waxesand the BET constants, and there were no significantdifferences between BET constants measured using isolated waxesor intact leaves. This indicates that the surfaces of the waxesare made up mainly of methylene and methyl groups, while mostfunctional groups appear not to contribute significantly tosurface sorption of PCP. This conclusion is supported by theobservation that the BET constant measured on silanized glasshaving a pure CH_3–surface was 5·7, which was notsignificantly different from 6·5. Specific leaf surfacecontact areas calculated from the BET constant and the areaof an adsorbed PCP molecule ranged from 1·6 (Hordeumvulgare) to 137 (Abies koreana). This means that the areas ofcontact between aqueous solutions and surface waxes were 1·6to 137 times larger than the apparent surface areas, which mightbe the consequence of a fractal leaf surface morphology. A leafsurface contact area greater than unity can result only froma rough surface that is wetted by the solution. It is argued,that interspecific differences in the rates of cuticular permeationmay be largely due to differences in leaf surface contact areasand not to differences in cuticular permeabilities. Consequently,differences in the rates of cuticular permeation can be interpretedonly if the leaf surface contact area that is effective in transportis known. Key words: Cuticular transport, epicuticular wax, fractality, specific leaf surface contact area, plant cuticle     

Characterization of the Topography and Wettability of English Weed Leaves and Biomimetic Replicas

The topography and wettability of the underside of English weed （Oxalis pes-caprae） leaves and of their biomimetic replicas are investigated. Polyvinyl siloxane molds were cast from the leaves and then filled with an epoxy pre-polymer to produce replicas. The particular topographical structures of leaves and replicas were evaluated by optical microscopy and Scanning Electron Microscopy （SEM） analysis. The static wettability of leaves and replicas was assessed by contact angle measurements, while the dynamic wettability was characterized by estimating contact angle hysteresis and studying the dynamic behavior of impacting water droplets. A smooth glass slip and its replica were used as control surfaces. The replica moulding method used was able to transfer the characteristic pattern of irregular 100 μm - 200 μm × 60 μm convex papillae interspersed with stomata of the original leaf to the epoxy replicas. The static contact angle of 143°± 3° and the contact angle hysteresis of 2~ indicate that the underside of the English weed leaf is close to superhydrophobic. The lower contact angles （130° ± 4°） and higher hysteresis （31°） observed for the replica when compared with the original leaves were associated to an inaccurate replication of the chemistry and structures of the three-dimensional wax projections covering the plant surface. Also, trichomes in the original leaves could not be accurately reproduced due to their flexibility and fragility. Differences in wetting behavior were also evident from droplet impact experiments, with rebound regimes prevailing in the original leaves and regimes characterized by higher adhesion and larger dissipation predominating in the replicas. Nevertheless, the morphological features of the leaf transferred to the replica were sufficient to promote a clear hydrophobic behavior of the replica when compared with the smooth epoxy reference surface.     

Effects of experimental warming on growth,biomass allocation,and needle chemistry of <Emphasis Type="Italic">Abies faxoniana</Emphasis> in even-aged monospecific stands

The response and adaption mechanisms of seedlings under long-term warming have remained largely unknown. In this study, we investigated the effects of warming for 6 years on growth, and needle carbon, nitrogen, chlorophyll, and carbohydrate levels in a coniferous tree species, Abies faxoniana. Seedlings were grown in even-aged monospecific stands under ambient and warming (ambient +2.2°C) temperature in climate control chambers. Warming caused statistically significant increases in the specific leaf area, leaf area ratio, root biomass, leaf biomass, branch biomass, stem biomass, and total mass of the seedlings, and reduced the root/shoot ratio. Warming also increased total chlorophyll concentrations, specific chlorophyll pigments, and Chlorophyll a/b ratios in both studied needle age classes. In addition, C/N ratios of current-year and 1-year-old needles increased by warming. In contrast, warming decreased the levels of N, sugar, cellulose, and starch in needles, while warming had no effect on the height, stem diameter, needle mass ratio, root mass ratio, and root/needle ratio. We conclude that warming increases branch growth and changes needle chemistry, which enhances the light capture potential of seedlings.     

Leaf surface properties of Norway spruce needles exposed to sulphur dioxide and ozone in an open-air fumigation system at Liphook

Samples of current-year and 1-year-old foliage were taken from Norway spruce (Picea abies (L.) Karst.) trees in April 1991, 4 months after a 3–4 year controlled fumigation with O₃ and SO₂ in the open at Liphook, south-east England. Trees were grown in seven plots, and treated in a factorial design with three levels of SO₂ and two levels of O₃ (ambient and c. 1.3 × ambient), with an extra ambient air plot. All statistical analyses were made on plot means. Leaf wettability, as measured by the contact angle of water droplets, was significantly affected by needle age and by SO₂ treatment (P≤0–05. in older needles, decreasing with increasing SO₂ concentration. There was no effect of O₃ on wettability, and no effect of any treatment on amounts of surface wax extracted by immersion of needles in chloroform. Electrolyte leakage rates from detached current-year needles were not affected by prior exposure to O₃, but decreased significantly (P= 0.034) with increasing exposure to SO₂. There was no detectable effect of fumigation on the rate of water loss from detached needles. Similarly, there was no effect of fumigation on the dry weight/fresh weight ratio of needles. The total sulphur content of needles increased significantly (P≤0.0001) with exposure to SO₂ and with needle age. Amounts of water-extractable sulphate, however, varied greatly among plots, but with no pattern with respect to fumigation treatment. It is concluded that leaf wettability and electrolyte leakage rates may be good indicators of the persistent effects of SO₂ on Norway spruce growing in the open air, and that the observed changes in leaf surface properties in response to SO₂ fumigation have implications for the processes, both biotic and abiotic, that occur on leaf surfaces.     

Impact of wastewater fouling on contact angle

Capillary dependent systems are highly influenced by surface fouling and may degrade as material surface properties change. In anticipation of a spacecraft microgravity fluids management system exposed to highly variable wetting conditions, the impact of urine wastewater fouling on capillary contact angle was examined. The results indicate that, in general, surface fouling can decrease the contact angle when crystalline structures or biofilms form. Small crystalline growth on the order of 10 μm can lower advancing contact angles θ_adv by approximately 30°, while biofilm growth can lower it by approximately 15°. Vacuum drying of fouled surfaces increased θ_adv by about 8°, and defects greater in height than 5% of the capillary length increased θ_adv by approximately 30°. These trends may indicate that promotion of wastewater fouling may improve the performance of capillary dependent fluids management systems. These results may also influence terrestrial technologies, including medical catheters and sustainable wastewater treatment systems exposed to wastewater fouling.     

Wetting characterisation of silicon (1,0,0) surface

Water droplets on bare silicon surfaces are studied to examine the wetting behaviour as a function of the surface energy and to parameterise water–silicon interactions in order to recover the hydrophobic behaviour measured by experiments. Two different wetting regimes characterised by a critical interaction strength value are observed. At a threshold value of the water–silicon interaction parameter, water molecules start penetrating into the first layer of silicon surface under thermally vibrating walls, resulting in two distinct wetting behaviours. Fixed (cold) silicon walls do not exhibit the two different wetting characteristics. Size effects are studied for nano-scale droplets, and line tension influence is observed depending on the surface wettability. Decrease in the droplet size increases the contact angle values for the low wetting cases, while contact angles decrease for smaller droplets on the high wetting surfaces. Considering the line tension effects and droplet size, ?_Si–O for water–silicon interactions to recover the hydrophobic behaviour of silicon surfaces is estimated to be 12.5% of the value predicted using the Lorentz–Berthelot mixing rule.     

Superhydrophobicity of Bionic Alumina Surfaces Fabricated by Hard Anodizing

Bionic alumina samples were fabricated on convex dome type aluminum alloy substrate using hard anodizing technique.The convex domes on the bionic sample were fabricated by compression molding under a compressive stress of 92.5 MPa.The water contact angles of the as-anodized bionic samples were measured using a contact angle meter (JC2000A) with the 3 μL water drop at room temperature.The measurement of the wetting property showed that the water contact angle of the unmodified as-anodized bionic alumina samples increases from 90° to 137° with the anodizing time.The increase in water contract angle with anodizing time arises from the gradual formation of hierarchical structure or composite structure.The structure is composed of the micro-scaled alumina columns and pores.The height of columns and the depth of pores depend on the anodizing time.The water contact angle increases significantly from 96° to 152° when the samples were modified with self-assembled monolayer of octadecanethiol (ODT),showing a change in the wettability from hydrophobicity to super-hydrophobicity.This improvement in the wetting property is attributed to the decrease in the surface energy caused by the chemical modification.     

Ultrastructure of <Emphasis Type="Italic">Eucalyptus grandis</Emphasis> × <Emphasis Type="Italic">E. urophylla</Emphasis> plants cultivated ex vitro in greenhouse and field conditions

The young and expanded leaf micromorphology and ultrastructure of Eucalyptus grandis 2 E. urophylla juvenile plants, cultivated in greenhouse and field conditions, were analyzed by scanning and transmission electron microscopy. In greenhouse leaves epicuticular wax needles covered the abaxial and adaxial surfaces. On the adaxial surface, the needles form an atypical arrangement in lines, mainly over the anticlinal wall of epidermis cells. After plant transfer to field conditions, the organization of epicuticular wax was altered forming amorphous layers on the adaxial leaf surface, in contrast to the abaxial surface, which maintained the wax needle cover. In both culture conditions the lamellar cuticle formed on the young leaves surface disappeared during leaf enlargement. The ex vitro environment induced the development of hypostomatic leaves. The dorsiventral organization of greenhouse leaves was replaced by an isobilateral arrangement in field conditions with concomitant aerial space reduction. Results suggest that those structural changes may be some of the strategies to avoid excessive plant transpiration during Eucalyptus hybrid plants' acclimatization.     

西安市常见绿化植物叶片润湿性能及其影响因素

利用接触角测定仪测定了西安市21种常见绿化植物叶片表面的接触角,探讨了叶片表面特性如蜡质、绒毛、气孔对接触角的影响。结果表明,植物叶片正背面、物种间的接触角差异均显著,叶片正面和背面接触角大小在40°～140°。接触角大小与变异系数呈负相关,可能由于接触角小的润湿叶片在不同的生境和位置下,受到环境条件的影响较大而出现大的变异;接触角较大的非润湿性叶片,环境物质持留时间较短,对叶片形态和组成影响较小,因而出现小的变异。植物叶片表面的接触角随蜡质含量的升高而增大。表皮蜡质去除后大部分叶片接触角明显降低,尤其是疏水性较强的银杏(Ginkgo biloba)、月季(Ro-sa chinensis)和紫叶小檗(Berberis thunbergii)。女贞(Ligustrum lucidum)正背面、加杨(Popu-lus canadensis)背面等亲水型的叶片蜡质去除后接触角反而增大。叶片绒毛的多少及其形态、分布方式对接触角具有重要的影响,不同的作用方式表现出润湿和不润湿的特征,人为将其去除可以增加叶片的润湿性。背面气孔密度与气孔长度、保卫细胞长度呈负相关;接触角则与气孔密度呈负相关,与气孔长度呈正相关。     

Surface tension measurements on filamentous broths

Summary The surface tension of microorganisms is an important parameter in biotechnological processes. In this work contact angles and surface tensions were determined for filamentous micro-organisms Streptomyces levoris and Aspergillus niger. The contact angles were 39.7° and 43.1°, and the surface tensions 59.0 mJ/m² and 57.3 mJ/m², respectively. The contact angles for the filamentous broths were measured with the axisymmetric drop shape analysis - contact diameter (ADSA-CD) method which is particularly well suited to rough surfaces. The contact angle results obtained were significantly larger than those earlier reported for bacteria.Visiting scientist from Helsinki University of Technology, Espoo, Finland.     

The ion concentration of dew condensed on Norway spruce (Picea abies (L.) Karst.) and Scots pine (Pinus sylvestris L.) needles

Summary Dew droplets collected with pipettes from coniferous needles were analysed for their ionic composition. Almost all samples of dew taken from Scots pine trees (Pinus sylvestris) showed significantly higher ion concentrations than those taken from Norway spruce trees (Picea abies). This can be explained by the micromorphology of the needle surface. The higher microscale roughness of the wax layer of a pine needle causes a more efficient flux of atmospheric aerosol particles compared to the spruce needle surface. Dew on coniferous needles is shown to be capable of maintaining pH values below 3 for several hours.     

Fabrication of a Bionic Needle with both Super-Hydrophobic and Antibacterial Properties

Many biological surfaces possess unusual micro-nano hierarchical structures that could influence their wettability, which provide new methods for the construction of novel materials. In this work, silver nanoparticles were successfully coated on the surface of stainless steel needle by a simple electroless replacement reaction process between the AgNO₃ solution and the activated stainless steel needle. After the replacement reaction, porous micro/nanostructures were formed on the surface of the stainless steel needle. By modifying long chains of thiol molecules, the stainless steel needle exhibited good super-hydrophobic property with a contact angle greater than 150°. Moreover, the silver coated stainless steel needle (bionic needle) showed strong antibacterial activity against the gram-negative bacterium Escherichia coli (E. coli). By calculating the area of the inhibition zone against E. coli formed on agar medium, the antibacterial activity of the bionic needle with the contact angle of 152° is much better than that with the contact angle of 138°. The as-prepared bionic needle with both super-hydrophobic and antibacterial properties has the potential to be applied in modern medical devices.     

Interfacial free energies of intact and reconstituted erythrocyte surfaces: Implications for biological adhesion

Model cell surfaces consisting of phospholipids or phospholipids and the erythrocyte membrane glycoprotein glycophorin have been formed at an oil/water interface. Interfacial free energies have been estimated from surface wetting by both hydrophobic and hydrophilic test droplets on both the model surfaces and on intact erythrocytes. The use of a dense fluorocarbon oil to form the oil/water interface facilitates analysis by minimising surface deformation by the test drop. Hydrophobic test droplets (polar hydrocarbon oils) show increasing contact angles (decreasing wetting) with increasing hydrophilicity (decreasing interfacial free energy) of the model interface. Hydrophilic test droplets (phase separated aqueous polymer systems) show the opposite behaviour, spreading more as the interfacial free energy is decreased. Both systems give similar estimates of the interfacial free energy. Glycophorin reproduces the wetting properties of intact cell surfaces by reducing the lipid-water interfacial free energy from 5·10^?3 J·m^?2 to 1·10^?6 J·m^?2. From molecular considerations it is concluded that ‘cell surface free energy’ is an ambiguous term; its magnitude depends on the location of the interface in question. Thus, in a thermodynamic analysis of interactions at biosurfaces (such as cellular adhesion, chemotaxis or membrane fusion), the interfacial free energies may vary by more than three orders of magnitude depending on the location of the particular interface.     

Phytohormones in needles of healthy and declining silver fir (Abies alba Mill.): I. Indole-3-acetic acid

 Levels of indole-3-acetic acid (IAA) were determined in needles from silver fir (Abies alba Mill.) trees in the northern Black Forest. IAA was quantified by gas chromatography (GC) as 1-heptafluorobutyryl-IAA-methylester (HFB-IAA-ME) using electron capture detection. Prior to GC analysis, extensive purification of needle extracts was performed employing two HPLC steps. Peak identity of HFB-IAA-ME was confirmed by combined gas chromatography-mass spectrometry in selected samples. Levels of IAA in needles belonging to different needle age-classes exhibited a cyclic seasonal pattern with highest concentrations in winter and lowest levels in spring when bud-break occurred. Such a cyclic seasonal pattern of IAA levels was also observed in needles from declining fir trees or fir trees suffering from a strong sulfur impact (S-impact) in the field due to a local SO₂ source. Levels of IAA increased with increasing needle age. This age dependency of IAA concentrations was most pronounced in late autumn when IAA levels were high and nearly disappeared in spring when IAA levels reached their minimum. In needles from declining fir trees or fir trees suffering from a strong S-impact in the field, IAA levels hardly increased with increasing needle age. It is suggested that in healthy trees high levels of IAA protect older needles from abscission and that the considerable losses of older needles of declining fir trees or of fir trees under S-impact are a consequence of the low levels of IAA found in older needles of such trees. Received: 4 May 1995 / Accepted: 29 August 1995     

Effects of Methanol on Wettability of the Non-Smooth Surface on Butterfly Wing

The contact angles of distilled water and methanol solution on the wings of butterflies were determined by a visual contact angle measuring system. The scale structures of the wings were observed using scanning electron microscopy, The influence of the scale micro- and ultra-structure on the wettability was investigated. Results show that the contact angle of distilled water on the wing surfaces varies from 134.0° to 159.2°. High hydrophobicity is found in six species with contact angles greater than 150°. The wing surfaces of some species are not only hydrophobic but also resist the wetting by methanol solution with 55% concentration. Only two species in Parnassius can not resist the wetting because the micro-structure （spindle-like shape） and ultra-structure （pinnule-like shape） of the wing scales are remarkably different from that of other species. The concentration of methanol solution for the occurrence of spreading/wetting on the wing surfaces of different species varies from 70% to 95%. After wetting by methanol solution for 10 min, the distilled water contact angle on the wing surface increases by 0.8°-2.1°, showing the promotion of capacity against wetting by distilled water.     

Needle CO2 exchange, structure and defense traits in relation to needle age in Pinus heldreichii Christ – a relict of Tertiary flora

 Pinus heldreichii Christ is a long-lived, slow-growing Tertiary relict from the Balkans. In this study we evaluated the physiological characteristics of eight needle-age classes of P. heldreichii grown at the Arboretum of the Institute of Dendrology in Kórnik, Poland. At the end of the growing season, current-year foliage had the highest rates of mass-based light-saturated net photosynthesis (A_sat) of 33.5 nmol CO₂· g^–1· s^–1. A_sat decreased with needle age, but older needle classes retained from approximately 62 to 26% of the current needles’ rate. The relationship between leaf N and chlorophyll a concentration among all needle-age classes was highly significant (r = 0.96, P = 0.0006). The variation in A_sat of 1- to 7-year-old needles was linearly related to needle N concentration (r = 0.98, P = 0.0001). Needle dark respiration rates among these needle age classes ranged from 0.8 to 2.2 nmol · g^–1· s^–1 and decreased with needle age and nitrogen concentration. Total phenols and glucose concentrations increased linearly with needle age. A similar pattern was observed in acid buffering capacity and the pH of tissue homogenates. The water content ranged from 62% for the current needles to 51% for the 6-year-old needles. Greater investment in leaf structural tissue and increased chemical defense is associated with higher structural cost of older needles and may reduce their photosynthetic activity. Significant declines in water and nitrogen content with needle age and an increase in content of phenolics is most likely a defense adaptation of P. heldreichii related to the species’ long-lived leaves. Received: 8 January 1997 / Accepted: 4 March 1997