Horticulture

Methylamine and ethylamine, the degradation products of glycine and alanine, are detected in increasingly high abundances with age in ancient sulfate samples up to ~40Ma. The comparison of these concentrations to their parent amino acids allow for the calculation of glycine and alanine decarboxylation rates and are assumed to be indicative of these rates in sulfate matrices. Because sulfate minerals have been detected in high abundance on Mars, these rates are extrapolated to Mars’ average surface temperatures to offer an estimate of the rates that might be expected on Mars. Chapter 4 analyzes modern sulfate samples from Southern Australia for amino acids and extrapolates these concentrations to equivalent bacterial concentrations . The low abundances of methylamine and ethylamine in these modern samples show consistency with previous findings . Chapter 5 focuses on a new analog to the Martian hematite blueberries that have been detected on Mars in the Meridiani Planum region. These ironstones are ubiquitous in San Diego county, and by analyzing samples from various deposits,nft hydroponic system they are dated using amino acid racemization based on the measured D/L-ratios and a sample calibration method. Similar concretions on Mars would show good preservation in such environments despite the fact that they’re iron rich.

The ironstone formation seems to be mediated by enhanced precipitation as well as the possibility of bacterially induced mineralization within these deposits. Chapter 6 investigate an extreme environment deep within a South African subterranean gold mine. These filtrates show extremely low bio-densities and is of interest because it might represent the extremely low levels of biomass and extremely slow turnover times that push the limits of analytical sensitivity. A simple steady-state model corroborates the long turnover times that have been reported for these samples. Two well known Mars analog sites comprise the next two chapters, rock samples from the Antarctic dry valley Deserts and surface soils from the Atacama Desert, Chile , which have been suggested to be the best terrestrial Mars soil analog . The fact that life can persist in these extreme climates is impressive enough, however the extremely cold samples from Antarctica show amazing preservation while the opposite is observed in the Atacama Desert surface and near-surface samples. The inferred cell counts based on total amino acid abundances within cryptoendolithic microbial life agree well with previous bio-density enumerations in similar environments. The Atacama Desert shows extremely degraded organic material at the surface and shows drastic variations in amino acid distributions and diagenetic state as a function of depth and surface micro-environment habitability. One of the premier instruments for advanced in situ Mars life detection experiments is the Urey – Mars Organic and Oxidant detector. Chapter 9 focuses on research and development of the instrument over the past 3 years, specifically the optimization of the extraction system for Mars exploration. Both laboratory and field experiment data is discussed in detail and exhibits efficient extraction of target bio-molecules, specifically amino acids and total organic carbon.

The use of sublimation as a second-stage extraction method for extract purification and concentration is evaluated for recoveries using various mineral matrices.The astrobiological studies have broad implications for the search for life on Mars over the next decade. Two missions will be launched within the next 6 years: NASA’s Mars Science Laboratory and the ESA’s ExoMars . The studies herein validate the importance of targeting amino acids in life detection studies, emphasize their importance as an indicator of bio-density, and demonstrate the potential for sequestration within Mars mineral deposits. Not to be dismissed is the fact that the detection limits of the Urey Mars Organic Detector are many orders of magnitude greater than necessary for the detection of amino acid biomarkers in some of the most uninhabitable places in the world such as the Atacama Desert. Sulfate minerals are highly abundant on Mars and our studies suggest that they can offer enhanced preservation on the Martian surface based on the estimated rates observed for in situ diagenetic reactions.Bacterial bio-density can be quantified by a number of traditional cell staining methods. Many of these methods target intact DNA, such as 4′,6-diamidino-2-phenylindole , acridine orange , or ethidium bromide. These methods target only intact nucleoid containing cells . Other staining methods, such as trypan blue, target cell membranes and will react with both living and dead cells. Cell staining can lead to erroneous bio-density calculation due to human error and interfering medium . Analyses of individual cellular molecular components can be used to accurately determine cell concentrations in natural samples. Two of these methods are cell enumerations based on total adenosine triphosphate and phospholipid fatty acid analyses, and these have been shown to be accurate at determining cell concentrations in natural samples . ATP is a nucleotide and a ubiquitous component of bacterial life while the majority of phospholipids and fatty acids are present as components within microbial cell walls. One unique aspect of PLFA analyses is that they can distinguish between different types of bacteria depending on the distribution of the target molecules. Similarly, any biomarker can be used to quantify bacteria such as amino acids or nucleobases.Quantifying total hydrolyzable amino acids yields accurate determination of the total protein content in bacterial colonies.

Traditional wet chemistry extraction and analytical methods for amino acid quantification involve acid hydrolysis followed by desalting, pre-column derivatization using a fluorescent chiral adduct, separation by reverse-phase high performance liquid chromatography , and quantification by fluorescence detection against standards of known concentration . Most amino acids are stable during traditional acid-hydrolysis methods as only the peptide bonds within proteins are cleaved during this treatment. The exception is tryptophan which is completely destroyed during acid hydrolysis while arginine, tyrosine, threonine, serine, methionine, and cysteine are degraded to a small degree during longer hydrolysis times .These degradation mechanisms involve the conversion of the carboxamide side groups to carboxyl groups through the incorporation of water and liberation of ammonia.Ortho-phthaldialdehyde/N-acetyl-L-cysteine was first used to derivatize amino acids by Aswad and later applied to amino acid quantification in geological samples . The fluorescent derivatizing chiral adduct is made by combiningOPA and NAC into an alkaline borate buffered solution to form a cyclic fluorescent derivative .This fluorescent chiral adduct reacts with primary amines to form fluorescent derivatives . The fluorogen reacts with all primary amines, so it targets all 20 protein amino acids except for proline. Highly specific fluorescence detection is accomplished at an excitation wavelength of 340 nm and an emission wavelength of 450 nm. This highly specific derivatization allows for low interference during quantification.In order to determine the amino acid composition and distribution of a typical bacterial culture,hydroponic nft system and to test the THAA method for cell enumeration, samples of cultured E. coli cells were run through traditional wet chemistry extraction and analytical protocols. This allowed for determination of the most important targets for the search for amino acid bio-signatures derived from bacterial proteins in the study of geological samples and for the purposes of life detection.Cultured E. coli cells were obtained and added to a sterilized crushed serpentine medium. Cell bio-densities were measured by traditional methods on the inoculated sample and a procedural blank growth medium that did not contain E. coli cells. The OD460 of the E. coli growth medium was measured to be 0.65 which corresponded to 6.5 x 109 E. coli cells in 10 ml of LB growth medium with a 5% measurement error. Because physiological variation may changes in cell size, capsule formation, or aggregation, small differences in the conversion between OD and total cell counts may be observed. For this reason, the total number of E. coli cells as determined from the OD reading was independently confirmed by measuring the mass of a solid E. coli pellet generated by overnight growth and centrifugation of a volume of LB medium identical to that used to inoculate the serpentine. If it is assumed that the E. coli cells were homogenously mixed into the 0.5-g crushed serpentine sample, a concentration of 1.3 x 1010 cells/g for the serpentine inoculated with E. coli was inferred. ~200 mg of the inoculated serpentine cell media and a serpentine growth medium blank were hydrolyzed and desalted according to the procedures of Zhao and Bada . The sample was vapor-phase hydrolyzed under 6M doubly-distilled HCl for 24 hours in a flame-sealed test tube after flushing with nitrogen. The hydrolyzed residue was loaded onto an equilibrated desalting column of ~2.5 mL of BioRad AG50W-X8 resin in a pasteur pipette. The sample was rinsed with ~6 column volumes of doubly-distilled water before eluting the amino acid fraction with 3mL of ~3M doubly-distilled ammonium hydroxide .

These fractions were concentrated on a vacuum centrifuge under 60°C heat into 1.5mL mini-eppendorf vials. These residues were resuspended into 100µL of ddH2O for derivatization and analysis by RP-HPLC. The OPA/NAC fluorescent derivative was prepared with chemicals purchased from Sigma. 4 mg of OPA was first dissolved into 300µL of methanol, and 250µL of borate buffer was added to the solution, followed by the addition of 435µL of double-distilled water. The last step is the addition of 15µL of 1M NAC solution . This derivatizing solution has a final concentration of ~0.03M OPA and ~0.015M NAC before they react. The final concentration of the cyclical derivative in the OPA/NAC solution is 0.015M OPA/NAC. The reaction between OPA/NAC and primary amines has been demonstrated to be linear over large concentration ranges for reaction with amino acids and biogenic amines . 10µL aliquots of diluted fractions of the desalted hydrolyzed E. coli extracts and procedural blanks were first dried down on a vacuum centrifuge at room temperature with 10uL of borate buffer to remove any residual ammonia from the NH4OH carried through from the desalting stage. These residues were brought up in 20µL of ddH2O and derivatized for 1 minute with 5µL of the 0.015M OPA/NAC solution. After this pre-column derivatization, the samples were separated by RP-HPLC and quantified with a fluorescence detector. The RP-HPLC setup utilizes an Hitachi L6200 Intelligent HPLC pumps, rheodyne sample injectors, coupled with a Phenomenex Luna-C18 RP-HPLC column and a Shimadzu fluorescence detector . Data sampling and analysis, including automatic and manual Gaussian peak integrations, were performed using Thermo Scientific Grams/AI software. Sample peak intensities were quantified against 100-1000x diluted commercial standards of known concentration , and the trace amounts of D-enantiomers ratio-normalized against racemic laboratory standards of similar concentration. The HPLC conditions included a stationary-phase buffer, 50mM sodium acetate solution with 8% methanol, with methanol as the mobile phase. Two gradient protocols were necessary to resolve the 16 amino acids extracted and purified by these methods . The traditional amino acid separation protocol developed over the last 20 years has been used in a variety of studies including those on natural samples , meteorites , and hydrolyzed bacteria . This RP-HPLC protocol was primarily developed to well separate the primary amino acids present in geological samples as well as their enantiomers. A slower methanol elution gradient was developed that was necessary to resolve the coeluting peaks of glycine and arginine, and to better resolve threonine as a shoulder of glycine and tyrosine from alanine. The trace amounts of D-enantiomers did not interfere with the peak separation as the D/L-enantiomer ratios were too small to be significant. This is expected of any extant bacterial community and the hydrolysis not harsh enough to cause a significant degree of racemization.The OPA/NAC fluorescent derivatizing reagent tags only primary amines, so proline does not react, tryptophan is completely destroyed during hydrolysis in 6N HCl for 24 hours, and asparagine and glutamine degrade to aspartic acid and glutamic acid during acid-catalyzed hydrolyis, respectively. This results in the derivatization of 16 of 20 total protein amino acids.Aspects of this study have been investigated before. Similar procedures were utilized by Glavin et al. to determine the amino acid composition in similarly treated fraction of hydrolyzed E. coli cultures, however they analyzed a more limited set of amino acids during their experiments. More recently, the recoveries of adenine from sublimed E. coli colonies were used to enumerate bacterial bio-density , so this study provides verification of procedures for THAA determination and accurate enumeration of cell bio-density.

Surveys conducted during the first two years of the study throughout the entirety of the six research blocks showed that the prevalence of PD in control vines actually declined slightly from the first to the second year , but not due to an increase in replanting efforts or vine death , Rather, this decline in prevalence likely reflects overwinter recovery of mild cases of the disease . Thus, the observed return of symptoms in most severely pruned vines does not appear to be explained by reinfection with X. fastidiosa after clearing of infection during the severe-pruning process. Our results indicate that the apparent effectiveness of severe pruning depended on the initial disease severity, and the effectiveness weakened over time. This suggests at least two constraints exist regarding the general utility of pruning as a PD management tool. First, severe pruning does not appear to be useful for mild cases of PD, as many of those same vines would recover from the infection over the winter . Second, there appears to be little value in pruning severely diseased vines; the high frequency of symptom return within a few years indicates that even severe pruning does not clear most vines of X. fastidiosa infection.

That leaves a statistically significant window with respect to intermediate severity cases,blueberry grow bag size which may benefit from severe pruning. The apparent benefit for this category of diseased vines would stem from infections that are not so localized that they are highly susceptible to natural recovery over the winter, but also not fully systemic such that the infection has developed below the pruning point . Reliable identification of this narrow class of diseased vines may require substantial experience with PD scouting, detailed record keeping, and an appreciation for variability in symptoms or infection dynamics based on grapevine cultivar and environmental conditions . Research in other bacterial plant pathosystems has evaluated the potential benefit of pruning and whether pruning extent is related to its effectiveness at clearing hosts of infection . A study of the citrus disease huanglongbing, associated with infection by Candidatus Liberibacter spp., evaluated two levels of pruning severity, neither of which showed promise as a disease management tool . In this pathosystem, it is plausible that a very protracted incubation period may undermine the effectiveness of pruning, because by the time the first symptoms are visible, the infection may have already moved throughout much of the tree. Collectively, our results are more similar to a study of citrus variegated chlorosis .

In this study, the presence of X. fastidiosa in plant tissues at different distances from symptomatic leaves was determined for varying levels of disease severity. X. fastidiosa was more widely distributed in trees with severe disease symptoms compared to those with early stage foliar symptoms. Although ColettaFilho et al. did not test whether pruning at various distances proximal to symptomatic leaves would eliminate X. fastidiosa infections, the current recommendation is to prune citrus material if early symptoms are present, and to not prune plants with severe disease symptoms . Citrus plant age is also an important consideration; Coletta-Filho and de Souza recommend that symptomatic citrus trees up to three-years-old be removed rather than pruned, whereas trees four-years-old or older should be pruned. We did not examine vine age as a factor in this study, but the biology of citrus and grape differ in terms of the overwinter recovery that can occur in grape and the apparently slower movement of X. fastidiosa in citrus compared to grape. Anecdotally, the two most mature plots in our study showed the most rapid return of disease, and the youngest plot showed the slowest return. More studies of the effect of vine age are needed before concluding that interactive effects of plant age and pruning differ between the PD and citrus variegated chlorosis pathosystems.The length of lifespan of a particular normal cell of any organism is predetermined. Similarly, the length of lifespan of all organisms is pre-determined by their genetic makeup and their external and internal environments and diet-related factors specific to an organism.

Therefore, the length of lifespan can be increased or decreased by manipulating the environment, diet and genetic factors only by small extent. The length of lifespan of the organisms can also be impacted by differential rates of senescence of cells and organs that ultimately lead to the death of the organisms. The differential rates of cellular senescence are influenced by several confounding factors, such as external and internal environments, diet and genetic factors. Because of these confounding factors that can impact rates of progression of degenerative changes in the organs, it is almost impossible to study aging in the absence of organ pathology. Based on numerous studies on aging in vertebrates and invertebrates, a recent informative review has suggested that oxidative stress theory of aging can only be applied to conditions in which age associated pathologies are included. Furthermore, it was suggested that in environment with minimal stress, oxidative damage plays little role in aging. This suggestion can be argued on the fact that little oxidative damage may take longer time to deregulate protective transcription factors, adaptive responses to stressors, and repair mechanisms, and thereby extending the lifespan of the organisms more than that produced by higher oxidative damage which can deregulate above biological functions in shorter time. Using vertebrate and invertebrate models, some major biochemical and genetic factors that are associated with aging processes have been identified. They include increased oxidative stress and chronic inflammation, decreased adaptive response to stressors, post-translational protein modifications, mitochondrial dysfunction, decreased of proteasome and lysosomal-mediated proteolytic activity, shortening of telomeres and transcriptional deregulation.

Among these, the theory of oxidative stress is most extensively investigated in various experimental models, using pharmacological agents, antioxidants, anti-inflammatory agents and deletion of one or more antioxidant enzymes as well as of mitochondrial complexes. Depending upon the experimental models, experimental designs, and substrate used to assay oxidative stress and criteria of oxidative stress, the role of oxidative stress in aging has been substantiated or questioned. We hypothesized that increased oxidative stress may be one of the primary early events that causes chronic inflammation, transcriptional deregulation, post-translational protein modifications, mitochondrial dysfunction, decreased of proteasome and lysosomal-mediated proteolytic activity and shortening of telomeres. Invertebrate models, such as Caenorhabditis eleganshas been extensively used to evaluate the role of oxidative stress in aging primarily due to shorter lifespan of about 3 days and ease of genetic manipulation. This review analyzes recent published studies on C. elegans on the role of oxidative stress in determining the length of lifespan by generating mutants that show suppression of mitochondrial function or lack of superoxide dismutase . Caenorhabditis elegans has been extensively used to investigate the role of oxidative stress in aging by measuring the length of lifespan. Mitochondria are considered the major sites for the production Reactive oxygen species ,blueberry box although ROS are also produced outside the mitochondria. In order to demonstrate the impact of oxidative stress, several mutants of C. elegans were generated. They include mutations in four clock genes , mutation in the iron sulfur protein of mitochondrial complex III, mutation in the gene NUO-6 and mutation in the gene daf-2. The effects of mutations on oxidative stress and lifespan are summarized in Table 1. The clk-1 gene encodes an enzyme that is necessary for the biosynthesis of ubiquinone that is required by the mitochondria to generate energy. Mutation in the clk-1 gene increases the life span by slowing down mitochondrial activity due to reduced availability of ubiquinone. This slowing of the electron transport chain would reduce oxidative stress. The role of reduced oxidative stress in extending the lifespan is further supported by the fact that overexpression of clk-1 gene in wild-type C. elegans increased mitochondrial activity and shortened the lifespan. A mutation in the iron sulfur protein of mitochondrial complex III causes low oxygen consumption, reduced oxidative stress and increased lifespan. Mutation in the daf-2 gene which codes for a member of insulin receptor family increased lifespan and enhanced resistance to oxidative stress. In this daf-2 mutant, expression of the SOD-3 gene, which encodes mitochondrial Mn-superoxide dismutase, was much higher than in the wild type. This implies that the increased levels of SOD-3 in the daf-2 mutant reduced oxidative stress and thereby increased lifespan. Mutation in the gene NUO-6 which encodes complex I of mitochondria increases life span of C. elegans by decreasing the mitochondrial function. Mutation in the age-1 increased lifespan by two folds. This mutant wormhad increased catalase and Cu/Zn SOD activities which may account for the increased resistance to the paraquat, a superoxide generating chemical.

The mutants C. elegans support the view that the levels of oxidative stress is one of the important determinant factors in determining the length of lifespan.Superoxide anions are produced enzymatically outside the mitochondria by different oxidases and nonenzymatically inside the mitochondria. SOD detoxifies superoxide to hydrogen peroxide , which is converted to water and oxygen by catalase. There are five superoxide dismutase isoforms SOD-1, SOD-2, SOD-3, SOD-4 and SOD-5 in C. elegans. However, in most organisms there are only 3 SODs. SOD-1 is present in the cytoplasm and represents the majority of SOD activity, whereas SOD-2 and SOD-3 are present in mitochondria. Increased levels of superoxide were observed in SOD deleted wild type worms or in ISP-1 and NOU-6 mutants. The effect of deletion of SOD on C. elegans lifespan is shown in Table 2. The impact of SOD deletion on the lifespan appears to be contradictory, depending upon the C. elegans model used. For example, SOD2 deletion markedly increased the lifespan of mutant clk-1 worms, but it decreased the lifespan of mutant isp-1worms. In addition, deletion of individual SOD genes from wild type C. elegans did not decrease the lifespan of these worms. This is in sharp contrast to other model, such as yeast, flies, and mice in which deletion of cytoplasmic or mitochondrial SOD caused decreased in the lifespan. In another study, it was demonstrated that the levels of superoxide were elevated in nou-6 mutant and isp-1 mutant and they lived longer than the wild type, however, the oxidative stress was low and overall levels of ROS did not change. From these results, it was concluded that elevation of superoxide is sufficient to increase the lifespan of these mutant worms. Based on these results, the role of oxidative stress in aging was questioned. It should be pointed out that if superoxide is precursor of ROS, the levels of ROS and oxidative stress should have been increased. This was not observed in the above study, suggesting that reduced oxidative stress possibly due to adaptive response by other antioxidant enzymes, such as catalase and glutathione peroxidase and improved repair mechanisms was responsible for the extension of lifespan of these mutant worms. In the same article, it was observed that addition of N-acetylcysteine and vitamin C individually abolished the effect of superoxide on life extension and other associated changes in nou-6 and isp-1 mutants. The antioxidant effect of NAC is mediated via glutathione, which in the presence of the high superoxide environment of mutant worms can be oxidized and then act as a pro-oxidant. Therefore, observed abolition of the effect of superoxide on life extension could be related to pro-oxidant effects of NAC and vitamin C. In order to assess the role of SOD on life extension further, a model of C. elegans was developed in which all five SODs were deleted, was established. The results showed that SOD 12345 worms were viable and exhibited a normal lifespan similar to that of wild-type despite increased sensitivity to multiple stressors. However, these SOD lacking worms showed reduced fertility, slow development, slower defecation cycle and decreased movement . From these results, it was concluded that SOD is dispensable for normal lifespan of C. elegans. This is in sharp contrast to mammals in which SOD is considered indispensable for survival. Thus, the results obtained on some genetic models of C. elegans cannot readily be extrapolated to the genetic models of mammals. If SOD is dispensable for the survival and lifespan of C. elegans as reported recently, overexpression of SOD should have no impact on the lifespan of these worms. On the contrary, it was reported that over expression of the major cytosolic Cu/Zn-SOD increased lifespan of wild type worms which was not related to reduced lipid oxidation or glycation.

We were unable to do this because of our study design, which did not examine seed set from single bee visits. Nevertheless, this is the first sunflower seed set study to detect an interspecific interactive effect at the community-level rather than at the individual-level. However, despite the importance of these interactive effects on sunflower yield, company was the factor that most strongly influenced seed set. Although there was little variation in head size between sunflower companies , using company as a classification may mask other differences, such as genetic differences between varieties and variation in field management techniques. By pairing control and hedgerow sites by company, variety and landscape context, we sought to minimize these potential differences,pe grow bag and the few differences in management practice were noted between companies. It is hypothesized that the effectiveness of field-edge vegetation re-diversification is maximized in landscapes that retain a small percentage of natural areas that can facilitate recolonization of restored habitats . The added benefits of diversification efforts may be minimal in complex landscapes with high proportions of natural habitat since ecosystem service providers are often already supported.

Diversification efforts may not support ecosystem providers in highly intensified landscapes with no remaining natural habitat, either because there are no source areas to colonize the new habitats or because the new habitats alone cannot support populations of ecosystem service providers . Although the landscape where we conducted our study constitutes a “cleared” landscape, and we did not detect landscape effects, other studies in the same location have found that hedgerows increase wild bee abundance, richness and population persistence and promote rare and/or more specialized species . Nevertheless we did not find evidence that these biodiversity benefits translated into higher rates of pollination services in adjacent sunflower crop fields. Although both wild bee richness and abundance were important factors contributing to sunflower seed set, these contributions may be attributable to factors other than hedgerows. For example, wild bee visitors to sunflower were predominately sunflower specialists; the amount of sunflower maintained in the landscape over time could therefore influence sunflower pollinator populations more strongly than hedgerow plantings that do not contain floral resources suitable for the specialists’ dietary requirements , as we found was true in the independent dataset. It is important to balance the conservation value of field-edge plantings with ecosystem service delivery objectives. While conservation and ecosystem service outcomes can be synergistic, win–win scenarios are challenging to achieve .

Hedgerows augment pollinator populations, which can be important for achieving wild bee conservation goals ; however, they may not be a “silver bullet” strategy for increasing crop pollination. Both the scale of the re-diversification effort relative to the farming system and the adjacent crop type could limit the effectiveness of hedgerow plantings. Hedgerows occupy <1% of our study landscape and contain 175 times less area than a typical average crop field in our study area. The intensity of bloom in hedgerows is also minimal in comparison to the hundreds of thousands of blooms in a single MFC field . Increasing the size of hedgerows relative to fields or introducing a suite of diversification techniques could increase the effectiveness of re-diversification efforts .Alternately, the configuration of habitat could impact pollinator populations. For example, when Morandin and Winston examined the optimal spatial distribution of a MFC, canola , they found that both profits and pollination services would be maximized if a central field was left fallow or allowed to revert to semi-natural habitat. The size, configuration and quality of habitat may all interact to influence pollinator communities . The benefits of field-edge diversifications may also differ based on crop identity and landscape context . For example, sunflower has easily accessible florets that attract both generalist and specialist pollinators. However, in systems where flowers have specific requirements, such as high bush blueberry that requires buzz-pollination, the identity of pollinator species may be of more importance .

Further, species-specific responses to habitat features may differ. Carvell et al. found bumble bees had differential responses to wildflower patch size and landscape heterogeneity, indicating that local and landscape habitat factors can also interact with one another, and with crop-specific attributes, to affect crop pollination. In a tropical region, Carvalheiro et al. found that wildflower plantings worked in concert with natural habitat to heighten mango production. There are a paucity of studies on the ecosystem service benefits from field-edge plantings, therefore the complex range of factors, including farming type, crop system, landscape context, and region , influencing their performance is still relatively unknown .Molecular networking1 , introduced in 2012, was one of the first data organization approaches to visualize the relationships between tandem mass spectrometry fragmentation spectra. In molecular networking, relationships between similar MS/MS spectra are visualized as edges. As MS/MS spectral similarity implies chemical structural similarity1 , chemical structural information can thus be represented as a network and chemical relationships can be visualized. This approach forms the basis for the web-based mass spectrometry infrastructure, Global Natural Products Social Molecular Networking2 which sees ~200,000 new accessions per month. Molecular networking has successfully been used for a range of applications in drug discovery, natural products research, environmental monitoring, medicine, and agriculture.

To tap into the chemistry of complex samples through metabolomics, a subset of MS/MS spectra can be annotated by spectral library matching or by using in silico approaches. While molecular networking facilitates the visualization of closely related molecules in molecular families, the inference of chemical relationships at a dataset-wide level and in the context of diverse sample metadata requires complementary representation strategies. To address this need, we developed an approach that uses fragmentation trees4 and machine learning5 to calculate all pairwise chemical relationships. These chemical relationships are represented as a chemicaltree that can be visualized in the context of sample metadata and molecular annotations obtained from spectral matching and in silico annotation tools. We show that such a chemical tree representation enables the application of various tree-based tools, originally developed for analyzing DNA sequencing data, for exploring mass-spectrometry data. Here, we introduce Qemistree software that constructs a chemical tree based on predicted molecular fingerprints from MS/MS fragmentation spectra10 . Molecular fingerprints are vectors where each position encodes a sub-structural property of the molecule, and recent methods allow us to predict molecular fingerprints from tandem mass spectra. In Qemistree, we use SIRIUS16 and CSI:FingerID to obtain predicted molecular fingerprints. Users can first perform feature detection to generate a list of observed ions with associated peak areas and MS/MS fragmentation spectra,growing bags referred to as chemical features henceforth, to be analyzed by Qemistree . Only chemical features with MS/MS data are included; features with only MS1 are not considered. SIRIUS then determines the molecular formula of each feature using the isotope and fragmentation patterns and estimates the best fragmentation tree explaining the fragmentation spectrum. Subsequently, CSI:FingerID operates on the fragmentation trees using kernel support vector machines to predict molecular properties . We use these molecular fingerprints to calculate pairwise distances between chemical features and hierarchically cluster the fingerprint vectors to generate a tree representing their chemical structural relationships. Although alternative approaches to hierarchically cluster features based on cosine similarity of fragmentation spectra exist19–21, we use molecular fingerprints predicted by CSI:FingerID for this. Previous work has shown that CSI:FingerID outperforms other tools for automatic in silico structural annotation. Therefore, we leverage it to search molecular structural databases to provide complementary insights into structures when no match is obtained against spectral libraries. Subsequently, we use ClassyFire23 to assign a 5-level chemical taxonomy to all molecules annotated via spectral library matching and in silico prediction . Phylogenetic tools such as iTOL24 can be used to visualize Qemistree trees interactively in the context of sample information and feature annotations for easy data exploration. The outputs of Qemistree can also be plugged into other workflows in QIIME 2 or in R, Python, etc. for system-wide metabolomic data analyses.

In this study, we apply Qemistree to perform chemically informed comparisons of samples in the presence of technical variation such as chromatographic shifts that commonly affect mass spectrometry data analysis. Additionally, we exemplify the use of a tree-based representation to visualize and explore chemical diversity using a heterogeneous collection of food products. Qemistree can be used iteratively to incorporate multiple datasets without the need for cumbersome reprocessing , allowing for large-scale dataset comparisons. Qemistree is available to the microbiome community as a QIIME 2 plugin and the metabolomics community as a workflow on GNPS2 . The chemical tree from the GNPS workflow can be explored interactively using the Qemistree GNPS dashboard. . To verify that molecular fingerprint-based trees correctly capture the chemical relationships between molecules, we designed an evaluation dataset using four distinct biological specimens: two human fecal samples, a tomato seedling sample, and a human serum sample. Samples were prepared by combining them in binary, tertiary, and quaternary mixtures in various proportions to generate a set of diverse but related metabolite profiles . Untargeted tandem mass spectrometry was used to analyze the chemical composition of these samples and obtain fragmentation spectra. The mass spectrometry experiments were performed twice using different chromatographic elution gradients, causing a retention time shift between the two runs . Processing the data of these two experiments with traditional LC-MS-based pipelines leads to the same molecules being detected as different chemical features in downstream analysis. Figure 1 shows the analysis of pure samples to demonstrate this. In Extended Data Figure 4, we highlight how these technical variations make the same samples appear chemically disjointed. Using Qemistree, we mapped each of the spectra in the two chromatographic conditions to a molecular fingerprint, and organized these in a tree structure . Because molecular fingerprints are independent of retention time shifts, spectra are clustered based on their chemical similarity. It is noteworthy that the structural information from chemical features with spectral library matches or other forms of annotation could also be used to compare the chemical composition of samples across different mass spectrometry runs. Qemistree improves upon this by enabling the use of all MS/MS spectra with molecular fingerprints for downstream comparative analyses, by not constraining analysis to the chemical features with spectral matches only. This tree structure can be decorated using sample type descriptions, chromatographic conditions, spectral matches obtained from molecular networking in GNPS , and any other chemical annotations. Figure 1 shows that similar chemical features were detected exclusively in one of the two batches. However, based on the molecular fingerprints, these chemical features were arranged as neighboring tips in the tree regardless of the retention time shifts. This result shows how Qemistree can reconcile and facilitate the comparison of datasets acquired on different chromatographic gradients. On the southwestern Oregon Coast lies Coos Bay. This bay is important to the economy of Oregon; functioning as its southernmost deep water port . It has also historically been a center of trade and commerce among the Indigenous American peoples of the southwestern Oregon Coast. It is home to the eponymous Hanis and Miluk Coos peoples, as well as the Siuslaw. It has also frequently hosted peoples of the Tututni, Alsea, Lower Umpqua, and Coquille tribes. It is the languages of these first two peoples, the Hanis and the Miluk, that is the focus of this paper: hanis kuukwiisand miluk kuukwiis .According to oral history, the Miluk and Hanis people came to settle in what is now known as Coos Bay between 12,000 and 15,000 years ago, with archaeological evidence of permanent habitation at least 3,300 years ago . As Whereat points out, it is unlikely that there will be significantly older archaeological evidence found due to the natural geography and the nature of the ebb tide in Coos Bay. Starting in 1850 with the Oregon Land Donation Act, the Hanis, Miluk, and Siuslaw peoples were forced to cede their lands to the United States. This was followed by forced internment, conquest, and occupation of their lands that continue until this day . Both languages are currently near-silent, with only a few community members that know some words, phrases, and stories, though there is not yet language fluency at this time. The Miluk and Hanis peoples were confederated by President Reagan into the Confederated Tribes of Coos, Lower Umpqua, & Siuslaw Indians . In recent years, there has been a push towards language reclamation, led by tribal councilor Enna Helms and Patricia Whereat, Troy Anderson, and Dr. Lawrence Morgan.

Moreover, recombination could also occur at genomic regions other than the antibiotic insertion site, as the whole genomic DNA was used as donor to generate these recombinants. However, due to the bias of the method used here that selected recombinants acquiring antibiotic resistance, recombination that occurred at other regions in the genome may not have been present in the recombinants selected for assessment in this study. In addition, only the minimum size of recombination events could be estimated based on existing polymorphisms. Nonetheless, by targeting various genomic regions, it was confirmed that recombination occurred at multiple regions. Future studies by optimizing the selection procedures for recombinants in the context of pathogenicity to plants could reveal changes in virulence due to IHR in X. fastidiosa. Interestingly, IHR was bidirectional, meaning that both subspecies could act as both donor and recipient for one another. The evidence from field observation of X. fastidiosa disease emergence in new plant species and the detection of IHR in strains isolated from these infections by MLST/MLSA and from confirmation of natural competence in habitats mimicking natural environments to the experimental validation of IHR suggests that IHR is occurring in nature and may have broader evolutionary implications in X. fastidiosa disease dynamics.

In conclusion, X. fastidiosa strains showed extensive natural competence abilities and the recombination potential differed among strains. Moreover, plastic plant pot intersubspecific recombination occurred readily between X. fastidiosa subsp. fastidiosa and subsp. multiplex strains. These results emphasize the importance of quarantine measures to limit the introduction of novel genotypes of X. fastidiosa in areas with pre-existing infection. Moreover, measures to isolate host plants of different subspecies may be required to prevent mixed infections, minimizing the risk of generating novel and virulent genotypes of X. fastidiosa by recombination.Plasmids pAX1.Cm and pKLN61 were used from previous studies. Plasmids pMSRA-Km and pMOPB-Km were prepared as described earlier and are being characterized for another study in our laboratory . Briefly, about 800 bp long upstream and downstream fragments flanking open reading frames of methionine S-S-oxide reductase and outer membrane protein , respectively, were PCR amplified from the Temecula1 genomic DNA. The upstream and downstream fragments were digested using Asci restriction enzyme , were ligated, and were cloned into pJET1.2/blunt cloning vector, and a kanamycin-resistant cassette was inserted between the two fragments. All the plasmids were transformed into E. coli EAM1competent cells that express X. fastidiosa DNA methylase . Plasmids were prepared from the overnight cultures of EAM1 using an extraction kit , and concentration was adjusted to100 ng/µl . Aliquots were stored at _20
C until use. Natural competence assays were performed in PD3 agar plates.

The recipient strains were adjusted to OD600 of 0.25 in PD3 broth. Ten microliters of this suspension were spotted onto PD3 agar plates and 1 µg of plasmid in a 10-µl volume was added to the spots. Following incubation at 28
C for about 3 days , spots were suspended in 1 ml of PD3 and serial dilutions were plated in the respective antibiotic PW plates in triplicates, depending on which antibiotic cassette each plasmid carried, and PW plates without antibiotics. After 2 to 3 weeks of incubation at 28
C, CFUs were enumerated for recombinants and total viable cells , followed by calculation of recombination frequency as the ratio of the number of recombinants to total viable cells. For a given experiment, at least three repetitions were performed per strain, and the experiments had two to six biological replicates. For each strain, spots without the addition of plasmids were included as controls for every experiment. Genomic incorporation of the antibiotic-resistant marker from the donor plasmid was confirmed by PCR as previously described .To compare flanking region DNA sequence homology among recipient X. fastidiosa strains with respect to each donor plasmid, up- and downstream flanking regions of the antibiotic insertion sites of plasmids pAX1.Cm, pKLN61, pMSRA-Km, and pMOPB-Km were obtained. Up-and downstream sequences homologous to each plasmid region were obtained from the genomes of strains WM1-1, Temecula1, Temecula1*, BB08-1, AlmaEM3, BBI64 , and EB92-1 . The genomes were sequenced using Illumina Miseq and PacBio sequencing systems, and resulting reads after quality trimming were mapped to the Temecula1 reference genome, using the Geneious map to reference algorithm . The two up-and downstream sequences were concatenated, were aligned using the Muscle Multiple Sequence Alignment tool in Geneious, and percent identity between each of the donor plasmids and recipient strains was determined.Commerce via global trade and transport provides a mechanism for introduction of invasive species to new territories, extending pest habitats outside of their native regions . Invasive species threaten biodiversity, habitat, nutritious food, clean water, resilient environments, sustainable economies, and human health . Agricultural production systems are continuously challenged by invasive species that attack high-value crops, thereby significantly hampering the ability of food industries to maintain profitability . The geographic range of agricultural crops provides the potential for invasive species to colonize regions on a global scale .

Factors that aid expansion include short life cycle, fast growth rate, high plasticity, and resiliency to a wide range of environmental conditions . Such factors are drivers of rapid evolutionary change, population increase, and global colonization . Practitioners and stakeholders should aim to implement new strategies to manage such new invasive species in agricultural production . Drosophila suzukii Matsumura is an invasive species native to Southeast Asia. Passive transportation is the main reason of the dispersal of this species . It was first detected in North America and Europe in 2008 , and later in South America in 2013 , and Northern Africa in 2017 . The long-serrated ovipositor of D. suzukii enables it to oviposit inside fresh fruit, which creates a challenging management problem . Emerged larvae burrow within fruit pulp rendering fruit unmarketable . When D. suzukii became established in the U.S. during 2008, the total annual revenue losses for the West Coast berry and cherry industries were estimated at over $500 million . Currently the situation is not changed in term of economic impact . This particular insect is challenging to manage due to its high dispersal potential, ability to survive and adapt to harsh environmental conditions, and ability to attack a wide host range. For these reasons, D. suzukii is a key pest of these fruit industries worldwide. In the last decade, conventional insecticide uses on affected crops significantly increased to manage D. suzukii fruit damage. Typically used insecticides include spinosyns, pyrethroids, and organophosphates . Intensive use of insecticides poses a tremendous risk to non-target organisms such as pollinators, natural enemies, and humans . In addition, frequent insecticide applications likely resulted in resistance development . These factors require development of an IPM program that includes alternatives to conventional insecticides for managing D. suzukii. Non-insecticidal control methods including cladding, irrigation, netting, mulching, pruning, monitoring and mass trapping have been implemented against D. suzukii . While each method provides some relief to D. suzukii pressure,nursery pots they provide limited reductions in crop damage . Behavioral control of D. suzukii on susceptible fruit indicated promise for industry adoption. The food-grade gum possesses tactile and odorant cues resulting in reduced egg infestation. The food grade gum makes use of physical properties to mimic fruit, resulting in D. suzukii laying their eggs in a soft gel-like substrate, instead of the fruit itself. The food grade gum is a mixture of food-grade ingredients which is highly attractive to D. suzukii and competes with the ripening fruit throughout the season . To the best of our knowledge, the food-grade gum modifies various D. suzukii behaviors, ultimately resulting in a significant decrease in fruit damage. The product diverts D. suzukii away from ripening fruit, which results in significant retention of the pest, keeping it away from fruit. Third, the food-grade gum acts as an egg sink. Since the D. suzukii eggs laid in this medium cannot develop, this translates in a substantial reduction of the pest population growth . The aim of this work was to determine the potential of the food grade gum to reduce D. suzukii damage in large-scale commercial open-field and screen house fruit production units on blueberry, cherry, raspberry, blackberry, and wine grape. The hypothesis was that food-grade gum would reduce D. suzukii damage in small fruit, tree fruit and grapes under semi-field and small-scale field conditions.

These studies were conducted during 2019 and 2020 in California and Oregon in the western United States.In all field trials, GUM dispensers were placed at least 27 meters away from untreated control plots to minimize volatile plume interaction between treatments. In the current study, cotton pads were used to apply ~1.8 g of GUM on each dispenser at the rate of 124 dispensers per hectare under commercial production conditions . Cotton pads were placed directly on the ground close to irrigation drippers to provide adequate daily moisture. Earlier work illustrated that dispensers have a field longevity of 21 days and for this reason, dispensers were therefore deployed 1 to 4 times depending on the duration of crop ripening and susceptibility. In three trials , egg laying data were collected in buffer plots that were located between UTC and GUM plots to determine the active range of released volatiles beyond treated areas. This design was implemented based on the assumption that volatiles from treatment plots may be blown or diffuse beyond treatment plots. Berries were brought to the laboratory to determine number of eggs in fruit for each of the plots using a dissecting microscope. All soft or damaged fruits were excluded when assessing presence of eggs. In some cases, at first fruit color, laboratory-reared D. suzukii flies were released in each plot with the intent to create a relatively even pest pressure in all plots. Colonies of D. suzukii used in field studies consisted of seasonally collected wild adults from multiple field sites in the Willamette Valley, Oregon, and Oxnard, California. Collected adults were released into plastic cages and reared at 24°Cand 70% relative humidity, with a 16:8 h photoperiod before being released in the respective field trials. Flies were constantly provided with water and artificial diet that served as both a food source and an oviposition medium. Before their use in experiments, all flies were allowed to mate for 8 d in mixed-sex cages. A replicated field trial on drip-irrigated Pinot noir wine grape was conducted in Yamhill County, Oregon, USA from 10 to 18 October 2019 on ~2.6 hectares. Vines were spaced at 1.5 by 5 m, and trellised on a standard four wire trellis system, supporting a ~2 m canopy. Rows were oriented along a north-south direction on an east facing slope. Three treatments , were included with ~0.056 ha plots. No pesticides were applied during the experimental period. Here, there were 28 GUM and buffer plots each and 18 UTC plots. GUM dispensers were applied on 10 October and ten berries were collected from each plot on this date. Sampling dates were 11, 14, and 18 October 2019.Trials were conducted in a commercial sweet cherry orchard located at the Mid-Columbia Agricultural Research and Extension Center , Hood River, Oregon, USA. A 1.12-hectare orchard was divided into twelve plots . UTC, buffer, and GUM plots were replicated four times. The GUM dispensers were deployed on day 0 . No insecticides were applied to the orchard for the duration of the experiment. Here, an additional 200 mated 8- to 12-day-old D. suzukii were released in the center of each plot on a weekly basis on 23 June, and 1, 8, and 15 July 2020 . Data were collected for 35 days from 16 June through 22 July 2019. Because of relatively large canopy size of cherry trees compared to the other crops, ten cherries were collected from the lower , middle , and upper portions of the central two trees in each plot weekly.Trials were conducted in 1.8 hectare of high bush blueberry plants . The experiment ran from 6 October to 15 October 2020. There were three treatment levels i.e., UTC , buffer , and GUM. The GUM plots were located directly next to the buffer, followed by UTC plots of equal size. Plots were each ~0.05 hectares . Spinosad was applied on 6 October on the UTC and buffer areas. Insecticide application and GUM deployment occurred only on 6 October. On 8, 10, 13 and 15 October, one fruit sample consisting of 10 berries was collected from each of the 36 plots.

Paper sprayed with Tangle Trap sticky coating was placed a) at the base of the plants with a ring of sticky tape around the base of the stem and partially on the stem of the plant to capture any insects crawling down, and b) extending from the base of the plant horizontally outward above the pot surface to ensure complete coverage of the area covered by the plant canopy . This experiment was replicated on a single potted plant over time on 7 dates . Data were analyzed using Fisher’s exact test using SAS 9.2 . At our planned field trial site that would later be used in the B. bassiana trial, pupation emergence cages were used to sample insects moving off foliage towards pupation sites and later emerging out of the soil after pupation. Cages were made from Schedule 40 white PVC pipe with a diameter of 10.2 cm with cages cut to a height of 5.1 cm. The cage was then topped with a double-sided sticky card cut to fit,plastic flower buckets wholesale which was fixed into place with two elastic bands. Four lines of four cages were pushed into the soil to a depth of approximately 1 cm immediately adjacent to each other at the base of a blueberry plant and oriented in a cardinal plane to determine which direction showed the most thrips activity.

The four adjacent cages in a particular plane were used to assess thrips movement in the understory of the blueberry plant in each directional. The study was replicated on 5 plants on a single date and conducted just prior to the commencement of the field trial. Data were analyzed with a nested ANOVA using SAS 9.2. In a greenhouse trial, Mycotrol O ® was applied directly to the soil surface as raw spores and compared to the same product colonized onto millet seed, also using soil application. Millet seed colonization used the Stanghellini and El-Hamalawi method as described below. The colonized millet seed, when allowed to imbibe water and incubate in the laboratory, can support 1.0 x 106 conidia/seed . Based on Stanghellini et al. with modification, we held the GHA colonized millet seed in containers such that the seed mat was at a depth of no greater than 2.54 cm. The seeds were wet with the consistency of very thin slurry and were gently stirred three times per day for four days to ensure they imbibed water properly so that mycelial growth and sporulation would occur. Sporulation was confirmed by slide mounting random sections of mycelia and checking for condia formation under the microscope.

Once spores were initially observed, the seed was held an additional three days so that sporulation could continue before use of the colonized seed in the field study. Mycotrol O® was applied in the maximum recommended field rate for high thrips levels of 2.84 L of material in 378.5 L of water.The colonized millet seed was tested in the greenhouse to determine if late second instar citrus thrips would become infected if they crawled over or through the seed when it was placed at the base of a laurel sumac seedling. A single small laurel sumac seedling, about ~10 cm tall, was placed into each of ten, 9.5 x 9.5 x 18 cm styrene cages with 6 cm diam air holes on all four sides that were covered with ultra fine mesh screening . Small holes were made in the bottom of the container and covered with pebbles to allow for drainage, then soil was added to a depth of 7.62 cm and the top of the container was covered with a removable lid. The base of each plant was completely surrounded by either B. bassiana colonized millet seed or with uncolonized seed . A minimum of 20 late second instar thrips were released onto the leaves of each plant, and were left until enough time had passed for the thrips to molt to the propupal stage.

The seedling was then cut at the soil line and examined for pupating thrips; the removable lid of the cage was sprayed with Tangle Trap sticky coating to collect any emerging adults after 5 days so infection could be measured. The study was replicated on 5 dates . Data were analyzed using 1-way ANOVA with time as a factor and means were separated using Tukey’s Least Significant Difference test using SAS 9.2. To determine the optimum number of colonized millet seeds needed for close to 100% infection when thrips were seeking pupal refuges off the plant, varying amounts of colonized seed were evaluated in a greenhouse trial based on the size of the seed once it had imbibed water and sporulation had occurred. After water inhibition, nine seeds completely filled one square cm of soil surface. There was a 0.5 cm buffer area around all sides of the cage, which was kept clear of seed to provide a 9 x 9 cm grid of seed on the soil surface below the plant. All but two leaves were plucked from the seedling. Small holes weremade in the bottom of the container, which was covered with pebbles to allow for drainage. The 9 x 9 cm2 grid was created from wire screen and differing amounts of sporulating seed or seed alone were placed on the light imprint made from the wire screen on the soil surface. Two replicate seedlings per treatment were set up per date in a complete block design . Plants were watered every third day. A minimum of 20 late second instar thrips were placed onto the leaves of the plant, and were left until enough degree-days had passed for the thrips to molt to the propupal stage, typically about 5 days. The seedling was then cut at the soil line and examined for pupating thrips; the removable lid was sprayed with Tangle Trap sticky coating to collect any emerging adults after another 5 days. Data were analyzed using a 3-way ANOVA with density of seed , application of B. bassiana , and date as factors . Unrecovered insects were counted as missing data and were not included in the analysis. The commercial blueberry test site selected was located north of Bakersfield in Delano, CA. The trial began in August of 2008 and was conducted post blueberry harvest. The V. corymbosum varieties contained within the test area were, ‘Santa Fe’, ‘Jewel’, and ‘Star’. The most susceptible variety of blueberry to citrus thrips damage grown at the test site was the ‘Star’ variety and ‘Star’ was used consistently for evaluation of thrips numbers for all aspects of the trial . Our cooperator was interested in alternatives to traditional pesticides as the farm regularly was dealing with extremely high citrus thrips populations. For example, in 2008 the grower sprayed 5-10 times per field , rotating with traditional chemicals to reduce thrips impact on the subsequent year’s fruit set. Irrigation in all fields took place via drip irrigation with one water delivery emitter per line at each plant base , but additionally, one portion of the blueberry field was equipped with 360° overhead sprinklers. This irrigation setup provided the ideal situation to test B. bassiana under two watering regimes.

The commercially available GHA strain is formulated to be mixed with water and for application via chemigation or as a foliar spray. The label states that no surfactant is needed to keep the spores in suspension. However, agitation alone in the 1,892.7 L holding tank was not sufficient to keep the material from precipitating,black flower buckets therefore 312.3 ml of Silwet L-77 was added to the tank mix. Mycotrol O® was applied directly to the soil surface with a gas-powered sprayer with a hand spray gun equipped with an adjustable flow meter. The dimensions of the plots were used to calculate the amount of material needed for both B. bassiana formulations . Plants in the test field were spaced every 0.92 m down each row, 3.35 m between each row, and each row was about 165 meters in length. Our studies were conducted in an 18-row section of a 4.04 ha field. The overhead sprinklers were spaced every 7 meters in the row and were located every other row for 12 rows. We chose to investigate the effectiveness of the B. bassiana colonized millet seed versus a Mycotrol O® soil application under two watering regimes, drip-line alone versus drip-line with overhead sprinkler, because B. bassiana conidia are highly subject to desiccation. Comparing the soil drench in both irrigation types with the colonized millet elucidated the effectiveness of the treatments when compared to the control. The blocks were laid out in a 3 x 2 factorial design, with each block consisting of most of five rows of blueberries , each being 27.4 m long . The berm used to grow blueberries at the commercial farm was 1.21 meters wide and each plot was 27.4 meters long. The spacing between adjacent rows was 3.35 m, while the spacing between the plants down a row was approximately 0.92 m with 30 plants per treatment plot . These dimensions result in 0.157 ha treated with raw spores but because the top of the berm was where thrips activity was evident and would be sampled, only 36% of the soil surface area was treated. The Mycotrol O® label states that the maximum field rate is 6.9 L/ha mixed in 935.3 L/ha water. We therefore chose to apply the entire 6.9 L of Mycotrol O® in 378.5 L of water per ha directly to the berm with no application between the rows, which resulted in 100% of the per ha rate of product being applied to 36% of the area and allowed the maximum amount of active ingredient to be applied to the area that would have almost all thrips activity . Our field trial was intended to determine the extent to which B. bassiana might fit into a program projected to both control citrus thrips effectively and provide rotation among available chemistries so as to reduce thrips resistance evolution.

Thus, we felt it was important to operate under the best possible conditions for thrips infection by Mycotrol O® , regardless of financial considerations, i.e. application of product at the maximum label rate in the area where thrips were most likely to be active. The amount of millet seed used in the field trail was calculated based on the area of the berm to be treated and likewise with the Mycotrol O® treatment, only 36% of the total field area was treated. The amount of seed used was one colonized seed/ 2 cm2 over an area of 576 m2 ; the fact that 0.45 kg of seed was needed per 840 cm2 resulted in the application of 3.40 kg of colonized millet seed for the 8 treated plots . Every other plant within the middle ten plants of the middle row of each plot were sampled with pupation emergence cages . These cages were placed tight against the base of each set of canes on the east side . With 5 cages per block and 4 replicate blocks per treatment, a total of 20 cages sampled thrips pupation per treatment over two sample periods, i.e. for two consecutive 3-day periods after the Mycotrol O® soil drench. The treatments were: no B. bassiana with and without overhead sprinkler; colonized millet seed with and without overhead sprinkler; and a soil drench of Mycotrol O® with and without overhead sprinkler . In total, data were collected from 240 emergence cages over the duration of the trial . The colonized millet seed was set to imbibe water and allowed to sporulate for three days before application and was applied using a hand fertilizer applicator . Four days post application of the millet seed, the soil drench of Mycotrol O® was applied and pupation emergence cages were placed in the field and left out for 3 days . After three days, the sticky cards from each emergence cage were collected and replaced with new cards and the traps were switched to the next plant on the east side. These traps were left in the field to sample thrips for another 3 days . Because the traps were placed out every other plant, this ensured that all of the middle ten plants were sampled over the two, 3-day sampling periods .

Only 39 cases needed to be top coded to 20 servings. The individual fruit and vegetable items for all 39 cases were examined carefully to make sure that they consisted of believable number of servings, albeit large numbers reported, and all 39 were accepted to remain part of the data file for analysis.The General RDD sample in File-1 was weighted to 1990 Census data for California adults using race, age group, and gender dimensions to emulate the standardized weighting of the CDPS surveys—all of which were weighted to the 1990 Census. Weighting within each of the three race/ethnic groups within each month’s sample, the ANOVA model was used to examine the year-month interaction term and variation among months for White, African American, and Latino groups. This last analysis produced the conclusions for this study with regard to seasonality in fruit and vegetable intake.As part of a nested design, three brief questions were asked to a randomized subset of survey respondents to see how well these might perform compared to the longer, more complex set of CDPS recall questions. This short form of the intake questions are referred to in this study as the “SF3.” For this analysis of the SF3,25 liter pot plastic approximately 57% of the File-1 sample was used due to prior random allocation.

These respondents were administered the SF3 questions ahead of the usual CDPS dietary intake questions. The SF3 captured the following: the number of servings of fruit not including 100% fruit juices ; the number of servings of 100% fruit juice ; the number of servings of vegetables ; and, the total number of servings of fruit and vegetables . Analyses were performed comparing each respondent’s SF3 answers and the respondent’s corresponding number of servings calculated from the CDPS intake responses. The initial analytical step examined Pearson correlations to see if they were positive and relatively high for this type of measurement . If true, results were then examined using a paired t-test to see if there was any consistent bias in the relationship between the SF3 and the CDPS questions. Paired t-tests were performed using a two-tailed significance level of 0.05. Looking at the 6,650 cases in File-1 and the 4,031 low-income cases in File-2 , the year-month interaction is significant for the total servings of fruit and vegetables for File-1 and File-2 . In File-1 this is not the case for servings of fruit . The observed pattern of servings of fruit intake seen in Exhibit 7 shows the month of July with the highest intake at 2.5 servings and November with the lowest intake at 2.0 servings.

Since the year-month interaction is significant for servings of vegetables , a direct interpretation of the monthly variation is not possible. In File-2 there is significant year-month interaction for both servings of fruit and vegetables . These findings indicate that the patterns of intake across the 12 months is different for each of the two years of data collection , and thus it is not feasible to combine the monthly samples from these two years to interpret variation among the months using all the cases in each file. The General RDD component of File-1 , a sample that does not have any race/ethnic over-samples included and thus very much like the CDPS general population sample, has no significant year-month interaction for the total servings of fruit and vegetables. This is also true, as might be expected, for the servings of fruit and the servings of vegetables, separately. Thus, it is the Targeted RDD component of File-1 consisting of the “all-incomes” over-samples of Latinos and African Americans where significant interaction is found for total fruit and vegetables , total fruit , and total vegetables . Given the findings for the General RDD component, the pattern of intake across the 12 months is not significantly different for each of the two years of data collection, and therefore, it is possible to combine the monthly samples from these two years to interpret variation among the months. However, the significant year-month interaction for the Targeted RDD component precludes a direct interpretation for the “all-incomes” oversamples of Latinos and African Americans.

The results for variation among months in the General RDD sample suggest that there is no significant difference among months for the total servings of fruit and vegetables. However, the p-value is exactly 0.05 and is right on the boarder of being statistically significant. Variation among months for the total servings of fruit and for the total servings of vegetables are each not significant. Exhibit 8 shows the average number of servings per month for each of the twelve months of the year for total fruit and vegetables, for fruit, and for vegetables in the General RDD sample after combining the data for Year-1 and Year-2. These results and monthly patterns in the General RDD sample are virtually unchanged even after controlling for race, ethnicity, and gender . Since the sample is designed to examine fruit and vegetable intake for Whites, African Americans, and Latinos, results are generated for each in both File-1 and File-2 . Exhibit 9 shows the results of the ANOVA Ftests, looking first if any year-month interaction exits, and if none exists, looking for significant variation among months as an indication of seasonal effects. No seasonality is found for Whites when looking at all income levels for total fruit and vegetables, or for fruit and vegetables separately. This is true for low-income Whites for servings of fruit and for servings of vegetables, but significant year-month interaction for the total number of servings of fruit and vegetables precludes any direct interpretation of monthly variation. No seasonality is found for African Americans when looking at all income levels for the total of fruit and vegetables or for fruit and vegetables separately . This finding is also true for low-income African American, with the exception of significant monthly variation for servings of fruit . It is not possible to interpret directly the results either for Latinos of all-incomes or for File-2 low-income Latinos, since there is significant year-month interaction for the total of fruit and vegetables as well as for fruit and vegetables separately.

An examination of the monthly pattern for Years 1 and 2 separately shows each year to be very different; however, the pattern within each year is similar for the Latinos in File-1 and in File-2. The very different patterns for each of the years are the cause for the significant year-month interaction. To demonstrate this, the pattern for the total number of servings of fruit and vegetables for Year 1 and Year 2 and for File-1 and File-2 Latinos is shown in Exhibit 10. Observe that March and April, June and July, and September, October, and November appear different between the two years. There is no explanation for these different patterns. When looking only at the low-income cases found in File-1, the picture changes slightly . For low-income Whites, no year-month interaction is now detected as it was shown when looking at all income Whites. This is also true for servings of fruit for lowincome African Americans. or African Americans , they may be harder to detect given the smaller sample sizes of these low-income groups. A year-month interaction for total fruit and vegetables is also not detected. For both Whites and for African Americans , no variation among months is found for the total servings of fruit and vegetables or for fruit and vegetables separately. For Latinos,25 litre plant pot with a large sample size of 1,040, year-month interaction for total fruit and vegetables is not detected and no significant variation among months is found for the total servings of fruit and vegetables . However, significant variation among months is found for servings of fruit. Exhibit 12 shows the observed highest months of fruit intake to beFebruary and June with the lowest observed months to be March, May, and November. It is not possible to easily determine variation among months for the servings of vegetables due to significant year-month interaction . To gain additional insight into the low-income Latino patterns, the data are examined by the two levels of Latino acculturation: high and low. Exhibit 13 shows that there is no significant variation among months for Latinos with high acculturation whether the data are examined for all incomes , or for those with low-income. Among Latinos with low acculturation, however, it is not possible to easily interpret the data for the all-incomes groups due to significant year-month interaction . For the low-income, low-acculturation Latinos, there is no significant variation among months for total fruit and vegetables . For servings of fruit, both the all-incomes and low-income Latinos show significant variation among months if they are low acculturated.

Exhibit 14 shows the observed average servings of fruit per month for low-acculturation Latinos in the all-incomes group and the low-income group . The months of February and June show the highest averages, while March, May, and November show the lowest averages of fruit intake. These observations for individual months for both lowincome acculturation groups are the same as seen in Exhibit 11 for all low-income Latinos regardless of acculturation level. The CDPS data for the eight bi-annual surveys conducted since 1989 are each weighted to the 1990 Census for California. The cases are weighted for gender, age group, and race dimensions. Since the General RDD sample in this study is similar to the CDPS general population sample, i.e., it includes the entire adult population , this study’s General RDD sample data have been weighted using the identical weighting approach used in the CDPS. In Exhibit 15, using the weighted General RDD sample data , no two groups are statistically different from each other. In an examination of the variation among months, Exhibit 18 shows the results for the overall population of all adults as well as the results for Whites, African Americans, and Latinos. Among all adults, variation among months is found to be significant for the total servings of fruit and vegetables consumed. However, this variation is not significant either for servings of fruit alone or for servings of vegetables alone. Exhibit 19 shows the monthly averages across the year. It is found that the average number of servings for total fruit and vegetables in the month of July is significantly higher than the number of servings in January , a difference detectable due to the relatively large sample size. However, July is found to be not significantly higher than any other month in the year. There is no significant variation among months found either for servings of fruit or for servings of vegetables for the adult population. For Whites, there are no significant differences in the variation of number of servings among months for the total of fruit and vegetables or for either fruit or vegetables alone. The observed monthly patterns for Whites are shown in Exhibit 20. African Americans show significant variation among months for the number of servings of total fruit and vegetables and for servings of vegetables . Despite the pattern for servings of fruit being very close to that of vegetables, no significant difference is detected for servings of fruit . Exhibit 21 shows the wide variation observed among the months of the year. Even with the smaller African American sample, significant differences between individual months for total servings of fruit and vegetables are seen, with the month of December higher than June and January and none of the other months being different from each other. Although the variation among months for servings of vegetables is found to be significant, the follow-up procedure could not detect any significant differences between individual months. The small AfricanAmerican sample, 177 cases distributed over 12 months , needs to be considered when interpreting these findings. Latinos show no significant differences in the variation of number of servings among months for the total of fruit and vegetables or for either fruit or vegetables alone. The observed monthly patterns for Latinos are shown in Exhibit 22. The large sample sizes used in this study reveal that patterns across the months of the year can differ from year to year, as indicated by the significant year-month interactions found for the overall samples of both File-1 and File-2.

Based on the preferred effective Ks, the highest θc deviation between the RZRT model and HYDRUS is one day. While this is a reasonable error for the hard pan characteristics tested here, it is based on one limited example and cannot be regarded as representative. Using effective soil hydraulic parameters to represent highly-contrast layered soil as a uniform soil profile is a complex problem which was beyond the scope of our simplified analysis. Hence, the hard pan analysis implemented in the RZRT learning tool can be used as a first approximation only. Obviously, in fields with a massive hard pan layer, deep percolation is limited, and implementation of Ag-MAR project is not recommended. The drainage curves for all USDA soil texture classes provide an initial estimation of soil suitability for Ag-MAR . The crops almond, walnut, alfalfa, and grape in Fig. 4 with flooding tolerance of 2, 7, 14, and 21 days, respectively, represent the four tolerance classes in Table 3. Since crops have different flooding tolerances, soil drainage largely controls the effective flooding duration. The slow drainage rate combined with the low flooding tolerance that some perennial crops have, make clayey soils, as expected, unsuitable for Ag-MAR. The clay loam soil with shallow-rooted high-tolerance crops, might be suitable for Ag-MAR in terms of drainage duration, however, its ability to transfer large quantities of water is hindered by its relatively low hydraulic conductivity questioning its Ag-MAR suitability. The silty clay loam is an exception of the clayey soils,30 plant pot as it can be used for Ag-MAR with moderate- and high-tolerance crops.

The reason for this exception is the low sand content combined with the silt and clay proportions, which results in a higher critical water content as aggregated soils have higher θccompared to structureless sandy soils . For the same reason, loam soil with lower θc can be used only for Ag-MAR if shallow-rooted moderate- and high-tolerance crops are considered. According to the drainage curves, silt loam and sandy loam soils are suitable for all crops, excluding minimal-tolerance crops, while sand, silt and loamy sand soils are suitable for all crops that have minimal flooding tolerance. For most Ag-MAR sites with contrasting soil layering along the root zone, the model prediction will be less accurate, and a procedure of parameter averaging may be necessary to improve the model performance. Similar model limitations that are related to the homogenous soil profile assumption, are expected at sites with substantial soil heterogeneity, as our results show for the Stoner gravelly sandy loam . A reasonable estimation of the hydraulic parameters is another limitation that should be considered. For example, according to the soils tested in this work, when using fitted and average unfitted hydraulic parameters, twap is overestimated by 0.2 and 1.7 days, on average, respectively . The model assumption of rigid porous media with constant hydraulic properties poses another difficulty, especially under Ag-MAR conditions where the soil can be waterlogged for relatively long periods . Changes in soil structure and hydraulic properties during prolonged flooding were reported in paddy soils due to clay swelling ; however, shrink-swell dynamics are more prominent in clayey soils , which are potentially less suitable for Ag-MAR. The use of hydraulic parameters from a soil database can lead to moderate or poor predictions compared to soil-specific hydraulic parameters.

The shape parameter m and the scale parameters θr, θs and Ks control the drainage curves and therefore are the most important parameters in the RZRT model. For improved accuracy, these parameters should be evaluated insitu at the designated Ag-MAR site. θr and θs can be estimated by the gravimetric method and Ks of the upper soil by various field infiltration tests . Estimating m can be attained by fitting of the SWRC or the particle size distribution ; however, these methods are time-consuming, so it is recommended to obtain m from a soil database, the literature, or to use a pre-defined m based on soil texture. Lastly, and probably most importantly, the parameter estimation of θc can change markedly depending on the method it was first evaluated with . Moreover, the concept of a constant θc is an oversimplification used in the model, because θc is a function of biotic and abiotic parameters, which vary spatially and temporally for a specific soil texture. Indeed, our data indicate that during intervals of AgMAR flooding and drainage, different θc can be obtained even for the same location in the soil . This is probably related to changes in soil respiration after flooding is initiated as well as SWRC hysteresis . Te idea that one should be eating healthy to stay healthy is not a debate. Numerous studies show how particular foods individualistically effect human health, but none thus far, to our knowledge, have investigated about the combined impact of a specific diet on the human body as a whole. It is critical for us to understand which kinds of things we should eat and the ways in which their collective consumption will impact our bodies. According to Dr. Tomas J. Carlson, a distinguished pediatrician and ethnobotany researcher, choosing foods from every color in the rainbow is the key to good health.Each fruit and vegetable gets its natural color from the chemical composition of the exclusive phytonutrient in it.Interestingly, the presence of one molecule in one fruit/ vegetable does not necessarily reflect the same color in another type of fresh produce.

For instance, although the rich red color in most red fruits and vegetables is naturally derived from the phytonutrient lycopene, most berries such as strawberries and raspberries do not contain lycopene. Instead, they contain brightly colored chemicals called anthocyanins, which are made in plants during ripening season through the joining of a molecule of a sugar with a molecule of their colorless “anthocyanidin” precursors.Anthocyanins are also found in raspberries, which are high in dietary fiber and vitamin C and have a low glycemic index because they contain 6% fiber and only 4% sugar per total weight.Higher quantities of fiber in the fruit, when consumed, helps lower the levels of low-density lipoprotein or the ‘unhealthy’ cholesterol to enhance the functionality of our heart and potentially induce weight loss. Te exact pigment that anthocyanins reflect is partly dependent on the variance in acidity or alkalinity in different plants. Because of the relatively high pH of the tissues in blueberry plants,grow raspberries in a pot these chemicals turn blue in color during the ripening process of the fruit.Recent research in the Journal of Nutrition suggests that the abundant antioxidant properties in wild blueberries contributes to the reduction in the development of such disorders as Alzheimer’s Dementia and cognitive loss.A type of antioxidants selectively found in yellow and orange colored foods are called cryptoxanthins. In a study conducted by Bovier et al., it is shown that the combination of the beta form of these carotenoids with other sources of nutrients such as lutein and zeaxanthin in carrots, oranges, and corn leads to improved visual processing speed with regular consumption in young healthy subjects.While green produce mainly derives its pigmentation from chlorophyll, its white counterparts get their natural color from anthoxanthins, favonoid pigments that exhibit antioxidant properties. Among green fruits and vegetables, broccoli stands apart as the most nutritious because of the special combination in which its 3 glucosinolate phytonutrients are found. Tis “dynamic trio” makes what are called Isothiocyanates , the detox-regulating molecules in broccoli that enhance vitamin A in the form of beta-carotene.Many recent studies claim that the antioxidants in ITCs not only regulate metabolism and cholesterol levels when consumed but also act as cancer chemopreventive phytochemicals.Fruits that are on the same level as broccoli with regards to health in the white-produce family are bananas. Japanese Scientists reveal that the high amounts of vitamin B6, manganese, potassium and fiber in the ripened versions of these fruits can help prevent high blood pressure, protect against atherosclerosis, and improve immunity levels in regular eaters.Despite an enormous amount of scientific knowledge and evidence for the overall beneficial effect of a single fruit/vegetable and/or phytonutrient at a time on human health, no study so far, to our knowledge, has been able to conclusively link the validity of these claims to the whole human body. This offers the opportunity for one to test the combined impact of eating a colorful diet on humans through a systematic study. The purpose of our investigation is to apply a more holistic approach to the study of how the human body is effected as a result of a diet that is composed of all the colors of the rainbow. In other words, in addition to exploring the individual food stuf’s role in improving health, we want to analyze the outcome of the regular incorporation of a whole pack of colorful foods into one’s meals. Consequently, this study can serve to reveal the effect, if any, of a continued and rigorous diet consisting of all colors of the rainbow on the physical and mental health of a randomized sample of the adult human population in a given demographically comparable community.

The current study is a small-scale secondary application of some of the methods used in a previously conducted study that has been reported elsewhere.The primary study used a randomized controlled trial to compare the effect of daily consumption of probiotic versus low-fat conventional yogurt on weight loss in healthy obese women; the outcomes tested were changes in anthropometric measurements . In our study, we measured hand grip strength and stress levels in addition to some of the parameters mentioned that were tested in the primary study. We created a Rainbow Diet Pack that consisted of the following fruits and/or vegetables in the respective quantities: raspberry , orange , baby carrots , corn broccoli foret , blueberry , and banana . The choice of each kind of fresh produce was based on the specifc nutritional facts and molecular composition of the phytonutrients in each . As per the personal choice of its members, the intervention group received daily administration of RDP during a 10-wk intervention period. Measurements were taken of both the study and the control groups twice: at baseline and at the end of the intervention period. Our study design was in accordance with the Declaration of Helsinki.Twenty-four normally healthy human adult volunteers who belonged to the same demographic identity and had similar dietary backgrounds were recruited by word-of-mouth from the local community of the student investigators and screened for health. A total of eight were chosen to participate. Individuals were eligible for the study if they were nonsmokers, free of known disease, not allergic to items in RDP, not taking medications and were identifed as being healthy according to the following criteria: body mass index between 18.5 to 24.9 kg/m2 and a self-report of no diseases/illnesses in the previous 6 months. Randomization A computerized random number generator was used to assign individuals chosen to participate to either the control or the intervention group. At the end of the baseline screening, a message containing the participant’s number assignments was sent via email to the participants. Participants and the student investigator were aware of group assignment during the intervention phase. Participants were not aware of other participants who have agreed to be in the study. Before analysis, the primary investigator received an anonymized data set and was no longer aware of group assignment post data collection; no data can be traced back to the individual participant.The aim of this study was to assess the effects of eating a diet consisting of all the colors of the rainbow in the form of an RDP once a day on weight loss, stress levels, and other indexes of health in normally healthy volunteers during a 10 wk intervention program. We found that consumption of RDP as lunch may result in positive changes in waist circumference, weight loss and stress levels as measured during the program. This was despite fnding no signifcant diferences in observed hand grip strength between the study and control groups. In spite of evidence for the beneficial effects of eating various naturally colorful produce on obesity and health, to our knowledge, this was the frst randomized controlled trial that investigated the effect of consuming the RDP as a whole on weight loss and stress levels in healthy human subjects.

Water samples were analyzed for their stable isotopic compositions using Los Gatos DLT- 100 laser isotope analysers for Dorset and Wolf Creek, a Los Gatos Liquid Water Isotope Analyzer for Bruntland Burn and Dry Creek, and a Picarro L2130-I for Krycklan. The precision of the liquid water stable isotope analysis is reported to be better than ±0.1 ‰ for δ18O and ±0.4 ‰ for δ2 H. All isotope data are given in delta-notation in reference to the VSMOW. At all sites – apart from Dry Creek – direct water-vapor equilibration analysis was used to sample the bulk soil water isotopic composition from the soil . The accuracy of the direct water-vapor equilibration method was ±0.3 ‰ for δ18O and ±1.1 ‰ for δ2 H. For a detailed description of the procedure, we refer to Sprenger et al. . Bulk soil water isotopic compositions at DC were sampled using cryogenic extraction at 100°C under vacuum of < 30 millitorr over 40 minutes, as described by McCutcheon et al. . We are aware that different methods of soil water extraction have been a major focus of research in the past few years, with no definitive agreement on a standard method . While differences between cryogenic extraction and the direct water-vapor method have been reported in laboratory experiments ,container raspberries previous work by the authors has found the direct equilibrium method to be a reliable method for extracting bulk soil water from sandy soils giving similar results to cryogenic extraction .

Source water apportionment of plant xylem: To quantify the potential source of vegetation water from different soil depths and over a range of time periods, a modification of the ellipsoid method was utilized for the gymnosperms and angiosperms at soil depths in 10 cm increments up to 40 cm. All soil samples deeper than 40 cm were lumped together. The 40 cm cut off was chosen due to fewer sites sampling below 40 cm and a large decrease in the temporal resolution of sampling which could otherwise skew results. Due to soil water fractionation resulting in deviation from the local meteoric water line, the data are not well represented in an ellipsoid shape such as that employed in Amin et al. . Therefore a minimum polygon area was used to encompass the data points. Based on Equation 5, we derived the sw-excess of xylem isotope data. For sw-excess less than 0 ‰, the xylem data plot below the soil water line of the corresponding soil water isotopes sampled on the same day. Thus, the sw-excess can serve as an indicator for deuterium fractionation between the uptake time at the root-soil interface and the measured xylem water. We acknowledge that the soil water line is not necessarily solely a product of evaporative enrichment and that seasonal variability of the stable isotope compositions of the precipitation can affect how much soil waters deviate from the LMWL . However, the process of how the “soil water line” developed is not important here, since we used the regression to describe the isotopic compositions of potential water sources for vegetation at the time of sampling.

The influence of site characteristics on soil and xylem isotopic samples was evaluated using Spearman rank correlation. Site characteristics, mean annual temperature , elevation , aridity index , annual precipitation , and latitude , were correlated to xylem and soil water δ2 H , δ18O , and the corresponding sw-excess , and lc-excess to a significance level of 0.001. Isotopic compositions of all soil depths, vegetation species and sampling times were bulked for each site to assess an overall trend of soils and vegetation in relation to climate indices. Statistical analysis of isotopes in precipitation, bulk soil water, angiosperms, and gymnosperms was conducted at each site using the Wilcoxon signed-rank test to test the statistical similarities of median values of the datasets. This allowed for a two-sided probability test without the assumption of normality. The datasets were tested to the 95% confidence limit using all available data .Plant water and soil water data from the five sites are plotted in Figure 2. For both soils and xylem, the sites occupied partially overlapping regions showing a general gradient from highly isotopically depleted at Wolf Creek, the coldest of our sites in Canada, to the more isotopically enriched waters at Bruntland Burn at the temperate/boreal transition in Scotland. For each site there was a substantial range of variability in soil and xylem water isotope composition over the course of the sampling year. Most soil and xylem samples plotted below the GMWL, although xylem waters were generally more 2 H-depleted at each site, which was also evident from the lc-excess data . Samples from Dry Creek and, in particular, Wolf Creek showed the greatest divergence from the GMWL.

These two sites slightly obscured an otherwise clear relationship between plotting position along the GMWL and the mean annual temperature gradient through Krycklan, Dorset and Bruntland Burn. Despite this, the isotopic ratios of δ2 H and δ18O in soils and xylem water correlate positively with air temperature, annual precipitation and aridity index, and negatively with elevation and to a lesser extent latitude . At all sites, substantial isotopic differences were apparent between xylem and soil water isotopes, and between angiosperms and gymnosperms . Gymnosperms generally plotted further from the GMWL . Soil waters at each site generally tracked precipitation and snowmelt inputs being more 2 H- and 18O-depleted in winter/spring and more enriched in summer; evidence of evaporative fractionation was also most evident in the more 2 H- and 18O-enriched summer soil water samples. The soil water data are shown relative to the sampling dates for each site in Figures S2 to S6 in the Supplementary material; also see Sprenger et al. for more detail. Soil water δ2 H data were significantly different from precipitation at Dry Creek, Dorset and Wolf Creek, while soil water δ18O differed from precipitation at Bruntland Burn and Dorset . Bruntland Burn, Krycklan and Dorset showed the greatest visual deviation of xylem δ2 H samples from soil water, while the most southern site, Dry Creek, and the most northern site, Wolf Creek, showed smaller differences between the xylem and soil water isotopes for δ2 H . However, at all sites the δ2 H characteristics of both angiosperms and gymnosperms were significantly different from soil water . Angiosperm xylem water δ18O at all sites, apart from Krycklan, was significantly different from soil water δ18O; whereas significant differences for gymnosperms were apparent only for Dorset and Bruntland Burn. Xylem water isotopic characteristics differed between angiosperms and gymnosperms at some sites. For δ2 H, they were significantly different for Krycklan, Bruntland Burn and Dry Creek, while for δ18O they were different for Dorset and Dry Creek . Snowmelt plotted on the LMWL and was more depleted for 18O than almost all measured soil and xylem waters,draining pots although a substantial number of xylem samples at Dry Creek, Krycklan and Wolf Creek were more depleted in 2 H but plotted off the LMWL . Similarly, at the four sites where groundwater samples were collected, the mean isotopic composition of groundwater fell on the LMWL but plotted towards the more depleted end of the range of soil water samples. This reflects the generally higher recharge of groundwater by depleted water following the spring melt at Dry Creek, Krycklan, and Wolf Creek ; and during winter rainfall at Bruntland Burn . Isotopic composition of groundwater at all sites showed limited temporal variation, indicating the volume of annual recharge is small relative to groundwater storage. Groundwater was generally more strongly depleted in 18O than xylem waters for both angiosperms and gymnosperms, although at each site a substantial proportion of xylem samples were more depleted in 2 H.The minimum boundary polygon analysis quantifies the degree to which xylem water for both angiosperms and gymnosperms overlaps bulk soil water sources at different depths. The use of the spatially bulked data for soils and vegetation at each site was necessary to provide a sufficient number of samples for the development of encompassing polygons.

This may lead to larger estimated polygon areas and a greater estimated overlap of soil and xylem water, although the effect is much less marked than for the ellipse method of Amin et al. . This may provide insight into the sources of xylem water, although the proportion that cannot be ascribed to soil water sources is equally informative regarding the need to hypothesise and identify other causal reasons. Distinct inter-site differences emerged in terms of the overall overlap of xylem and soil water isotopic composition . For Bruntland Burn, soil water had a 77% overlap for angiosperms, but only 6% for Gymnosperms. At Dorset, like Bruntland Burn, angiosperms showed a much higher degree of overlap than gymnosperms . At Dry Creek, almost all xylem water in both angiosperms and gymnosperms overlapped soil water at almost all profile depths. Of all sites, Krycklan had the lowest degree of overlap with only 27% for angiosperms and 0% of gymnosperms . Finally, while Wolf Creek had only angiosperms present as willow and birch shrubs, a 99% overlap between xylem water and soil water was evident. The depth dependent overlap of xylem and soil water isotopic composition showed differences between depths, with higher overlap tending to be in shallow soil depths for most sites. For Bruntland Burn, there was 72% and 55% overlap between angiosperms and soil water at 0-10 cm and 10-20 cm depths, respectively, but only 9% and 3% for gymnosperms. Dorset was the only site with the greatest overlap occurring in deeper soil, with overlaps of 34%, 28%, 24%, 59% and 31% for 0-10, 10-20, 20-30, 30-40, and >40cm, respectively. The gymnosperms at Dorset had a similar deviation to that of angiosperms, with much smaller overlaps of 4%, 7%, 8%, 18%, and 7%, for 0-10, 10-20, 20-30, 30-40, and >40cm, respectively. Depth-dependent overlap of soil and angiosperms at Dry Creek was high through all soil layers with the greatest overlap in the near-surface soils . Gymnosperms at Dry Creek had a similarly high overlap of 78%, 55%, 86%, 72% and 86 % for 0-10, 10-20, 20-30, 30-40, and >40cm, respectively. At Krycklan, the upper two soil depths had approximately the same overlap for angiosperms , with a moderate decrease to 12% in the 20-30 cm soils. None of the soil water at any depth overlapped the gymnosperm samples. Wolf Creek angiosperms showed a high overlap in the upper two soil depths with a more substantial decrease in deeper soils .The general patterns of the pooled data sets for the entire study year mask differences in the degree to which seasonal variations in the isotopic composition of xylem water can be ascribed to soil water data collected on the same day or integrated over increasing monthly time windows to capture antecedent conditions. However, as described in section 2.2, soil water boundary polygons for increased averaging periods can also be calculated to estimate the overlap relative to xylem. Depth dependent overlaps are shown in Figure S7 and Figure S8, respectively. At Bruntland Burn, a longer time window of soil water isotopes explained a greater degree of variation in xylem water isotopic composition for angiosperms . Bulked soil water samples collected on the same day provided 80% and 87% of overlap in spring and autumn, respectively, but only 4% in summer. Increasing this window to 3 months increased overlap to 90%, 38% and 87% in spring, summer, and autumn, respectively. The spring and summer bulked soil and xylem water overlap increased to 100% and 58%, respectively, with a 6 month window. For gymnosperms, same day sampling provided no overlap in spring and summer, and only 7% in autumn . For a 3 month window, overlap increased to 20% in spring, but only 3% in summer and 7% in autumn. For a 6 month window, the autumn overlap increased to 13%. There were marked seasonal differences between angiosperms and gymnosperms at Dorset. For angiosperms, bulked soil and xylem water overlapped for same day sampling 100% in spring, 0% in summer and 20% in autumn . This increased to 20% in summer for a 3 month averaging window and 47% in summer for a 6 month average. The overlaps were much lower for gymnosperms; same day sampling showed bulked soil and xylem water overlaps of only 13% in spring, 2% in summer and 7% in autumn .

Data were summarized separately for each of the two years. For each year, a Chi square goodness of fit test was used to examine whether or not there were equal levels of a damage type among three treatment categories within a variety. If there was a significant difference in damage among the three treatments in a variety, then Fisher’s Exact tests were used to compare each pair of treatments. Tests were considered significant at the p < 0.05 level. This study examined the impact from feeding by two Leptoglossus species, L. clypealis and L. zonatus, on developing almonds in four varieties during the growing season. Almonds of each variety were caged with equal numbers of adults of each species to determine the potential impact. Early in the growing season, leaf footed bugs fed by puncturing their stylets into the developing almond accessing the jelly-like immature kernel. In both years, almond drop in the controls was low,blueberry pot size while almond drop in cages with Leptoglossus adults was twice as high. The first month of the study in April had higher levels of almond drop than in the later weeks of the study in May. The time in April corresponded to weeks when the almond shell could be punctured mechanically and indicated that the developing almonds were more vulnerable than in May, when the shell had hardened.

Overall, almond drop during the season was lower from L. clypealis than from L. zonatus. This may be due to the smaller size of L. clypealis compared to L. zonatus adults. Monterey had higher levels of almond drop from L. zonatus in both years than the other three varieties. Feeding by leaffooted bugs also caused significant damage to almonds in the form of nut strikes, kernel necrosis and shriveled kernels. The final damage observed at almond harvest was higher overall from feeding associated with L. zonatus than L. clypealis. Other studies have found significant crop damage from the presence of Leptoglossus clypealis and Leptoglossus zonatus. For example, feeding by L. zonatus in physic nut increased fruit abortion. A study of L. clypealis on pistachio found that when one adult L. clypealis fed for 48 h, the number of fruits dropped was higher on average than in controls. L. zonatus feeding in citrus resulted in spots on the outer citrus rind and feeding on satsuma mandarin by 1–3 adults for 14 days resulted in 37.5–100 percent of premature fruit abortion. Studies such as these demonstrate the need for controls, to determine the natural level of fruit abscission, and which damage symptoms can be attributed to insect feeding.Developing almonds may be more susceptible than mature almonds to feeding damage from Hemiptera, as has been observed in pistachios. In the present study, the almond shell could be punctured by an insect pin until the end of April but by May, the external almond shell became too firm to puncture and almonds were presumed no longer susceptible to bug feeding. The majority of almond drop in the four varieties occurred in the first few weeks of observations.

After the fifth observation week of caging Leptoglossus on branches, the percent almond drop decreased relative to the early weeks of observations and remained minimal. This agrees with an early study by Haviland, where almonds were artificially damaged by puncturing, and more almond drop occurred in April than in May. Our study was a no-choice test, where bugs were caged with only one almond variety; when offered one variety and no choice, the levels of almond drop were similar among the four varieties. However, in a natural field-study where almond drop was assessed after a Leptoglossus infestation, higher levels of almond drop were observed in Fritz . When provided with a choice of varieties, L. clypealis appeared to prefer to feed on Fritz. Almonds on the trees that were fed on by bugs later in their development may not drop but can suffer downgrades or rejects at harvest as observed for other crops. In pistachios, Leptoglossus sp. was capable of causing feeding damage to the pistachio kernel even after the hardening of the shell. Michalides et al. found that the large bugs including Leptoglossus clypealis were able to cause pistachio kernel damage until late June, when nut development was at the final stage. A late season feeding study of large bugs in pistachios found that kernel necrosis could range up to 20%. The vulnerability of almonds later in the growing season has not been fully investigated. Leptoglossus may cause feeding injury to almonds which are well developed, and thus might be monitored and controlled throughout the growing season. In other systems, fruit size and shell hardness has been found to influence the level of insect feeding damage.

Feeding on physic nut by Leptoglossus zonatus resulted in larger fruits having more seed abortion and feeding resulted in more shriveled or damaged nuts; similarly, some varieties of olives with larger fruits had a higher infestation rate by the olive fruit fly, Bactrocera oleae. In hazelnut, shell thickness was not correlated with damage by brown marmorated stink bugs, Halyomorpha halys, and the authors suggested that shell thickness should not be a criterion to select cultivars resistant to H. halys. Follett et al. reported that husk and shell thickness in macadamia nut do not primarily determine susceptibility of fruits to Nezara viridula. Hull width could relate to the susceptibility of the almond to feeding damage by the two leaf footed bug species. Although there was a significant difference in hull widths between Nonpareil and Fritz, there was no relationship between hull width and almond drop. In addition, the hull width of Nonpareil was significantly thicker than Fritz, but hull strikes on Nonpareil and Fritz from L. clypealis and L. zonatus did not vary significantly, and nut strikes were also not higher on Fritz. This suggests that damage from Leptoglossus feeding on these two varieties was not influenced by hull width . In 2015, higher levels of damage were observed in all four varieties compared to 2014, but again did not relate to the hull width differences . The final assessment of damage from L. zonatus and L. clypealis included hull strikes, nut strikes, kernel necrosis and shriveled kernels. Insects probing the hull results in hull strikes and suggest that the internal almond kernel may be damaged. Nut strikes, almond kernel necrosis and shriveled kernels can lead to downgrades or unsellable product. In both years of this study, the cages with both Leptoglossus species typically had higher levels of hull strikes and nut strikes than in the control cages. In 2014, L. clypealis feeding did not result in significant kernel necrosis compared to controls in any variety,plant raspberry in container but L. zonatus had higher levels of kernel necrosis than controls in Monterey and Carmel; in 2015, kernel necrosis was consistently higher in L. zonatus cages than in controls. In 2014, the percent of shriveled kernels at harvest did not vary between the controls and the L. clypealis cages in any variety. However, the percent of shriveled kernels was higher in L. zonatus cages than controls in two varieties, Monterey and Carmel, while in 2015, the cages with L. zonatus resulted in higher levels of shriveled kernels in Nonpareil and Monterey. Research has similarly found that insect feeding damage varies among almond varieties as well as in varieties of other crops such as in apple, blueberry and olives. Hull strikes that are characterized by a spot on the external portion of the almond could correspond directly to internal damage such as nut strikes or almond damage. The number of external hull strikes was similar to the number of internal nut strikes and damaged nuts for L clypealis. However, for L. zonatus in both years, the number of hull strikes recorded was often lower than the number of nut strikes or damaged nuts. Hull strikes could be used as a proxy to estimate the number of almonds with internal damage such as nut strikes or kernel necrosis which might later be unsellable . Evidence of hemipteran probing and feeding has been used as a proxy for feeding damage in other systems. Nut strikes are expected to occur due to feeding injury from sucking insects where insects probe their mouth parts into fruits during feeding.

In 2014, nut strikes on the four varieties due to L. clypealis was an average of 15%, while nut strikes from L. zonatus averaged 24.75%; in 2015, L. zonatus similarly had higher numbers of nut strikes than in controls. In contrast to the relationship found between hull strikes and other kernel damage from Leptoglossus feeding, in other crops, the presence of holes, spots, or mouth part stylet sheaths on a fruit has not always been found to translate directly into fruit damage. For example, Wiman et al. reported that in blueberry, external fruit probing by Halyomorpha halys indicated by a high numbers of stylet sheaths had very little internal damage as measured necrosis and discoloration. However, in apples, the punctures by the mullein bug were related to internal damage, which is more similar to what was observed in this study. Interestingly, some kernel necrosis was observed in control cages of all the varieties in both years. In 2014, the controls in each variety had few or no almonds with hull strikes yet had some level of kernel necrosis. A similar pattern was observed in 2015 as well. In cages with feeding by Leptoglossus there was a higher level of kernel necrosis than in controls. It should be noted that Leptoglossus feeding does not account for the kernel necrosis observed in the controls, but perhaps another agent such as a pathogen could be responsible. Studies such as the one herein with caged insects help to determine the damage symptoms which can be attributed to insect feeding. Similarly, a study of pistachios used caged bugs to determine which damage symptoms could be attributed to feeding. Pathogens may be responsible for a percentage of kernel necrosis observed in controls in this study, but this would need to be investigated. The final damage parameter, shriveled kernels, was also observed in all the controls, similar to the pattern observed for kernel necrosis. It should again be noted that this is a natural type of damage which occurs in the crop, and it is not only induced by feeding of L. clypealis and L. zonatus. However, in L. zonatus cages, two varieties had levels of shriveled kernels which exceeded the controls. Drosophila suzukii, also known as the spotted wing drosophila , is a vinegar fly originating from Southeast Asia. SWD was first detected in North America in August 2008 in Santa Cruz County, California, where it was observed infesting strawberries and caneberries.1,2 In 2009, SWD was detected in Washington, Oregon, and Florida. By 2010, SWD was detected in Utah, Mississippi, North Carolina, South Carolina, Wisconsin, and Michigan in the United States, and Alberta, Manitoba, Ontario, and Quebec in Canada.Recent trapping indicates that SWD can be found in virtually any region of North America where host fruit are available. A coincidental invasion of SWD with a genetically distinct population has also been observed in Europe, with initial detections in both Spain and Italy in 2008, followed by its spread throughout the continent.In North America, SWD is primarily a pest of berries and cherries. In Europe, it is reported to also damage a number of stone fruits and grapes. Unlike native vinegar flies in North America and Europe, female SWD possess a serrated ovipositor that can pierce the skin of healthy, soft-skinned fruits to lay eggs. These eggs quickly develop into larvae, which consume the fruit and render it unmarketable. The only other Drosophila species known to oviposit in sound, marketable fruit is Drosophila pulchrella Tan. This species is native to Japan.1 Growers have attempted to mitigate crop damage risk by applying additional insecticide, harvesting more frequently, performing field sanitation, and implementing trapping programs to detect SWD populations. These management practices are costly and many growers still face significant yield losses from SWD infestations.Raspberry producers are perhaps the most affected by SWD’s invasion among California commodities, although producers of blueberries and cherries have experienced substantial losses too. Strawberry producers have experienced lower damage rates and primarily on the lower-value fruit produced for processing.

We developed a Bayesian framework that permitted quantitative integration of borehole flow meter data and cross hole geophysical information for estimating the hydraulic conductivity distribution at each pixel along the tomographic cross sections. Figure 2 illustrates the estimates of hydraulic conductivity along two vertical cross sections at the Oyster site, obtained using the Bayesian estimation framework and radar velocity tomograms. Hydraulic conductivity data from an electromagnetic borehole flow meter log are superimposed on top of the estimates obtained using radar tomographic data within the Bayesian framework. Comparison of the estimated hydraulic conductivity field and tracer breakthrough data suggested that the tomographic estimates were extremely useful for helping to reduce the ambiguity associated with interpreting bacterial and chemical transport data collected during tracer tests. Even though this site was fairly homogeneous and it had extensive borehole control ,planting blueberries in a pot it was difficult to capture the variability of hydraulic conductivity using borehole data alone sufficiently to ensure reliable transport predictions.

By comparing numerical model predictions with tracer test measurements at the Oyster Site, Scheibe and Chien found that conditioning the models to the geophysical estimates of hydraulic conductivity significantly improved the accuracy and precision of the model predictions relative to those obtained using borehole data alone. This study suggested that the geophysical based methods provided information at a reasonable scale and resolution for understanding field-scale processes. This is an important point, as it is often difficult to scale the information gained at the laboratory scale or even from discrete well bore samples for use at the remediation field scale.In addition to the importance of hydrogeological heterogeneity in controlling contaminant and bacterial transport, geochemical heterogeneity plays a large role in reactive flow and transport. In the same general area as the study discussed above, we used crosshole ground penetrating radar amplitude data to estimate lithology and sediment Fe and Fe. In this study, we developed a sampling-based Bayesian approach to fuse the diverse geochemical, lithological, and geophysical datasets into an integrated interpretation. The geophysical data do not sense the geochemical parameters directly, but the geophysical attribute is sensitive to lithology, which is in turn sensitive to sediment geochemistry.

Our developed estimation approach exploited this mutual dependence to estimate lithology and sediment geochemical parameters using geophysical data. Figure 3a illustrates the 2-D geophysical attribute field obtained from inversion of GPR amplitude data. Figures 3b-3d illustrate the mean values of the estimated Fe and Fe distributions; variances associated with these estimates are available but are not shown in the figure. Cross-validation exercises revealed that the estimates obtained using the geophysical data were accurate and greatly improved the 2-D identification of the geological and geochemical properties. This study represents perhaps the highest resolution field-scale characterization of geochemical properties performed todate. Although geophysical methods have been used to estimate hydrological parameter probability density functions with very high resolution , this level of detail may not always be necessary to adequately describe the controls on transport. Many studies suggest that spatial variations of lithological changes tend to be closely correlated with hydrological parameters and with geochemical parameters. As such, if an understanding of the relationships between lithofacies, hydrological and geochemical parameters is available, field-scale mapping of lithofacies may provide sufficient information about the controls on transport over large spatial scales. Figure 4 illustrates how geophysical methods have been used with sparse hydrological data and with stochastic estimation techniques to estimate the probability of being within hydraulically important units.

The top image illustrates the probability of being within a transmissive fracture zone at the NABIR Field Research Center at the Oak Ridge National Laboratory in Tennessee obtained using seismic velocity tomographic and flow meter data within a Monte Carlo markov Chain approach. This fracture zone is the ongoing focus of a Uranium bio-stimulation experiment at this site . The middle image shows the probably of being within a sandy lense at the Hanford 100H site in WA, and was obtained using seismic and radar tomographic data and a discriminant analysis technique. Finally, the lower figure illustrates the probability of being within a sandy lithofacies at the DOE Bacterial Transport Site in Oyster, VA , which was estimated using radar velocity data within a Bayesian estimation approach.Remediation approaches, such as in situ chemical manipulation and bio-stimulation, often induce dynamic spatiotemporal transformations in subsurface systems, such as the dissolution and precipitation of minerals, gas evolution, redox variations, biofilm generation, and changes in permeability and porosity. The limited understanding of bio-geochemical-hydrological processes and the inadequacy of conventional approaches for characterizing or monitoring these processes hinders our ability to guide contamination remediation. We have investigated the capability of time-lapse geophysical datasets for remotely detecting changes in hydrological-biogeochemical properties during bio-stimulation at both the laboratory and the field scale. Using column-scale experiments, we have tested the sensitivity of different geophysical methods to reaction products that occur during bio-stimulation, such as gasses, precipitates and biofilms during nitrate and sulfate reduction. As a consequence of the greater energy available from nitrate reduction, a majority of nitrate in a groundwater system is reduced prior to reduction of the less energetic manganese, iron, and sulfate. Thus, the onset of denitrification is an important indicator for determining the redox state of the aquifer that is being perturbed, and advanced techniques to detect this onset and monitor associated processes in a remote and accurate manner are needed. We conducted laboratory based experiments to investigate the sensitivity of radar attributes for detecting N2 gas generated during denitrification as the gas replaces the original pore water. The key steps in the experimental design included packing and inoculating sand columns with Acidovorax sp. , providing nutrients to the microbes to stimulate denitrification, and measuring the hydraulic and geophysical responses during the bio-stimulation experiment.

TDR measurements were made along the length of the column and over time to estimate the spatiotemporal variations in dielectric constant during the stimulation experiment. We used a volumetric averaging/mixing model to represent the effective dielectric measurement as a function the individual components that contribute to the measurement,raspberries in pots including the dielectric constants of N2 , of water, and of the sand grains. Figure 5 illustrates the estimated percent of the pore space that was filled by N2 gas as a function of time after the bio-stimulation experiment began for three different TDR measurement locations. This figure illustrates how the pore water started to be significantly replaced by gas after about thirteen days. Gas was first detected by probe #1, or the top probe in the column. The apparent ‘jumps’ in the estimated evolved N2 gas are associated with the pressure release procedure. With time, all of the probes sensed the presence of a significant volume of gas. At the end of the experiment, we estimated that approximately 22% of the pore spaces were filled with N2 gas for the bottom third of the column, about 21% of the pore spaces were filled with gas for the middle third of the column, and that 31% of the pore spaces were filled with N2 gas at the top third of the column, yielding an average estimated gas saturation over the entire column of 24.6%. The experiment suggested that the radar velocity may be a good indicator of the onset and extent of denitrification that occurs during bio-stimulation. Our laboratory and field-scale experiments have also indicated that the geophysical methods hold good potential for detecting some amendments as they are introduced into an aquifer and as they distribute over time. We have conducted geophysical imaging in support of a Cr bio-remediation effort that is being performed at the 100H Site at the Hanford Reservation in Hanford. Here, HRC, which is a slow-release polylactate was injected into a sandy portion of a saturated aquifer to reduce and immobilize Cr. Field tomographic data were collected before and after HRC injection. Figure 6 illustrates some of the field-estimates obtained using radar and seismictomographic data at the site. The top image in Figure 6 illustrates the hydraulic conductivity zonation in the stimulation zone, obtained using a statistical approach with radar and seismic tomographic data together with borehole flow meter data. Although the entire section is characterized as being ‘sandy’ using conventional data, there are areas within the section that are more permeable . The subsequent images in Figure 6 show the changes in electrical conductivity as a function of time after HRC was injected into the aquifer, obtained using radar amplitude and velocity information following Peterson.

We find that the HRC, which is electrically conductivity upon injection due to the release of lactic acid when mixed with the groundwater, originally distributes near the injection area. However, very soon after injection , the lactic acid redistributes itself into the higher hydraulic conductivity section, and migrates down gradient. After 9 days, the change in the electrical response due to the lactate injection is minimal compared to the change in the conductivity associated with the metals precipitation around the HRC reaction front . The bottom image in Figure 6 shows that the seismic energy is completely attenuated in the vicinity of the HRC. These images illustrate the utility of high-resolution geophysical methods for imaging the amendment location and redistribution as a function of heterogeneity. The geophysical responses to HRC that we observed at the field scale were similar to the responses observed in the laboratory.In 2007, 311 million kg of pesticide active ingredients were used in agriculture in the United States . California, the state with the largest agricultural output, uses 25% of all U.S. agricultural pesticides, or 84.5 million kg annually. Although recent studies have demonstrated widespread organophosphate pesticide exposures in the general U.S. population, including pregnant women and children, exposures are often higher in agricultural populations . Among pregnant women in the Center for the Health Assessment of Mothers and Children of Salinas study, all of whom lived in an agricultural region, and many of whom either worked in agriculture or lived with people who did, OP urinary metabolites or dialkylphosphate levels were ∼40% higher than those in a representative sample of U.S. women of childbearing age . We observed adverse associations in the CHAMACOS study between prenatal maternal DAP concentrations and children’s performance on the Bayley Scales of Infant Development at 2 y , measures of attention at 5 y , and on the Wechsler Intelligence Scale for Children at 7 y . Several studies in other populations have similarly reported adverse associations of prenatal exposure to OP pesticides and child neurodevelopment , but few studies have examined the effects of other potentially neurotoxic pesticides on child cognitive development. Populations residing in agricultural areas may be exposed to a complex mixture of neurotoxic pesticides through pesticide drift and para-occupational exposures . Some of these pesticide classes—including OPs as well as carbamates— share atleast one mode of action, depression of acetylcholinesterase , and there isin vitro evidence that there may be additive inhibitory effects from exposure to certain pesticide mixtures . Furthermore, although biomarkers such as DAP metabolites are an important tool for assessing exposures to pesticides, several challenges limit the utility of pesticide biomarkers in epidemiologic analyses. For example, many pesticides have a short halflife in the body, and biomarkers reflect only very recent exposures . In addition, no biomarkers are available for some pesticides, leaving only environmental concentrations or modeling to characterize exposure. Since 1990, all agricultural pesticide applications in California have been compiled in the uniquely comprehensive Pesticide Use Reporting database. In several studies, PUR data have been shown to correlate with pesticide levels in various media. For example, we have shown significant associations between nearby use of specific pesticides based on the PUR data and levels in house dust , and moderate to strong associations between agricultural use of malathion, chlorpyrifos, and diazinon with community air samples . In addition, several epidemiologic studies conducted in California have shown that higher nearby agricultural pesticide use is associated with various adverse health outcomes, including OP and fungicide use with Parkinson disease , OP and pyrethroid use with autism and birthdeffects , organochlorine pesticide use with autism , and the use of carbamates and the neonicotinoid imidacloprid with neural tube deffects in children .