Due to a loss of samples, our K uptake and DOC datasets remain incomplete and will therefore not be discussed in the results; the extent of the recovered K uptake and DOC data is presented in Table 3 along with ammonium, nitrate, and phosphate tree replicates. A complementary mixed effects model using individual trees as an error term, in addition to the model formula above, was used to assess the effects of fertilization on nutrient uptake. For this method outliers with a Z-score of 3.5 or greater were removed from each treatment group and the remaining data were analyzed as described for a standard ANOVA except with an error term. This analysis precluded the use of p-scores as the package does not give them. Post-hoc analysis, as described below, is similarly non-applicable to this examination, therefore discussion of differences in results between the mixed effects and ANOVA models will remain limited. A Tukey Honestly Significant Differences post-hoc test was used as a post-hoc examination of the ANOVA results. Tukey’s HSD tests all pairwise comparisons, while controlling the probability of making one or more Type I errors, producing a conservative measure of significant differences between treatment combinations. Tukey’s HSD is used for post-hoc interpretation of significant ANOVA results in order to determine what differences between treatment groups are resulting in ANOVA significance. ANOVA/ANCOVA and Tukey’s post-hoc are both suitable for datasets with unequal variance in sample sizes. Our data was slightly unbalanced in the pseudoreplication across individual trees resulting in slight differences in plot and pseudoreplicate measurements. Tree replicates, and root pseudoreplicates, are shown by the treatment groups in Table 4; with slightly fewer pseudoreplicates evidenced at the 10X concentration in the N- and K-only addition plots.

Tree and plot replication, along with mean uptake rates and standard deviation for each nutrient uptake by treatment,roll bench are shown in Table 5. All analyses conducted in R .Both the full ANCOVA and simplified ANOVA models showed significant effects of an N*K interaction on the uptake rates of ammonium, nitrate, and phosphate. Significant effects on the uptake rates of each nutrient, and DOC exudation, are shown in Table 5 with the results from each the model tested, and an asterisk noting incomplete datasets. Ammonium uptake was significantly affected by an N*K interaction in both models , with model results improving noticeably with the removal of DBH as a covariate. Nitrate uptake was very significantly affected by and N*K interaction in both models , with a marginally significant effect of P addition on uptake in the full model. Phosphate uptake was significantly affected at the P<0.05 level by an N*K interaction in each model. Potassium uptake was significantly affected by an N addition, and N*K interaction effect, while DOC was significantly affected by a P*K interaction; however, as these results are based on incomplete datasets, their significance will be reported in Table 6 but not mentioned in later discussion. P-values for individual predictor variables in the ANCOVA and ANOVA models, along with their model statements, are given in Table 7, Table 8, and Table 9, for ammonium, nitrate, and phosphate respectively. P-values of individual model predictors for K uptake and DOC exudation are presented in Tables 10 and 11 respectively. DBH had no effect on nutrient uptake and model results improved when it was removed as a covariate suggesting that it is not a strong determinant of nutrient uptake; and therefor that seedling studies may be accurate estimations of mature tree uptake rates for Tetragastris panamensis and other tropical species. The DBH of the sampled trees ranged from 84-564 mm, with a mean of 186.36 and a standard deviation of 108.04. A one-sided t-test of mean uptake rates of NH4+ and NO3-, regardless of treatment, showed no significant differences at p=0.05 between the two forms of N. A Tukey Honestly Significant Differences test was used as a conservative post hoc test to explore what differences between treatment groups were potentially responsible for the significant interaction.

The Tukey HSD post-hoc was applied using the simplified model formula using only N and K as predictors of nutrient uptake; this compared the means of all combinations of N and K fertilized plots to each other and corrected for multiple comparisons in order to determine which treatment combinations were significantly different. Treatment combinations are expressed as the absence or presence of either N or K, with each combination of treatments on the left side of the dash being compared to those on the right. The adjusted p-values for Tukey’s HSD are given for each nutrient in Table 12 with an asterisk denoting a significant difference in nutrient uptake between treatment groups. A Tukey HSD plots indicate significant differences between groups as a sideways boxplot where no overlap occurs between the whiskers and the zero line – the 95% confidence interval that a group’s true mean is significantly different from the zero line, the mean of all groups. The relative difference in uptake rates between plots can be assessed from plots of Tukey’s HSD; ammonium uptake was significantly lower in N and K fertilized plots versus uptake in N fertilized as indicated by the non-overlapping boxplot and center line in Figure 1. No other significant differences between individual treatment groups was observed for ammonium, however boxplots of ammonium uptake rates with illustrate the antagonistic relationship between N and K on uptake . Nitrate uptake showed a significant increase in N-only fertilized plots relative to control, and a decrease in N and K fertilized plots compared to N-only fertilized plots . This shows a negative interaction between the presence of N and K on nitrate uptake with single nutrient fertilization producing greater increases in uptake than fertilization with both nutrients . The same pattern of seemingly greater uptake rates with single nutrient addition is observed for phosphate in Figure 5. Phosphate uptake showed a nearly significant decrease in uptake in N and K fertilized plots compared to N-only fertilized plots . There were no significant differences observed between treatments in the Tukey’s HSD for phosphate however the overall N * K interaction effect was significant in the ANOVA model. Although non-significant, a consistent pattern was observed in the differences between treatment groups for all nutrients : notably that the addition of N and K in combination reduces uptake rates compared to N addition alone.

Additionally, N appears to consistently,commercial greenhouse supplies although not-significantly, increase nutrient uptake relative to control plots. This suggests an negative relationship between N and K where addition of either nutrient singly is likely to increase nutrient uptake rates while an interaction between N and K depresses uptake of all nutrients. A linear mixed effects model of nutrient uptake showed that solution concentration, along with N and P, were most likely to affect nutrient uptake rates instead of the N*K interaction. ANOVA F-values for the mixed linear effects model of phosphate uptake are shown in Table 13 and suggest that concentration and N were the dominant influences on uptake, while t-values indicate K interactions with N or P may be qualitatively different than other fertilization effects. ANOVA results for NH4+ uptake show that only concentration produced a large effect on NH4+ uptake while no other nutrient appeared to strongly influence uptake . In contrast, NO3- uptake appeared to be much more sensitive to the effects of N and P, and their interactions, than NH4+ . T-values for K interactions are also evidenced for NH4+ and NO3- uptake suggesting that K is producing a universal effect on the uptake of macro-nutrients, however the dynamics and drivers of this potential effect are uncertain.Nutrient solution-depletion methods have examined the uptake of N, as NH4+ and NO3-, more extensively than other nutrients, however, few studies have directly addressed the effects of altered relative N availability on nutrient uptake. Uptake rates of mature in situ spruce and maple trees both showed significantly greater uptake of NH4+ than NO3- in all seasons; with the early growing season showing the lowest uptake rates for NH4+ and NO3-, 0-6 umol/g dw*hr and -5 – 0.5 umol/g dw*hr respectively, while uptake rates in the late fall showed an increase in NH4+ uptake up to 25 umol/g dw*hr . Mature beech trees evidenced uptake rates of 0.25-1.5 umol/g fresh weight * hr in two studies that did not record concurrent NO3- uptake , while mature loblolly pine showed NH4+ uptake rates of 1-4 umol/g dw*hr which were significantly higher than those of NO3- . Seedlings of two maple species exposed to the same solution-depletion method showed uptake rates of NH4+ between 2 and 10 umol/g dw*hr, for external NH4+ concentrations <100 umol/L, while NO3- uptake was consistently lower . While the preference for NH4+ in midlatitude species has been widely observed only one study of NH4+ uptake rates across an N deposition gradient provides evidence for the effect of N availability on uptake rates. Using a labeled isotope modification of the depletion method uptake rates of beech, spruce, pine and birch across an N deposition gradient showed greater N uptake in sites with higher N deposition .

A compilation of reported uptake ranges is shown in Table 16 for comparison with our results. Our data show similar uptake rates to those of NH4+ in mid-latitude species, with the indication that N uptake may be stimulated with N addition, however, our data showed only marginally significant differences in the uptake rates of NH4+ versus those of NO3- for mature Tetragastris panamensis. Demand for N and P have also been inferred by measurements of nutrient concentrations in plant tissues and changes in root biomass. Tropical tree seedlings grown under experimental water and nutrient regimes showed leaf N:C ratios significantly increased in response to low soil water content and N fertilizer addition . In contrast, leaf P:C ratios were only moderately affected by nutrient availability and water content, suggesting greater sensitivity of nutrient uptake to N availability . In the same factorial fertilization experiment this study was conducted in N addition increased fine root biomass in the top five cm .Tissue nutrient concentrations in a tropical pioneer species showed that the transpiration rate of the plant provided a significant control on uptake and varied with N availability, not P . Our results suggest that N availability plays a significant role in the uptake of nutrients, by its interaction with K as well as the seemingly increased uptake rates of all nutrients relative to control and NK plots when added alone. Uptake of NO3-appeared to be more significantly affected by nutrient availability than that of NH4+ , following observations of mid-latitude forests that demand each form of N may be sensitive to soil N conditions. Our data support the interpretation that these forests are at least partially limited by N and that the availability of N may influence nutrient acquisition via effects on transpirational control of nutrient uptake. P uptake studies in the literature are rare for tropical and non-crop species thus interpretation of our results relies on context by other ecological studies inferring nutrient demand and cycling. In our study site FRB increased with P addition but decreased with simultaneous P and N addition . An N by P factorial fertilization experiment in a secondary tropical forest in China found that P addition increased P concentrations throughout the plant, although P concentrations decreased when N and P were added simultaneously . A meta-analysis of root phosphatase production in relation to soil fertility found that N fertilization increased phosphatase exudation, promoting the uptake of P, while P addition suppressed phosphatase production.These studies suggest that addition of P induces a greater demand for nutrients, however specifically not for additional P, an effect which decreases when N is abundant. With a simultaneous increase in root biomass and decrease in phosphatase production, the addition of P appears to promote nutrient acquisition by altering C investment in root tissue instead of phosphatase C-exudation in order to increase long-term passive nutrient acquisition. Our study does not allow a determination of changes in total nutrient uptake due to changes in root biomass, however, our results show that P did not affect the active uptake rates of any nutrients, despite P uptake being significantly affected by the availability of N and K. A pattern of N limitation underlying potential P limitation is also indicated by studies of above ground nutrient and C cycling dynamics.

Ghosh and Mukherji compared the effects of two nonionic surfactants and rhamnolipid biosurfactant at 10X CMC on mineralization of pyrene in liquid culture and found that the rhamnolipid amendment resulted in decreased mineralization compared to the unamended control due to the preferential degradation of the biosurfactant over pyrene, causing a decreased specific growth rate and pyrene utilization by P. aeruginosa. Bezza and Chirwa also observed a decrease in PAH degradation in the rhamnolipid and nutrient supplemented liquid culture treatments because of preferential microbial consumption of the biosurfactant. Most of the studies examining the preferential utilization of surfactants by PAHdegrading microorganisms were accomplished in liquid culture, and relatively few studies have examined this phenomenon in a soil matrix that would have a more realistic representation of  bio-remediation practices. For example, while nonionic surfactants are less likely to adsorb to soil surfaces compared to cationic surfactants due to the negative charge of the soil, some of the Brij-35 or rhamnolipid biosurfactant added to the mineralization assays was likely sorbed to the soil . Lladò et al. concluded that rhamnolipid biosurfactant applied to a microbial consortium resulted in increased PAH biodegradation in liquid culture; however, the same rhamnolipid biosurfactant application did not improve PAH biodegradation after 200 d when applied to a soil matrix. Maslin and Maier suggested that rhamnolipid biosurfactant was used as a preferential carbon source over phenanthrene by the native soil microbial populations when the rhamnolipid was present at higher concentrations. Deschênes et al. observed that the biodegradation of three-ringed PAHs was not affected by the addition of rhamnolipid biosurfactant at concentrations below and above the CMC; however,fodder system trays four-ringed PAHs anthracene, and chrysene had a 4–6 week lag phase before biodegradation commenced in aged PAH-contaminated soil.

The enumeration of viable M. vanbaalenii PYR- 1 CFUs in the rhamnolipid-amended treatments had a similar pattern compared to the treatments amended with glucose, an easily degraded monosaccharide, with substantial growth at the beginning of the incubation, followed by significant decrease in CFUs as the carbon substrate used for microbial growth was depleted. Yu et al. also observed rapid PAH-degrading bacteria growth using rhamnolipid biosurfactant up to 1000 mg L−1 , which resulted in a 40-fold increase in the maximum bacteria population as compared to the unamended control. Bezza and Chirwa concluded that the addition of a lipopeptide biosurfactant produced by Bacillus cereus SPL-4 resulted in a significant increase in the number of CFUs in PAHcontaminated soil during a 64-d incubation. While nontoxic to M. vanbaalenii PYR-1, Brij-35 at both surfactant concentrations showed increased resistance to degradation as compared to the rhamnolipid biosurfactant based upon viable M. vanbaalenii PYR-1 CFUs. In the Brij-35 10X CMC-amended culture, viable CFUs were significantly lower compared to the Brij- 35 0.1X CMC treatments after 14 d of incubation, potentially due to the limited bio-availability of the surfactant in the micellar phase at 10X CMC as compared to the monomeric surfactant phase at 0.1X CMC . In another study, Zhang et al. showed that the small radius size and structure characteristics of the micellized nonionic surfactants could prevent close contact between the microorganism and surfactant, resulting in limited degradation. Previous studies also showed preferential degradation of biosurfactants compared to synthetic surfactants by microorganisms. Zeng et al. compared the microbial degradation of a nonionic surfactant , cationic surfactant , anionic surfactant , and biosurfactant and observed that the rhamnolipid biosurfactant was the most readily biodegradable among all surfactants in the study. Mohan et al. compared the biodegradability of Triton X-100 and rhamnolipid biosurfactant as sole carbon sources to Vibriocyclotrophicus sp. nov., a PAH-degrading microorganism, and showed that the rhamnolipid was readily biodegradable under aerobic conditions while Triton X-100 was only partially biodegradable. Based on results from the 14C-pyrene mineralization experiment and the effect of surfactants on cell growth, the amendment of Brij-35 or rhamnolipid biosurfactant at the highest rate was not toxic to the PAH-degrading soil microorganisms.

Therefore, it may be concluded that in this study, the rhamnolipid biosurfactant was preferentially used by the PAH-degrading soil microorganisms and only after the eventual depletion of the biosurfactant, the mineralization of 14C-pyrene in both soils would start. This wasconsistent with the observation that the lag time of pyrene mineralization increased with increasing rhamnolipid amendment rate .Compared to the native sandy loam soil treatments, the native clay soil treatments had a significantly shorter lag period in the unamended and all surfactant-amended treatments, except for rhamnolipid at the high rate . The differences between the clay and sandy loam soil treatments in 14C-pyrene mineralization by the native microbes may be attributed to the higher TOC in the clay soil. The quantity and quality of soil organic matter has been previously shown to regulate the soil microbial community diversity and activity, as organic matter acts as energy and nutrient sources for heterotrophic soil microorganisms, stimulating microbial biomass growth and activity . Additionally, the finer silt and clay fractions could provide more sites for bacteria or organic matter attachment, leading to a larger microbial biomass in the clay aggregate and better utilization of 14C-pyrene . Bioaugmentation of M. vanbaalenii PYR-1 significantly increased the mineralization of 14C-pyrene in both PAH-contaminated soils . Because of the rapid mineralization, bioaugmentation of M. vanbaalenii PYR-1 in soils with or without surfactant resulted in a shortened lag period before 14C-pyrene mineralization commenced as compared to all corresponding soil treatments without the bacterial augmentation . There were no differences in the length of lag period between the clay and sandy loam bioaugmentation treatments, except for the medium rate rhamnolipid treatment where the clay soil had a shorter lag period than the sandy loam soil . Additionally, bioaugmentation of M. vanbaalenii PYR-1 in both soils resulted in an increased biodegradation rate in all soil treatments as compared to the native soils, except for the rhamnolipid-amended treatments at medium and high rates . The bioaugmentation of M. vanbaalenii PYR-1 in the clay soil treatments resulted in greater biodegradation rates compared to the bioaugmented sandy loam soil treatments, except for treatments amended with rhamnolipid biosurfactant at themedium and high rates . The addition of Brij-35 at the low and medium rates in the bioaugmented clay soil treatments resulted in a higher biodegradation rate, with the rate constant at 3.88 d−1 and 4.55 d−1 , respectively, as compared to the bioaugmented clay soil without the surfactant . However, after 25 d of incubation, the microbes in the native clay soil amended with Brij-35 surfactant at all three levels had undergone rapid 14C-pyrene mineralization and were not significantly different as compared to the bioaugmented clay soil treatments amended with Brij-35 .

This suggests that the bioaugmentation of PAH degrading microbes may not be necessary for treating PAH-contaminated soils in environments where the native soil microbial community is capable of degrading PAHs and in situations where PAHs are readily bioavailable. After 50 d of incubation, there were no significant differences between the unamended bioaugmented soil treatments, and the bioaugmented soil amended with Brij-35 at all levels . This observation may be partially attributed to the production of surface-active trehalose containing glycolipids by M. vanbaalenii PYR-1 . It is worth noting that the mixture of the biologically produced glycolipids by M. vanbaalenii PYR-1 with the rhamnolipid biosurfactant or Brij-35 surfactant did not result in increased 14C-pyrene mineralization. In a previous study, mixtures of synthetic surfactants such as sodium dodecyl sulfate, Tween-80, Triton X-100, and Brij-35 had a synergistic effect,aeroponic tower garden system causing a lower CMC as well as a significant PAH solubility enhancement in the mixed surfactant systems, leading to increased biodegradation of phenanthrene . Mycobacterium vanbaalenii PYR-1 has been extensively studied in pure cultures for the elucidation of mechanisms of PAH degradation; however, few studies have examined PAH biodegradation by M. vanbaalenii PYR-1 in contaminated soils where microbe survival and growth may be a limiting factor due to various environmental and microbial variables such as microbial competition with native soil populations, nutrient availability, moisture content, soil pH, and soil temperature . In the current study, the survival and PAH-degradation enhancement of M. vanbaalenii PYR-1 bioaugmentation can be clearly seen in the unamended and rhamnolipid-amended treatments by the significant decrease in lag period and total 14C-pyrene mineralization . Ghaley et al. also observed rapid PAH mineralization in pyrene-contaminated soils bioaugmented with M. vanbaalenii PYR-1 with a similar lag period and total pyrene mineralization . A similar Mycobacterium sp. also exhibited effective degradation potential for PAHs, resulting in enhanced 14C-pyrene mineralization in three different petroleum contaminated soils . Polycyclic aromatic hydrocarbons are a class of fused-ring aromatic compounds and 16 PAH compounds are designated as priority pollutants by the U.S. EPA because of their known or suspected toxicity and genotoxicity as well as frequent environmental occurrence . Bioremediation, the utilization of microorganisms to biologically degrade hazardous organic compounds to levels below concentration limits established by regulatory authorities, is considered an effective technique to remediate soils contaminated with PAHs . In a 2007 U.S. EPA report on contaminant treatment technologies, 37 out of 145 PAH remediation projects bio-remediation applications . However, biological treatment of PAHcontaminated soils is dependent upon the presence and degradation activity of soil microbes capable of transforming the priority pollutants .

Additionally,  bio-remediation efficacy for PAH-contaminated soil may also be limited by the bioavailability of soil-bound PAHs due to their physical and chemical properties, resulting in low aqueous solubility and high solid-water distribution ratios that promote PAH accumulation in the solid phases of the terrestrial environment . Surfactant-amended bio-remediation has been proposed to increase the rate of PAH desorption from the soil to the aqueous phase through micellar solubilization and/or by direct modification of the soil matrix . In recent years, interest inand feasibility of biosurfactant-enhanced bio-remediation has increased because rhamnolipid biosurfactants offer several advantages over their synthetic counterparts such as increased stability in pH and temperature extremes and environmental compatibility, while still offering similar PAH desorption effects . Under situations where PAH bioavailability is limited, surfactants and biosurfactants have been shown to increase transport of PAHs from the soil matrix into the aqueous phase, resulting in increased bioavailability to PAH-degrading microorganisms and enhance PAH biodegradation . However, surfactants may also negatively affect the PAH-degrading activity of the soil microbial community in PAH-contaminated soils . For example, some surfactants, especially anionic surfactants, can bind to peptides, enzymes, and DNA and change the biological function of soil microorganisms . Conversely, surfactants can also be preferentially utilized as carbon and energy sources for growth by PAH-degrading microorganisms, resulting in a decrease in metabolism of the target contaminant, thus inhibiting PAH biodegradation . However, the effects of surfactant amendments on the PAH degradation capacities of the soil microbial community has been rarely examined. A better understanding of the soil microbial community dynamics in response to amendment of different surfactants is valuable for successful surfactant-enhanced  bio-remediation of PAH-contaminated soils. We previously reported that amendment of the synthetic surfactant Brij-35 enhanced pyrene mineralization in soils, while amendment of rhamnolipid biosurfactant significantly inhibited the mineralization and also increased the lag period before mineralization commenced . As the differences in pyrene degradation likely resulted from changes in the soil microbial communities due to surfactant application, we hypothesized that the PAH-degrading soil microorganisms that maintained a high relative abundance could be identified by analysis of 16S rRNA gene sequences. The objectives of this study were to evaluate the effects of Brij-35 and rhamnolipid amendment on the native soil microbes associated with PAH mineralization and to determine whether such effects were dose dependent. The 16S rRNA gene highthroughput sequencing and phylogenetic investigation of communities by reconstruction of unobserved states were employed to analyze the shifts in soil microbial taxa due to the presence of surfactants and to assess the bacterial species and functional genes responsible for PAH biodegradation.For each sample, 2 g soil was spiked with 10 mg kg-1 pyrene according to Brinch et al. . An additional 8 g soil was added to the treated soil and then mixed over a 5-d period. Following pyrene spiking, 10 mL sterilized minimal basal salts solution was added to the native soil treatment, followed by the addition of 10 mL surfactant solution resulting in initial amendment rates of 14, 140, and 1,400 µg g-1 for rhamnolipid biosurfactant and 21.6, 216, and 2,160 µg g-1 for Brij-35, respectively.

Interestingly, the B.napus plants grown on fritted clay exhibited odd growing patterns, with certain leaves exhibiting a shriveled curled leaf phenotype in both well-watered and drought conditions. Furthermore, the sand-like quality ofthe substrate caused compaction post-watering, leading to decreased root growth as compared to soil. Replicating the low moisture assay done for our A.thaliana mutants would be more appropriate to look at drought-tolerant phenotypes in our B.napus. Evaluation of PP2C levels at the end of the drought assay would allow us to have better insight on gene expression of these negative regulators, and the effectiveness of our knockdown. Similarly as proposed for A.thaliana mutants, assessment of stomatal conductance through petiole feeding of ABA, and stomatal density would be important to give us insight on whether our plants are more ABA-responsive when fully grown, and if there are any stomatal development was affected by the knockdown. The Fritted clay approach towards looking into drought tolerance of B.napus plants was shown to give a uniform stress to the rd29a-mediated PP2C knockdown lines, however, issues of both high seedling mortality rates and lack of real-world applications lead me to believe that the drought assay developed for A.thaliana plants is a better method. We were able to generate ABA responsive B.napus rd29a-mediated PP2C knockdown lines, however the expression levels ofthe knockdown between lines stiil needs to be assessed. Finally, testing the same ABA responsive lines in the drought assay we developed for A.thaliana plants would allow us to observe if the enhanced ABA response would lead to increased drought tolerance.The use of copper-based nanoparticles in agriculture as fungicides and bactericides is increasing rapidly due to their relatively low toxicity and higher efficiency in delivering the active component .

There are numerous copper containing pesticides on the market, e.g. copper sulfate , cuprous oxide , copper hydroxide ,hydroponic channel and nano copper . The U.S. Department of Agriculture maintains an official list of synthetic substances that can be used for organic farming. According to this list, copper-based materials are allowed for use in organic crop production.However, more and more evidence indicates that copper-based nanoparticles induce phytotoxicity in various plants, such as bean,lettuce,alfalfa,cilantro,cucumber.The adverse impact include decreased root and shoot elongation, disturbed mineral nutrients homeostasis, decreased photosynthesis rate, inhibited antioxidant enzyme activities.So far the molecular mechanism underlying those physiological changes is not well understood. In recently years, “omics” have become a promising methodology for studying plant responses to abiotic and biotic stress.Transcriptomics-based gene expression and proteomics-based protein production have been applied to evaluate the changes of plants to external stressors at the molecular level.Unlike transcriptomics and proteomics, which reveal what might be happening in plant tissues, metabolomics profiling can tell what already happened. Metabolites are the end product of gene expression, and the changes of metabolites are regarded as ultimate responses of plant to stress.Thus, environmental metabolomics is becoming a powerful tool to investigate the response of plants to various stressors, e.g., water, light, temperature, and high levels of metals.Recently, Pidatala et al. employed LC-MS/ MS based metabolomics to elucidate the stress response mechanism of lettuce to lead. They observed several key metabolic pathways, including sugar and amino acid metabolism, that were disturbed by lead.Our recent study,applying GC-TOF-MS based metabolomics and PLS-DA multivariate analysis, also revealed the profile of metabolites in root exudates was significantly altered by nCu.More recently, we determined that nCu altered the nutritional supply of cucumber fruit,using 1 H NMR and GC-MS based metabolomics. Those studies demonstrate that metabolomics is a powerful tool to investigate the stress response of plant to contaminants.

Therefore, the present study was designed to generate a mechanistic understanding of the effects of nCu on cucumber plants by applying untargeted GC-TOF-MS based metabolomics. Fruit metabolic profiling provides information on low molecular weight metabolites, which not only directly reflects fruit nutrient levels, but also generates a more comprehensive understanding of the metabolic network and the biological pathways impacted by NPs.ICP-MS based metallomics were also performed to supply the elemental changes under nCu stress. All those techniques provide a comprehensive insight into the overall stress response mechanism of cucumber plants to nCu, which helps understand their long-term impact in terrestrial environments.Cucumber seeds were purchased from Seed Savers Exchange . The soil was collected from the Natural Reserve System of UC Santa Barbara . The soil composition is shown in Table S1.NCu was suspended in nanopure water and sonicated for 30 minutes before being applied to the soil. The final concentration of nCu in soil was 0 , 200 , 400 and 800 mg kg−1 . These concentrations are within the range of those predicted for biosolids applied to soils or due to the application of copper-based nanopesticides.Each treatment had four replicates. In each replicate, pairs of cucumber seedlings were grown in 3.0 L Poly-Tainer containers. The cucumber plants were grown from April 2, 2015 until harvest on June 28, 2015. The temperature in the greenhouse was controlled to be 25.5–30.0 °C during the day and 17.7–18.9 °C at night. At harvest, the fresh weight of root, stem, leaf and fruit from each treatment was recorded. In addition, the weight of each mature fruit from each treatment was recorded. Only the matured fruits were selected for metallomics and metabolomics analysis. The number of matured cucumber fruits from each treatment were 6 , 5 , 3 and 6 ; fruits from each treatment were split in two parts, to use for metabolomics and for metallomics analyses.At harvest, the cucumber tissues were oven-dried for 7 days at 60 °C. The oven-dried tissues were ground to powder and digested with HNO3 and H2O2 using a microwave oven system .

The digestion method was based on EPA 3051.Standard reference materials, NIST 1547 and 1570a , were also digested and analyzed as samples. The recoveries for all elements were between 90% and 99%.7 The mineral nutrient elements were analyzed using inductively coupled plasma-mass spectrometry .At harvest, the fresh cucumber fruits were immediately placed in liquid N2, and lyophilized . The freeze-dried fruit samples were delivered to the Genome Center Core Services at University of California Davis to analyze metabolites via gas chromatography time-of-flight mass spectrometer-mass spectrometry . A description of the analytical method has been reported previously.Sample pretreatment was done similar to Fiehn et al.Partial least-squares discriminant analysis was run based on GC-TOF-MS data using online resources . PLS-DA is a supervised clustering method, which uses a multiple linear regression technique to maximize the separation between groups and help to understand which variables carry the class separating information.More details regarding data acquisition, data processing and data reporting are provided in the ESI.†Biomass, and mineral nutrients data were analyzed by oneway ANOVA, and group means were compared by conducting Tukey’s Honestly Significant Difference test using IBM SPSS Statistics 22. A probability of p ≤ 0.05 was considered to be significant. Photosynthesis rate, transpiration rate, stomatal conductance, and water use efficiency were analyzed with anested ANCOVA in which the independent factors were treatment; with replicates nested within a treatment,hydroponic dutch buckets and leaf temperature as a covariate. Leaf temperature was used as a covariate because gas exchange is often very sensitive to air temperature.Photosynthesis is one of the physiological processes most sensitive to environmental stresses.In order to monitor the physiological changes during development without damaging the plant tissue, photosynthetic rate , transpiration , stomatal conductance and instantaneous water use efficiency of cucumber leaves were measured 30 days after sowing. Fig. 1 shows that photosynthetic rate decreased in all nCu treatments compared to the control, but only the low and medium group were statistically significant . Previous studies showed that nano copper depleted PSII action centers, leading to photoinhibition25 and disruption of the repair cycle.In addition, stomatal conductance and transpiration rates tended to increase in nCu treatments compared to the control. The actual mechanism for Cu to increase stomatal conductance and transpiration rate is still unknown. One possible explanation is that Cu can trigger the formation of reactive oxygen species through redox process cycling between Cu+ and Cu2+.It is not known if nCu by itself can trigger ROS. Previous work has shown that ROS open the stomata, increasing stomatal conductance and therefore the transpiration rate.WUEi is defined as the ratio of the rate of carbon assimilation to the rate of transpiration. The decline in carbon assimilation rate and the increase in transpiration rates in response to exposure to nCu resulted in a statistically significant decline in WUEi .Previous studies demonstrated that nCu has comparatively higher solubility than its oxidized forms, and is sensitive to aqueous matrix pH.Therefore, we hypothesized that Cu would bio-accumulate in cucumber tissues, including fruits. As shown in Fig. 2 and Table S2,† Cu concentrations in all tissues in nCu treatment groups were significantly higher than that in the control , which indicates Cu is taken up by the roots and translocated even to the fruits. In both control and nCu treated plants, Cu was most abundant in the roots, although there was significantly more bioaccumulation in the nCu exposed plants.

This is consistent with previous reports that copper is sequestered primarily in the root compartment.It is noteworthy that the translocation factors of nCu/Cu ions in control plants is 0.33, but it decreased to 0.21, 0.22 and 0.23 in the plants exposed to 200, 400 and 800 mg kg−1 nCu, respectively. In the control treatments, plants uptake ionized Cu from the soil. One would expect a similar translocation factor in the nCu treated group if ionized Cu was translocated in a similar manner. The reduced translocation rate suggests that cucumber plants grown in nCu treated soils not only took up Cu ions, but also nCu. But we believe that most of the nCu just adsorbed on the root surface. Confocal microscopy and μ-XRF evidenced that ZnO and CeO2 NPs are mainly distributed on the outer layer of epidermis cells or trapped in epidermis cell walls; only a small portion of NPs could penetrate the epidermis cell wall and translocate to upper plant tissues via the transpiration stream.In addition, high ionic strength in the soil aqueous matrix probably accelerated the aggregation of nCu.Moreover, nCu or released Cu ions may complex with soil clay minerals or organic matter.All these factors reduced the possibility of small sized and free nCu existing in soil. Thus, Cu in the stems, leaves and fruits is likely from solubilized Cu leached from nCu. In order to evaluate the ability of nCu for releasing Cu ions into soil, additional experiments were conducted : soil was spiked with 200 mg kg−1 of CuCl2·2H2O and nCu separately. Cucumber seedlings were allowed to grow in these spiked soil samples for 10 days, and water soluble Cu 42 was determined via ICP-MS after removing the plants on day 10. After 10 days, the water-extractable Cu in the control , 200 mg kg−1 CuCl2 and 200 mg kg−1 nCu treatment was 1.8, 43 and 24 mg kg−1 , respectively.Thus, within 10 days nCu released a considerable amount of ionized Cu into the soil for plant uptake. These significant differences in bioavailable Cu between exposure to copper salts or nCu highlight the challenge in conducting comparisons. Simply exposing plants to “equivalent” amounts of copper may lead to very different results.Root, stem, and leaves supply and transport mineral nutrients. Results showed that nCu did not disturb any mineral nutrient homeostasis in these tissues except Fe. The Fe concentration in root and leaves were significantly decreased by nCu at all concentrations . In roots, Fe decreased by 67%, 54%, and 66% compared to the control, when exposed to 200, 400 and 800 mg kg−1 nCu, respectively. In leaves, Fe decreased by 56%, 43% and 54% compared to the control. In addition, Fe concentrations in stems also tended to decrease when exposed to nCu, but this was not statistically significant. Previous studies demonstrated a competitive relationship between Cu and Fe uptake: it was observed that in Arabidopsis thaliana and Cucumis sativus, a low Fe supply led to increased Cu concentration in leaves, while high Cu supply lowered leaf Fe concentration.The authors found that the presence of Cu affected the activity of ferric reductase. At high Cu supply, ferric reductase activity was inhibited. This led to decreased demand for Fe by the plant and subsequently less Fe accumulation.

We evaluated mean JJAS precipitation in the GHA during each of the five classes of seasons.At the compound of the Debrebirkan Selassie church in Gondar, Northwest Ethiopia , 5 mm diameter cores were obtained from Juniperus procera trees in May 2007 as part of a larger sampling campaign . Successful cross-dating between 32 trees from five sites in the North Gondar zone was achieved by comparison of the wood anatomy directly on the surface of the samples and skeleton plotting . Cross-dating was evaluated using the computer program COFECHA and the annual nature of the tree rings was confirmed by AMS radiocarbon dating . Oxygen isotope ratios were measured on one tree core in a pilot study to obtain a preliminary insight into the potential environmental sensitivity of this isotopic variable in tree rings. Slivers were cut from absolutely dated annual growth rings representing years 1905–2003. Lignin was oxidized by the in situ generation of chlorine dioxide and hemicelluloses were hydrolyzed from the resulting holocellulose to yield a-cellulose . After sample homogenization, 0.30–0.35 mg of dry a-cellulose was weighted into silver vessels and pyrolysed over glassy carbon at 1,090 C. Oxygen isotope ratios were measured using a PDZ Europe 20-20 mass spectrometer interfaced to a Europa ANCA GSL elemental analyzer at Swansea University.Analytical precision was typically 0.3% as was illustrated by a repeat analysis of samples from 1910 . Two influential variables on tree-ring d18O are the d18O of precipitation and the amount of isotopic enrichment of leaf water that occurs during transpiration . Leaf-water enrichment is driven by the water vapor pressure deficit of the atmosphere under a wide range of environmental conditions.

To test the impact of evaporative enrichment from leaves, we statistically regressed the tree-ring d18O record against monthly and multi-month VPD,rolling bench as estimated by NCEP2 reanalysis. A positive relationship between treering d18O and VPD would suggest evaporative enrichment is an important driver of inter annual tree-ring d18O variability. A non-positive relationship would suggest tree-ring d18O variability is dominated by variations in d18O of precipitation. We compared monthly NCEP2 reanalysis data to treering d18O to test relationships between tree-ring d18O and variables suspected of influencing the source of precipitation in the GHA. Variables chosen were anti-cyclonic circulation of atmospheric moisture over the Gulf of Guinea and STIO. A good inverse proxy for anti-cyclonic moisture transports is cloud cover. For each month and range of months, we calculated the mean cloud frequency over the southern Gulf of Guinea and western STIO . We then found the range of months when this cloud-cover correlated most strongly with tree-ring d18O. Exploratory analysis revealed that another good anti-cyclonic moisture transport index is the sum of meridional moisture transports over southern Africa to the east of the Gulf of Guinea and zonal moisture transports in the western STIO . We repeated the analysis described above for this alternate index. For visualization of the more general circulation features associated with variability of tree-ring d18O, we also compared tree-ring d18O to the circulation fields evaluated throughout this study.JJAS precipitation declined substantially throughout much of the GHA during the 1948–2009 period. Figure 2a is a time series of standardized JJAS precipitation totals in the GHA region considered in this study, outlined in red in Fig. 2b, c. Declines during the 1960s–1980s occurred in concert with the well-known reductions throughout the Sahel . GHA precipitation decreased by approximately 1 standard deviation during the 1950–1989 period , corresponding to decreases of over 30 mm per decade throughout much of the Ethiopian Highlands, over 60 mm per decade in western Sudan, and little change in southern Sudan, northern Uganda, and western Kenya.

Following the 1980s, JJAS precipitation continued to decline in the GHA while there was no decline in precipitation throughout much of the Sahel region west of Sudan . During 1990–2009, mean GHA precipitation was reduced by more than 0.5 standard deviation units compared to 1970–1989. Exceptions were observed in some areas in northeastern Ethiopia and northern Sudan, where post-1989 precipitation increased by over 0.5 standard deviation units. Notably, Fig. 14 in ‘‘Appendix’’ shows a similar spatial structure in recent precipitation trends for the MAMJ season. The similarity in spatial patterns of precipitation trends during JJAS and MAMJ suggests the same mechanism may be suppressing precipitation during both seasons despite the fact that the circulation patterns that cause precipitation during these two season are quite different. Since station coverage has decreased over the past 20 years , we considered 10 alternate gridded estimates of JJAS precipitation in addition to our CHGCLIM data. For the GHA and Sahel regions, Table 1 lists each additional dataset’s rate of change in mean JJAS precipitation total comparing the 1970–1989 to the 1990–2009 period. Eight of the 10 alternate datasets agree with CHG-CLIM that JJAS precipitation did not increase in the GHA after the 1970–1989 period. For the Sahel region, 7 of 10 alternate datasets agree that 1990–2009 totals exceeded 1970–1989 totals. In addition, 9 out of 10 alternate datasets agree with CHG-CLIM that precipitation in the GHA either declined more or recovered less than in the Sahel. Notably, not all precipitation datasets cover the entire 1970–2009 period. If the negative post-1970 precipitation trend indicated by most datasets is real, datasets beginning in 1979 may indicate artificially low precipitation totals for 1970–1989 and datasets ending before 2009 may indicate artificially high totals for 1990–2009. Despite this bias, 5 of 6 datasets not covering the entire 1970–2009 still agree on reductions in GHA precipitation. Finally, while the TRMM 3B43 data have limited temporal coverage compared to the other data sets, they indicate a substantial decline in precipitation over the GHA and no change in the Sahel from 1998 to 2009 .

Although missing data surely hinder the accuracyof interannual GHA precipitation estimates for all gauge based datasets, particularly for the southern Sudan region , the broad consensus from the data considered here is that recovery from the drought in the 1970s and 1980s has been at least partially suppressed in the GHA relative to the Sahel.NCEP/NCAR and ECMWF reanalysis datasets suggest that JJAS vapor transports into the GHA primarily originate from the Atlantic Ocean . Transports into the GHA are almost entirely confined to the lower troposphere below 700 hPa and are likely recycled through the rainforests of the Congo Basin on their way from the Atlantic . Moisture in the Congo Basin appears to mainly originate from the Gulf of Guinea, driven by strong subtropical surface highs over the southern Atlantic and Indian Oceans and a strong low over northern Africa. Moisture is transported from the Congo Basin toward the GHA by low level westerly and southwesterly winds, drawn into the region by thermally driven low surface pressures within the area of the Inter-Tropical Convergence Zone to the north and east into the GHA. There is substantial inter annual variability in the amount of moisture entering the GHA via this pathway. South of the center of the North African surface low, deep convection and enhanced moisture transports from the south and west drive monsoonal precipitation in the Sahel and GHA . For the Sahel,grow table hydroponic the primary driver of inter annual and longer-term variability of JJAS precipitation is the north-south displacement of upper-level jet features, zone of maximum convection, and southern boundary of the thermal low over northern Africa . Wetter years occur when these features are displaced to the north. This rule was apparent in the northern GHA. From 1953 to 1988, northern GHA precipitation correlated very well with the SLP gradient between Sudan and the southeastern coast of the Mediterranean Sea . The rainiest seasons occurred when SLP was anomalously high in Sudan and anomalously low to the north in Egypt and surrounding countries . Variability in this SLP gradient explains inter annual swings in JJAS precipitation as well as the Sahel-like decline in JJAS precipitation from the mid-1950s through mid- 1980s over the GHA. For the southern GHA, JJAS precipitation correlated much more strongly with Bombay SLP during 1953–1988 . This corroborates the findings of Camberlin , who, along with Hoskins and Rodwell , hypothesized that diabatic heating associated with the Indian monsoon supported an intensified tropical easterly jet across Africa and a low-level response across Africa that involved ridging across the Sahara at *25 N, surface lows around 12 N, and westerly wind toward the GHA. NCEP/NCAR and ECMWF reanalyses support the idea that enhanced westerly transports across tropical Africa contribute to enhanced GHA precipitation . Figures 6 and 7 show correlation between various aspects of NCEP/NCAR atmospheric circulation and JJAS precipitation in the northern and southern GHA, respectively . Northern GHA precipitation is correlated with a strong North African surface low and cyclonic southwesterly winds toward the GHA . Correlation with southwesterly wind velocity is not confined to the lower troposphere where the vast majority of water vapor is transported, but instead extends throughout all altitudes below the TEJ . Northern GHA precipitation is positively correlated with an enhanced TEJ . Both of these results are consistent with observations of a relatively weak African Easterly Jet and strong TEJ during wet monsoon seasons in the Sahel region . While correlations between reanalysis atmospheric circulation and JJAS precipitation in the southern GHA were weaker than for the northern GHA , precipitation records for both regions correlate with moisture transport traveling from the Gulf of Guinea and across the Congo Basin .

A main difference between drivers of north versus south GHA precipitation is that southwesterly cyclonic circulation is not necessary to deliver moist air to the southern GHA. Instead, southern GHA precipitation correlates with purely westerly flow across Africa between the surface and 500 hPa. This westerly flow is associated with enhanced surface low-pressure over western India and the western Indian Ocean north of Madagascar , consistent with the results of the SLP analysis. Southern GHA precipitation also correlated with ascending motion over western Africa and on the eastern boundary of the STIO . Correlations with ascending motion were particularly strong over the STIO and weaker over the GHA in the ECMWF analysis. In both reanalyses, correlation with ascending motion was generally confined to the mid- and upper-troposphere between 600 and 200 hPa. The correlation fields indicate that enhanced convection in rainy seasons corresponds with an enhanced TEJ, as was the case for northern GHA precipitation. Previous work has shown that an enhanced TEJ contributes to enhanced instability throughout the atmospheric column during the north African summer monsoon .From 1989 to 2009, the SLP gradient between Sudan and the southern Mediterranean coast strengthened and Bombay SLP declined . According to the strong statistical relationships between SLP and GHA precipitation, these trends should be associated with increasing JJAS precipitation throughout the GHA region . The fact that GHA precipitation did not rebound following the 1980s suggests that a secondary factor worked to suppress it and alter the previously established relationships with SLP. To diagnose these altered relationships, we evaluate trends in atmospheric circulation and moisture transports in recent decades. Figure 8 shows linear trends in atmospheric circulation and moisture transports from 1979 to 2009, according to the NCEP2 reanalysis. Alternatively, see Fig. 18 in ‘‘Appendix’’ for differences between post- and pre-1988 periods rather than trends . Striking features in both figures are strong negative trend in convection and atmospheric water vapor content within and around the GHA. These negative trends appear to be linked to changes in SSTs and convection over the STIO that have been previously associated with negative precipitation trends over the GHA during boreal spring . In an analysis of the MAMJ season, Williams and Funk found that a westward extension of the Indian-Pacific tropical warm pool has led to a westward extension of the convective branch of the tropical Walker circulation over the STIO, and consequently, a westward migration of the western descending branch of the Walker system into northern Africa. Dynamically, increased SST in the STIO has driven large increases in convection and precipitation over the STIO, and the increase in the amount of diabatic energy released during precipitation has led to increased divergence of DSE in the overlying mid- and upper-troposphere. This intensified outflow of mid- and upper-tropospheric DSE from the STIO has caused increased subsidence over northern Africa and decreased moisture transports into the GHA for at least the past 30 years.

Citrus plants and forestry species were set at the same planting line. Row spacing between lines was 5 meters and between plants 2 meters. It was observed that plants with up to 70% shading around noon showed reduction in the development of symptoms of HLB with values between 5% affected canopy and 8% , while in without shading presented 18% of the canopy with symptoms. The incidence of the disease was also lower in citrus plants in the shaded areas , while in the area without shading was 97%. The data were subjected to analysis of variance and, when detected significant difference, were compared by Duncan.Huanglongbing caused by the bacterium Liberibacter asiaticus is the most devastating disease of citrus in the United States today. Current methods for identifying infected trees include scouting for symptoms in the field and detection of Las by quantitative PCR in samples from suspected trees. However, because infected trees may appear asymptomatic and come up negative with qPCR, given the typically focal nature of the infection, there is a great and urgent need for additional detection methods. We describe here an indirect detection method based on Las-induced systemic changes in the microbiota of citrus trees following infection with Las but prior to symptom formation. More specifically, the method entails swabbing citrus leaves, extracting microbial DNA from the swabs, PCR amplifying the bacterial 16S and fungal ITS regions, sequencing using the Illumina platform, and analyzing the data using QIIME,dutch bucket hydroponic a microbial community analysis pipeline. A combination of ordination techniques and taxon abundance rules are then used to call a tree positive, negative, or suspect.

The method has been successfully used to distinguish infected trees from non-infected ones in greenhouse experiments at UC Davis and under field conditions in Texas. Our method is currently being optimized for deployment under California field conditions. This work proposes a simulation model that evaluates the impacts on production and on the citrus plantations depending on the level of infection and the spread of huanglongbing . We used the Individual Based Modeling methodology. In this model, we considered the invasion of psyllids from neighboring orchards and scenarios where the orchard performs the sanitary management, i.e. roguing symptomatic citrus plants and replanting healthy trees. The productivity of citrus trees was calculated through the model defined by Bassanezi RB and Bassanezi RC , which made possible to obtain the orchard yield depending on the incidence of HLB. The productivity and costs depend on the age of the plant, the sanitary management and the epidemiological states. To analyze the impact on orchard yield was made a simplified calculation of cash flow. The sanitary management is guided by the Normative Instruction of Brazilian Ministry of Agriculture, Livestock and Food Supply, which recommend to the citrus farmers to perform the inspection in an orchard every 3 months and roguing symptomatic plants.It is known that the primary means of attraction and reproduction of psyllids is through the shoots of plants, in which the deposition of eggs is made only in young plant tissues. Older plants and young can have different levels of flush, what results in different levels of attraction to the vectors of diseases. The simulation results show an increase of the yield and a lower disease incidence with the sanitary management in long-term. Other contribution of this work is the easy way to change the many parameters and scenarios in the model.

Hamlin and Valencia oranges were each harvested twice per year for two growing years in Lake Alfred, FL and processed into cold pressed oils. During each harvest, both asymptomatic and symptomatic fruit were sampled. There was one sampling early in each orange’s harvest season and a second sampling late in the harvest season. The oils underwent several quality tests including US Pharmacopeia mandated physiochemical tests ; taste panels; aroma panels; and qualitative and quantitative gas chromatography. Overall results showed both symptomatic and asymptomatic Hamlin oils had aldehyde contents below the US Pharmacopeia minimum for both harvest years. For Hamlin oranges harvested in the early season harvest for the 2015- 2016 season, there were significant differences between symptomatic and asymptomatic oils for aldehyde content, specific gravity, UV absorbance, and optical rotation. There were no significant differences between solutions made with Hamlin asymptomatic and symptomatic oils in taste panels. However, there were significant differences between early season Hamlin asymptomatic and symptomatic oils in aroma panels. Several differences were seen between asymptomatic and symptomatic Valencia oils for the US Pharmacopeia tests for both the 2014-2015 and 2015-2016 harvest seasons. Additionally, late season symptomatic Valencia oils exceeded the US Pharmacopeia maximum for specific gravity for both years. There were no significant differences between solutions made with Valencia asymptomatic and symptomatic oils in taste panels. However, there were significant differences between late season Valencia asymptomatic and symptomatic oils in aroma panels. Quantitative gas chromatography showed that multiple compounds important to orange aroma and flavor which are significantly different between asymptomatic and symptomatic samples for both Hamlin and Valencia oils. These compounds include decanal, linalool, citronellal, citronellol, neral, and geranial. The aroma panel results and decrease in concentration of these important orange aroma compounds show that HLB may have a negative effect on cold pressed Hamlin and Valencia oils. The Asian Citrus Psyllid Diaphorina citri Kuwayama, was detected on March 2006, on citrus plants in the residential area of Ciudad Obregon Sonora, one year later it was observed in Guaymas and Hermosillo, Sonora.

Since its discovery in Ciudad Obregon, surveys were carried out in order to determine its regional dispersion and migration to citrus commercial plantations. In 2010 an area-wide strategic management plan was established. The main objective was to keep ACP populations at the minimum level and lower the risk of HLB establishment in the state of Sonora. This plan continues until now and it has been successful in keeping ACP populations at very low levels and no HLB on citrus plants, have been detected. The plan is based in a coordinated effort and mandatory actions in commercial plantations including weekly surveys by yellow sticky traps and tap sampling, two area-wide insecticide applications are made one in the dormant stage and other in the fall , besides during the growing season if monitoring indicates that an orchard is over the regional ACP population mean it is considered as hot spot and recommended for control. Citrus plants in rural and residential areas are also monitored and sprayed to reduce the ACP population at the regional level. Results of the area-wide management plan are considered satisfactory after ten years of the first detection of ACP in Sonora. Considerable reduction in ACP populations has been achieved and not HLB plants have been detected on the surveys realized. However, in the last two years HLB infected Psyllids were detected in rural areas of Southern Sonora and an aggressive action plan was implemented controlling ACP populations and eliminating citrus plants in the area were these infective Psyllids were observed. Further surveys and psyllid analysis for HLB have not shown any more infected insects or citrus plants in the area. Surveillance activities play an integral part in disease prevention and control, and underpin the three main stages of disease mitigation: the prevention of entry and establishment of exotic pathogens; the detailed investigation of more established pathogens; and the monitoring of disease control measures. As with any disease mitigation measure, it is important that surveillance activities are planned, implemented,dutch buckets system and evaluated using scientifically valid approaches. This process of “survey validation” allows us to ensure that surveillance schemes are performing satisfactorily , and allows appropriate adjustments to be made to improve survey performance and efficiency. Whilst survey validation is best achieved using a systematic methodology, the range of different surveillance aims in different settings means that this methodology must also be flexible. One way to achieve a suitable balance of structure and flexibility is to identify specific attributes of the surveillance aims as part of the validation process, which can then be used to determine which precise validation approaches are most appropriate. Although HLB is a threat to the citrus industry throughout the USA, its impact upon the industry to date at the state level has varied. In Florida, HLB is thought to be well established throughout all commercial orchards; in Texas, it has been reported in some orchards but at lower levels than in Florida; and in California, the disease has not yet been detected in commercial orchards. As a result of this variation, the surveillance activities in place differ between the three states. We use a recently developed survey validation framework to evaluate these HLB surveillance activities. Since our approach allows the different epidemiological scenarios in each state to be explicitly accounted for within the context of a standardised framework, strengths and weaknesses of the different surveillance schemes can be better identified. Many citrus production areas are currently threatened by HLB. Whilst minimising the risk of spread of the HLB bacterium and insect vector are important strategies for reducing the risk of HLB entry, there will always be a potential risk of introduction. This means that effective early detection surveillance activities – focussed at detecting Las infection at an early stage – must be in place. Our previous work has demonstrated that the epidemiology of a pathogen should be considered when developing surveillance strategies.

The presence of insect vectors as well as host plants in the HLB pathosystem adds another layer of complexity to the issue of surveillance, since it leads to the question of whether to sample from hosts, vectors, or both. Although this question has considerable implications for the design of the surveillance strategy, many surveillance schemes focus on sampling from host plants, with vectors sampled more opportunistically. To investigate this issue further, we have developed a statistical model of host and vector sampling and linked this to a mathematical model of pathogen transmission through the HLB pathosystem as a whole. The resultant model allows us to quantify the relative sampling efforts and/or costs required from hosts and vectors in order to detect a specified incidence of infection in either. From this, we demonstrate that the overall incidence at first detection is minimised when samples are exclusively collected from either hosts or vectors but not from both. As well as identifying whether hosts or vectors should be sampled, our method gives a numerical output which indicates how robust this decision is to changes in sampling costs. This has potential for use as a simple tool to determine where best to place sampling resources whilst accounting for both epidemiological issues and economic constraints. Sweet oranges have little resistance to HLB, a disease presumably caused by Canadidatus Liberibacter asiaticus . As HLB has become widespread in Florida over ten years, we have very carefully followed the extensive collections of our breeding materials including mutants and germplasm accessions within the UF-CREC citrus breeding program. We have found that two 13 year-old Valencia clones and one Kansu orange are apparently tolerant to HLB. All multiple propagations of three clones have been rejuvenated by continual vigorous growth of new shoots with few foliar and fruit symptoms. In this study, we used comparative transcriptional and anatomical analyses to evaluate gene expression and anatomical differences between three tolerant Valencia types and a susceptible standard Valencia. Underlying tolerance mechanisms revealed by RNAseq and anatomical analysis will be described. The results potentially lead to identification of key genes and the genetic mechanism in irradiated Valencia to restrain disease development. The psyllid Diaphorina citri is the vector of the bacteria associated with huanglongbing , which is the most destructive citrus disease worldwide. Chemical control is the primary tactic against this insect. However, alternative methods are important to achieve a more effective control in an integrated pest management programs. Thus, this research was carried out to assess the influence of different kaolin formulations on the settling and probing behavior of D. citri. In both studies, two wettable powder kaolin formulations were sprayed three times at different concentrations on sweet orange plants. In the experiment to assess the settling behavior, three concentrations of both formulations were tested. A non-choice test was performed, where 16 adult psyllids were released in a cage with seedlings of the same treatment, and the number of psyllids/plant at different time intervals was counted. For the probing trial, the electrical penetration graph technique was used.

The portal includes user-friendly manual curation tools to allow the research community to continuously improve the knowledgebase as more experimental research is published. Bulk downloads are available for all genome and annotation datasets from the FTP site.The inability to culture the huanglongbing -associated pathogen Candidatus Liberibacter asiaticus , has provided significant challenges for understanding the molecular mechanisms controlling disease progression. To provide insight on how CLas manipulates citrus during infection, we used a comparative proteomics approach to identify dynamic changes in the phloem proteome of navel sweet oranges. Navels were mock or graft inoculated with the California CLas strain HHCA. At ten months post inoculation, crude phloem was extracted from uninfected and infected navel trees. Differentially expressed proteins were identified using label-free quantitative mass spectrometry. We identified various protease classes such as glycosidases, serine proteases, cysteine proteases, and aspartic proteases as significantly induced. Homologous proteins act as immune-related proteases in Arabidopsis, tomato, and maize and are targeted by diverse pathogens to promote infection. The over expression or deletion of specific proteases in crops such as maize, tomato and potato have shown to have enhanced resistance and susceptibility to pathogens, respectively. To identify whether the identified proteases are also induced in citrus field samples, we acquired uninfected and infected samples from a citrus grove in Texas. We used mass spectrometry to quantify protease abundance. Additionally,hydroponic grow system we assayed for activity using activity-based protein profiling . ABPP permits the quantification and identification of active proteases.

Our current data demonstrates that the abundance and activity of several proteases is increased in infected samples. A specific group of proteases display enhanced abundance but no altered activity during infection. Based on these observations, we hypothesize that CLas suppresses citrus defense responses by targeting host protease activity. Identifying the role of citrus proteases will contribute to the development of HLB biomarkers and specific proteases could be manipulated to enhance resistance to CLas. Huanglongbing is a widespread and devastating citrus disease. The HLB-associated pathogen, Candidatus Liberibacter asiaticus is transmitted by the Asian citrus psyllid, a piercing-sucking insect. The Asian citrus psyllid feeds on citrus phloem and can acquire CLas by feeding on an infected tree. In this study, we used mass spectrometry and quantitative proteomics to identify proteins that were differentially expressed in navel phloem during infection. Multiple differentially expressed proteins were proteases and we were able to identify the same classes of proteases as differentially expressed in infected navel trees sampled from the field in Texas. These proteins have the potential to serve as biomarkers for HLB as well as targets that can be manipulated for disease control. We have also investigated the genetic diversity and conservation of predicted secreted proteins present in CLas. We analyzed eight currently available genome sequences and sequenced two new strains from China and Florida. Using stringent criteria, we identified a group of CLas secreted proteins that are conserved across all sequenced strains. Collectively, the completion of this research has enabled a greater understanding of how the plant responds to CLas infection as well as identified plant and bacterial components with promise for pathogen detection and control. Up to now, cultivation of citrus greening pathogens has been attempted in many laboratories. “Candidatus Liberibacter asiaticus ” is known as a fastidious bacterium that is unculturable in many traditional culture media. To facilitate basic research and establish definitive diagnosis, we are developing a new selective culture medium for Las and the other related species.

At first, we examined whether Las can multiply in some traditional media and Las specific media reported previously. Living Las cells were extracted from the infected citrus leaves according to our procedure which was developed in our previous reports. The multiplication of Las cells was evaluated from the copy number of Las DNA amplified using real-time PCR. The results indicated that almost existing media do not support the multiplication of Las cells enough. Thus we explored constitutions of a new Las selective medium. By comparing with other bacterial genomes, inability of Las genome to produce essential metabolic pathway was predicted. Then based on this prediction, we have developed a culture medium by adding the compounds to replenish the shortage of nutrients for Las. And a new medium could induce the multiplication of Las cells. But multiplication was very slow and there was no visible typical colony on a solid medium. In spite of lack of visible colonies, after 14 days of incubation, Las cells were detected by in situ hybridization. Moreover, RNA-seq, RT-PCR and partially DNA sequencing could be performed from the invisible agents on the medium. Towards establishing the practical and reproducible method of cultivation, we continue to improve the medium. Response of Candidatus Liberibacter asiaticus to antibiotic treatment in vitro was demonstrated experimentally using a new medium. Primarily, a metabolic pathway analysis of Ca. L. asiaticus genome was carried out using KEGG database. Comparative approaches between Ca. L. asiaticus and closely-related bacterial species allowed us to identify a group of genes that do not remain in the pathogen’s genome and select essential nutrimental components for the preparation of a new culture medium, including sugar, amino acids, fatty acids and vitamins that cannot be synthesized by the pathogen. Quantification of Ca. L. asiaticus was investigated for a month using real time PCR assays and in situ hybridization. Ca. L. asiaticus became detectable two weeks after initiating incubation and thereafter. Very small colony-like patterns appeared in the media, but the further growth was not observed. Furthermore, in-vitro assays to examine pathogen’s responses to eight antibiotics was conducted using the new medium. The results showed that Ca. L. asiaticus did not proliferate in the culture medium supplemented with polymyxin B. On the other hand, supplement of tetracycline or oxytetracycline in the medium resulted in accelerating the growth of the pathogen.

The mode of action of tetracycline and oxytetracycline on Ca. L. asiaticus remained unknown. In short, this study suggests that a culture-dependent approach may give us vital insights into further understanding of the behavior of Ca. L. asiaticus and contribute to the screening of potential agents and chemicals for citrus greening disease management. Development of methods to fight Huanglongbing , caused by “Candidatus Liberibacter asiaticus” , has been hampered by lack of ability to culture CLas in the lab, which precludes the testing of gene functions that may be involved in CLas viability or virulence. Identification of such gene functions will have an enormous impact on the ability to control CLas and eventually allow development of integrated strategies to stop HLB spread by the Asian citrus psyllid and the resulting economic devastation to citrus agriculture. Development of the ability to culture CLas in vitro will open up new avenues of research that are guaranteed to provide a game change in knowledge of CLas biology and transmission, including a culturing platform necessary for development of an amenable genetic system. The Ca. Liberibacter Culturing Consortium was established to develop such an in vitro culture system and make it available to the citrus industry and academic research community by establishing a system for host cell-free culture of CLas, providing standard operating procedures for culture of CLas to the research community, and providing cultures through standard repositories. Progress to date in these efforts will be reported.Candidatus Liberibacter asiaticus , the most prevalent causal agent of huanglongbing citrus disease in North America and Asia, is a phloemlimited and unculturable bacterium. However, during the past few years,ebb flow table genomes of several Liberibacter species were sequenced suggesting that members of this genus have suffered a significantly genome reduction. Comparative genomics of Las has allowed the prediction of the putative effector proteins, potentially involved in virulence. In this scenery, where small secreted protein could play crucial roles between plant and pathogen interaction, effector proteins identification is a complex objective. Plant defense can be triggered by the host perception of conserved pathogen-associated molecular patterns . Pathogens in turn can suppress PAMP-triggered immunity, causing disease. Identification of Las effector proteins is a key step to understand the mechanisms by which Las disrupt the host response. To provide a functional basis for this hypothesis, eight of these sequences were cloned and expressed fused to green or red fluorescent proteins under the control of 35S promoter. Agrobacterium-mediated transient expressions were performed in Nicotiana tabacum and N. benthamiana leaves. Bacterial protein expressions were monitored over a 10-day period by fluorescence and confocal laser scanning microscopy. All tested genes shown expression in plant cells, and revealed different subcellular distribution patterns respect to the control. CLIBASIA_04560 encoded a hypothetical protein which is localized in nucleus when it is transiently expressed in N. benthamiana. More interesting, this protein triggers an increase of H2O2 production respect to the control without necrotic or cell death phenotype in N. benthamiana after 3 dpi.

Now, we are studying if Clibasia_04560 may act as an effector protein modulated the energetic metabolism in citrus plants. Asian citrus psyllid is a known vector of Huanglongbing disease that is an imminent threat to the California citrus industry. At present, controlling the psyllid vector is the best management strategy to delay the spread of HLB, while disease management tools are developed. In recent years ACP finds from trap catches have increased in the San Joaquin Valley especially in residential areas and at packinghouses and juice plants. Researchers have attributed HLB spread in Florida to human-assisted transport of infected psyllids and infected plant material including bulk citrus. ACP quarantine boundaries and HLB quarantine boundaries have been established in California and preharvest treatments are required to ship insect-free bulk citrus between quarantined zones to mitigate the spread of ACP and eventually HLB. It is clear from the finds at juice plants and packinghouses that current methods of orchard disinfestation are not reducing psyllids sufficiently and psyllids are being moved in bulk citrus In the context of identifying a postharvest treatment to control ACP after harvest and before shipping to packinghouses, University of California Researchers in collaboration with USDA-ARS and Fruit Growers and Supply have begun testing the potential of fumigation on Asian Citrus Psyllid to develop a treatment plan for disinfesting bulk citrus. We discuss the prospects for use of postharvest fumigation to disinfest bulk citrus prior to shipping to other regions of the state. Asian citrus psyllid feeding behaviors play a significant role in the transmission of the phloem-limited Candidatus Liberibacter asiaticus bacterium that causes the economically devastating citrus greening disease. Recent studies have shown a fibrous ring of thick-walled sclerenchyma around the phloem in mature citrus leaves that is more prominent on the lower compared with the upper side. We performed Electrical Penetration Graph studies on ACP adults placed on lower or upper surfaces of young or mature Valencia orange leaves. Feeding sites on the same leaf tissues were then sectioned and examined by epifluorescence microscopy. Based on the EPG recordings and histological correlations, we found that thick-walled fibrous sclerenchyma located around the phloem of mature citrus leaves significantly reduced phloem ingestion by psyllids placed on the lower leaf surface compared with ingestion from the upper surface of mature leaves or on young leaves. The longest duration of phloem ingestion was observed from the upper side of young flush leaves that had the least developed sclerenchyma. Bouts of phloem salivation , however, were significantly longer on mature leaves compared with young flush. ACP adults made consecutive phloem feeding attempts on the lower side of mature leaves and those bouts resulted in unsuccessful or shorter periods of phloem ingestion. ACP adults also made more frequent and longer bouts of xylem ingestion on mature leaves compared with psyllids placed on young leaves. Our results support the hypothesis that the presence of a thick, well-developed fibrous ring around phloem tissues of mature leaves acts as a barrier to frequent or prolonged phloem ingestion by ACP from citrus leaves. This may have an important role in limiting or preventing CLas acquisition and/or transmission by ACP, and could be used for identification and development of resistant citrus cultivars. Phytophagous insects including Asian citrus psyllids use multiple sensory modalities [vision, olfaction, contact chemoreception, gustation , perception of auditory or vibrational stimuli] to locate host plants or conspecifics.

With the recent use of chemical nerve agents such as sarin, there is continued interest on the part of many governments in stockpiling BuChE as a countermeasure. Currently BuChE is purified from outdated blood supplies; however, the high cost of this route and its low supply limit its utility. It has been estimated that extraction of BuChE from plasma to produce 1 kg of enzyme, which would yield small stockpile of 2,500 400- mg doses, might require extraction of the entire US blood supply. Large amounts of the enzyme are required for effective prophylaxis because of the 1 : 1 enzyme/substrate stoichiometry needed for protection against OP agents. Not surprisingly, recombinant routes have been explored and the enzyme can in fact be produced by microbial fermentation, animal cell culture, and transgenic goats and stably or transiently expressed in Nicotiana, albeit at modest levels of 20–200 mg/kg fresh weight biomass, with yield improvements being the target of ongoing research. The bacterial product is nonfunctional and the mammalian cell culture products do not have the plasma 1/2 needed for prophylaxis and may be difficult and expensive to scale, as discussed by Huang et al.. Goatmilk produced BuChE can be obtained at 1–5 g/L milk, but consists mostly of dimers, is undersialylated and has short plasma 1/2. While expression yields are impressive, transgenic animal sources face challenges of herd expansion to satisfy emergency demand, as well as potential adventitious agent issues, and these challenges need further definition. Furthermore, of these options, only plant-based bio-synthesisyields an enzyme that is sialylated and appears to reproduce the correct tetrameric structure of the native human form in sufficient yield to be commercially attractive; hence, the plant-based route became the basis for our modeling exercise. Not surprisingly,grow hydroponic the plant route for BuChE manufacture is also the subject of continued DARPA interest and support .

BuChE can be produced stably in recombinant plants or transiently in nonrecombinant plants by viral replicons delivered by agrobacterial vectors introduced into the plants via vacuum-assisted infiltration. Relative to stable transgenic plants, the advantages of speed of prototyping, manufacturing flexibility, and ease of indoor scale-up are clearly differentiating features of transient systems and explain why this approach has been widely adopted in the manufacture of many PMP . In our analysis of BuChE, we used expression yields from several sources that evaluated various Agrobacteriummediated expression systems, including Icon Genetics’ magnICON expression technology. Magnifection should be familiar to most readers of this volume as it has been applied in R&D programs throughout the world and its features have been the topic of multiple original studies and reviews ; therefore, the method is not described here in further detail. Likewise, the process of vacuumassisted infiltration has been described in detail by Klimyuk et al., Gleba et al., and others and is not further explained here.For BuChE, we modeled the use of an N. benthamiana transgenic line modified to express the mammalian glycosylation pathway, beginning with a mutant host lacking the ability to post translationally add plant-specific pentoses but with the ability to add galactosyl and sialic acid residues to polypeptides, based on work recently reported by Schneider et al.. Use of this host obviates the need to enzymatically modify the plant-made polypeptide in vitro after recovery to ensure the presence of correct mammalian glycan, a procedure that could substantially increase the cost of the AI. A glycanengineered host can be produced in two ways, by stable transformation or via use of multi-gene agrobacterial vectors. The feasibility of sialylation via the latter approach was shown recently by Schneider et al.for BuChE. Although there is an extra element of time required to develop a stable transgenic host compared to the transient modification of a pathway, the availability of a transgenic plant obviates the need to manufacture several Agrobacterium vectors carrying the genes for the product and two binary vectors carrying genes for the sialylation pathway; a procedure that would require additional capital and operational investments to generate multiple inocula in large scale.

Therefore, for modeling upstream processes, we assumed that transgenic seed was available and that the resultant BuChE would have mammalian glycans and form tetrameric structures, and hence its biological activity and plasma half-life would be comparable to the native human enzyme.To model downstream purification of BuChE, we assumed harvest and extraction at 7 days after inoculation. Biomass disruption was by homogenization, followed by filtration and clarification, as generally described, but with modifications required for scale-up as indicated in Results and Discussion. Purification of the enzyme was by procainamide affinity chromatography. In the overall process, plant growth, inoculation, and product accumulation steps occur indoors in controlled environments, and extraction, clarification, and final purification of BuChE take place in classified suites, so that manufacturing and release of the enzyme can be compliant with FDA cGMP guidance for human therapeutics. Design premises for this process, specific assumptions used in modeling, and resultant cost calculations are presented .Cellulases currently under evaluation in bioethanol programs are all produced by microbial fermentation. Despite decades of research on lowering cellulase manufacturing costs, these enzymes still account for 20–40% of cellulosic ethanol production costs. Hence, lowering the cost of the bio-catalyst is critical to the eventual adoption of bio-fuel processes that utilize renewable plant biomass feed stocks without competing with food or feed supplies. An alternative to fermentation produced cellulases is the production of these enzymes in crop plants, with the ultimate goal of producing cellulases at commodity agricultural prices. This process concept was modeled to estimate enzyme and ethanol costs produced by this approach. Should such a process for cellulases prove economically viable, it might encourage the production of other cost-sensitive PMB as well as bio-materials, food additives, and industrial reagents.Scale requirements and cost limitations of cellulases for biofuel applications constrained us to model production to open fields, with minimal indoor operations. We initially surveyed two scenarios for inducing production of cellulases in field-grown plants. The first was adaptation of the typical agroinfiltration method. Nomad Bioscience has reported successful substitution of the agroinfiltration step with “agrospray,” a technique in which a suspension containing the Agrobacterium inoculant is admixed with a small amount of surfactant and sprayed onto the leaves of host plants. This approach eliminates the necessity to grow plants in containers , a requirement imposed by the mechanics of the vacuum infiltration treatment in current procedures.

Concomitantly, it also eliminates the cost of setting up and operating commercial-scale vacuum chambers, robotic tray manipulators, biomass conveyer systems, and so forth. Thus, this new approach should enable large-scale field inoculation of plants with agrobacteria and the production of biologics with more favorable economics. While we modeled the costs of producing cellulases via the agrospray approach, the sheer volume of enzymes needed for commercial-scale cellulosic ethanol processes necessitated a large investment in inoculum production infrastructure, including multiple fermentation trains and associated processing equipment. Further, the most efficient method of inoculating large areas was by aerial spraying, a procedure that not only entailed higher cost but that would also face regulatory uncertainties over spraying GM bacteria. We opted instead for an alternative model using transgenic N. tabacum plants, each line of which carries an ethanol-inducible gene for one component enzyme of the cellulase complex. Synthesis of the cellulase is triggered by application of a dilute solution of ethanol onto the leaves, a process that has been demonstrated in small scale using a double-inducible viral vector.We assumed that the dilute ethanol solution would be applied via ground irrigation systems that are currently used in agricultural practices,mobile grow rack instead of aerial tankers. It was also assumed that the ethanol would be taken off as a side stream from the associated ethanol production facility that uses the cellulase enzymes. In so doing, we obviated the need to produce multiple inocula of GM bacteria and deliver them via aerial spraying. We were also able to model higher biomass density as well as higher expression yields of the enzymes in planta. These changes resulted in multiple economic benefits and were therefore adopted in our calculations.Issues that are important in PMP, such as mammalian-like glycosylation or other post translational modifications, high purity, or specific formulation, are not relevant in the manufacture of cellulases and hence we modeled the use of conventional Nicotiana species in the production of the several enzymes necessary for complete saccharification of feed stock.The use of agricultural crops to produce enzymes at low cost has been suggested. In this case study, we modeled the use of stable transgenic N. tabacum varieties, each modified to express one cellulase protein upon induction with dilute ethanol. The process is based on inducible release of viral RNA replicons from stably integrated DNA proreplicons. A simple treatment with ethanol releases the replicon leading to RNA amplification and high-level protein production. To achieve tight control of replicon activation and spread in the non-induced state, the viral vector has been deconstructed, and its two components, the replicon and the cell-to-cell movement protein, have each been placed separately under the control of an inducible promoter. In greenhouse studies, recombinant proteins have been expressed at up to 4.3 g/kg FW leaf biomass in the ethanol-inducible hosts, but seed lines for field application have yet to be developed. In our modeling, we assumed that each transgenic line would have been already field tested and available for implementation. We also assumed that large-scale stocks of each transgenic seed would need to be produced and have included this unit operation in our cost calculations. Because cellulases are needed in different ratios to effect saccharification of different feed stocks, we assumed that seeds would be mixed at the appropriate ratios and that the seed mixtures would be planted directly in the field. At maturity, what one would expect is a field of plants representing all the needed cellulase classes in the appropriate ratio for the intended feed stock.

The current method of hydroponic cultivation of seedlings for transplantation to open fields, a common commercial tobacco cultivation practice to ensure germination and plants with good leaf size and quality, was substituted by direct seeding for more favorable economics. For example, traditionally tobacco may be grown at 12,000–16,000 plants/ha depending on variety. Higher-density seedling production for nontraditional uses of tobacco has been reported, targeting planting densities of over 86,000 plants/ha. While transplanting ensures germination and quality, there is an economic limit to the scale at which it can be deployed with highly cost-sensitive AI, leading to interest in direct seeding practices. Experimental high density cultivation studies via direct seeding have reported 400,000 to over 2 million plants/ha and biomass yields exceeding 150 mt/ha. Our modeling included these higher-density practices to determine economic impact.In contrast to typical PMP products, the cellulases would not be extracted after accumulation; rather, the plants would be mechanically harvested and transported to a centralized facility for silaging and storage. Since the cellulase enzymes need to be continuously supplied to the saccharification process in the bio-ethanol plant and the harvested tobacco is only available for a limited period during the year, the silage inventory would increase during the tobacco-harvesting period and would decrease during the fall/winter. Cellulase activity in the ensilaged biomass is expected to be stable during the off-season storage. For feedstock conversion, cellulase-containing biomass would be mixed with pretreated lignocellulosic feed stock under controlled conditions to effect saccharification. Although not considered in this economic analysis, this feed stock replacement could also reduce corn stover feed stock requirements and associated costs. After separation of solids, the sugar solution would be fermented conventionally into ethanol, followed by distillation. The overall process we modeled is based on the US National Renewable Energy Laboratory process described by Humbird et al, with substitution of fungal cellulase production in the NREL model by the cellulases stored as silage described herein. Design premises for this process, specific assumptions used in modeling, and the resultant cost calculations are presented .Process flow sheets for rBuChE production are shown. The seeding and indoor growth of N. benthamiana is shown in Figure 1.Figure 2 shows the agrobacterial seed train and production fermentor , the vacuum infiltration system , and the plant incubation facility for the infiltrated plants .

Whereas William Kent may have ‘leaped the fence, and saw that all nature was a garden’, to jump or destroy the wall of a horticultural expo is to typically find the periphery of a city, complete with its own implied social delineations. It is in this context that the dissolution of the physical and psychological frame of the institutionalized expo itself— rather than the frames of the individual gardens within—that is the more potent force in contemporary landscape and urbanism.During the past decade, grocery stores have expanded into underserved urban communities with growing awareness of ‘food deserts’ and their associations with urban health disparities and economic disinvestment.Even though scholars contest whether physical access to grocery stores truly improves food accessibility and health , policy advocates continue to promote grocery stores as the ultimate solution to urban food deserts. Federal, state, and local governments have followed suit with the creation of incentives and tax breaks for food retailers that locate in underserved urban communities.Despite numerous studies on food deserts and their potential impacts, there are few explorations of the prescribed “solution” of grocery stores. Moreover, recent studies on the impacts of new and recently closed urban grocery stores tend to focus on resident behavioral changes . Thus, scholars and policymakers alike have limited understandings of the broader neighborhood implications of grocery stores newly introduced into underserved urban communities. This dissertation addresses this gap by analyzing how local organizations and agencies pursue grocery development in response to neighborhood issues. By exploring local processes, I aim to understand whether governments should continue to incentivize urban grocery stores and if so, under what conditions. As such, the ultimate goal of this dissertation is to understand the conditions for successful grocery development across contexts. Using a comparative case study approach, I analyze the historical drivers, planning processes, and outcomes of recently completed ground-floor grocery stores in two distressed San Francisco Bay Area communities: a Fresh and Easy Neighborhood Market in San Francisco’s Bayview Hunters Point and the Mandela Foods Cooperative in Oakland’s West Oakland. Despite their marked differences, both stores were cited in national advocacy efforts around the federal Healthy Food Financing Initiative . In 2005, British supermarket magnate, Tesco created the Fresh and Easy chain primarily for untapped urban markets and health and cost-conscious consumers in California, Arizona, and Nevada.

In 2006, amidst massive grocery closings citywide, former San Francisco Mayor Gavin Newsom,flower pot wholesale the Office of Economic and Workforce Development, and the Department of Public Health coordinated Fresh and Easy’s entry into San Francisco’s distressed Bayview Hunters Point neighborhood. As the first new grocery store in the neighborhood in decades, Fresh and Easy represented a convergence of previously disparate efforts around public health, economic and workforce development. As such, the City’s vision of grocery development was consistent with the rhetoric of federal food access programs. Yet ultimately, national and global capitalist forces led to the store’s demise. In September 2013, Tesco sold the Fresh and Easy chain to a private equity firm, following two years of massive profit losses. The Bayview Hunters Point location was one of numerous stores that were closed as a result. Despite similar underlying socio-economic conditions, West Oakland’s first coordinated grocery store efforts took a markedly different form. Beginning in the late 1990s, West Oakland residents and activists created farmers markets, mobile grocery stores, and community gardens—all with the goal of establishing a full service cooperative grocery store. For the next decade, environmental justice activist Dana Harvey led efforts to realize this vision. Following financial hurdles and disputes with developers over retail space, Harvey and her team of consultants negotiated a 2300 square foot ground floor retail space. In June 2009, the worker-owned Mandela Foods Cooperative and café opened for business. As of 2013, Harvey is exploring possibilities for expansion with funding support from the California Fresh Works program. Through an institutional analysis of the planning processes, I find that both Fresh and Easy and Mandela Foods reflect distinctive neighborhood revitalization legacies, critical moments of institutional capacity building, localized versions of national policy narratives, and the role of charismatic leaders in grocery store implementation. While national narratives shape the rhetoric of urban grocery development, ultimately local context dictates how food access issues are defined, who addresses them, and how. In Bayview, a city-led grocery agenda entailed top-down planning processes that aligned with the corporate model of the Fresh and Easy store. Ultimately, collaborations across government agencies and a systematic role of the Department of Public Health failed to sustain the community’s first new grocery store. In West Oakland, an activist-defined grocery agenda produced grassroots planning processes consistent with the community-based model of the Mandela Foods Cooperative. Conflict and community protests eventually won out—albeit for a scaled-down version of West Oakland activists’ original vision. These findings suggest that federal grocery incentive programs should: 1) maintain a broad framework that enables local communities to define food access problems and their solutions on a case-by-case basis, 2) encourage diverse solutions not limited to grocery stores and supermarkets, and 3) emphasize community reinvestment goals.As mentioned in the beginning of this chapter, urban grocery stores are now on federal, state, and local policy agendas. Much of this current momentum is the result of an expansive body of research on ‘food deserts,’ which has pointed to urban grocery stores as a comprehensive solution to food access, health disparities, and economic disinvestment.

As early as the 1970s, government agencies and researchers profiled the phenomenon of “supermarket flight” due to deindustrialization and the consolidation of the grocery industry . The combined impacts of these trends contributed to the continued “suburbanization” of grocery stores with that of housing and employment centers. Already since the 1960s, Community Development Corporations helped reintroduce grocery stores and other urban retail into underserved areas with expanding federal programs for neighborhood revitalization . Urban grocery stores entered more squarely into federal policy agendas with the push for “inner city revitalization” beginning in the 1990s and new evidence of the “urban grocery gap” . By the late 1990s and early 2000s, researchers conducted the first detailed accounts of the socio-economic and health impacts of lacking urban grocery stores in the UK. This research had a resounding impact on domestic research, recasting the “urban grocery gap” as the phenomenon of food deserts. In the U.S., the visceral metaphor of the ‘food desert’ effectively shifted discourses on the function of urban grocery stores from economic reinvestment to health promotion. The greater extent of food desert research that followed suggested a positive correlation between the lack of grocery stores and poor health outcomes in poor urban communities . A separate but related body of literature linked food environments more generally to growing rates of childhood obesity in the U.S. . These studies directly informed the first comprehensive federal efforts focused dually on health promotion and retail revitalization, namely First Lady Michelle Obama’s Let’s Move childhood obesity initiative and the Healthy Food Financing Initiative discussed at the beginning of this chapter. Already since the 1990s, community-based organizations nationwide addressed urban food access issues through “community food security” initiatives—alternative modes of food production and consumption ranging from farmers markets, produce stands, mobile grocery stores, and community gardens. Overall, policy and practice were built on similar assumptions about the contributions of new food retail outlets on the health and livelihoods of disenfranchised urban residents. However, researchers, policymakers, and journalists—with varied technical backgrounds and political leanings—continued to contest the claims of research and policies that purported the promise of urban grocery stores. For example,berry pots some researchers found that areas defined as food deserts are not devoid of food stores as the term would suggest. Others found that areas otherwise defined as food deserts can in fact be replete with corner markets and ethnic food stores that meet culturally preferred food needs .

Even in spite of this evidence, scholars have agreed that poor communities tend to lack stores that provide greater fresh food options . Furthermore, disinvested communities become targets for fast food establishments, convenience stores, and liquor stores, which may contribute to poor diets and health disparities . As such, some prefer to characterize underserved communities as ‘food swamps’ as opposed to food deserts to emphasize the ubiquity of “low quality” food options . These debates came to a head in late 2011 and early 2012, when The Economist and The New York Times highlighted several longitudinal studies claiming that the introduction of grocery stores into poor communities had no effect on health outcomes and that cost was more of a barrier to healthy food access than physical access . While these suffer from methodological shortcomings, even proponents of urban grocery stores urged policymakers to fully consider non-physical means of improving urban food access. This includes greater attention to nutrition education, food affordability and the role of food assistance programs, and efforts to expand healthy food offerings at corner markets and liquor stores in underserved areas . This raises the question—should grocery stores be developed in poor communities at all? Even the critics say yes. It is an undisputed fact that poor communities tend to lack grocery stores, by their very nature of being under-resourced, disinvested areas. The issue is the definition of the problem and how it points to a rather simple solution that is ultimately inconsequential to the broader issues faced by poor communities. For example, community activists and researchers argue that perhaps the root of the problem is the use and operationalization of the term ‘food desert,’ a term that may not fully capture food access issues as experienced by disenfranchised residents. Along these lines, researchers argue that a purely spatial definition of food deserts fails to capture the broader political-economic factors that contribute to inequitable access to resources in poor communities. For example, Donald argues that singularly “built environment solutions” to food access issues fail to consider the problem of food cost—healthier food items cost more due to agricultural subsidies that favor the production and distribution of low-cost, highly processed, “unhealthy” food. argues that the mere designation of food deserts constitutes ‘neoliberal paternalism’—the assumption that the plight of urban communities are ultimately only solved through top-down market solutions .Scholars cast urban grocery stores as either a normative solution or a faulty “build-it-and-theywill-come” approach that requires redefinition and reassessment. Still, the practical reality is that grocery incentive programs continue to expand at local and state levels. Leading corporate retailers such as Walmart, Target, Kroger, and SuperValu continue to reenter urban markets by creating “urban format” versions of their largely suburban supermarket chains. Community based organizations are pursuing their own alternatives to chain supermarkets through corner store conversions, food cooperatives, mobile grocery stores, and farmers markets. In sum, urban grocery stores will continue to be developed in under served urban areas if not for current policies but due to industry and urban trends. What does this mean for future research? Shannon argues for research that “questions the naturalizing language of market relations that positions low-income communities as just another emerging market” . On a practical level, Donald calls for studies that explore: 1) before/after assessments of communities where new grocery stores are introduced, 2) how the changing grocery retail environment shapes local implementation, and 3) the institutional context and “localized geographies” of food access issues. In line with planning scholars and critical geographers, this dissertation reinforces the idea that future research must account for the complexity of food access problems and its solutions. First, this dissertation provides a detailed account of grocery planning processes to complement spatial and quantitative studies of food deserts. The greater extent of literature on urban grocery stores has involved assessments of the phenomenon of food deserts—namely evaluations of the “problem” of lacking food deserts and its possible impacts. In light of recent critiques, scholars have made strides in redefining food deserts and how food accessibility more generally might be measured. While there is ample evidence to suggest the “problem” of lacking grocery stores exists, there are few detailed accounts16 of how local communities and municipalities themselves define the problem and choose to address it through grocery development. Second, this dissertation explores grocery store development as a set of processes instead of as a finite solution.

Since all three variants showed similar binding kinetics in BLI experiments, we hypothesized that the differences in EC50 may result from rCMG2-Fc stability differences between variants during the 8-h cell incubation period at 37°C. With a less stable variant, the fraction of functional rCMG2-Fc decays over time, together with the fact that CMG2 and PA binding is reversible , resulting in a higher EC50 compared to more stable variants. The observed binding to PA was the same before and after incubation at 37°C for APO and Agly variants . For the ER variant, the fraction of functional rCMG2-Fc decayed over time, and a significant drop was observed for the overnight incubation sample , which explains the higher EC50 for the ER variant in the TNA . GnGnXF, MAN8, and Agly rCMG2-Fc glycoforms were each independently simulated for 100 ns. Figure 8 shows the initial and final conformations of all glycoforms from the simulations. The images at t = 0 are all aligned by the Fc CH2 domain; the images at t = 100 ns are each independently aligned to best illustrate macro-structural orientation. For all simulations, we see the core structure of the protein remains intact. However, there is significant variability in the macro-structural orientation of the final structures’ CMG2 and Fc domains. The final GnGnXF and Agly structures exhibit significantly contracted linkers with respect to the final MAN8 structure, where the linker is fully extended. All glycoforms retain accessibility of the PA binding site after simulation. This is in agreement with the BLI , where all three variants have similar ka and kd, and the functional ELISA , where all three variants have control curves with similar absorbance level. To quantitatively characterize the macro-structural differences between the GnGnXF, MAN8, and Agly glycoforms, we report the center of mass distance between the CMG2 and Fc domains in Figure 9. Among the glycoforms, MAN8 has the highest COM distance with the narrowest spread at around 7.2 nm, GnGnXF and Agly have lower COM distances with wider spreads, roughly centered around 6 nm,vertical plant rack with the Agly COM spread being the widest.

The significantly higher COM of MAN8 than GnGnXF and Agly is visually consistent with the final conformations in Figure 8. The large spread in Agly rCMG2-Fc COM distance is largely due to refolding of the flexible residues in and near the LNK region. The COM distance temporal profile in Supplementary Figure S10 displays a gradual increase in Agly COM distance, which tapers off around 80 ns. Thus, the larger spread in Agly COM is due to conformational changes during the simulation, not a single conformation with increased flexibility. The root mean square fluctuation data in Supplementary Figure S11 show increased Agly RMSF in and around the LNK region during the first 50 ns and a reduction in Agly RMSF during the latter 50 ns, which is consistent with the trending exhibited in the Agly COM distance.The backbone root mean square deviation of ordered domains referenced from the initial and final conformations of the CMG2 and Fc regions for all three simulated glycoforms is shown in Figure 10. A low RMSD indicates protein folding transition is minor and the protein structure is stable. For each monomer, the ordered domain of the CMG2 region were defined as residues 10–181, while the ordered domains of the Fc region were defined as the CH2 and CH3 regions: residues 210–308 and 315–414, respectively. Each domain’s RMSD was fit and referenced independently, and subsequently averaged to produce the plots in Figure 10. Two RMSD profiles were averaged to generate the CMG2 RMSD, and four RMSDs were averaged to generate the Fc RMSD for each glycoform. All RMSDs are below 1.6 Å, indicating a generally conserved fold for all the ordered domains of each glycoform. This is further confirmed by the secondary structure data, provided in Supplementary Figure S12. In general, the RMSD from the final structure is lower than that of the initial, indicating conformational convergence is progressing throughout the simulations. The low RMSD and conserved secondary structure in all three simulated glycoforms indicate that there were no major refolding events; thus, the reduced activity in the TNA and the functional ELISA of the ER variant is likely not due to reduced fold stability. The Fc RMSD is noticeably lower than the CMG2 RMSD for the Agly and GnGnXF glycoforms, indicating minor fold transitions in the CMG2 domains of these glycoforms.The hydrophobic solvent accessible surface area distributions are reported in Figure 11.

The MAN8 glycoform has the highest amount of hydrophobic SASA in the simulation, while the aglycosylated has the lowest. Increased hydrophobic SASA likely yields a greater aggregation propensity . To elucidate which residues contribute most to the hydrophobic SASA difference between MAN8 and Agly, the average per-residue hydrophobic SASA was calculated, and the five residues with the greatest positive difference of MAN8 minus Agly hydrophobic SASA were obtained. These residues were Pro199, Pro201, Leu205, and Pro300 of one monomer, and Leu206 of the other monomer. All of these residues are located in close proximity to the N-terminal region of the Fc domain, shown in Figure 12. We see that these residues are highly spread out in the MAN8 structure, moderately spread out in the GnGnXF structure, and closely associating in the Agly structure. The increased SASA in the MAN8 glycoform is consistent with the existence of the high molecular weight band in the ER variant at ~250 kDa in the SDS-PAGE results .In this paper, experimental and computational techniques were employed to study the effects of N-glycosylation on the expression, structure, function, and stability for anthrax decoy protein rCMG2-Fc.N-glycosylation was found to strongly stabilize rCMG2-Fc in planta as both APO and ER variants have over two-fold higher expression level than the Agly variant, shown in Figure 1. The increase in expression level of glycosylated variants could be attributed to a decreased susceptibility of the APO and ER variants to proteases, where the steric hindrance of oligosaccharides inhibits proteolytic degradation of glycosylated rCMG2-Fc. From a manufacturing standpoint, producing glycosylated rCMG2-Fc would require less than half the production capacity of the aglycosylated form. Thus, when glycosylation is not detrimental, preserving natural N-glycosylation sites can enhance protein production. Alternatively, retaining the aglycosylated protein in the ER can also help improve yield, since the ER has fewer types of proteases than the apoplast . Furthermore, unlike the apoplast, the ER has chaperone proteins to provide folding support . It is also possible to enhance a glycoprotein’s function via modification of the N-glycosylation profile during expression. This can be achieved using subcellular targeting and/or the co-expression of glycan-processing enzymes or addition of enzyme inhibitors in the agroinfiltration buffer. For example, high mannose Fc N-glycosylation has been shown to enhance antibody-dependent cell-mediated cytotoxicity , which can be achieved by targeting the protein to the ER or addition of mannosidase I inhibitor to the agroinfiltration buffer or cell culture medium .

It is worth noting that N-glycosylation of Fc is not strictly required when Fc is fused to the target protein for the purpose of increasing circulation half-life . SDS-PAGE and Western blot confirmed that intact rCMG2-Fc was produced, with bands near 50 kDa . There is also a band around 250 kDa in the non-reducing SDS-PAGE for the ER variant , which may correspond to a high molecular weight protein aggregate. The hypothesis of increased aggregation propensity of the ER variant is supported by the hydrophobic SASA predicted from our MD simulations. MAN8 was found to have significantly higher hydrophobic SASA,growing strawberries vertical system with the five strongest contributing residues in the N-terminal region of the Fc domain. Protein aggregation might reduce PA binding capacity, as it is evident in the functional ELISA , which is later discussed in the rCMG2-Fc function section.The ability for rCMG2-Fc variants to sequester anthrax PA and prevent cell death was assessed using a cell-based TNA. Results are shown in Figure 5, where the ER variant has a statistically higher EC50 value than APO and Agly variants. The possibility of FcγR dependent toxin neutralization was ruled out as the EC50 values did not change upon FcγR blocking. This is likely because previous studies used antibodies or serum against PA that bind to PA but not necessarily blocks the its binding site to the anthrax cellular receptor CMG2. Thus, when incubated with cells, the antibody-bound PA can still bind to CMG2 and form prepore, resulting in LF and EF endocytosis. In this situation, Fc and FcγR could form an immune complex that is then sorted and degraded in the lysosome . In our experiments, rCMG2-Fc competitively inhibits binding between cellular receptor CMG2 and PA. This completely eliminates LF and EF internalization. The binding kinetics between rCMG2-Fc variants and PA were determined by BLI, results shown in Figure 6. The 37°C KD values were slightly lower than the 25°C KD values, but no appreciable difference in binding kinetics as a function of glycosylation was observed at 25 or 37°C. Considering the CMG2 domain is linked to Fc through a flexible linker, it is not surprising that the glycosylation of the Fc domain has minimal impact on the binding kinetics of the CMG2 domain with PA. Moreover, the sub-nanomolar affinity reported in this work is consistent with previous work on rCMG2 and PA binding kinetics , which is direct evidence that neither the fused Fc domain nor its glycosylation interferes with CMG2/PA binding kinetics. Even though the kinetics of all three variants were unaffected by glycosylation, it is possible that the fraction of functional protein changes over time.

The BLI experiments only characterized the interaction kinetics, which are independent of the fraction of functional rCMG2-Fc on the sensor tip. The hypothesis that the fraction of functional protein at 37°C is glycosylation-dependent was confirmed with the functional rCMG2-Fc ELISA, where the ER variant lost the most activity overnight, consistent with the ER variant having the highest EC50. However, the MD simulation data exhibit high fold stability in all glycoforms, according to the RMSD of ordered domains as well as the secondary structure . Thus, we hypothesize that the reduction in activity is not due reduced fold stability. Moreover, the MAN8 had the highest hydrophobic SASA among the three simulated glycoforms, indicating a higher aggregation propensity. The residues that contributed the most to the decreased hydrophobic SASA in the simulated Agly from the MAN8 were located in the N-terminal region of the Fc domain, just after the C-terminal region of the linker . This region is also more extended in the MAN8 glycoform, as shown in the final conformation and the COM distance distribution . This exposure of hydrophobic residues in the thinly extended N-terminal region of the Fc domain could facilitate the aggregation with other rCMG2-Fc or protein fragments. This could explain the reduced activity for ER variant in the functional ELISA and the 250 kDa band observed in the non-reducing SDS-PAGE gel for the ER variant . Fusion protein aggregation has been observed in the literature for another Fc fusion protein ALK1-Fc, where a high abundance of MAN5 glycoform was found as high molecular weight aggregates . It is worth noting that a recent study on high-mannose type IgG showed a decrease in protection factors of backbone amide nitrogen in the CH2 domain , which could also contribute to the reduced activity of the ER variant. Meanwhile, Lu et al., found that highmannose glycans have no detrimental effect on antibody stability and aggregate rate . The antibodies used in their study were IgG1 and IgG2, which has a molecular weight ~150 kDa. Since both the protein size and structure can affect protein stability, it is not surprising to see diverse protein stability results performed on different molecules. Within Fc-fusion proteins, structure can still vary depending on the fusion partner size and structure. However, we do expect Fc-fusion proteins with similar structure, molecular weight and conserved glycosylation site as rCMG2-Fc to likely exhibit similar behaviors. The APO and Agly variants had no significant difference in fraction of functional protein after being incubated overnight at 37°C. This result is in agreement with a previous study where human IgG1s stored at 37°C for 21 days had no difference in aggregation or fragmentation , suggesting the absence of glycans had no major impact on stability under physiological temperature.

For plants inoculated by the drop method, the number of culturable cells declined by 4 to 5 log CFU/leaf by day 2 to an average of 10 CFU/leaf . After inoculation by spray, the average number of culturable E. coli O157:H7 ATCC 700728 cells remained higher and ranged from 100 to 1000 CFU/plant, even on day 7 of the study .Although there were significantly higher numbers of culturable EC4045 cells than strain ATCC 700728 cells at 24 h after inoculation, the culturable amounts of both strains declined to equivalently low levels within 48 h on lettuce . Real-time PCR was used to quantify total E. coli O157:H7 cells on the lettuce. The number of E. coli O157:H7 cells remained equivalent to the inoculum cell density for 7 days, regardless of the strain or the inoculation method used . The addition of PMA prior to real-time PCR amplification revealed that viable cell quantities were equal to the numbers of total and culturable cells at the time of inoculation but then declined by an average of 100- fold within the first day. For both ATCC 700278 and EC4045 strains, the number of viable cells detected using PMA real-time PCR remained above the detection limit and was significantly higher than estimated by the standard plate count method .Lettuce plants in the field were spray inoculated with E. coli O157:H7 ATCC 700278 at a density of 106 CFU/plant. E. coli O157:H7 was not detected on uninoculated lettuce plants either by culture or by real-time PCR with or without PMA. Within 2 h after application onto the lettuce, culturable cell amounts declined by 2 and 4 log for plants inoculated in the spring and summer trials, respectively . Two days after inoculation, the amounts of culturable E. coli ATCC 700278 were reduced to the lower limit of detection by plating for most plants . At that time, E. coli O157:H7 was not retrieved by plating or enrichment for 3% and 13% of the spring and summer plants, respectively. In contrast to colony enumerations of E. coli O157:H7, total cell numbers determined by real-time PCR quantification were equivalent to the inoculum levels 2 h after application to lettuce plants.

Two days after application the numbers of E. coli O157:H7 estimated by real-time PCR decreased,vertical farming supplies on average, by 2 log CFU/plant and were below the limit of detection for six out of 24 plants tested for both field trials.Accurate measurements of pathogen amounts and viability are important for risk assessments and control mechanisms aimed at preventing outbreaks of food borne illness. This study was the first to apply a culture-independent method to quantify the number of E. coli O157:H7 cells on plants inoculated with the pathogen in the field. We also compared the utility of the method for quantifying virulent and avirulent pathogen survival on laboratory-grown lettuce. The first step in the development of culture-independent methods for bacterial enumeration is to ensure that the technique is optimized for the specific organism and environmental conditions of interest. While numerous assessment methods are available, we selected PMA combined with real-time PCR because this technique offered the possibility of rapid enumeration of viable cells in a variety of environments. For E. coli O157:H7 ATCC 700728, we first identified appropriate real-time PCR primers, PMA concentrations, and PMA incubation times with a goal of selective and sensitive detection of viable E. coli O157:H7 cells in the presence of high levels of dead E. coli O157:H7 cells. Among the results was the confirmation that PMA-mediated PCR-inhibition is incomplete when the product size is less than 200 bp. Although our tests on PMA real-time PCR revealed some potential short-comings with the method, most notably the interference of high quantities of inactivated cells and the relatively high numbers of cells required for detection, these parameters were within the range found in other PCR-based studies. E. coli O157:H7 strains ATCC 700728 and EC4045 were inoculated onto leaves of potted lettuce plants and incubated in a growth chamber under temperature and low humidity conditions that favored the decline of cell numbers in trends similar to those recorded in field trials performed in the Salinas Valley. This approach differs from most other laboratory studies that examined plants exposed to high moisture and temperatures supportive of pathogen growth. Within 2 days after inoculation on lettuce plants, the number of E. coli O157:H7 ATCC 700728 cells able to form a colony on TSA declined 1000-fold when the pathogen was initially applied at a level of 6 log CFU per plant or per leaf using an aerosolized spray and declined .The numbers of culturable cells remained similar for the subsequent 5 days, suggesting that the initial events after contact with the plant are crucial for determining the survival of the organism on lettuce.

In contrast with plate counts, real-time PCR targeting of the E. coli O157:H7 ATCC 700278 and EC4045 inoculants showed that the genomic DNA of the organisms remained on the lettuce in quantities nearly equivalent to the inoculum concentrations over the duration of the 7-day experiment. This finding confirmed that both strains adhered to the leaf surfaces and the recovery method was sufficient to remove the majority of the cell inoculants from the plant. Moreover, the results indicated the presence of high numbers of intact E coli O157:H7 cells on lettuce even when very few were able to form colonies on TSA. Exposure of E. coli O157:H7 suspensions to PMA prior to realtime PCR quantification facilitated the detection of cells with an intact cell membrane, and these cells were likely viable. Application of this method to E. coli ATCC 700728 and EC4045 recovered from growth chamber lettuce indicated that a significant fraction of the pathogen inoculants were viable, even though colony-based assessments indicated otherwise. This finding was particularly evident for E. coli O157:H7 inoculated onto leaves by direct drop inoculation. While an average of 5 log CFU per leaf were viable as measured by PMA real-time PCR, less than 1 log CFU per leaf were enumerated by culturing. These results are supported by previous studies reporting that E. coli O157:H7 enters into a viable but not culturable state on lettuce leaves incubated at cold temperatures. Similar outcomes were reported for other E. coli including E. coli O104:H4 in water or after exposure to toxic concentrations of copper. Once the stress condition was removed, a fraction of the cells recovered the capacity to grow in laboratory media, thereby indicating a potential of the E. coli O1O4:H4 cells to cause human disease. The formation of VBNC bacterial cells on plants also was previously described for Listeria monocytogenes on parsley. The number of viable L. monocytogenes cells was 1 to 2 log higher than the culturable cells recovered from parsley grown in greenhouses at 20uC under low relative humidity ; growth of the VBNC cells was not restored on the plants when the RH was increased to 100%. Although we did not examine for E. coli O157:H7 recovery from the VBNC-like state,vertical lettuce tower future efforts might investigate whether those cells can recover and resume growth either under growth-conducive conditions on lettuce or after removal of the cells from the plants and prior to or after plating for viable cell enumeration.

Overall, the toxigenic strain EC4045 survived in similar quantities as ATCC 700728 on lettuce. A recent study showed that certain lineages of E. coli are more commonly associated with plants and presumably have evolved the capacity to tolerate plant associated environments better than E. coli isolated from other sites. Because we only compared two strains, subsequent investigations should examine multiple attenuated and virulent O157:H7 strains isolated from different sites for their capacity to colonize and persist on lettuce under field relevant conditions. Survival of E. coli O157:H7 on lettuce also was measured in two field studies. Culturable amounts of strain ATCC 700728 declined shortly after inoculation onto plants in the field, as we reported previously. Rates of cell decline were similar to E coli O157:H7 on lettuce in the growth chamber directly inoculated in drops with a pipette. Real-time PCR estimates of E. coli O157:H7 ATCC 700728 in lettuce leaf washes showed that this strain was present on the plants immediately after inoculation and 2 h later in quantities equivalent to the inoculum levels. Importantly, the rapid decline in culturable E. coli during the first hours after application onto plants in the field was not due to an inability to remove the organism from the lettuce or from dispersal and lack of strain attachment. Rather, it appears that the majority of the E. coli cells in the inoculum either died shortly after application or entered a VBNC state. In contrast to the growth chamber experiment results, the numbers of E. coli O157:H7 cells were below detection by real time PCR within 2 days after inoculation onto field lettuce. These findings suggest that the E. coli O157:H7 cells and genomic DNA were degraded rapidly. Environmental parameters such as solar radiation, heat, and water stress could be responsible for the differences in the stability of E. coli O157:H7 DNA in the field compared with the laboratory. Alternatively, cell maintenance might depend on other microorganisms on the leaves. Also, it is notable that different lettuce cultivars were used in the field and growth chamber studies, which may have impacted survival. The potential for cultivar-dependent effects was shown for E. coli O157:H7 on lettuce cultivars grown under axenic conditions in the laboratory.

Because the E. coli O157:H7 ATCC 700728 DNA was degraded on field-grown plants within 2 days after inoculation, it is unlikely that the organism developed a VBNC state, particularly over longer time scales . However, this possibility could not be directly addressed using the PMA realtime PCR assay in the field trials. Viable cell amounts measured by culturing and PMA real-time PCR were in agreement immediately after application of ATCC 700728 onto laboratory grown lettuce, but PMA-mediated detection was impaired on plants from the field. For those plants, the viable cell number estimates for strain ATCC 700278 were 10-fold lower as measured by PMA real-time PCR than by colony enumeration and total cell numbers estimated by real-time PCR. These differences might have been due to the higher turbidity or opacity of the lettuce plant washes from the field samples, thereby preventing light from penetrating the suspension during the PMA photo inactivation step. This interference would prevent the inactivation of free PMA, resulting in sufficient quantities of the compound to bind genomic DNA released from viable cells during the subsequent DNA extraction and amplification steps. In addition, the PMA real-time PCR assay was unable to detect low numbers of cells. Attempts to detect the ATCC 700278 strain after concentrating the leaf washes were unsuccessful . Similarly, PMA real-time PCR was found to be more reliable for viable cell detection in diluted wastewater than in pooled and concentrated wastewater samples. Such factors strongly limit the overall usefulness of this approach for field-grown plants. However, this method is informative for examination of E. coli O157:H7 on ‘‘cleaner’’ plants grown in the growth chamber and not exposed to the biotic conditions that are common outdoors. In conclusion, this study illustrates the similarities and differences between controlled studies of human pathogens on plants in a growth chamber and experiments examining the population dynamics of pathogens on plants under production-like conditions in the field. By applying relevant environmental conditions and droplet inoculation in the growth chamber, we were able to more closely mimic the rapid decline in E. coli O157:H7 culturability that was observed after inoculation of this organism onto lettuce plants in the Salinas Valley. Culture-independent assessments confirmed that the pathogen remains on the plant long after application. However, field studies showed that at least for the majority of E. coli O157:H7 cell inoculants, the loss in culturability was most likely due to cell death rather than an inability to form colonies on standard laboratory media. Hence, this work confirmed our observations that low numbers of E. coli O157:H7 persist on lettuce grown in the Salinas Valley and variations in pathogen survival among individual plants are dependent on other unknown factors .Ethnically diverse populations are disproportionately exposed to hazardous environmental materials by virtue of living in close proximity to toxic waste materials.