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Biofilm formation on abiotic surfaces in fresh produce processing facilities may play a role in foodborne outbreaks by providing protective microniches for pathogenic bacteria. Our previous study showed that a strain of Ralstonia insidiosa isolated from a fresh produce processing plant could enhance the incorporation of Escherichia coli O15:H7 in biofilms under various environmental conditions. These results raised the concern that R. insidiosa might have the ability to incorporate other foodborne pathogens and promote their survival and growth in biofilms. To test this hypothesis, 6 strains of Shiga toxin producing E. coli , 2 strains of Salmonella , and 6 strains of Listeria monocytogenes were examined for dual-species biofilm formation with R. insidiosa . A significant increase in biomass formation was observed in 7 of the 14 R. insidiosa -pathogen combinations, while significantly enhanced incorporation of pathogenic cells into biofilms was seen in 12 of the 14 R. insidiosa -pathogen combinations. The synergistic interactions between R. insidiosa and the tested foodborne pathogens seemed dependent on intimate cellular contact between the two strains. Overall, this study showed that R. insidiosa could enhance the incorporation of biofilms of different types of foodborne pathogenic bacteria and should be considered a bridging bacterium for biofilm formation in various food processing environments. [ABSTRACT FROM AUTHOR]

Radish (Raphanus sativus var. longipinnatus) microgreens were produced from seeds inoculated with Escherichia coli O157:H7 by using peat moss-based soil-substitute and hydroponic production systems. E. coli populations on the edible and inedible parts of harvested microgreen plants (7 days postseeding) and in growth medium were examined. E. coli O157:H7 was shown to survive and proliferate significantly during microgreen growth in both production systems, with a higher level in the hydroponic production system. At the initial seed inoculation level of 3.7 log CFU/g, E. coli O157:H7 populations on the edible part of microgreen plants reached 2.3 and 2.1 log CFU/g (overhead irrigation and bottom irrigation, respectively) for microgreens from the soil-substitute production system and reached 5.7 log CFU/g for those hydroponically grown. At a higher initial inoculation of 5.6 log CFU/g seeds, the corresponding E. coli O157:H7 populations on the edible parts of microgreens grown in these production systems were 3.4, 3.6, and 5.3 log CFU/g, respectively. Examination of the spatial distribution of bacterial cells on different parts of microgreen plants showed that contaminated seeds led to systematic contamination of whole plants, including both edible and inedible parts, and seed coats remained the focal point of E. coli O157:H7 survival and growth throughout the period of microgreen production. [ABSTRACT FROM AUTHOR]

Low temperature scanning electron microscopy (LT-SEM) has revealed anatomical details suggesting that Osperalycus and Gordialycus (Acariformes: Nematalycidae) have an unusual feeding apparatus that is hypothesized to be specialized for feeding on the fluid contents of small microorganisms (diameter < 5 μm). Both mite genera have a feeding strategy that appears to involve picking up small microorganisms and placing them onto the subcapitulum for puncturing. However, they have slightly different variants of the same basic rupturing mechanism. Whereas Gordialycus has evolved expansive and convergent rutella to hold the microorganisms in place while pushing chelicerae into them, Osperalycus has evolved a pouch into which a microorganism is inserted. The rutella reinforce this pouch while the chelicerae break up the microorganism. Both types of mouthpart apparatus seem to be adapted to minimize waste, an appropriate specialization given the organically impoverished habitats in which these mites live. [ABSTRACT FROM AUTHOR]

Located at the anterior end of the turkey hen's vagina are numerous discrete tubular invaginations of the surface epithelium, collectively referred to as the sperm storage tubules (SSTs). After mating or artificial insemination, sperm ascend the vagina, enter the SSTs, and over the ensuing days and weeks, gradually exit the SSTs and are transported to the anterior end of the oviduct to fertilize a daily succession of ova. Little is known regarding the cellular and molecular mechanisms responsible for sperm subsistence in the lumen of the SST. In this study, the origin of microvillus blebs (MvBs) on the apical tips of SST epithelial cells was examined, and their possible role in sperm survival was discussed. Regardless, if sperm are present or not, transmission electron microscopy revealed two types of microvilli differentiated by the presence or absence of pleomorphic unilaminar MvBs localized to their apical tips. Although some MvBs appeared to be discharging their contents into the SST lumen, others appeared to have pinched off the microvillus stem. When SSTs contained clusters of densely packed sperm, the sperm heads of those sperm adjacent to the SST epithelial cell surface were surrounded by the microvilli. Associated with the plasmalemma of sperm throughout the SST lumina were membrane fragments and small vesicles (30–130 nm in diameter), some of which appeared to have fused with sperm. It is concluded that the MvBs are a form of shedding vesicle released from the SST epithelial cell microvilli by apocrine secretion. On the basis of observations described herein and those of other authors, it is suggested that the MvBs contribute to sustained sperm storage in the SSTs by (1) supplying metabolic substrates used by resident sperm, (2) serving as fusogenic vehicles providing exogenous macromolecules that reversibly suppress sperm functions associated with fertilization (decapacitation?) and stabilize the sperm plasmalemma, and (3) acting as transport vesicles actively transporting fluid from the SST epithelial cells to the SST lumen. [ABSTRACT FROM AUTHOR]

Biofilm-forming bacteria resident to food processing facilities are a food safety concern due to the potential of biofilms to harbor foodborne bacterial pathogens. When cultured together, Ralstonia insidiosa, a strong biofilm former frequently isolated from produce processing environments, has been shown to promote the incorporation of Escherichia coli O157:H7 into dual-species biofilms. In this study, interactions between E. coli O157:H7 and R. insidiosa were examined under different incubating conditions. Under static culture conditions, the incorporation of E. coli O157:H7 into biofilms with R. insidiosa was not significantly affected by either low incubating temperature (10°C) or by limited nutrient availability. Greater enhancement of E. coli O157:H7 incorporation in dual-species biofilms was observed by using a continuous culture system with limited nutrient availability. Under the continuous culture conditions used in this study, E coli O157:H7 cells showed a strong tendency of colocalizing with R. insidiosa on a glass surface at the early stage of biofilm formation. As the biofilms matured, E coli O157:H7 cells were mostly found at the bottom layer of the dual-species biofilms, suggesting an effective protection by R. insidiosa in the mature biofilms. [ABSTRACT FROM AUTHOR]

Similar to phytopathogens, human bacterial pathogens have been shown to colonize the plant phylloplane. In addition to environmental factors, such as temperature, UV, relative humidity, etc., the plant cultivar and, specifically, the leaf blade morphological characteristics may affect the persistence of enteropathogens on leafy greens. This study was conducted to evaluate the effect of cultivar-dependent leaf topography and the role of strain phenotypic characteristics on Escherichia coli O157:H7 persistence on organic spinach. Spinach cultivars Emilia, Lazio, Space, and Waitiki were experimentally inoculated with the foodborne E. coli O157:H7 isolate EDL933 and its isogenic mutants deficient in cellulose, curli, or both curli and cellulose production. Leaves of 6-week-old plants were inoculated with 6.5 log CFU per leaf in a biosafety level 2 growth chamber. At 0, 1, 7, and 14 days, E. coli O157:H7 populations were determined by plating on selective medium and verified by laser scanning confocal microscopy. Leaf morphology (blade roughness and stoma density) was evaluated by low-temperature and variable-pressure scanning electron microscopy. E. coli O157:H7 persistence on spinach was significantly affected by cultivar and strain phenotypic characteristics, specifically, the expression of curli. Leaf blade roughness and stoma density influenced the persistence of E. coli O157:H7 on spinach. Cultivar Waitiki, which had the greatest leaf roughness, supported significantly higher E. coli O157:H7 populations than the other cultivars. These two morphological characteristics of spinach cultivars should be taken into consideration in developing intervention strategies to enhance the microbial safety of leafy greens. [ABSTRACT FROM AUTHOR]

Abstract: Picris divaricata Vant., a plant species native to subtropical China, was recently identified as the first Cd/Zn hyperaccumulator from Asteraceae. P. divaricata was grown from wild collected seed for 4months in a series of pH adjusted test soils with added Zn levels 0–7000mgkg−1 and Cd levels 0–150mgkg−1. Plants did not hyperaccumulate Zn (threshold >3000μgg−1) and weakly hyperaccumulated Cd with little or no dose–response. P. divaricata has multicellular simple trichomes concentrated on the leaf margins and midrib. X-ray analysis showed that Zn was concentrated in larger trichomes and epidermal cells adjacent to the trichome but virtually absent in other leaf tissues. Within the trichomes, Zn was localized in ovate spots around the tips of individual cells. These tips and other locations in the trichome cell contained black electron dense material when examined with transmission electron microscopy, some of which was identified as SiO2. Silicon and Mn were concentrated in the same areas as Zn. Si has been previously associated with alleviating Zn, Mn and Cd toxicity. Our results support this observation and further investigation is warranted. Calcium and P were concentrated in the distal tips of trichomes, similar to patterns previously observed for calcicole plants grown in elevated Ca soils. Overall, nonsecretory trichomes from many plant families may have a common origin as tissues adapted to handle a variety of environmental metals. [Copyright &y& Elsevier]

Irrigation water and washing water have been inferred to be associated with contamination of fresh fruits and vegetables with pathogenic microorganisms infectious for humans. The objective of the present study was to determine whether apples experimentally contaminated with Cryptosporidium oocysts represent a food safety concern. Laser scanning confocal microscopy revealed no morphological changes in Cryptosporidium parvum oocysts attached to apples alter 6 weeks of cold storage. suggesting that oocysts might remain viable and possibly infectious during prolonged storage. Mice were fed apple peels from experimentally contaminated apples to determine whether oocysts had remained infectious on apples stored for 4 weeks. All mice developed cryptosporidiosis. To evaluate the strength of oocyst attachment to apples, washing methods that have been reported to be helpful for recovery of oocysts from various foodstuffs were evaluated, except that the intensity of washing was increased in the present study. None of the tested washing methods succeeded in completely removing oocysts from the apple peel. The most efficient removal (37.5%) was achieved by rigorous manual washing in water with a detergent and by agitation in an orbital shaker with Tris-sodium dodecyl sulfate buffer. Glycine and phosphate-buffered saline buffers had no effect on oocyst removal. Scanning electron microscopy revealed that some oocysts were attached in deep natural crevices in the apple exocarp and others were attached to the smooth surface of the peel. Some oocysts were closely associated with what appeared to be an amorphous substance with which they might have been attached to the apple surface. [ABSTRACT FROM AUTHOR]

Pathogen contamination of fresh produce significantly impacts public health and consumer confidence in eating fresh fruits and vegetables. In tomato fruits, studies have shown that the stem scar plays an important role in pathogen infiltration. However, the exact mechanisms and pathways for pathogen movement inside tomatoes are not well documented. This study examined in detail the pathway of pathogen internalization in tomato fruit. Tomatoes were infused with potassium iodide and examined with Xradia Bio MicroCT at 0.5×∼4×. Micro CT images revealed that infiltrated potassium iodide moved along the vascular bundles inside the fruit. In separate trials, tomato fruits were further infiltrated with red fluorescent microspheres and E. coli O157:H7 -pGFP. Stem scars and core tissues were subsequently excised after fruit surface disinfection and observed using a Zeiss 710 laser confocal microscope, or stomached, cultured, and enumerated for infiltrated E. coli O157:H7. Populations of internalized E. coli O157:H7 were confined in xylem vessels and concentrated immediately beneath the stem-scar, with a sharp decline in population with vertical distance from the stem-scar. Our observations suggest that 1) vascular bundles, especially the xylem vessels in tomato fruit, play a critical role in pathogen internalization, and 2) pathogen internalization is a passive, rather than active process, as the infiltration and movement of both microspheres (non-living) and E. coli O157:H7 cells (living) behaved similarly inside tomatoes. This study presents visual evidence of the critical role of vascular bundles in pathogen internalization in tomato fruit using Micro CT and Confocal Laser Microscopy. The finding is important for developing science-based food safety practices, interventions in controlling pathogen internalization, and new tools for probing pathogen-plant interactions. [ABSTRACT FROM AUTHOR]

The Shiga toxigenic Escherichia coli O104:H4 isolated during the 2011 European outbreak expresses Shiga toxin 2a and possess virulence genes associated with the enteroaggregative E. coli (EAEC) pathotype. It produces plasmid encoded aggregative adherence fimbriae I (AAF/I) which mediate cell aggregation and biofilm formation in human intestine and promote Shiga-toxin adsorption, but it is not clear whether the AAF/I fimbriae are involved in the colonization and biofilm formation on food and environmental matrices such as the surface of fresh produce. We deleted the gene encoding for the AAF/I fimbriae main subunit (AggA) from an outbreak associated E. coli O104:H4 strain, and evaluated the role of AAF/I fimbriae in the adherence and colonization of E. coli O104:H4 to spinach and abiotic surfaces. The deletion of aggA did not affect the adherence of E. coli O104:H4 to these surfaces. However, it severely diminished the colonization and biofilm formation of E. coli O104:H4 on these surfaces. Strong aggregation and biofilm formation on spinach and abiotic surfaces were observed with the wild type strain but not the isogenic aggA deletion mutant, suggesting that AAF/I fimbriae play a crucial role in persistence of O104:H4 cells outside of the intestines of host species, such as on the surface of fresh produce. [ABSTRACT FROM AUTHOR]

Blue mold caused primarily by Penicillium expansum and to a lesser extent other Penicillium spp. is the most destructive disease of stored apples in the US and worldwide. It was recently shown that resistance to blue mold exists in wild apple germplasms, Malus sieversii , from Kazakhstan and in other species from different regions maintained as a collection in Geneva, NY. We initiated studies to determine the durability and the mechanism(s) of resistance to P. expansum in select wild apple accessions. Wound responses (up to 96 h in 24 h intervals), affecting P. expansum infection, and related cytological changes were determined in accessions with varying levels of resistance. In general, the more resistant the accession, the quicker the wound response that prevented the fungus from infecting tissue and causing decay. No decay developed on immune apple accessions, even when inoculated immediately after wounding at the inoculum concentration of 10 5 conidia/mL. On a moderately resistant accession, a 24 h interval between wounding and inoculation was sufficient to avert decay. Reactive oxygen species (ROS) were detected at high levels immediately after wounding in the immune as well as susceptible accessions. Callose and lignin/suberin appears to play a minor role in resistance responses. Our results indicate the presence of a high level of durable resistance/immunity in the wild apples which is governed by several mechanism(s). This presents a new challenge for explaining the observed resistance and at the same time creates an opportunity for exploiting these resistant mechanisms in breeding programs to incorporate resistance to fruit decays into commercial cultivars. [ABSTRACT FROM AUTHOR]

Abstract: The objectives of this study were to create interspecific hybrids between Cyrtanthus elatus and C. sanguineus and to confirm the hybrid origin of the progeny based on morphological characters and using molecular markers. The tip of the leaves, the shape and size of cells, and stomata distribution in abaxial and adaxial surface of leaf epidermis were analyzed by low temperature scanning electron microscope (LT-SEM). Molecular markers generated from random amplification polymorphic DNA (RAPD) and single nucleotide polymorphisms (SNPs) analysis of internal transcribed spacer 1 and 2 of rRNA gene (ITS 1, 2) were also analyzed to confirm hybrid status. Putative C. elatus × C. sanguineus hybrid A 3 resembled C. elatus and C. sanguineus ‘Pringle Bay’ (PB)בFred Meyer’ (FM) B 23 and was similar to C. sanguineus at flowering. However, the hybrid origin of hybrid A 3 could not be confirmed based on the morphological characteristics of flowers, the tip of the leaves, and cell structure and surface images in both abaxial and adaxial images of LT-SEM. Based on RAPD markers, putative hybrid A 3 and other putative hybrids that did not flower (A 4, A 5, A 13 and D 39, D 40, and D 42) can be considered as a hybrid based on the presence of both species-specific bands for parental taxa. SNP markers of ITS 1, 2 region also revealed that other putative hybrids of A and D that did not flower during the evaluation can be interspecific hybrids of C. elatus and C. sanguineus. Therefore, the use of SNP markers of ITS 1, 2 is considered a more accurate tool to characterize hybrid origin than morphological characteristics and RAPD markers. [Copyright &y& Elsevier]

Abstract: The importance of reactive oxygen species (ROS) in plant defense responses against certain pathogens is well documented. There is some evidence that microbial biocontrol agents also induce a transient production of ROS in a host plant which triggers local and systemic defense responses to pathogens. The ability of biocontrol agents used to control postharvest diseases to induce defense-related oxidative responses in fruits, however, has not been explored. Here we show that the yeast antagonists, Metschnikowia fructicola (strain 277) and Candida oleophila (strain 182) generate greater levels of super oxide anion (O2 −) on intact fruit surfaces (poor in nutrients) then those applied on a nutrient-poor agar medium. Even though yeast antagonists show a high level of O2 − on nutrient-rich media, when applied on fruits around wounds (areas abundant in nutrients) accumulation of O2 −, as detected by nitro blue tetrazolium staining, occurred much more rapidly on the latter. Using laser scanning confocal microscopy we observed that the application of M. fructicola and C. oleophila into citrus and apple fruit wounds correlated with an increase in H2O2 accumulation in host tissue. In citrus fruit, the level of H2O2 around inoculated wounds increased by 4-fold compared to controls (wounds inoculated with water) as early as 18h after inoculation. Yeast continued to stimulate H2O2 production in citrus fruit up to 66h after inoculation and H2O2 levels were still 3-fold above the control. Living yeast cells were detected in fruit wounds at this time point indicating the ability of M. fructicola to tolerate host ROS, which has been reported to be an intrinsic characteristic of efficient yeast antagonists. Similar increase in H2O2 accumulation around yeast-inoculated wounds was observed in apple fruit exocarp. The present data, together with our earlier discovery of the importance of H2O2 production in the defense response of citrus flavedo to postharvest pathogens, indicate that the yeast-induced oxidative response in fruit exocarp may be associated with the ability of specific yeast species to serve as biocontrol agents for the management of postharvest diseases. [Copyright &y& Elsevier]

The setae of mites are not regarded as secretory structures, yet in the flat mite genus Raoiella , each developmental stage presents droplets of fluid associated with the tips of their dorsal setae. To understand the origin of this fluid, the ultrastructure of the dorsal setae is investigated in females of Raoiella bauchani Beard & Ochoa and the invasive pest species Raoiella indica Hirst using scanning and transmission electron microscopy techniques. The dorsal setae are barbed along their entire length and have either a broadened plumose or a flat spatulate tip. Ultrastructurally, they present the typical features of mechanoreceptors, but have a "hollow" axis represented by a protoplasmatic core containing dendritic branches. This combination of ultrastructural characters indicates that the setae might be multimodal receptors: acting as both mechanoreceptors and contact chemoreceptors. The epidermal cells that underlie the setal sockets are columnar and have an ultrastructure that suggests they have a glandular function. Moreover, these cells present regular microvilli apically and form extracellular cuticular canals, containing epicuticular filaments, that are connected with the microvilli proximally and which open via pores onto the surface of the setal base distally. This arrangement indicates that the secretion from the microvilli passes into the canals and is then conducted to pores at the base of the seta, where it then accumulates and moves up the setal shaft, along the longitudinal grooves of the barbs. Based on similar arrangements in some insect taxa, the organization of the structures here observed in Raoiella suggests the passage of a non-polar, water insoluble, lipoid fluid through the cuticle, the function of which is still obscure. • Dorsal setae in the genus Raoiella could act as both mechanoreceptor and contact chemoreceptors. • Structure of epidermal cells underlying the dorsal setae suggests an active secretory role. • These secretory cells are connected to extracellular tubule cuticular structures. • Tubule structures open via pores on the side walls of the setal sockets. • Presence of epicuticular filaments suggests an involvement in the transport mechanisms of waterproofing lipoid materials. [ABSTRACT FROM AUTHOR]

One of the great unanswered questions with respect to biological science in general is the absolute necessity of docosahexaenoic acid (DHA, 22:6n-3) in fast signal processing tissues. N-6 docosapentaenoic acid (n-6DPA, 22:5n-6), with just one less double bond, group, is fairly abundant in terrestrial food chains yet cannot substitute for DHA. Gradient temperature Raman spectroscopy (GTRS) applies the temperature gradients utilized in differential scanning calorimetry (DSC) to Raman spectroscopy, providing a straightforward technique to identify molecular rearrangements that occur near and at phase transitions. Herein we apply GTRS and DSC to n-6DPA and DHA from −100 to 20 °C. 20 Mb three-dimensional data arrays with 0.2 °C increments and first/second derivatives allowed complete assignment of solid, liquid and transition state vibrational modes, including low intensity/frequency vibrations that cannot be readily analyzed with conventional Raman. N-6DPA and DHA show significant spectral changes with premelting (−33 and −60 °C, respectively) and melting (−27 and −44 °C, respectively). The CH2 (HC CH) CH2 moieties are not identical in the second half of the DHA and DPA structures. DPA has bending (1450 cm −1 ) over almost the entire temperature range. In contrast, DHA contains major CH 2 twisting (1265 cm −1 ) with no noticeable CH 2 bending, consistent with a flat helical structure with a small pitch. Further modeling of neuronal membrane phospholipids must take into account torsion present in the DHA structure, which essential in determining whether the lipid chain is configured more parallel or perpendicular to the hydrophilic head group. [ABSTRACT FROM AUTHOR]

Biofilm formation is an important strategy for foodborne bacterial pathogens to survive in stressful environments such as fresh produce processing facilities. Bacterial cell aggregation strongly promotes the initiation of microcolonies and the formation of biofilms on abiological surfaces. We previously showed that Ralstonia insidiosa , an environmental bacterial species frequently isolated from fresh produce facilities, may serve as a “bridge bacterium” that strongly enhanced the incorporation of several foodborne bacterial pathogens into dual species biofilms. While the R. insidiosa strain exhibited moderate cell aggregation in liquid culture, co-culturing Listeria monocytogenes with R. insidiosa resulted in significant augmentation of cell aggregation. Electron microscopy indicated that L. monocytogenes cells were initially attracted to the R. insidiosa aggregates and formed large dual species aggregates that were predominately composed of L. monocytogenes cells. The predominant presence of L. monocytogenes in the dual species aggregates was also confirmed by differential plating. These findings suggest that bridge bacteria such as R. insidiosa play critical roles in the survival of foodborne bacterial pathogens, such as L. monocytogenes and Escherichia coli , by promoting multispecies biofilm formation. The implications of such bridge bacteria on food safety need to be further evaluated. [ABSTRACT FROM AUTHOR]

• Varroa destructor mites use a muscularized pharynx for fluid uptake. • The pharyngeal pump operates at a mean cycle repetition rate of 4.5 cycles per second. • Feeding is characterized by numerous, short tandem events of pharyngeal pumping. • Events are grouped into feeding bouts separated by movement or long quiescence. Feeding behaviors and biomechanics of female Varroa destructor mites are revealed from AC-DC electropenetrography (EPG) recordings of mites feeding from Apis mellifera honey bee pupae and histology of mite internal ingestion apparatus. EPG signals characteristic of arthropod suction feeding (ingestion) were identified for mites that fed on pupae during overnight recordings. Ingestion by these mites was confirmed afterwards by observing internally fluorescent microbeads previously injected into their hosts. Micrographs of internal ingestion apparatus illustrate the connection between a gnathosomal tube and a pharyngeal lumen, which is surrounded by alternating dilator and constrictor muscles. Inspection of EPG signals showed the muscularized mite pharyngeal pump operates at a mean repetition rate of 4.5 cycles/s to ingest host fluids. Separate feeding events observed for mites numbered between 23 and 33 over approximately 16 h of recording, with each event lasting ~10 s. Feeding events were each separated by ~2 min. Consecutive feeding events separated by either locomotion or prolonged periods of quiescence were grouped into feeding bouts, which ranged in number from one to six. Statistical analyses of EPG data revealed that feeding events were prolonged for mites having lower pharyngeal pump frequencies, and mites having prolonged feeding events went unfed for significantly more time between feeding events. These results suggest that mites may adjust behaviors to meet limitations of their feeding apparatus to acquire similar amounts of food. Data reported here help to provide a more robust view of Varroa mite feeding than those previously reported and are both reminiscent of, as well as distinct from, some other acarines and fluid-feeding insects. [ABSTRACT FROM AUTHOR]