Location of Escherichia coli O157:H7 on and in apples as affected by bruising, washing, and rubbing.

Confocal scanning laser microscopy (CSLM) was used to determine the location of Escherichia coli O157:H7 cells on the surface and in tissue of bruised Red Delicious cv. apples. Undamaged and bruised apples were inoculated by immersing in a suspension of E. coli O157:H7 cells transformed with a plasmid that encodes for the production of a green fluorescent protein. Apples were then washed in 0.1% (wt/vol) peptone water and/or rubbed with a polyester cloth and examined to determine if these treatments removed or introduced cells into lenticels, cutin, and cracks on the skin surface. Optical slices of the apples obtained using CSLM were examined to determine the depth at which colonization or attachment of cells occurred. Populations of E. coli O157:H7 on the surface of apples were determined to assess the effectiveness of washing and rubbing in physically removing cells. The location of cells on or in undamaged and bruised areas of apples that were not washed or rubbed did not differ significantly. However, washing apples resulted in an approximate 2-log reduction in CFU of E. coli O157:H7 per cm2 of apple surface. On unwashed apples, cells were detected at depths up to 30 microm below the surface. No E. coli O157:H7 cells were detected at locations more than 6 microm below the surface of washed apples. Cells that remained on the surface of rubbed apples appeared to be sealed within naturally occurring cracks and crevices in waxy cutin platelets. These cells may be protected from disinfection and subsequently released when apples are eaten or pressed for cider production.

Chemical composition of the Prunus laurocerasus leaf surface. Dynamic changes of the epicuticular wax film during leaf development.

The seasonal development of adaxial Prunus laurocerasus leaf surfaces was studied using newly developed methods for the mechanical removal of epicuticular waxes. During epidermal cell expansion, more than 50 microg leaf(-1) of alkyl acetates accumulated within 10 d, forming an epicuticular wax film approximately 30 nm thick. Then, alcohols dominated for 18 d of leaf development, before alkanes accumulated in an epicuticular wax film with steadily increasing thickness (approximately 60 nm after 60 d), accompanied by small amounts of fatty acids, aldehydes, and alkyl esters. In contrast, the intracuticular waxes stayed fairly constant during development, being dominated by triterpenoids that could not be detected in the epicuticular waxes. The accumulation rates of all cuticular components are indicative for spontaneous segregation of intra- and epicuticular fractions during diffusional transport within the cuticle. This is the first report quantifying the loss of individual compound classes (acetates and alcohols) from the epicuticular wax mixture. Experiments with isolated epicuticular films showed that neither chemical conversion within the epicuticular film nor erosion/evaporation of wax constituents could account for this effect. Instead, transport of epicuticular compounds back into the tissue seems likely. Possible ecological and physiological functions of the coordinate changes in the composition of the plant surface layers are discussed.

Viability loss of neem (Azadirachta indica) seeds associated with membrane phase behaviour.

Storage of neem (Azadirachta indica) seeds is difficult because of their sensitivity to chilling stress at moisture contents (MC) > or =10% or imbibitional stress below 10% MC. The hypothesis was tested that an elevated gel-to-liquid crystalline phase transition temperature (Tm) of membranes is responsible for this storage behaviour. To this end a spin probe technique, Fourier transform infrared microspectroscopy, and electron microscopy were used. The in situ Tm of hydrated membranes was between 10 degrees C and 15 degrees C, coinciding with the critical minimum temperature for germination. During storage, viability of fresh embryos was lost within two weeks at 5 degrees C, but remained high at 25 degrees C. The loss of viability coincided with an increased leakage of K+ from the embryos upon imbibition and with an increased proportion of cells with injured plasma membranes. Freeze-fracture replicas of plasma membranes from chilled, hydrated axes showed lateral phase separation and signs of the inverted hexagonal phase. Dehydrated embryos were sensitive to soaking in water, particularly at low temperatures, but fresh embryos were not. After soaking dry embryos at 5 degrees C (4 h) plus 1 d of further incubation at 25 degrees C, the axis cells were structurally disorganized and did not become turgid. In contrast, cells had a healthy appearance and were turgid after soaking at 35 degrees C. Imbibitional stress was associated with the loss of plasma membrane integrity in a limited number of cells, which expanded during further incubation of the embryos at 25 degrees C. It is suggested that the injuries brought about by storage or imbibition at sub-optimal temperatures in tropical seeds whose membranes have a high intrinsic Tm (10-15 degrees C), are caused by gel phase formation.

Living under a "dormant" canopy: a molecular acclimation mechanism of the desert plant Retama raetam.

Desert plants are exposed to a combination of environmental stress conditions, including low water availability, extreme temperature fluctuations, high irradiance and nutrient deprivation. Studying desert plants within their natural habitat may therefore reveal novel mechanisms and strategies that enable plants to resist stressful conditions. We studied the acclimation of Retama raetam, an evergreen stem-assimilating desert plant, to growth within an arid dune ecosystem. Retama raetam contained two different populations of stems: those of the upper canopy, exposed to direct sunlight, and those of the lower canopy, protected from direct sunlight. During the dry season, stems of the upper canopy contained a very low level of a number of essential proteins, including the large and small subunits of rubisco, ascorbate peroxidase and the D1 subunit of the reaction centre of photosystem II. However, RNA encoding these proteins was present; cytosolic transcripts were associated with polysomes, while chloroplastic transcripts were not. Upon water application, as well as following the first rainfall of the season, these "photosynthetically suppressed" stems recovered and accumulated essential proteins within 6-24 h. In contrast, stems of the lower canopy contained the essential proteins throughout the dry season. We suggest that R. raetam uses an acclimation strategy of "partial plant dormancy" in order to survive the dry season. "Dormancy", as evident by the post-transcriptional suppression of gene expression, as well as the suppression of photosynthesis, was induced specifically in stems of the upper canopy which protect the lower canopy by shading.

Attachment of Escherichia coli O157:H7 to the surfaces and internal structures of apples as detected by confocal scanning laser microscopy.

Confocal scanning laser microscopy (CSLM) was used to demonstrate the attachment of Escherichia coli O157:H7 transformed with a plasmid encoding for green fluorescent protein (GFP) to the surface and within the internal structures of nonwaxed Red Delicious cv. apples. Apples at 2 or 25 degrees C were inoculated with an E. coli O157:H7 cell suspension at 2 or 25 degrees C. The effect of a negative temperature differential (cold inoculum, warm apple), a positive differential (warm inoculum, cold apple), and no differential (warm inoculum, warm apple), in combination with a pressure differential (atmospheric versus 10,130 Pa), on the attachment and infiltration of cells was determined. CSLM stereo images of external surfaces of apples subjected to all combinations of test parameters showed preferential cellular attachment to discontinuities in the waxy cuticle on the surface and to damaged tissue surrounding puncture wounds, where the pathogen was observed at depths up to 70 microm below the skin surface. Attachment to lenticels was sporadic but was occasionally observed at depths of up to 40 microm. Infiltration through the floral tube and attachment to seeds, cartilaginous pericarp, and internal trichomes were observed in all apples examined, regardless of temperature differential during inoculation. The pressure differential had no effect on infiltration or attachment of E. coli O157:H7. Image analysis to count cells at various depths within tissues was used to quantitatively compare the extent of infiltration into various apple structures as well as the effects of the temperature differential. Puncture wounds harbored greater numbers of the pathogen at greater depths than did other sites examined. Attachment or infiltration of cells was greater on the intact skin and in lenticels, russet areas, and the floral tube of apples inoculated under a negative temperature differential compared to those inoculated under no temperature differential. The results suggest that E. coli O157:H7 attached to internal core structures or within tissues of apples may evade decontamination treatments. Interventions designed to deliver disinfectants to these locations or to remove viable cells of E. coli O157:H7 and other pathogens from apples by other means need to be developed and validated.

Brittleness of twig bases in the genus Salix: fracture mechanics and ecological relevance.

The twig bases within the genus Salix were investigated. Brittleness of twig bases as defined in the literature neither correlates with Young's modulus nor with growth strains, which were measured for S. alba, S. fragilis and S. x rubens. For the species S. alba, S. appendiculata, S. eleagnos, S. fragilis, S. purpurea, S. triandra, S. viminalis, and S. x rubens, fracture surfaces of broken twigs were investigated and semiquantitatively described in terms of 'relative roughness' (ratio of rough area of fracture surface over whole area of fracture surface). The relative roughness clearly corresponds with the classification into brittle and nonbrittle species given in the literature. An attempt was made to quantify brittleness with mechanical tests. The absolute values of stress and strain do not correlate with the brittleness of the twig bases as defined by the relative roughness. However, the 'index stress' (ratio of stress at yield over stress at fracture) or the 'index strain' (ratio of strain at yield over strain at fracture), correlate well with the relative roughness. The graphic analysis of index stress against index strain reveals a straight line on which the eight species are ordered according to their brittleness. Depending on growth form and habitat, brittle twig bases of willows may function ecologically as mechanical safety mechanisms and, additionally, as a propagation mechanism.

Grapevine fleck virus-like viruses in Vitis.

Two sets of degenerate primers for the specific amplification of 572-575 nt and 386 nt segments of the methyltransferase and RNA- dependent RNA polymerase cistrons of members of the genera Tymovirus and Marafivirus and of the unassigned virus Grapevine fleck virus (GFkV) were designed on the basis of available sequences. These primers were used for amplifying and subsequent cloning and sequencing part of the open reading frame 1 of the genome of GFkV, Grapevine asteroid mosaic-associated virus (GAMaV) and of another previously unreported virus, for which the name Grapevine red globe virus (GRGV) is proposed. Computer-assisted analysis of the amplified genome portions showed that the three grapevine viruses are phylogenetically related with one another and with sequenced tymoviruses and marafiviruses. The relationships with tymoviruses was confirmed by the type of ultrastructural modifications induced in the host cells. RdRp-specific degenerate primers were successfully used for the aspecific detection of the three viruses in crude grapevine sap extracts. Specific virus identification was obtained with RT-PCR using antisense virus-specific primers.

Heterologous expression in Aspergillus nidulans of a Trichoderma longibrachiatum endoglucanase of enological relevance.

An Aspergillus nidulans transformant expressing the Trichoderma longibrachiatum endoglucanase 1 gene (egl1) has been constructed. The extracellular production of EGL1 in different culture media has been studied, and a medium has been found in which EGL1 is the predominant extracellular protein produced. The enzymatic properties of the heterologously produced EGL1 are very similar to those of the native enzyme. Grape maceration in the presence of culture filtrate enriched in EGL1 resulted in increased release of aroma precursors, particularly in the case of aromatic grapes. Cryoscanning electron microscopy of the flesh of grapes treated with EGL1-enriched culture filtrate revealed degradation of the cell wall matrix.

Visualization of harpin secretion in planta during infection of apple seedlings by Erwinia amylovora.

Erwinia amylovora is a Gram-negative pathogenic bacterium that infects pear and apple trees as well as other plants from the Rosaceae family. E. amylovora pathogenicity is dependent on a functional Hrp type III secretion system. Harpin, a protein playing a major role in virulence, has been shown to be exported in vitro via the type III secretion apparatus. The data presented here focus on harpin detection in planta after infection of apple seedlings with the wild-type strain CFPB 1430. Using a specific harpin antiserum, harpin was not detected inside the host plant cells, but was found associated with the bacteria and secreted. The extracellular localization of harpin is in agreement with the physiological effects induced by purified harpin when applied as an exogenous elicitor. Harpin was not found associated with the host plant cell wall, a result that weakens its postulated role in cell wall loosening. A differential labelling was observed at the bacterial level: for some bacteria, harpin was exclusively cytoplasmic, whereas in others, it appeared as small patches over the bacterial outer membrane or associated with extracellular linear structures. All the bacteria present within the same area were similarly labelled, suggesting co-ordination in the secretion process. All observations suggest that harpin is synthesized in the bacterial cytoplasm and that secretion occurs from this cytoplasmic pool upon sensing of a plant or bacterial signal.

Cytological comparison of leaves and stems of Prunus avium L. shoots cultured on a solid medium with agar or gelrite.

An axillary proliferating clone of Prunus avium L. was subcultured every four weeks on solid MS medium with agar as the gelling agent. Vitrification (hyperhydricity) of shoots was induced in one four week cycle with the same medium except that agar was replaced by gelrite. During culture on the vitrifying medium, the water content of the shoots progressively increased with a parallel decrease in chlorophyll content. Cytological differences between the leaves and stems of the vitrified and normal shoots were detected by light and electron (both transmission and scanning) microscopy. Leaves of vitrified shoots were characterized by lower number of chloroplasts in the palisade parenchyma and by a defective cuticle. The stems of vitrified shoots had a less developed and lignifled xylem tissue, lacked sclerenchymatic areas and showed hypertrophy of the cortical parenchyma. More intense vacuolar activity with evaginations of the chloroplast envelope into the vacuole was noted in cells of vitrified leaves.

Water permeability of plasma membranes of cultured rice, grape, and CH27 cells measured dielectrically.

The capacitance of suspensions of cultured rice cells (Oryza sativa L. ssp. japonica), grape cells (Vitis sp.), and CH27 cells originated from murine B-cell lymphoma was measured in the frequency range of 0.2 to 10 MHz. The relationship between the increase in capacitance caused by the presence of cells at 0.4 MHz, delta C, and the cell density was linear. Measurement of capacitance was useful in measurement of transitional changes in cell volume under external osmotic stress when sucrose was added. From the course of volume changes with such stress, the water permeabilities of the plasma membrane, Lp, were measured to be 0.015, 0.020, and 0.090 pm/(s.Pa) at 25 degrees C, for rice cells, grape cells, and CH27 cells, respectively. The smaller Lp for plant cells seemed to explain why preservation of plant cells by freezing is more difficult than for animal cells. From the temperature dependence of Lp, the apparent activation energies were calculated to be 12.0 +/- 2.9 and 13.0 +/- 5.2 kcal/mol for rice cells and CH27 cells, respectively.

Subcellular location of polyphenol oxidase in apples.

The location of polyphenol oxidase (PPO) in cells of apple fruit was examined by immunohistochemistry and subcellular fractionation. In mature apple fruits, where vacuoles occupy most of the cells, PPO was detected immunochemically near the cell walls with use of anti-apple PPO antibodies. In cells of immature fruits and tissue culture, PPO was detected in organelles other than the vacuoles, probably in plastids. The plastid fraction was purified by density gradient ultracentrifugation, and the activities of PPO and marker enzymes of plastids were the highest in the plastids. Most apple PPO was in plastids, as are other plant PPOs, and some of the protein was solubilized and proteolyzed during ripening and storage.