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Consumers appreciate leafy green vegetables such as baby leaves for their convenience and wholesomeness and for adding a variety of tastes and colors to their plate. In Western cuisine, leafy green vegetables are usually eaten fresh and raw, with no step in the long chain from seed to consumption where potentially harmful microorganisms could be completely eliminated, e.g., through heating. A concerning trend in recent years is disease outbreaks caused by various leafy vegetable crops and one of the most important foodborne pathogens in this context is Shiga toxin-producing Escherichia coli (STEC). Other pathogens such as Salmonella, Shigella, Yersinia enterocolitica and Listeria monocytogenes should also be considered in disease risk analysis, as they have been implicated in outbreaks associated with leafy greens. These pathogens may enter the horticultural value network during primary production in field or greenhouse via irrigation, at harvest, during processing and distribution or in the home kitchen/restaurant. The hurdle approach involves combining several mitigating approaches, each of which is insufficient on its own, to control or even eliminate pathogens in food products. Since the food chain system for leafy green vegetables contains no absolute kill step for pathogens, use of hurdles at critical points could enable control of pathogens that pose a human health risk. Hurdles should be combined so as to decrease the risk due to pathogenic microbes and also to improve microbial stability, shelf-life, nutritional properties and sensory quality of leafy vegetables. The hurdle toolbox includes different options, such as physical, physiochemical and microbial hurdles. The goal for leafy green vegetables is multi-target preservation through intelligently applied hurdles. This review describes hurdles that could be used for leafy green vegetables and their biological basis, and identifies prospective hurdles that need attention in future research.

Despite the overruling impact of light in the phyllosphere, little is known regarding the influence of light spectra on non-phototrophic bacteria colonizing the leaf surface. We developed an in vitro method to study phenotypic profile responses of bacterial pure cultures to different bands of the visible light spectrum using monochromatic (blue: 460 nm; red: 660 nm) and polychromatic (white: 350–990 nm) LEDs, by modification and optimization of a protocol for the Phenotype MicroArray™ technique (Biolog Inc., CA, USA). The new protocol revealed high reproducibility of substrate utilization under all conditions tested. Challenging the non-phototrophic bacterium Pseudomonas sp. DR 5–09 with white, blue, and red light demonstrated that all light treatments affected the respiratory profile differently, with blue LED having the most decisive impact on substrate utilization by impairing respiration of 140 substrates. The respiratory activity was decreased on 23 and 42 substrates under red and white LEDs, respectively, while utilization of one, 16, and 20 substrates increased in the presence of red, blue, and white LEDs, respectively. Interestingly, on four substrates contrasting utilization patterns were found when the bacterium was exposed to different light spectra. Although non-phototrophic bacteria do not rely directly on light as an energy source, Pseudomonas sp. DR 5–09 changed its respiratory activity on various substrates differently when exposed to different lights. Thus, ability to sense and distinguish between different wavelengths even within the visible light spectrum must exist, and leads to differential regulation of substrate usage. With these results, we hypothesize that different light spectra might be a hitherto neglected key stimulus for changes in microbial lifestyle and habits of substrate usage by non-phototrophic phyllospheric microbiota, and thus might essentially stratify leaf microbiota composition and diversity.

Understanding the mechanisms of Phytophthora capsici sporangial dissemination is paramount to understanding epidemic initiation and development. Direct laboratory observations showed P. capsici sporangial dispersal occurred in water with capillary force, but did not occur in response to wind or a reduction in relative humidity. Atmospheric sporangial concentrations were monitored under field conditions using a volumetric spore sampler in a commercial cucurbit field and in an experimental setting where copious sporangia were continuously available in close proximity to the spore trap. Dispersal was infrequent (0.7% of total hours monitored) during sampling in a commercial field; 14 sporangia were detected during a 7.5-week sampling period. In the experimental field situation, dispersal occurred in 4.6% of the hours sampled and 438 sporangia were impacted onto tapes during a 7-week sampling period. Airborne sporangial concentrations were positively associated with rainfall at both sites, but not vapor pressure deficit. Furthermore, in the experimental field situation, wind speed was not significant in regression analysis. Wind speed was not measured in the commercial field. Hence, both direct laboratory observations and volumetric spore sampling indicate that dispersal of sporangia via wind currents is infrequent, and sporangia are unlikely to be naturally dispersed among fields by wind alone.

Differential protection of plants by Enterobacter cloacae was studied by investigating early sensing and response behavior of Pythium ultimum sporangia toward seeds in the presence or absence of E. cloacae. Ten percent of P. ultimum sporangia were activated within the first 30 min of exposure to cucumber seeds. In contrast, 44% of the sporangia were activated as early as 15 min after exposure to corn seeds with over 80% sporangial activation by 30 min. Germ tubes emerged from sporangia after 2.5 and 1.0 h in the cucumber and corn spermospheres, respectively. Seed application of the wild-type strain of E. cloacae (EcCT-501R3) reduced sporangial activation by 45% in the cucumber spermosphere, whereas no reduction was observed in the corn spermosphere. Fatty acid transport and degradation mutants of E. cloacae (strains EcL1 and Ec31, respectively) did not reduce sporangial activation in either of the spermospheres. Although wild-type or mutant strains of E. cloacae failed to reduce seed colonization incidence, pathogen biomass on cucumber seeds was reduced in the presence of E. cloacae strains EcCT-501R3 and Ec31 by 4 and 8 h after sowing, respectively. By 12 h, levels of P. ultimum on cucumber seeds treated with E. cloacae EcCT-501R3 did not differ from levels on noninoculated seeds. On corn seeds, P. ultimum biomass was not affected by the presence of any E. cloacae strain. When introduced after sporangial activation had occurred, E. cloacae failed to reduce P. ultimum biomass on cucumber seeds compared with that on nontreated seeds. Also, increasing numbers of sporangia used to inoculate seeds yielded increased pathogen biomass at each sampling time. This indicates a direct link between the level of seed-colonizing biomass of P. ultimum and the number of activated and germinated sporangia in the spermosphere, suggesting that E. cloacae suppresses P. ultimum seed infections by reducing sporangial activation and germination within the first 30 to 90 min after sowing.

Chytridiomycosis and ranavirosis are 2 emerging infectious diseases that have caused significant global amphibian decline. Although both have received much scrutiny, little is known about interactions between the 2 causative agents Batrachochytrium dendrobatidis (Bd) and ranavirus (Rv) at the individual host and population levels. We present the first longitudinal assessment of Bd, Rv, and co-infections of a temperate amphibian assemblage in North America. From 2012 to 2016, we assessed the temporal oscillations of Bd, Rv and co-infection dynamics in a sample of 729 animals representing 13 species. Bd, Rv, and co-infected amphibians were detected during all 5 yr. Bd, Rv, and co-infection prevalence all varied annually, with the lowest instances of each at 2.1% (2013), 7.9% (2016), and 0.6% (2016), respectively. The highest Bd, Rv, and co-infection prevalence were recorded in 2012 (26.8%), 2016 (38.3%), and 2015 (10.3%), respectively. There was no association between Bd or Rv infection prevalence and co-infection, either when assessing the entire amphibian assemblage as a whole (odds ratio 1.32, 95% CI: 0.83-2.1, p = 0.29) or within species for amphibians that were more numerically represented (n > 40, p > 0.05). This suggests neither Bd nor Rv facilitate host co-infections within the sampled host assemblage. Instead, the basis for co-infections is the spatiotemporal distribution of both pathogens. Despite lack of interplay between Bd and Rv in this population, our study highlights the importance of considering numerous pathogens that may be present within amphibian habitats in order to properly anticipate interactions that may have direct bearing on disease outcomes.

Background: The phyllosphere is subjected to fluctuating abiotic conditions. This study examined the phenotypic plasticity (PP) of four selected non-phototrophic phyllosphere bacteria [control strain: Pseudomonas sp. DR 5-09; Pseudomonas agarici, Bacillus thuringiensis serovar israeliensis (Bti), and Streptomyces griseoviridis (SG)] regarding their respiration patterns and surfactant activity as affected by light spectrum and nutrient supply.Methods: The PP of the strains was examined under four light regimes [darkness (control); monochromatic light-emitting diodes (LED) at 460 nm (blue) and 660 nm (red); continuously polychromatic white LEDs], in the presence of 379 substrates and conditions.Results: Light treatment affected the studied bacterial strains regarding substrate utilization (Pseudomonas strains > SG > Bti). Blue LEDs provoked the most pronounced impact on the phenotypic reaction norms of the Pseudomonas strains and Bti. The two Gram-positive strains Bti and SG, respectively, revealed inconsistent biosurfactant formation in all cases. Biosurfactant formation by both Pseudomonas strains was supported by most substrates incubated in darkness, and blue LED exposure altered the surface activity profoundly. Blue and white LEDs enhanced biofilm formation in PA in highly utilized C-sources. Putative blue light receptor proteins were found in both Pseudomonas strains, showing 91% similarity with the sequence from NCBI accession number WP_064119393.Conclusion: Light quality–nutrient interactions affect biosurfactant activity and biofilm formation of some non-phototrophic phyllosphere bacteria and are, thus, crucial for dynamics of the phyllosphere microbiome.

The aim of this study was to determine the temporal release of fatty acids and sugars from corn and cucumber seeds during the early stages of seed germination in order to establish whether sugars found in exudate can prevent exudate fatty acid degradation by Enterobacter cloacae. Both saturated (long-chain saturated fatty acids [LCSFA]) and unsaturated (long-chain unsaturated fatty acids [LCUFA]) fatty acids were detected in corn and cucumber seed exudates within 15 min after seed sowing. LCSFA and LCUFA were released at a rate of 26.1 and 6.44 ng/min/seed by corn and cucumber seeds, respectively. The unsaturated portion of the total fatty acid pool from both plant species contained primarily oleic and linoleic acids, and these fatty acids were released at a combined rate of 6.6 and 0.67 ng/min/seed from corn and cucumber, respectively. In the absence of seed exudate sugars, E. cloacae degraded linoleic acid at rates of 29 to 39 ng/min, exceeding the rate of total fatty acid release from seeds. Sugars constituted a significant percentage of corn seed exudate, accounting for 41% of the total dry seed weight. Only 5% of cucumber seed exudate was comprised of sugars. Glucose, fructose, and sucrose were the most abundant sugars present in seed exudate from both plant species. Corn seeds released a total of 137 g/seed of these three sugars within 30 min of sowing, whereas cucumber seeds released 0.83 g/seed within the same time frame. Levels of glucose, fructose, and sucrose found in corn seed exudate (90 to 342 g) reduced the rate of linoleic acid degradation by E. cloacae to 7.5 to 8.8 ng/min in the presence of either sugar, leaving sufficient concentrations of linoleic acid to activate Pythium ultimum sporangia Our results demonstrate that elevated levels of sugars in the corn spermosphere can prevent the degradation of LCUFA by E. cloacae, leading to its failure to suppress P. ultimum sporangial activation, germination, and subsequent disease development.