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     

Wetting of the upper needle surface of Abies grandis: influence of pH, wax chemistry and epiphyllic microflora on contact angles

Wetting of the upper needle surface of Abies grandis Lindl. by aqueous solutions of different pH values was investigated. With increasing needle age, contact angles decreased significantly from about 75° on current-year needles to values lower than 30° on 4-year-old needles. On older needles, contact angles were significantly lower, by more than 10°, when aqueous solutions of pH9-0 were used compared with those of pH3-0. On the surfaces of older needles, contact angle titrations were carried out, contact angles being measured with aqueous solutions covering a pH range from 3.0 to 11.0. Measured titration curves showed clear inflection points around pH 7.0, indicating the existence of ionizable carboxylie groups in the interface between needle surface and atmosphere. The evidence seems convincing that the pronounced pH dependence of wetting is mainly due to the presence and/or activity of epiphyllic micro-organisms, whereas the cuticular wax composition of Abies grandis needles does not appear to contribute significantly to this phenomenon. Thus, the results presented here allow the general conclusion that changes of contact angles measured on leaf surfaces may not always be due to changes in the leaf surface chemistry and/or the fine structure of leaf surface waxes, but may also be due to increased amounts of epiphyllic micro-organisms significantly altering the leaf surface wetting properties.     

Effect of Maillard reaction products on the growth of selected food-poisoning micro-organisms

The activity of the Maillard reaction products (MRP) prepared by heating (15 h at 90°C) a solution of 1·71 mol/l glucose and 2·05 mol/l glycine at pH values 6·0 and 8·8, against food-poisoning micro-organisms, including Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, Salmonella enteritidis and Aeromonas hydrophila , was investigated. High and low pH MRPs strongly inhibited A. hydrophila , whereas Staph. aureus and L. monocytogenes were slightly inhibited by the high pH MRPs only and Salmonella strains were resistant to both.     

Epiphytic microorganisms on strawberry plants (Fragaria ananassa cv. Elsanta): identification of bacterial isolates and analysis of their interaction with leaf surfaces

Epiphytic bacteria were isolated from strawberry plants cultivated in the field or in the greenhouse in order to investigate their interaction with leaf-surface transport properties. Colonization of lower leaf sides was higher on field-grown plants, whereas upper leaf sides were more densely colonized on plants cultivated in the greenhouse. Fungal isolates significantly contributed to total microbial biomass on leaf surfaces of greenhouse-grown strawberry plants, whereas these organisms were rarely abundant on field-grown plants. Microscopic investigations of bacteria in the phyllosphere revealed that the highest densities of bacteria were observed on living trichomes, which obviously provide a source of nutrients. Isolated strains were characterized by colony morphology, microscopy and histochemistry. About 324 isolated bacterial strains were grouped into 38 morphotypes. Of the morphotypes, 12 were identified by 16S rRNA gene sequencing. Dominating bacteria belonged to the genus Pseudomonas, Stenotrophomonas, Bacillus and Arthrobacter. Cuticular water permeability of isolated cuticular membranes and intact leaf disks was measured before and after treatment with one of the most prominent epiphytic bacteria, Pseudomonas rhizosphaerae. Results showed that cuticular transpiration was significantly increased by P. rhizosphaerae. This shows that leaf-surface properties, such as cuticular water permeability, can be influenced by bacteria, leading to improved habitable conditions in the phyllosphere.     

Attributes of fresh gilt-head seabream (Sparus aurata) fillets treated with potassium sorbate, sodium gluconate and stored under a modified atmosphere at 0±1°C

The development of microbial populations on fillets of Mediterranean gilt-head sea bream ( Sparus aurata ) treated with potassium sorbate, sodium gluconate or a combination of both and stored under a modified atmosphere (MA) of 40% CO₂, 30% O₂ and 30% N₂ at 0±1 °C for about 30 d was studied. The pH of aqueous solutions of the preservatives was adjusted to 6·0 with HCl. The preservatives were applied by dipping. The use of sorbate plus gluconate was more effective than sorbate alone. Gluconate had a positive effect on the growth of Gram-positive micro-organisms. Changes in the concentrations of glucose, glucose-6-phosphate, ammonia, acetic acid, trimethylamine-nitrogen and sorbate were also monitored.     

Role of leaf surface sugars in colonization of plants by bacterial epiphytes

The relationship between nutrients leached onto the leaf surface and the colonization of plants by bacteria was studied by measuring both the abundance of simple sugars and the growth of Pseudomonas fluorescens on individual bean leaves. Data obtained in this study indicate that the population size of epiphytic bacteria on plants under environmentally favorable conditions is limited by the abundance of carbon sources on the leaf surface. Sugars were depleted during the course of bacterial colonization of the leaf surface. However, about 20% of readily utilizable sugar, such as glucose, present initially remained on fully colonized leaves. The amounts of sugars on a population of apparently identical individual bean leaves before and after microbial colonization exhibited a similar right-hand-skewed distribution and varied by about 25-fold from leaf to leaf. Total bacterial population sizes on inoculated leaves under conditions favorable for bacterial growth also varied by about 29-fold and exhibited a right-hand-skewed distribution. The amounts of sugars on leaves of different plant species were directly correlated with the maximum bacterial population sizes that could be attained on those species. The capacity of bacteria to deplete leaf surface sugars varied greatly among plant species. Plants capable of supporting high bacterial population sizes were proportionally more depleted of leaf surface nutrients than plants with low epiphytic populations. Even in species with a high epiphytic bacterial population, a substantial amount of sugar remained after bacterial colonization. It is hypothesized that residual sugars on colonized leaves may not be physically accessible to the bacteria due to limitations in wettability and/or diffusion of nutrients across the leaf surface.     

Design of a Temperature Gradient Incubator

S ummary . A temperature gradient incubator heated and cooled by circulating liquids is described which is suitable for incubating micro-organisms in liquid media and on solid media in roll tubes. The maximum deviation from a linear temperature gradient is 0·35% of the range (0·13°) even when used at temperatures up to 30° above ambient. The temperature of any set of sample wells is uniform to within 0·07° (3·5% of the difference between adjacent rows).     

The effect of temperature, pH and medium in a surface adhesion immunofluorescent technique for detection of Listeria monocytogenes

A rapid surface adhesion-based immunofluorescence technique was used to detect Listeria monocytogenes from inoculated culture systems. The effect of culture type (pure, mixed and meat), pH (7·00, 6·40, 4·76 and 3·13), acids (citric and HCl) and temperature (25°, 30° and 37°C) on the adhesion of Listeria to the polycarbonate membrane used in this technique was determined. It was found that pH had a significant effect ( P < 0·05) with higher numbers of Listeria adhering at low pH values (4·76). Culture type was also important with significantly higher numbers of Listeria ( P < 0·05) adhering to membranes immersed in meat cultures than in pure or mixed cultures. This effect was seen at 30°C but not at 25° or 37°C. The total viable count (TVC) on the membrane was unaffected by pH but temperature had an influence with optimum adhesion occurring at 25°C. The reasons for observed differences and their implications for the surface adhesion immunofluorescent rapid method are discussed.     

Leaf Water Repellency as an Adaptation to Tropical Montane Cloud Forest Environments

Adaptations that reduce water retention on leaf surfaces may increase photosynthetic capacity of cloud forests because carbon dioxide diffuses slower in water than air. Leaf water repellency was examined in three distinct ecosystems to test the hypothesis that tropical montane cloud forest species have a higher degree of leaf water repellency than species from tropical dry forests and species from temperate foothills-grassland vegetation. Leaf water repellency was measured by calculating the contact angle of the leaf surface and the line tangent to a water droplet through the point of contact on the adaxial and the abaxial surface. Leaf water repellency was significantly different between the three study areas. The hypothesis that leaf water repellency is higher in cloud forest species than tropical dry forests and temperate foothills-grassland vegetation was not confirmed in this study. Leaf water repellency was lower for cloud forest species (adaxial surface = 50.8°; abaxial surface = 82.9°) than tropical dry forest species (adaxial surface = 74.5°; abaxial surface = 87.3°) and temperate foothills-grassland species (adaxial surface = 77.6°; abaxial surface = 95.8°). The low values of leaf water repellency in cloud forest species may be influenced by presence of epiphylls and loss of epicuticular wax on the leaf surfaces.     

高寒草地甘肃臭草茎-叶性状的坡度差异性

茎与叶的生长形态决定植物与外界环境的物质交换能力, 茎叶的异速生长模式对认识植物表型可塑性及其调节机理具有重要意义。在祁连山高寒退化草地, 利用ArcGIS建立研究区域的数字高程模型(DEM), 并提取样地坡度数据, 采用标准化主轴估计(SMA)方法, 研究了不同坡度甘肃臭草(Melica przewalskyi)种群茎与叶的生长。结果表明: 随着坡度增大, 甘肃臭草茎干质量、叶干质量、叶面积均呈逐渐减小趋势, 叶片数呈增加趋势; 甘肃臭草叶干质量的增长速度显著大于茎干质量的增长速度, 叶面积与茎干质量近等速增长; 不同坡度间的比较显示, 随着坡度变陡甘肃臭草茎干质量与叶干质量异速斜率显著减小(p < 0.05), 陡坡上的甘肃臭草若要生成与缓坡样地中相同的叶生物量需要投入更多的茎生物量, 茎干质量与叶面积的y轴截距显著减小(p < 0.05), 即相同的茎干质量投入下, 较大坡度的甘肃臭草叶面积投入显著降低, 趋向于减小叶面积增加叶数量。坡度梯度上甘肃臭草加快了茎的相对生长速率而减小了在叶面积上的投入, 体现了不同坡度甘肃臭草茎-叶生物量分配机制及资源利用策略, 同时说明高寒草地中小叶更具生境适应性。     

Canopy cover and leaf age affect colonization by tropical fungal endophytes: Ecological pattern and process in Theobroma cacao (Malvaceae)

Fungal endophytes inhabit healthy tissues of all terrestrial plant taxa studied to date and are diverse and abundant in leaves of tropical woody angiosperms. Studies have demonstrated that plant location and leaf age influence density of endophyte infection in leaves of tropical forest trees. However, ecological factors underlying these observations have not been explored in detail. Here, we establish that foliar endophytes of a tropical tree (Theobroma cacao, Malvaceae) are transmitted horizontally and that endophyte-free seedlings can be produced for experimental manipulation by protecting aerial tissues from surface wetting. At Barro Colorado Island, Panama, we used transects of endophyte-free seedlings to determine the importance of several factors (canopy cover, abundance of aerial and epiphytic propagules, leaf age, leaf chemistry, leaf toughness and duration of exposure to viable air spora) in shaping colonization by endophytic fungi. Endophytes colonized leaves of T. cacao more rapidly beneath the forest canopy than in cleared sites, reflecting local abundance of aerial and epiphytic propagules. The duration of exposure, rather than absolute leaf age, influenced endophyte infection, whereas leaf toughness and chemistry had no observed effect. Endophytes isolated from mature T. cacao grew more rapidly on media containing leaf extracts of T. cacao than on media containing extracts from other co-occurring tree species, suggesting that interspecific differences in leaf chemistry influence endophyte assemblages. Together, these data allow us to identify factors underlying patterns of endophyte colonization within healthy leaves of this tropical tree.     

Development and application of a simple filter paper imprinting technique for the detection and enumeration of colonies of ureolytic micro-organisms

A rapid, simple and inexpensive method has been devised to determine the proportion of colonies of ureolytic organisms in cultures of complex microbial populations. Spiral plated cultures displaying well separated colonies are tested for ureolytic micro-organisms by imprinting the colonies onto filter papers impregnated with a solution of 1 mol/l urea and phenol red (0·1% w/v) in 0·1 mol/l phosphate buffer (pH 6·8). A rapid colour change indicates ureolytic activity. The proportion of ureolytic colony-forming units in cultures of saliva specimens from 90 school children ranged from less than 1% to 40% (mean 9·9%± 7·7). Saliva and dental plaque specimens from 16 adult subjects were also tested and the occurrence of urease-positive organisms was substantially less in plaque (3·6%± 3·7, range 0·1–12) than saliva (18·7%± 13·8, range 1·3–51). The predominant ureolytic oral species was Streptococcus salivarius , 75 (54·7%) of 137 tested isolates being urease-positive.     

Factors affecting the survival of Salmonella and Escherichia coli in anaerobically fermented pig waste

The survival of Salmonella typhimurium was investigated in acidogenic, anaerobically fermented pig wastes and in synthetic media, each containing volatile fatty acids (VFA). Salm. typhimurium survived at pH 6·8, but not at pH 4·0, when incubated at 37°C for 24 h in either fermented or synthetic medium containing VFA. The minimum inhibiting concentration of VFA for Salm. typhimurium after 48 h incubation at 30°C at pH 4·0 was 0·03 mol/l and for Escherichia coli it was 0·09 mol/l. Fermented pig wastes in a digester, maintained at pH 5·9, were inoculated with Salm. typhimurium and then incubated at 37°C for 24 h. The pH was adjusted to either 4·0 or 5·0 and after a further 48 h at 30°C, Salm. typhimurium survived at pH 5·0 but not at pH 4·0. It was concluded that pH is critical in determining the survival of this organism in acidogenic anaerobically fermented pig waste.     

From epiphyte to tree: differences in leaf structure and leaf water relations associated with the transition in growth form in eight species of hemiepiphytes

Stranglers must undergo a transformation in growth form from epiphyte to tree to become reproductive mature and thus require developmental and/or physiological plasticity to cope with radical changes in their rooting environment. Differences in leaf structure and water relations between epiphytic-phase and free-standing individuals were marked in the five Ficus species examined. Epiphytic Ficus had several-fold higher specific leaf area (cm₂ g^?1) and 2- to 4-fold lower stomatal densities than conspecific trees. Osmotic potentials at full saturation were, on average, 0-6 MPa higher (less negative) and the bulk modulus of elasticity approximately 50% lower in epiphytic plants than in conspecific trees. This resulted in leaves of epiphytic and tree individuals losing turgor at approximately the same relative water content, hut at a substantially higher leaf water potential in the epiphytic plants. In contrast, differences in leaf structure and water relations between epiphytes and trees of Clusia minor and Coussapoa villosa were small. In greenhouse experiments, alteration of the water and nutrient supply to epiphytic F. tuerckheimmii plants did not lead to significant changes in leaf structure.     

The physico-chemical basis of leaf wettability in wheat

Summary Wild type wheat (Triticum aestivum L.) and three mutant lines that have reduced glaucousness on the flag leaf sheath have been examined for variations in glaucousness, contact angles, wax chemistry and wax morphology. On the sheath and culm, organs that are glaucous in the wild type, increasing glaucousness is correlated with increasing contact angles, an increasing proportion of -diketones plus hydroxy--diketones in the was and an increasing proportion of wax tubes. Organs that were non-glaucous in all four lines, namely both surfaces of the vegetative leaves and the adaxial surface of the flag leaf, had high contact angles, a dense covering of wax plates and waxes rich in primary alcohols but devoid of -diketones and hydroxy--diketones. The abaxial surface of the flag leaf was the most complex of the organ surfaces studied. In the wild type the glaucousness of the sheath continued onto this surface for 1–2 cm and this was correlated with the other characters studied as it was on the sheath. In the mutants, however, the tubes were absent. Flat ribbons of varying widths, a new wax structure in wheat, as well as various types of plates were found instead. These structures continued to the flag leaf tip and were also present on the abaxial surface of the wild type flag leaf. Changes in contact angle at the tip could not be correlated with the other measured parameters.     

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.     

Changes in the strength of attachment of micro-organisms to surfaces following treatment with disinfectants and cleansing agents

Suspensions of Pseudomonas aeruginosa and Staphylococcus epidermidis , and biofilms established (16 h) on submerged glass and stainless steel (216 2B) coupons, were exposed to sodium hypochlorite (0·02% or 0·015% w/v), Dodigen (0·0015% w/v or 0·0006% w/v), sodium dodecylsulphate (6% w/v or 0·1% w/v) and Tween-80 (6% w/v) for 5 min at 20 °C. Survival was assessed by viable counts and blot succession. Biofilm bacteria were significantly less susceptible to these biocides than were planktonic cells, but their attachment to the surfaces was loosened by such treatments. Treatment with the non-ionic surfactant, Tween-80, however, strengthened the attachment of Staph. epidermidis to stainless steel. Such effects on attachment strength, which are species and surface dependent, have profound implications on post-treatment cleansing and possible re-contamination of product in clean-in-place (CIP) systems.     

Effects of sporulation pH on the heat resistance and the sporulation of Bacillus cereus

Spores of Bacillus cereus ATCC 7004, 4342 and 9818 were obtained in nutrient agar at several pH from 5·9 to 8·3. The optimum pH for sporulation was around 7, but good production of spores was obtained in the range 6·5–8·3. With all three strains, D -values clearly dropped with sporulation pH, decreasing by about 65% per pH unit. z -Values were not significantly modified ( P > 0·05) by this factor. Mean z -values of 7·13 °C ± 0·16 for strain 7004, 7·67 °C ± 0·04 for 4342 and 8·80 °C ± 0·64 for 9818 were obtained.     

Effect of heat treatment on the proteolytic activity of mesophilic bacteria isolated from goats' milk cheese

Strains of mesophilic lactococci and lactobacilli isolated from goats' milk cheese were grown to maximum density in milk at 30°C, pH 6·5. They were subsequently cooled to 12°C and then heated at 50°, 52° and 54°C (holding time, 15 s). The micro-organisms tested were Lactococcus lactis subsp. lactis IFPL 60, IFPL 22 and IFPL 359, Lactobacillus casei subsp. casei IFPL 731 and Lactobacillus plantarum IFPL 3, isolated from raw goats' milk cheese. The heated cells presented lower viability and acidification capacity than unheated cells. After heat treatment at 50°C, all the test strains effected practically no reduction in pH of milk (6 h), except for Lactococcus lactis subsp. lactis IFPL 60, which reduced pH to 5·9 as compared to 4·9 attained by the unheated controls. After treatment, proteolytic, aminopeptidase and dipeptidase activities of cell-free extracts decreased to a lesser extent than the number of viable cells with acidifying ability. The results suggest that these strains, if treated at 50°C, may be suitable as extra sources of important ripening enzymes in cheese making.     

Production and stability of pediocin N5p in grape juice medium

The production and stability of pediocin N5p from Pediococcus pentosaceus , isolated from wine, were examined in grape juice medium. Maximum growth and higher titre (4000 U ml^-1) were observed at a initial pH of 7·5 and 30°C. The activity of the inhibitory substance was stable between pH values from 2·0 to 5·0 at 4° and 30°C. At pH 10·0 it was completely inactivated. When submitted to 30 min at 80°, 100° and 115°C, maximal stability was observed at pH 2·0. Ethanol up to 10% did not affect pediocin activity at acid pH, nor did 40–80 mg 1^-1 SO₂, independently or combined with different ethanol concentrations, affect inhibitory activity.