Epiphytic bacteria: development of a method for determining respiring bacteria on leaves

A method was developed for studying the total numbers and the proportion of active bacteria on leaf surfaces. It involves staining gelatin impressions of leaves treated with an electron transport system indicator, 2-( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyl tetrazolium chloride (INT). The method is rapid, inexpensive and allows simultaneous observation of numbers, ecology and respiratory activity of epiphytic bacteria. The total numbers of epiphytic bacteria for four species of aquatic plants varied between 0.6 to 10.2 times 10⁶/cm². The proportion of active bacteria on leaves of aquatic plants ranged from 2.2 to 42.9%. The method was also applied to a comparison of surface fouling of glass slides and aquatic leaf surfaces, indicating significant differences in numbers of bacteria but little difference in the proportion active on the two surfaces.     

Foliar water uptake: a common water acquisition strategy for plants of the redwood forest

Evaluations of plant water use in ecosystems around the world reveal a shared capacity by many different species to absorb rain, dew, or fog water directly into their leaves or plant crowns. This mode of water uptake provides an important water subsidy that relieves foliar water stress. Our study provides the first comparative evaluation of foliar uptake capacity among the dominant plant taxa from the coast redwood ecosystem of California where crown-wetting events by summertime fog frequently occur during an otherwise drought-prone season. Previous research demonstrated that the dominant overstory tree species, Sequoia sempervirens, takes up fog water by both its roots (via drip from the crown to the soil) and directly through its leaf surfaces. The present study adds to these early findings and shows that 80% of the dominant species from the redwood forest exhibit this foliar uptake water acquisition strategy. The plants studied include canopy trees, understory ferns, and shrubs. Our results also show that foliar uptake provides direct hydration to leaves, increasing leaf water content by 2–11%. In addition, 60% of redwood forest species investigated demonstrate nocturnal stomatal conductance to water vapor. Such findings indicate that even species unable to absorb water directly into their foliage may still receive indirect benefits from nocturnal leaf wetting through suppressed transpiration. For these species, leaf-wetting events enhance the efficacy of nighttime re-equilibration with available soil water and therefore also increase pre-dawn leaf water potentials.     

Methods for Examining the Microflora of Mouldy Hay

A comparison of four methods of estimating the microflora of mouldy hay showed that more micro-organisms were removed by washing samples than by blowing in a small wind tunnel. By contrast, actinomycete spores were estimated more efficiently from air using an Anderson Sampler than by plating diluted washings because fewer bacteria were blown than washed off the hay. Estimates of fungal spores were larger from counts in haemocytometer cells than by dilution plating but counts of actinomycetes and bacteria were higher by dilution plating. Fungi grew equally well from air or water suspensions and with the Cascade Impactor and Anderson Sampler their numbers were estimated with less variation than for the numbers of 'actinomycetes and bacteria'even on improved media and after correcting for viability, clumping and interference between colonies.     

Comparative surface-to-hand and fingertip-to-mouth transfer efficiency of gram-positive bacteria,gram-negative bacteria,and phage

AIMS: To determine the transfer efficiency of micro-organisms from fomites to hands and the subsequent transfer from the fingertip to the lip. METHODS AND RESULTS: Volunteers hands were sampled after the normal usage of fomites seeded with a pooled culture of a Gram-positive bacterium (Micrococcus luteus), a Gram-negative bacterium (Serratia rubidea) and phage PRD-1 (Period A). Activities included wringing out a dishcloth/sponge, turning on/off a kitchen faucet, cutting up a carrot, making hamburger patties, holding a phone receiver, and removing laundry from the washing machine. Transfer efficiencies were 38.47% to 65.80% and 27.59% to 40.03% for the phone receiver and faucet, respectively. Transfer efficiencies from porous fomites were <0.01%. In most cases, M.luteus was transferred most efficiently, followed by phage PRD-1 and S. rubidea. When the volunteers' fingertips were inoculated with the pooled organisms and held to the lip area (Period B), transfer rates of 40.99%, 33.97%, and 33.90% occurred with M. luteus, S. rubidea, and PRD-1, respectively. CONCLUSIONS: The highest bacteral transfer rates from fomites to the hands were seen with the hard, non-porous surfaces. Even with low transfer rates, the numbers of bacteria transferred to the hands were still high (up to 10(6) cells). Transfer of bacteria from the fingertip to the lip is similar to that observed from hard surfaces to hands. SIGNIFICANCE AND IMPACT OF THE STUDY: Infectious doses of pathogens may be transferred to the mouth after handling an everyday contaminated household object.     

Foraging strategies of caterpillars

Summary An analysis of the foraging behaviors of several species of palatable and unpalatable lepidopterous larvae indicates that palatable caterpillars partition their time between feeding and behaviors that could be related to escape visually oriented predators. Depending on the species, palatable caterpillars do all or several of the following: 1) restrict themselves to the underside of leaves at all times, 2) restrict foraging to night-time, 3) commute to and from their feeding area on leaves, 4) move from the unfinished leaf to a distant leaf after a feeding bout, thus removing themselves from the evidence of their eating, 5) snip off partially-eaten leaves after feeding on them. The less palatable, or unpalatable, caterpillars do not snip off partially-eaten leaves, feed from leaves leaving tattered edges, and are often exposed resting and feeding on the leaf surfaces in direct sunshine. I conclude that some caterpillar foraging behaviors may have evolved under the selective pressure of visually-oriented predators that use leaf-damage as a cue in their searching behavior.     

Patterns of unobstructed splash dispersal

Unobstructed splash dispersal patterns were measured in the absence of rain over mown grass using a fluorescent tracer, and a colorimetric method was used indoors in still air. When drops fell into a thin horizontal water film 0–1 mm deep, the volume of the incident drops dissipated as splash droplets was similar to the volume splashed from the film, irrespective of the distance of fall of the drops. Drop size, angle of inclination and distance of fall had significant effects on the volume of drops splashed from an inclined surface. The effects of rigidity, inclination and nature of surface were found to be significant when drops impacted onto surfaces with or without a wax covering and either rigidly or loosely supported. When splash- and dry-air-dispersed Lycopodium spores were simultaneously released, many more splashed spores were caught close to the source, but the dispersal gradient of splashed spores was steeper than that of dry-air-dispersed spores. Splash-dispersed spores were caught on slides, cylinders and rotorods but trap efficiency could not be evaluated.     

High population of Sphingomonas species on plant surface

A unique microbial community exists on upper leaf surfaces because strong irradiation by the sun restricts and selects growing micro-organisms. The micro-organisms on plant aerial parts are possibly able to control plant pathogens and to promote plant growth as do some of the micro-organisms on plant roots. Sphingomonas species on plant tissues (seed, leaf and flower) were detected and enumerated by the amplification of a specific part of the small subunit rRNA gene. Sphingomonads were found on 26 plant species belonging to 11 families. The maximum population number of Sphingomonas was 10⁸ g⁻¹ (wet weight) plant tissue. The maximum population ratio of Sphingomonas to the total aerobic bacteria was 58% when it was counted on glucose-nutrient agar plates. Consistent differences in population number and population ratio were not found among seeds, leaves and flowers. The role of sphingomonads on plant aerial parts is discussed.     

BACTERIAL CANKER OF STONE-FRUITS: IV. INVESTIGATION OF A METHOD FOR MEASURING THE INOCULUM POTENTIAL OF CHERRY TREES

Pseudomonas mors-prunorum Wormald, the organism causing bacterial canker of stone-fruits, was present on healthy cherry leaves during the autumn in numbers sufficient to suggest that they were the main source of inoculum for the infection of stems and branches. Quantitative estimates of the pathogen could be obtained by shaking leaves in water and plating out the washings. This method was examined in detail as a possible means of comparing the inoculum potentials on different cherry varieties.
The numbers of the pathogen varied considerably between leaves and between branches, but not between trees of the same variety. It was estimated that a sample of 192 leaves, eight from each of twenty-four different branches would be adequate to show differences between varieties if sufficiently replicated in time. The accuracy of a twenty-four branch sample was tested by comparing parallel samples from two separate groups of trees of the same variety. There was a highly significant correlation between the numbers of bacteria obtained from the two series of samples. From the error variance in an analysis of variance, it was calculated that five successive samples would show a significant difference between two such groups of trees if one exceeded the other by 54%.
It was necessary to wash leaves for 4 hr. or more, depending on temperature, to recover all the bacteria from the leaf surfaces. At temperatures lower than 27° C. however, bacterial growth intervened before this stage was reached. At laboratory temperatures a period of 4 hr. washing was found to be most suitable for comparing varieties and gave 80–90% recovery of bacteria without errors due to growth.
A characteristic flora of non-parasitic bacteria was observed on cherry trees, coexisting with the pathogen on the leaf surfaces. The fungus Pullaria pullulans was also extremely abundant.     

Acumen function in leaves and its vertical distribution in a tropical rain forest of Costa Rica

Water retention on the leaf surface can be maladaptive to the plant because it increases the colonization of epiphylls and interferes with the physiologic processes of the leaf, diminishing the photosynthetic capacity. To test if leaf driptips facilitate leaf drying after rainfall in a tropical rain forest of Costa Rica, we (1) experimentally measured the capacity to retain water on leaf surfaces of 30 plant species before and after driptip removal, and (2) analyzed the development of driptips along forest strata. We expected leaf driptips to be less developed in the upper strata due to the environmental conditions of the canopy (i.e., high solar radiation, strong winds and low relative humidity), which favor the natural drying of leaves. The presence of driptips increased 100% the water run off capacity of leaves in all the analyzed species. Also, the development of leaf driptips was smaller in canopy species than in understory species. Additionally, they became less developed in canopy species as trees increased in height. These results support the hypothesis that the adaptive role of driptips is to facilitate the drying of leaf surfaces.     

Degradation of phenanthrene on plant leaves by phyllosphere bacteria

The activity of phyllosphere bacteria in the degradation of phenanthrene was investigated as a mechanism for the removal of atmospheric phenanthrene after its deposition on plant leaves. Initially, leaf samples of six plant species were collected from two roadsides in Bangkok to determine the presence of phenanthrene-degrading bacteria. The numbers of phenanthrene-degrading phyllosphere bacteria were varied and ranged from 3.5 x 10(4) to 1.95 x 10(7) CFU/g, in which the highest number was found from Ixora sp. Further studies were carried out in the laboratory by spraying phenanthrene on Ixora sp. leaves and then monitoring the amount of deposited phenanthrene and number of phenanthrene-degrading bacteria after incubation. The results showed that the amount of phenanthrene was significantly reduced on leaves containing phenanthrene-degrading bacteria. These were detected along with a rapid increase in the number of bacteria on leaves. The results indicated that many phyllosphere bacteria could utilize phenanthrene to support their growth and thereby reduce the amount of deposited phenanthrene on leaf surfaces. Several phenanthrene-degrading bacteria were later isolated from the leaves and identified with a high 16S rDNA sequence similarity to the genera Pseudomonas, Microbacterium, Rhizobium, and Deinococcus.     

Factors affecting seed rain beneath fleshy-fruited plants

We investigated factors affecting seed rain beneath nine fleshy-fruited fruiting plant species growing in a 1-ha plot of planted Pinus thunbergii in central Japan. We tested whether the numbers of seeds and seed species dropped by birds beneath fruiting plants were correlated with the number of fruits removed by birds from the plants. Most of fruiting plant species with high fruit removal had significantly high seed rain. Both the numbers of seeds and seed species dropped were significantly, positively correlated with the number of fruits removed across for all fruiting plant species. Therefore, fruit removal predicted the difference among heterospecific fruiting plants in seed rain. We also tested whether the number of fruits removed from fruiting plants by birds was related with fruit crop size, fruit size, and height of the plants, and the numbers of fruits and fruit species of neighboring plants near the plants. Most of fruiting plant species with high fruit crop size had significantly high fruit removal. The number of fruits removed was significantly, positively correlated with both the fruit crop size and the number of neighboring fruits across the nine fruiting plant species. However, the effect of the neighboring fruit density on fruit removal was lower remarkably than that of fruit crop size. Therefore, fruit crop size best predicted the differences among heterospecific fruiting plants in fruit removal. We suggest that fruiting plant species with high fruit crop size and high fruit removal contribute to intensive seed rain beneath them. This revised version was published online in June 2006 with corrections to the Cover Date.     

A Note on Scanning Electron Microscopic Assessment of Stomacher Action on Chicken Skin

Scanning electron microscopy was used in conjunction with normal microbiological procedures to evaluate the stomacher as a means of recovering bacteria from chicken skin. Stomaching removed most bacteria from the skin and viable counts of skin micro-organisms obtained by this method were not significantly different from those obtained by blending. The stomacher represents a useful alternative to blending techniques commonly used to assess contamination of poultry carcasses.     

Mucilages and polysaccharides in Ziziphus species (Rhamnaceae): localization, composition and physiological roles during drought-stress.

The drought-tolerant tree species Ziziphus mauritiana Lamk. and Z. rotundifolia Lamk. were shown to have similar high mucilage concentrations (7-10% dry weight) in their leaves, with large numbers of mucilage-containing cells in the upper epidermis and extracellular mucilage-containing cavities in the leaf veins and stem cortex. The main sugar constituents of the water-soluble mucilage extract were rhamnose, glucose and galactose. During drought-stress in two independent studies, foliar mucilage content was unaffected in both species, but glucose and starch contents declined significantly in crude mucilage extracts from droughted leaves. Enzymatic hydrolysis of the mucilage extract using alpha-amylase and amyloglucosidase released glucose, indicating that a mucilage-associated water-soluble glucan, with alpha-1,4- and alpha-1,6-linkages, may exist which was extracted together with the mucilage. From the current data, it is not possible to localize the glucan to determine whether or not it is associated with mucilage-containing cells. Data from pressure-volume analyses of drought-stressed and control leaves showed that, in line with their similar mucilage contents, the relative leaf capacitance isotherm (change in relative water content per unit change in water potential) was similar in both species. During drought-stress, reduced relative capacitance resulted from osmotic adjustment and decreased wall elasticity. Data suggest that in Ziziphus leaves, intracellular mucilages play no part in buffering leaf water status during progressive drought. In Ziziphus species, growing in environments with erratic rainfall, the primary role of foliar mucilage and glucans, rather than as hydraulic capacitors, may be as sources for the remobilization of solutes for osmotic adjustment, thus enabling more effective water uptake and assimilate redistribution into roots and stems prior to defoliation as the drought-stress intensified.     

Mammal Browsers and Rainfall Affect Acacia Leaf Nutrient Content,Defense, and Growth in South African Savannas

Five sets of herbivore exclosures situated in mesic and semi‐arid savannas in Hluhluwe‐iMfolozi Park, South Africa were used to investigate the effects of mammal browsers and savanna type on plant traits relating to leaf nutrient content, defense, and growth in seven Acacia species. Mostly, browsing did not significantly affect leaf nutrient content but for a few species (i.e., increasing foliar N and P, decreasing C/N, and total polyphenols). Browser effects on structural defenses tended to be more pronounced than for leaf nutrient content and chemical defenses, particularly for semi‐arid species, resulting in longer, thicker, and denser spines, and a lower bite size index on browsed plants for most semi‐arid species. Browsing had no significant effect on growth rates for all species. Secondly, we investigated the effect of savanna type (mesic vs. semi‐arid) on the same set of plant traits and growth rates. A trade‐off in defense strategy was evident where mesic species had lower quality leaves and invested more heavily in growth and chemical defenses, while semi‐arid species generally had higher nutrient content leaves and invested more in structural defenses and higher levels of ramification. These findings suggest that the previously documented trade‐off in plant growth, resprouting ability and architecture between herbivore versus fire‐adapted savanna woody species can possibly be extended to include browse quality and defense type.     

Effects of rainfall on the use of foliar analysis for diagnosing boron toxicity in field-grown wheat

The effect of rain on foliage elemental composition, especially B, was assessed using samples of wheat collected at three harvests from a field trial conducted in soil containing excessive levels of B. Moderate rainfall substantially decreased both the B concentration and content of whole shoots and young leaves. The change in B concentration due to rain suggests that foliar analysis is unreliable for diagnosing B toxicity. For the other elements examined (Ca, Cu, Mg, Mn, Mg, P, S, Zn), rainfall had little effect.     

The role of marginal hydathodes in foliar water absorption

Summary

Marginal epithem hydathodes may, in some conditions, absorb water. A toothed leaf margin can be regarded as a water-holding structure. It is suggested that the lack of hydathodes in north-temperate evergreens and in the leaves of rain forest trees is related to the need for such species to reduce foliar water absorption. The high proportion of deciduous species with hydathodes in woodland vegetation is thought to be related to the summer potential soil water deficit in Britain, leaf-absorbed water supplementing water from the soil. The term ‘potomorphic’ is suggested for leaves with water absorbing structures.     

Self-propulsion of dew drops on lotus leaves: a potential mechanism for self cleaning

This study shows that condensation on the hierarchically structured lotus leaf can facilitate self-propulsion of water droplets off the surface. Droplets on leaves inclined at high angles can be completely removed from the surface by self-propulsion with the assistance of gravity. Due to the small size of mobile droplets, light breezes may also fully remove the propelled droplets, which are typically projected beyond the boundary layer of the leaf cuticle. Moreover the self-propelled droplets/condensate were able to remove contaminants (eg silica particles) from the leaf surface. The biological significance of this process may be associated with maintaining a healthy cuticle surface when the action of rain to clean the surface via the lotus effect is not possible (due to no precipitation). Indeed, the native lotus plants in this study were located in a region with extended time periods (several months) without rain. Thus, dew formation on the leaf may provide an alternative self-cleaning mechanism during times of drought and optimise the functional efficiency of the leaf surface as well as protecting the surface from long term exposure to pathogens such as bacteria and fungi.     

Gasometric method of water deficit measurement in leaves

A gasometric method was developed for measuring water deficit in leaves. For a leaf at full turgor the amount of water penetrating into the tissue after removing the air from intercellular spaces by means of a vacuum pump, is equal in volume to the gas removed from the intercellular spaces. In a leaf with a water deficit the amount of the infiltrating water is greater than the removed gas volume by the amount egual to the water deficit. Determination of the volumes of the gas removed and penetrating water enables water deficit, if any, to be calculated. Comparative measurements carried out on five plant species confirmed the correctness of the method suggested. Reduction of the measuring time allowed to eliminate completely the sources of errors associated with the growth of tissue and loss of dry weight during respiration.     

Inoculum Sources for the Spread of Leaf Scald Disease of Sugarcane Caused by Xanthomonas albilineans in Guadeloupe

Leaf scald of sugarcane, caused by Xanthomonas albilineans, is thought to be spread mainly in infected cuttings and transmitted on infested cutting implements. Several observations made in Guadeloupe indicated that other means of spreading also occur. The dispersal of the pathogen outside sugarcane was investigated with plants inoculated by an antibiotic-resistant marked strain of X. albilineans and with plants naturally infested with wild strains of the pathogen. The bacteria were isolated in water droplets (rain or dew) on the surface of sugarcane leaves at dawn. It was also detected on the surface of dry leaves during the day by leaf imprinting onto a selective culture medium. The bacteria were much more frequently isolated from the surface of symptomatic leaves than from symptomless ones. Aerial dispersal of X. albilineans was investigated by placing Petri dishes containing selective culture medium between sugarcane plants but without direct contact with the leaves. The pathogen was isolated in four out of 270 dishes which were randomly set 3–14 h in a diseased field. These results indicated that the pathogen exuded from the leaves and then was spread by aerial means (rain, insects, …) or by leaf contact. The bacteria were also found in roots and rhizospheric soil of infested sugarcane stools suggesting that X. albilineans could be transmitted by root to root contact or by the soil. Finally, isolations of the pathogen in sugarcane inflorescences were positive. So, fuzz transmission may also occur.     

Inoculum Sources for the Spread of Leaf Scald Disease of Sugarcane Caused by Xanthomonas albilineans in Guadeloupe

Leaf scald of sugarcane, caused by Xanthomonas albilineans, is thought to be spread mainly in infected cuttings and transmitted on infested cutting implements. Several observations made in Guadeloupe indicated that other means of spreading also occur. The dispersal of the pathogen outside sugarcane was investigated with plants inoculated by an antibiotic-resistant marked strain of X. albilineans and with plants naturally infested with wild strains of the pathogen. The bacteria were isolated in water droplets (rain or dew) on the surface of sugarcane leaves at dawn. It was also detected on the surface of dry leaves during the day by leaf imprinting onto a selective culture medium. The bacteria were much more frequently isolated from the surface of symptomatic leaves than from symptomless ones. Aerial dispersal of X. albilineans was investigated by placing Petri dishes containing selective culture medium between sugarcane plants but without direct contact with the leaves. The pathogen was isolated in four out of 270 dishes which were randomly set 3-14 h in a diseased field. These results indicated that the pathogen exuded from the leaves and then was spread by aerial means (rain, insects,…) or by leaf contact. The bacteria were also found in roots and rhizospheric soil of infested sugarcane stools suggesting that X. albilineans could be transmitted by root to root contact or by the soil. Finally, isolations of the pathogen in sugarcane inflorescences were positive. So, fuzz transmission may also occur.