The power of light: Impact on the performance of biocontrol agents under minimal nutrient conditions.

Background: The spectral distribution of light (different wavelength) has recently been identified as an important factor in the dynamics and function of leaf-associated microbes. This study investigated the impact of different wavelength on three commercial biocontrol agents (BCA): Bacillus amyloliquefaciens (BA), Pseudomonas chlororaphis (PC), and Streptomyces griseoviridis (SG). Methods: The impact of light exposure on sole carbon source utilization, biofilm formation, and biosurfactant production by the selected BCA was studied using phenotypic microarray (PM) including 190 sole carbon sources (OmniLog®, PM panels 1 and 2). The BCA were exposed to five monochromatic light conditions (420, 460, 530, 630, and 660 nm) and darkness during incubation, at an intensity of 50 µmol m-2 s-1. Results: Light exposure together with specific carbon source increased respiration in all three BCA. Different wavelengths of light influenced sole carbon utilization for the different BCA, with BA and PC showing increased respiration when exposed to wavelengths within the blue spectrum (420 and 460 nm) while respiration of selected carbon sources by SG increased in the presence of red light (630 and 660 nm). Only one carbon source (capric acid) generated biosurfactant production in all three BCA. A combination of specific wavelength of light and sole carbon source increased biofilm formation in all three BCA. BA showed significantly higher biofilm formation when exposed to blue (460 nm) and green (530 nm) light and propagated in D-sucrose, D-fructose, and dulcitol. PC showed higher biofilm formation when exposed to blue light. Biofilm formation by SG increased when exposed to red light (630 nm) and propagated in citraconic acid. Conclusion: To increase attachment and success in BCA introduced into the phyllosphere, a suitable combination of light quality and nutrient conditions could be used.

Seed inoculation with antagonistic bacteria limits occurrence of E. coli O157:H7gfp + on baby spinach leaves.

BACKROUND: During the last decades, outbreaks of foodborne illnesses have increasingly been linked to fresh and/or minimally processed fruit and vegetables. Enterohemorrhagic Escherichia coli was the causal agent for major outbreaks in Europe with leafy green vegetables and sprouts. To improve food safety, microbial antagonism has received attention during recent years and could be one of the solution to prevent contamination of food borne pathogens on fresh produce. Here we investigate the antagonistic effect of three bacterial strains (Pseudomonas orientalis, P. flavescens and Rhodococcus sp.) isolated from spinach leaves against E. coli O157:H7gfp + under laboratory and greenhouse conditions. RESULTS: Our results shows that significantly less culturable E.coli O157:H7gfp + were retrieved from the spinach canopy subjected to antagonist seed treatment than canopy inoculation. Seeds inoculated with Rhodococcus sp. significantly reduced growth of E. coli O157:H7gfp + compared with the other antagonists. The result from the in vitro study shows a significant reduction of growth of E. coli O157:H7gfp+, but only after 44 h when E. coli O157:H7gfp + was propagated in a mixture of spent media from all three antagonists. CONCLUSIONS: The antagonistic effect on phyllospheric E.coli O157:H7gfp + observed after seed inoculation with Rhodococcus sp. might be an indication of induced resistance mechanism in the crop. In addition, there was a small reduction of culturable E.coli O157:H7gfp + when propagated in spent media from all three antagonists. Nutritional conditions rather than metabolites formed by the three chosen organisms appear to be critical for controlling E. coli O157:H7gfp+.

Blue Light Inhibits E. coli, but Decisive Parameters Remain Hidden in the Dark: Systematic Review and Meta-Analysis.

Blue light (400-500 nm) alleviates overexposure risks associated to UV light and has therefore gained increased interest in multiple applications. This meta-analysis deals with decontamination of E. coli through the use of blue light based from nine recent publications identified via a systematic literature search. In these studies, various pathogenic and non-pathogenic E. coli strains grown in nutritional broths were exposed to wavelengths ranging from 395 to 460 nm. Five meta-analyses were performed using Cochrane's software for meta-analyses (Review Manager): one including all studies to estimate the effect of E. coli reduction and four subgroup-analyses considering reported intensities, wavelengths, exposure dose as well as serovars/pathovars. Random effects models were used. All included studies used colony-forming units to estimate the impact of E. coli reduction. None of the included studies involved an organic matrix (e.g., skin, food related surface). Exposure to blue light had a significant and large reducing effect on viable counts of E. coli. However, substantial heterogeneity across studies was observed. Among subgroups, reported intensity and wavelength showed the clearest impact on E. coli reduction. With respect to the reported exposure dose, the picture across the spectrum was scattered, but effect sizes tend to increase with increasing exposure dose. Substantial heterogeneity was also present with respect to all serovar/pathovar subgroups among the included studies. The present body of reports does not display a strong basis for recommendation of relevant intensities, wavelengths and exposure doses for superficial blue light decontamination in medical or food safety contexts. A serious shortcoming in most studies is the absence of a clear documentation of inoculum preparation and of study parameters. We suggest improvement for study protocols for future investigations.

Dining in Blue Light Impairs the Appetite of Some Leaf Epiphytes.

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.

High-throughput method for detection and quantification of lesions on leaf scale based on trypan blue staining and digital image analysis.

BACKGROUND: Field-grown leafy vegetables can be damaged by biotic and abiotic factors, or mechanically damaged by farming practices. Available methods to evaluate leaf tissue damage mainly rely on colour differentiation between healthy and damaged tissues. Alternatively, sophisticated equipment such as microscopy and hyperspectral cameras can be employed. Depending on the causal factor, colour change in the wounded area is not always induced and, by the time symptoms become visible, a plant can already be severely affected. To accurately detect and quantify damage on leaf scale, including microlesions, reliable differentiation between healthy and damaged tissue is essential. We stained whole leaves with trypan blue dye, which traverses compromised cell membranes but is not absorbed in viable cells, followed by automated quantification of damage on leaf scale. RESULTS: We present a robust, fast and sensitive method for leaf-scale visualisation, accurate automated extraction and measurement of damaged area on leaves of leafy vegetables. The image analysis pipeline we developed automatically identifies leaf area and individual stained (lesion) areas down to cell level. As proof of principle, we tested the methodology for damage detection and quantification on two field-grown leafy vegetable species, spinach and Swiss chard. CONCLUSIONS: Our novel lesion quantification method can be used for detection of large (macro) or single-cell (micro) lesions on leaf scale, enabling quantification of lesions at any stage and without requiring symptoms to be in the visible spectrum. Quantifying the wounded area on leaf scale is necessary for generating prediction models for economic losses and produce shelf-life. In addition, risk assessments are based on accurate prediction of the relationship between leaf damage and infection rates by opportunistic pathogens and our method helps determine the severity of leaf damage at fine resolution.

Size Matters: Biological and Food Safety Relevance of Leaf Damage for Colonization of Escherichia coli O157:H7 gfp.

This study examined the biological and food safety relevance of leaf lesions for potential invasion of food pathogens into the plant tissue (internalization). This was done by determining the role of artificial leaf damage in terms of damaged leaf area on proliferation of E. coli O157:H7 gfp+. In a two-factorial experiment, unwashed fresh baby leaf spinach (Spinacia oleracea L.) was subjected to four damage levels (undamaged, low, moderate, high damage; factor 1) and three incubation intervals (0, 1, 2 days post-inoculation; factor 2). Individual leaves were immersed for 15 s in a suspension loaded with E. coli O157:H7 gfp+ (106 CFU × mL-1). The leaves were analyzed individually using image analysis tools to quantify leaf area and number and size of lesions, and using confocal laser scanning and scanning electron microscopy to visualize leaf lesions and presence of the introduced E. coli strain on and within the leaf tissue. Prevalence of E. coli O157:H7 gfp+ was assessed using a culture-dependent technique. The results showed that size of individual lesions and damaged leaf area affected depth of invasion into plant tissue, dispersal to adjacent areas, and number of culturable E. coli O157:H7 gfp+ directly after inoculation. Differences in numbers of the inoculant retrieved from leaf macerate evened out from 2 days post-inoculation, indicating rapid proliferation during the first day post-inoculation. Leaf weight was a crucial factor, as lighter spinach leaves (most likely younger leaves) were more prone to harbor E. coli O157:H7 gfp+, irrespective of damage level. At the high inoculum density used, the risk of consumers' infection was almost 100%, irrespective of incubation duration or damage level. Even macroscopically intact leaves showed a high risk for infection. These results suggest that the risk to consumers is correlated with how early in the food chain the leaves are contaminated, and the degree of leaf damage. These findings should be taken into account in different steps of leafy green processing. Further attention should be paid to the fate of viable, but non-culturable, shiga-toxigenic E. coli on and in ready-to-eat leafy vegetables.

Leaf mineral content govern microbial community structure in the phyllosphere of spinach (Spinacia oleracea) and rocket (Diplotaxis tenuifolia).

The plant microbiome is an important factor for plant health and productivity. While the impact of nitrogen (N) availability for plant growth and development is well established, its influence on the microbial phyllosphere community structure is unknown. We hypothesize that nitrogen impacts the growth and abundance of several microorganisms on the leaf surface. The bacterial and fungal communities of baby leaf spinach (Spinacia oleracea), and rocket (Diplotaxis tenuifolia) were investigated in a field trial for two years in a commercial setting. Nitrogen fertilizer was tested in four doses (basic nitrogen, basic + suboptimal, basic + commercial, basic + excess) with six replicates in each. Culture-independent (Illumina sequencing) and culture-dependent (viable count and identification of bacterial isolates) community studies were combined with monitoring of plant physiology and site weather conditions. This study found that alpha diversity of bacterial communities decreased in response to increasing nitrogen fertilizer dose, whereas viable counts showed no differences. Correspondingly, fungal communities of the spinach phyllosphere showed a decreasing pattern, whereas the decreasing diversity of fungal communities of rocket was not significant. Plant species and effects of annual variations on microbiome structure were observed for bacterial and fungal communities on both spinach and rocket. This study provides novel insights on the impact of nitrogen fertilizer regime on a nutrient scarce habitat, the phyllosphere.

Mixed blessings: A qualitative exploration of mothers' experience of child care and feeding in the rapidly urbanizing city of Addis Ababa, Ethiopia.

Many studies have drawn attention to the vital role mothers have in safeguarding the health and nutritional wellbeing of their children. However, little is known about mothers' experiences and the challenges they face in fulfilling this role in rapidly urbanizing cities in Africa. This study aims to explore child care and feeding practices of mothers with children under five years of age in Addis Ababa, Ethiopia. This qualitative study was conducted using a semi-structured interview guide. A total of thirty-six interviews were conducted with purposively selected participants. All interviews were audio recorded, transcribed verbatim and translated for analysis. We used a thematic analysis approach, which was guided by a resilience framework. The findings are presented as three major themes. 1) 'Mixed blessings-balancing motherhood's expectations'. While mothers identified positively with the social recognition and sense of fulfillment of being a 'good mother', they were ambivalent/torn about earning the necessary income from outside work and fulfilling their duties at home. 2) 'Instabilities due to rampant urban sprawl'. While women expressed a keen desire to balance work and motherhood, the disintegrating social capital, due to large in-migration, market fluctuations and abrupt/forced resettlements to new housing units had left mothers without support for childcare, stressed and exhausted. 3) 'Anchored by faith: a source of resilience to cope with adversities'. In the face of the multiple adversities, mothers cited their strong faith as their most reliable foundation for their resilience. In summary, the societal and environmental changes accompanying the rapid urbanization in low income settings makes combining child care and working outside the home very challenging for mothers. As a result they suffer from fatigue and feelings of isolation. Efforts to improve child feeding and care in urban low-income settings need to consider context appropriate strategies that support mothers with small children.

What Influences Urban Mothers' Decisions on What to Feed Their Children Aged Under Five-The Case of Addis Ababa, Ethiopia.

Mothers carry the prime responsibility for childcare and feeding in low-income countries. Understanding their experiences in providing food for their children is paramount to informing efforts to improve the nutritional status of children. Such information is lacking in Sub-Saharan Africa. To understand what influences urban mothers' food acquisition and their motivations for selecting food for their children, 36 in-depth interviews were carried out with mothers having children under five years of age. Interviews were conducted in the local language, audio-recorded, transcribed, and translated into English. Data were analyzed using thematic analysis which led to the identification of four major themes: mothers give-in to a child-driven diet; quick-fix versus the privilege of planning; keen awareness on food safety, nutrition, and diet diversity; and social, familial, and cultural influences. The findings indicate that child feeding practices are influenced by interlinked social and environmental factors. Hence, nutrition education campaigns should focus on targeting not only families but also their children. Attention should also be given to food safety regulations, as well as to the much-needed support of mothers who are struggling to ensure their children's survival in low-income countries.

Light spectrum modifies the utilization pattern of energy sources in Pseudomonas sp. DR 5-09.

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.

Escherichia coli O157:H7 reduction in hamburgers with regard to premature browning of minced beef, colour score and method for determining doneness.

This study investigated the effect of premature browning (PMB) on the survival of Escherichia coli O157:H7 in beef hamburgers after cooking with respect to interior colour of the hamburger and recommendations to cook hamburgers to a core temperature of 71 °C. Assessment of doneness by visual inspection or measurement of internal temperature was compared in terms of survival and the increased relative risk of illness due to PMB was estimated. At the last consume-by-day, hamburgers made from minced meat packaged in 80/20 O2/CO2 (MAP hamburger) and from meat minced at retail packaged in atmospheric condition (control hamburger) were inoculated with a gfp-tagged strain of E. coli O157:H7 (E. coli O157:H7gfp+). Hamburgers were cooked for different times during assessment of the core temperature every 30s and cut in halves after cooking. Doneness was evaluated based on visual judgement of the internal colour using a score chart (C-score) from 'uncooked' (score 1) to 'tan with no evidence of pink' (score 5). An alternative five point score chart (TCC-score) including texture of the meat, clarity of meat juice and internal colour was also developed. Enumeration of viable E. coli O157:H7gfp+ in cooked hamburgers was based on fluorescent colonies recovered from plates. Results showed that MAP hamburgers developed PMB when compared with controls (P=0.0003) and that the shortest cooking time for the highest C-score was 6 and 11 min for MAP and control hamburgers, respectively. The mean temperature in the MAP hamburger was then 60.3 °C. The TCC-score reduced the difference between MAP and control hamburgers. It was also shown that the survival of E. coli O157:H7gfp+ was highest in MAP hamburgers. The predicted absolute risks for illness were highest for MAP hamburgers for all C-scores and the relative risk associated with PMB increased with doneness. For a C-score of 4 (slightly pink) the predicted relative risk for illness was 300 times higher for MAP hamburger than for controls. A variable pathogen reduction was observed when cooking hamburgers to temperatures of 70-76 °C (the 5th and 95th percentile range was around 3.3 log CFU). The lower reductions, at the 5th percentile, may, depending on initial contamination levels, not be enough to ensure sufficient and safe inactivation of E. coli O157:H7. Efforts to inform consumers about PMB in minced meat packaged in high oxygen packages (≥60% O2) are needed with the aim to make consumers use thermometers correctly or at least not determine doneness based only on meat colour.

Simultaneous removal of nitrate and sulfate from greenhouse wastewater by constructed wetlands.

This study evaluated the effectiveness of C-enriched subsurface-flow constructed wetlands in reducing high concentrations of nitrate (NO) and sulfate (SO) in greenhouse wastewaters. Constructed wetlands were filled with pozzolana, planted with common cattail (), and supplemented as follows: (i) constructed wetland with sucrose (CW+S), wetland units with 2 g L of sucrose solution from week 1 to 28; (ii) constructed wetland with compost (CW+C), wetland units supplemented with a reactive mixture of compost and sawdust; (iii) constructed wetland with compost and no sucrose (CW+CNS) from week 1 to 18, and constructed wetland with compost and sucrose (CW+CS) at 2 g L from week 19 to 28; and (iv) constructed wetland (CW). During 28 wk, the wetlands received a typical reconstituted greenhouse wastewater containing 500 mg L SO and 300 mg L NO. In CW+S, CW+C, and CW+CS, appropriate C:N ratio (7:3.4) and redox potential (-53 to 39 mV) for denitrification resulted in 95 to 99% NO removal. Carbon source was not a limiting factor for denitrification in C-enriched constructed wetlands. In CW+S and CW+CS, the dissolved organic carbon (DOC)/SO ratios of 0.36 and 0.28 resulted in high sulfate-reducing bacteria (SRB) counts and high SO removal (98%), whereas low activities were observed at DOC/SO ratios of 0.02 (CW) to 0.11 (CW+C, CW+CNS). On week 19, when organic C content was increased by sucrose addition in CW+CS, SRB counts increased from 2.80 to 5.11 log[CFU+1] mL, resulting in a level similar to the one measured in CW+S (4.69 log[CFU+1] mL). Consequently, high sulfate reduction occurred after denitrification, suggesting that low DOC (38-54 mg L) was the limiting factor. In CW, DOC concentration (9-10 mg L) was too low to sustain efficient denitrification and, therefore, sulfate reduction. Furthermore, the high concentration of dissolved sulfides observed in CW+S and CW+CS treated waters were eliminated by adding FeCl.

Leaf microbiota of strawberries as affected by biological control agents.

The increasing use of biological control agents (BCAs) against Botrytis cinerea in strawberry raises the question of whether there are any undesirable impacts of foliar applications of BCAs on nontarget microorganisms in the phyllosphere. Therefore, our objective was to investigate this issue within a field study. Strawberry plants were repeatedly sprayed with three BCAs-namely, RhizoVital 42 fl. (Bacillus amyloliquefaciens FZB42), Trianum-P (Trichoderma harzianum T22), and Naturalis (Beauveria bassiana ATCC 74040)-to suppress Botrytis cinerea infections. Microbial communities of differentially treated leaves were analyzed using plate counts and pyrosequencing and compared with the microbial community of nontreated leaves. Plate count results indicate that the applied Bacillus and Trichoderma spp. survived in the strawberry phyllosphere throughout the strawberry season. However, no significant impacts on the leaf microbiota could be detected by this culture-dependent technique. Pyrosequencing of internal transcribed spacer ribosomal RNA and 16S RNA sequences revealed a change in fungal composition and diversity at class level after the introduction of T. harzianum T22 to the phyllosphere, whereas the bacterial composition and diversity was not affected by either this Trichoderma preparation or the other two BCAs. Our results suggest that pyrosequencing represents a useful method for studying microbial interactions in the phyllosphere.

Use of a passive bioreactor to reduce water-borne plant pathogens, nitrate, and sulfate in greenhouse effluent.

The goal of this study was to evaluate the use of passive bioreactors to reduce water-borne plant pathogens (Pythium ultimum and Fusarium oxysporum) and nutrient load (NO(-) 3 and SO(2-) 4) in greenhouse effluent. Sterilized and unsterilized passive bioreactors filled with a reactive mixture of organic carbon material were used in three replicates. After a startup period of 2 (sterilized) or 5 (unsterilized) weeks, the bioreactor units received for 14 weeks a reconstituted commercial greenhouse effluent composed of 500 mg L(-1) SO(2-) 4 and 300 mg L(-1) NO(-) 3 and were inoculated three times with P. ultimum and F. oxysporum (10(6) CFU mL(-1)). Efficacy in removing water-borne plant pathogens and nitrate reached 99.9% for both the sterilized and unsterilized bioreactors. However, efficacy in reducing the SO(2-) 4 load sharply decreased from 89% to 29% after 2 weeks of NO(-) 3-supply treatment for the unsterilized bioreactors. Although SO(2-) 4 removal efficacy for the sterilized bioreactors did not recover after 4 weeks of NO(-) 3-supply treatment, the unsterilized bioreactor nearly reached a similar level of SO(2-) 4 removal after 4 weeks of NO(-) 3-supply treatment compared with affluent loaded only with SO(2-) 4, where no competition for the carbohydrate source occurred between the denitrification process and sulfate-reducing bacteria activity. Performance differences between the sterilized and unsterilized bioreactors clearly show the predominant importance of sulfate-reducing bacteria. Consequently, when sulfate-reducing bacteria reach their optimal activity, passive bioreactors may constitute a cheap, low-maintenance method of treating greenhouse effluent to recycle wastewater and eliminate nutrient runoff, which has important environmental impacts.

Utilisation of carbon sources by pythium, phytophthora and fusarium species as determined by biolog® microplate assay.

This study examined the metabolic activity of pure cultures of five root pathogens commonly found in closed hydroponic cultivation systems (Phytophthora cryptogea (PC), Phytophthora capsici (PCP), Pythium aphanidermatum (PA), Fusariumoxysporum f.sp. radicis-lycopersici (FORL) and Fusarium solani (FS)) using sole carbon source utilisation in order to develop effective biocontrol strategies against these pathogens. Aliquots of 150 microL of the mycelial suspension were inoculated in each well of GN2 microtitre plates. On the basis of average well colour development and number of positive wells, the pathogens were divided into two groups, (i) PA and FORL and (ii) PC, PCP and FS. Group (i) was characterised by a short lag-phase, a rapid exponential phase involving almost all carbon sources offered and a long stationary phase, while group (ii) had a more extended lag-phase and a slower utilisation rate of the carbon sources offered. The three isolates in group (ii) differed significantly during their exponential phase. The lowest utilisation rate of carbon sources and number of sources utilised was found for PCP. Of the major group of carbon sources, six carbohydrates, three carboxylic acids and four amino acids were rapidly used by all isolates tested at an early stage. The carbon sources gentibiose, alpha-D-glucose, maltose, sucrose, D-trehalose, L-aspartic acid, L-glutamic acid, L-proline persisted to the end of the exponential phase.Moreover, similarities between the metabolic profiles of the tested pathogen and the those of the resident microflora could also be found. These findings are of great importance as regards the role of the resident microflora in the biocontrol.