Development and Application of Predictive Models for Survival, Growth, and Death of Enteric Pathogens in Leafy Greens Supply Chain

Leafy greens are essential part of a healthy diet. Because of their health benefits, production and consumption of leafy greens has increased considerably in the U.S. in the last few decades. However, leafy greens are also associated with a large number of foodborne disease outbreaks in the last few years. The overall goal of this dissertation was to use the current knowledge of predictive models and available data to understand the growth, survival, and death of enteric pathogens in leafy greens at pre- and post-harvest levels. Temperature plays a major role in the growth and death of bacteria in foods. A growth-death model was developed for Salmonella and Listeria monocytogenes in leafy greens for varying temperature conditions typically encountered during supply chain. The developed growth-death models were validated using experimental dynamic time-temperature profiles available in the literature. Furthermore, these growth-death models for Salmonella and Listeria monocytogenes and a similar model for E. coli O157:H7 were used to predict the growth of these pathogens in leafy greens during transportation without temperature control. Refrigeration of leafy greens meets the purposes of increasing their shelf-life and mitigating the bacterial growth, but at the same time, storage of foods at lower temperature increases the storage cost. Nonlinear programming was used to optimize the storage temperature of leafy greens during supply chain while minimizing the storage cost and maintaining the desired levels of sensory quality and microbial safety. Most of the outbreaks associated with consumption of leafy greens contaminated with E. coli O157:H7 have occurred during July-November in the U.S. A dynamic system model consisting of subsystems and inputs (soil, irrigation, cattle, wildlife, and rainfall) simulating a farm in a major leafy greens producing area in California was developed. The model was simulated incorporating the events of planting, irrigation, harvesting, ground preparation for the new crop, contamination of soil and plants, and survival of E. coli O157:H7. The predictions of this system model are in agreement with the seasonality of outbreaks. This dissertation utilized the growth, survival, and death models of enteric pathogens in leafy greens during production and supply chain.

Soil Health, Soil Biology, Soilborne Diseases and Sustainable Agriculture

Soil Health, Soil Biology, Soilborne Diseases and Sustainable Agriculture provides readily understandable information about the bacteria, fungi, nematodes and other soil organisms that not only harm food crops but also help them take up water and nutrients and protect them from root diseases. Complete with illustrations and practical case studies, it provides growers and their consultants with holistic solutions for building an active and diverse soil biological community capable of improving soil structure, enhancing plant nutrient uptake and suppressing root pests and pathogens.

Bacterial communities of the specialty crop phyllosphere: response to biological soil amendment use, rainfall, and insect visitation

Microorganisms in the plant rhizosphere, the zone under the influence of roots, and phyllosphere, the aboveground plant habitat, exert a strong influence on plant growth, health, and protection. Tomatoes and cucumbers are important players in produce safety, and the microbial life on their surfaces may contribute to their fitness as hosts for foodborne pathogens such as Salmonella enterica and Listeria monocytogenes. External factors such as agricultural inputs and environmental conditions likely also play a major role. However, the relative contributions of the various factors at play concerning the plant surface microbiome remain obscure, although this knowledge could be applied to crop protection from plant and human pathogens. Recent advances in genomic technology have made investigations into the diversity and structure of microbial communities possible in many systems and at multiple scales. Using Illumina sequencing to profile particular regions of the 16S rRNA gene, this study investigates the influences of climate and crop management practices on the field-grown tomato and cucumber microbiome. The first research chapter (Chapter 3) involved application of 4 different soil amendments to a tomato field and profiling of harvest-time phyllosphere and rhizosphere microbial communities. Factors such as water activity, soil texture, and field location influenced microbial community structure more than soil amendment use, indicating that field conditions may exert more influence on the tomato microbiome than certain agricultural inputs. In Chapter 4, the impact of rain on tomato and cucumber-associated microbial community structures was evaluated. Shifts in bacterial community composition and structure were recorded immediately following rain events, an effect which was partially reversed after 4 days and was strongest on cucumber fruit surfaces. Chapter 5 focused on the contribution of insect visitors to the tomato microbiota, finding that insects introduced diverse bacterial taxa to the blossom and green tomato fruit microbiome. This study advances our understanding of the factors that influence the microbiomes of tomato and cucumber. Farms are complex environments, and untangling the interactions between farming practices, the environment, and microbial diversity will help us develop a comprehensive understanding of how microbial life, including foodborne pathogens, may be influenced by agricultural conditions.

Factors driving the abundance of ixodes ricinus ticks and the prevalence of zoonotic I. ricinus-borne pathogens in natural foci

Environmental factors may drive tick ecology and therefore tick-borne pathogen (TBP) epidemiology, which determines the risk to animals and humans of becoming infected by TBPs. For this reason, the aim of this study was to analyze the influence of environmental factors on the abundance of immature-stage Ixodes ricinus ticks and on the prevalence of two zoonotic I. ricinus-borne pathogens in natural foci of endemicity. I. ricinus abundance was measured at nine sites in the northern Iberian Peninsula by dragging the vegetation with a cotton flannelette, and ungulate abundance was measured by means of dung counts. In addition to ungulate abundance, data on variables related to spatial location, climate, and soil were gathered from the study sites. I. ricinus adults, nymphs, and larvae were collected from the vegetation, and a representative subsample of I. ricinus nymphs from each study site was analyzed by PCR for the detection of Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum DNA. Mean prevalences of these pathogens were 4.0% ± 1.8% and 20.5% ± 3.7%, respectively. Statistical analyses confirmed the influence of spatial factors, climate, and ungulate abundance on I. ricinus larva abundance, while nymph abundance was related only to climate. Interestingly, cattle abundance rather than deer abundance was the main driver of B. burgdorferi sensu lato and A. phagocytophilum prevalence in I. ricinus nymphs in the study sites, where both domestic and wild ungulates coexist. The increasing abundance of cattle seems to increase the risk of other hosts becoming infected by A. phagocytophilum, while reducing the risk of being infected by B. burgdorferi sensu lato. Controlling ticks in cattle in areas where they coexist with wild ungulates would be more effective for TBP control than reducing ungulate abundance.

Changes In The Bacterial Community Of Soil From A Neutral Mine Drainage Channel.

Mine drainage is an important environmental disturbance that affects the chemical and biological components in natural resources. However, little is known about the effects of neutral mine drainage on the soil bacteria community. Here, a high-throughput 16S rDNA pyrosequencing approach was used to evaluate differences in composition, structure, and diversity of bacteria communities in samples from a neutral drainage channel, and soil next to the channel, at the Sossego copper mine in Brazil. Advanced statistical analyses were used to explore the relationships between the biological and chemical data. The results showed that the neutral mine drainage caused changes in the composition and structure of the microbial community, but not in its diversity. The Deinococcus/Thermus phylum, especially the Meiothermus genus, was in large part responsible for the differences between the communities, and was positively associated with the presence of copper and other heavy metals in the environmental samples. Other important parameters that influenced the bacterial diversity and composition were the elements potassium, sodium, nickel, and zinc, as well as pH. The findings contribute to the understanding of bacterial diversity in soils impacted by neutral mine drainage, and demonstrate that heavy metals play an important role in shaping the microbial population in mine environments.

Bacterial Diversity Assessment In Soil Of An Active Brazilian Copper Mine Using High-throughput Sequencing Of 16s Rdna Amplicons.

Mining activities pose severe environmental risks worldwide, generating extreme pH conditions and high concentrations of heavy metals, which can have major impacts on the survival of organisms. In this work, pyrosequencing of the V3 region of the 16S rDNA was used to analyze the bacterial communities in soil samples from a Brazilian copper mine. For the analysis, soil samples were collected from the slopes (geotechnical structures) and the surrounding drainage of the Sossego mine (comprising the Sossego and Sequeirinho deposits). The results revealed complex bacterial diversity, and there was no influence of deposit geographic location on the composition of the communities. However, the environment type played an important role in bacterial community divergence; the composition and frequency of OTUs in the slope samples were different from those of the surrounding drainage samples, and Acidobacteria, Chloroflexi, Firmicutes, and Gammaproteobacteria were responsible for the observed difference. Chemical analysis indicated that both types of sample presented a high metal content, while the amounts of organic matter and water were higher in the surrounding drainage samples. Non-metric multidimensional scaling (N-MDS) analysis identified organic matter and water as important distinguishing factors between the bacterial communities from the two types of mine environment. Although habitat-specific OTUs were found in both environments, they were more abundant in the surrounding drainage samples (around 50 %), and contributed to the higher bacterial diversity found in this habitat. The slope samples were dominated by a smaller number of phyla, especially Firmicutes. The bacterial communities from the slope and surrounding drainage samples were different in structure and composition, and the organic matter and water present in these environments contributed to the observed differences.

Toxicity Of Fungal-generated Silver Nanoparticles To Soil-inhabiting Pseudomonas Putida Kt2440, A Rhizospheric Bacterium Responsible For Plant Protection And Bioremediation.

Silver nanoparticles have attracted considerable attention due to their beneficial properties. But toxicity issues associated with them are also rising. The reports in the past suggested health hazards of silver nanoparticles at the cellular, molecular, or whole organismal level in eukaryotes. Whereas, there is also need to examine the exposure effects of silver nanoparticle to the microbes, which are beneficial to humans as well as environment. The available literature suggests the harmful effects of physically and chemically synthesised silver nanoparticles. The toxicity of biogenically synthesized nanoparticles has been less studied than physically and chemically synthesised nanoparticles. Hence, there is a greater need to study the toxic effects of biologically synthesised silver nanoparticles in general and mycosynthesized nanoparticles in particular. In the present study, attempts have been made to assess the risk associated with the exposure of mycosynthesized silver nanoparticles on a beneficial soil microbe Pseudomonas putida. KT2440. The study demonstrates mycosynthesis of silver nanoparticles and their characterisation by UV-vis spectrophotometry, FTIR, X-ray diffraction, nanosight LM20 - a particle size distribution analyzer and TEM. Silver nanoparticles obtained herein were found to exert the hazardous effect at the concentration of 0.4μg/ml, which warrants further detailed investigations concerning toxicity.

Convergence Of Soil Nitrogen Isotopes Across Global Climate Gradients.

Quantifying global patterns of terrestrial nitrogen (N) cycling is central to predicting future patterns of primary productivity, carbon sequestration, nutrient fluxes to aquatic systems, and climate forcing. With limited direct measures of soil N cycling at the global scale, syntheses of the (15)N:(14)N ratio of soil organic matter across climate gradients provide key insights into understanding global patterns of N cycling. In synthesizing data from over 6000 soil samples, we show strong global relationships among soil N isotopes, mean annual temperature (MAT), mean annual precipitation (MAP), and the concentrations of organic carbon and clay in soil. In both hot ecosystems and dry ecosystems, soil organic matter was more enriched in (15)N than in corresponding cold ecosystems or wet ecosystems. Below a MAT of 9.8°C, soil δ(15)N was invariant with MAT. At the global scale, soil organic C concentrations also declined with increasing MAT and decreasing MAP. After standardizing for variation among mineral soils in soil C and clay concentrations, soil δ(15)N showed no consistent trends across global climate and latitudinal gradients. Our analyses could place new constraints on interpretations of patterns of ecosystem N cycling and global budgets of gaseous N loss.

Biofilms Of Enterococcus Faecalis And Enterococcus Faecium Isolated From The Processing Of Ricotta And The Control Of These Pathogens Through Cleaning And Sanitization Procedures.

The biofilm formation of Enterococcus faecalis and Enterococcus faecium isolated from the processing of ricotta on stainless steel coupons was evaluated, and the effect of cleaning and sanitization procedures in the control of these biofilms was determined. The formation of biofilms was observed while varying the incubation temperature (7, 25 and 39°C) and time (0, 1, 2, 4, 6 and 8days). At 7°C, the counts of E. faecalis and E. faecium were below 2log10CFU/cm(2). For the temperatures of 25 and 39°C, after 1day, the counts of E. faecalis and E. faecium were 5.75 and 6.07log10CFU/cm(2), respectively, which is characteristic of biofilm formation. The tested sanitation procedures a) acid-anionic tensioactive cleaning, b) anionic tensioactive cleaning+sanitizer and c) acid-anionic tensioactive cleaning+sanitizer were effective in removing the biofilms, reducing the counts to levels below 0.4log10CFU/cm(2). The sanitizer biguanide was the least effective, and peracetic acid was the most effective. These studies revealed the ability of enterococci to form biofilms and the importance of the cleaning step and the type of sanitizer used in sanitation processes for the effective removal of biofilms.

Mineral Nutrition Of Campos Rupestres Plant Species On Contrasting Nutrient-impoverished Soil Types.

In Brazil, the campos rupestres occur over the Brazilian shield, and are characterized by acidic nutrient-impoverished soils, which are particularly low in phosphorus (P). Despite recognition of the campos rupestres as a global biodiversity hotspot, little is known about the diversity of P-acquisition strategies and other aspects of plant mineral nutrition in this region. To explore nutrient-acquisition strategies and assess aspects of plant P nutrition, we measured leaf P and nitrogen (N) concentrations, characterized root morphology and determined the percentage arbuscular mycorrhizal (AM) colonization of 50 dominant species in six communities, representing a gradient of soil P availability. Leaf manganese (Mn) concentration was measured as a proxy for carboxylate-releasing strategies. Communities on the most P-impoverished soils had the highest proportion of nonmycorrhizal (NM) species, the lowest percentage of mycorrhizal colonization, and the greatest diversity of root specializations. The large spectrum of leaf P concentration and variation in root morphologies show high functional diversity for nutritional strategies. Higher leaf Mn concentrations were observed in NM compared with AM species, indicating that carboxylate-releasing P-mobilizing strategies are likely to be present in NM species. The soils of the campos rupestres are similar to the most P-impoverished soils in the world. The prevalence of NM strategies indicates a strong global functional convergence in plant mineral nutrition strategies among severely P-impoverished ecosystems.

Effect of silicate fertilization on soil and on palisade grass plants under grazing intensities

Application of calcium silicate (SiCa) as soil acidity corrective was evaluated in a Rhodic Hapludox soil with palisade grass conducted under pasture rotation system with different grazing intensities. Experimental design was complete randomized blocks with four grazing intensities - grazing intensities were imposed by forage supply (50, 100, 150 and 200 kg t-1 of DM per LW) - in experimental plots with four replicates and, in the subplots, with seven doses of calcium silicate combined with lime: 0+0, 2+0, 4+0, 6+0, 2+4, 4+2 and 0+6 t ha-1, respectively. In the soil, it was evaluated the effect of four levels of calcium silicate (0, 2, 4 and 6 t ha-1) at 45, 90, and 365 days at three depths (0-10, 10-20 and 20-40 cm) and at 365 days, it was included one level of lime (6 t ha-1). For determination of leaf chemical composition and silicate content in the soil, four levels of calcium silicate (0, 2, 4 and 6 t ha-1) were evaluated at 45 and 365 days and at 45 days only for leaf silicate, whereas for dry matter production, all corrective treatments applied were evaluated in evaluation seasons. Application of calcium silicate was positive for soil chemical traits related to acidity correction (pH(CaCl2), Ca, Mg, K, H+Al and V), but the limestone promoted better results at 365 days. Leaf mineral contents were not influenced by application of calcium silicate, but there was an increase on silicate contents in leaves and in the soil. Dry matter yield and chemical composition of palisade grass improved with the application of correctives.

New methods to quantify NH3 volatilization from fertilized surface soil with urea

Gaseous N losses from soil are considerable, resulting mostly from ammonia volatilization linked to agricultural activities such as pasture fertilization. The use of simple and accessible measurement methods of such losses is fundamental in the evaluation of the N cycle in agricultural systems. The purpose of this study was to evaluate quantification methods of NH3 volatilization from fertilized surface soil with urea, with minimal influence on the volatilization processes. The greenhouse experiment was arranged in a completely randomized design with 13 treatments and five replications, with the following treatments: (1) Polyurethane foam (density 20 kg m-3) with phosphoric acid solution absorber (foam absorber), installed 1, 5, 10 and 20 cm above the soil surface; (2) Paper filter with sulfuric acid solution absorber (paper absorber, 1, 5, 10 and 20 cm above the soil surface); (3) Sulfuric acid solution absorber (1, 5 and 10 cm above the soil surface); (4) Semi-open static collector; (5) 15N balance (control). The foam absorber placed 1 cm above the soil surface estimated the real daily rate of loss and accumulated loss of NH3N and proved efficient in capturing NH3 volatized from urea-treated soil. The estimates based on acid absorbers 1, 5 and 10 cm above the soil surface and paper absorbers 1 and 5 cm above the soil surface were only realistic for accumulated N-NH3 losses. Foam absorbers can be indicated to quantify accumulated and daily rates of NH3 volatilization losses similarly to an open static chamber, making calibration equations or correction factors unnecessary.

Influence of soil properties on the abundance of plant species in ferruginous rocky soils vegetation, southeastern Brazil

Ferruginous "campos rupestres" are a particular type of vegetation growing on iron-rich primary soils. We investigated the influence of soil properties on plant species abundance at two sites of ferruginous "campos rupestres" and one site of quartzitic "campo rupestre", all of them in "Quadrilátero Ferrífero", in Minas Gerais State, southeastern Brazil. In each site, 30 quadrats were sampled to assess plant species composition and abundance, and soil samples were taken to perform chemical and physical analyses. The analyzed soils are strongly acidic and presented low fertility and high levels of metallic cations; a principal component analysis of soil data showed a clear segregation among sites due mainly to fertility and heavy metals content, especially Cu, Zn, and Pb. The canonical correspondence analysis indicated a strong correlation between plant species abundance and soil properties, also segregating the sites.

Mineralogical characterization of a highly-weathered soil by the Rietveld Method

The mineralogical characterization through mineral quantification of Brazilian soils by X-ray diffraction data using the Rietveld Method is not common. A mineralogical quantification of an Acric Ferralsol from the Ponta Grossa region, state of Paraná, Brazil, was carried out using this Method with X-Ray Diffraction data to verify if this method was suitable for mineral quantification of a highly-weathered soil. The A, AB and B3 horizons were fractioned to separate the different particle sizes: clay, silt, fine sand (by Stokes Law) and coarse sand fractions (by sieving), with the procedure free of chemical treatments. X-ray Fluorescence, Inductively Coupled Plasma Atomic Emission Spectrometry, Infrared Spectroscopy and Mössbauer Spectroscopy were used in order to assist the mineral identification and quantification. The Rietveld Method enabled the quantification of the present minerals. In a general way, the quantitative mineralogical characterization by the Rietveld Method revealed that quartz, gibbsite, rutile, hematite, goethite, kaolinite and halloysite were present in the clay and silt fractions of all horizons. The silt fractions of the deeper horizons were different from the more superficial ones due to the presence of large amounts of quartz. The fine and the coarse sand fractions are constituted mainly by quartz. Therefore, a mineralogical quantification of the finer fraction (clay and silt) by the Rietveld Method was successful.

Lessons from the Terra Preta de Índios of the Amazon region for the utilisation of charcoal for soil amendment

The potential of charcoal and of partially combusted organic waste to mimic the soil organic matter of the Terras Pretas de Índios (Amazonian Dark Earths) from the Amazon Region is discussed. These materials serve as soil conditioners and as sequesterers of carbon in recalcitrant and in reactive forms. Studies carried out by Brazilian and by international groups have contributed to the emergence of an awareness of the compositions and of the uses of these materials. In this contribution we report on chemical studies that are leading to the development of a scientific and technological awareness, and of innovations that will have value in finding novel uses in applications to soil of chars from organic wastes such as those from the biofuel industry, and from metallurgical and various coal plant residues.