Tag: hydroponic

Cold-induced sweetening in potato Storage at 4–8°C is desirable for extending potato shelf life, but cold-induced sweetening , that is, the degradation of starch to hexoses, occurs. These sugars are protective, however, sugared tissues will form carcinogenic acrylamide under high-temperature processing, blacken, and become bitter, leading to PLW. Manipulating the pathways that influence hexose levels, that is, starch biosynthesis and degradation, sucrose hydrolysis to hexoses, and glycolysis, has provided some protection against tuber CIS, and thus has the potential to reduce PLW. Postharvest chilling injury This describes the loss of quality and accelerated spoilage when tropical commodities are stored below 13°C. Postharvest chilling injury affects a wide range of species and results in extensive PLW. Chilling may also trigger higher starch content, which is degraded to replenish sugars for continued osmoprotection to abate PCI. An interesting observation is that starch degradation may slow down or even halt as cold storage progresses in tomato, banana, and apple fruit, and even if fruits are rewarmed, black plastic planting pots starch degradation does not resume. In bananas, TFs that regulate this mechanism have been identified. The biological rationale for this mechanism is unclear and should be investigated in more diverse species.

Postharvest physiological deterioration Cassava root deteriorates 72 h after harvesting due to postharvest physiological deterioration , a disorder that leads to losses of 20–30%. Silencing AGPase in cassava causes sugars to accumulate in the root. A positive correlation between low PPD and high sugar content was found in the transgenic cassava that extendedroot longevity, and was likely due to sugars serving as ROS scavengers. Salinity stress Saline soils trigger sugar accumulation in the leaves, which initially serve as osmoprotectants, but over time, inhibit leaf photosynthesis. The rapid conversion of sugars to starch in tomato fruit promotes sugar export from the leaf to the fruit by mass flow, thus relieving photosynthesis. The ‘extra’ starch stored in ‘salt-stressed’ green fruit is hydrolyzed during ripening, boosting sugar content and intensifying fruit sweetness post harvest.Pathogen infestation decreases post harvest quality, but recent findings showing that pathogen colonization is associated with changes in host carbohydrate levels, offer new avenues for disease mitigation. Starch may accumulate in the host as an early response after perceiving bacterial effectors or volatile organic compounds in some species. Such accumulation could physically contain the microbes in situ, thus reducing systemic spread. If the infestation becomes advanced, accelerated breakdown of the accumulated starch to sugars may provide the host with energy and carbon for the biosynthesis of protective antimicrobial compounds.

Starch accumulation may also be induced by the pathogen after infection, and may involve reprogramming carbon allocation between source and sink. This is seen with black Sigatoka disease in bananas, and in citrus greening and grapevine red blotch where phloem starch accumulation is part of the disease response. BSD in bananas not only alters starch metabolism in vegetative tissues, but also changes the physical and chemical characteristics of starch in the harvested fruit. These examples across host species and pathogen types illustrate that spatial–temporal changes in starch metabolism may be important to host disease response. The RF1 banana mutant with BSD resistance, was associated with high levels of sugars and starch accumulation. Thus, starch accumulation could be a critical factor that could be modulated pre- and post infection to reduce the damage caused by pathogens.The postharvest quality of sink tissues, for example, fruits, roots, and tubers, depends on carbon allocation from the source , and agronomic practices, environmental factors, and disease pressure can all influence this process. Reconfiguration of carbon allocation in response to development or stress , is mediated in part by the trehalose-6- phosphate-sucrose nonfermenting-1-related kinase 1 signaling pathway, a central energy hub that senses sugar status. The T6P-SnRK1 pathway regulates carbohydrate content in potato tuber , sweet potato, and various fruits. T6P-SnRK also regulates starch physicochemical properties, anthocyanin accumulation, and CIS in various species, which all have an impact on PLW.As shown in multiple examples, manipulating the carbohydrate profile of horticultural crops by biotechnology should impact postharvest quality and directly reduce PLW.

Increasing starch accumulation, modulating starch degradation, altering starch composition, or changing sugar content are key targets. The composite Figure 4 and accompanying Table 1 illustrate many genes and regulatory factors that influence these processes.Throughout this entry, we have pointed to many unanswered questions related to carbohydrate metabolism in horticutural crops and experimental approaches to address them. Here, we highlight additional steps that could yield new knowledge to improve produce for reduced PLW. 1) Determine the extent to which starch metabolism influences fruit quality. An unresolved question is if the amount, and the rate of sugars released from starch, influence the substrate pool, and the synthesis of downstream flavor compounds . 2) Bioengineer genes encoding carbohydrate enzymes and regulatory proteins for improved postharvest sensory and nutritional quality. Many of these enzymes are regulated at the post-transcriptional level. A deeper understanding of these regulatory mechanisms could be leveraged to introduce subtle adjustments to the carbohydrate composition of tissues. Fine-tuning the amount and spectrum of carbohydrates produced could be used to optimize shelf life or the flux toward specialized metabolic pathways. 3) Establish the spatial–temporal profile of starch in fruit tissues. A starch-to-sugar atlas in fruit, through development and in response to pre- and postharvest stress, would provide fundamental and high-resolution data on core energy and carbon metabolic processes. Such an atlas would serve as a baseline to identify targets for gene editing. 4) Determine the role of fruit photosynthesis, in determining fruit quality. Fruit chloroplasts have photosynthetic and CO2 fixation capacity, an internal source of CO2, and fruit chlorophyll correlates with fruit quality. Fruit photosynthesis was activated when source photosynthesis was impaired under drought stress, and was accompanied by starch accumulation. Yet, the role of fruit photosynthesis in fruit growth, carbohydrate production, and stress response is unclear. Data that clarify these potential relationships are needed as the first step to exploiting these processes for enhanced fruit quality. 5) Leverage data from Arabidopsis to study starch metabolism in leafy greens. There is a growing mechanistic understanding of starch metabolism in A. thaliana. These data could be applied to starch-rich leafy greens such as spinach, or those of the related Brassicaceae, that is, cabbage, kale, collards, and so on. This would largely fill the knowledge gap between the current models of starch metabolism and their real-world application toward crop improvement. 6) Identify factors controlling carbon allocation from source to sink for postharvest quality and shelf life. Although it is tempting to focus on the harvested product, source-sink relationships help determine produce size, abiotic and biotic stress responses, and quality attributes . Varying the expression of key genes, for example, T6P-SnRKs, SWEETs, INVs, and so on, combined with precision agronomical practices, could improve many of these critically important attributes that influence marketability .Carbohydrate biosynthesis and degradation denote changes in energy conversion and storage, with consequences for postharvest quality. Despite the multifaceted role of starch and sugars in plant tissues, rarely are these compounds seen beyond serving as bulk reserves for direct consumption. Here, we argue that their metabolic dynamism is pivotal to the physiology and quality of the harvested organ, which, through their effect on organ size, aroma, taste, flavor, texture, drainage pot and visual appearance, will reduce postharvest waste at the consumer end. Further, because carbohydrates are vital substrates for respiration, and act as ‘stress-protectants,’ they influence storage and shelf life, and as a result, postharvest loss.

We also show that carbohydrate movement from source tissues to the harvested organ should not be ignored when investigating produce quality. Finally, the identified genes, enzymes, and regulators involved in carbohydrate metabolism we present, offer opportunities for precision modification of postharvest attributes to reduce waste and loss.Plant phenology is a key determinant of plant success and ecosystem productivity. Furthermore, as phenological events are often triggered by environmental cues, especially temperature, the study of phenology is essential for predicting how species will respond to climate change. Over the past decade, there has been a concerted effort to incorporate phenological traits, including the onset and duration of individual phenological phases, into evolutionary ecology and climate change biology. Despite the importance of phenology to plant success, however, little is known about the phenological behavior of most species. In particular, the way in which different environmental factors serve as phenological cues across the majority of species remains a mystery. This is mainly due to the difficulty of acquiring the data necessary to identify specific environmental factors that drive phenological transitions for a given species. The collection of these data has traditionally required long-term field observations or manipulative experiments that are difficult to scale up such that they capture entire regions, communities, or plant clades. Efforts to collect species-level phenological data, therefore, have been pursued in only a relatively small number of species from a limited geographic distribution and often over short timescales, resulting in a substantial gap in our understanding of phenology . To address this gap, researchers have recently turned to the vast collections of plant specimens in the world’s herbaria for phenological information. Herbarium specimens can be viewed as records of the phenological status of an individual, population, or species at a given time and place . While the phenological information provided by an individual specimen is limited, many specimens can be used collectively to assemble a long-term picture of the phenological behavior of a region and the species that inhabit it. Expanded phenological information derived from large numbers of specimens can offer insight into two key ecological phenomena: long-term shifts in phenology at a given location over decades or even centuries; and how seasonal or interannual environmental variation cues phenological transitions. It is now being recognized that herbarium specimens provide a reliable method for estimating phenological sensitivity in plants . Furthermore, specimens offer unique attributes that have the potential to greatly expand our understanding of phenology. First, specimens offer a detailed history of phenological change, with many collections dating back centuries , before the modern influence of climate change. Second, given their diversity in both phylogenetic and geographic sampling, specimens offer the opportunity to study the evolution of phenological traits in a wide range of lineages and biomes as well as how phenological traits may shape patterns of diversity under future climate change. The pace of herbarium-based phenological research has accelerated rapidly over the past decade facilitated by the increasing availability of online digitized herbarium specimens. As more of these collections are digitized and climate change research continues to advance, it is now an appropriate time to evaluate the current state of herbarium based phenological research and discuss potential limitations, areas for improvement, and opportunities for future research.For hundreds of years, botanists and naturalists have collected and preserved plants as herbarium specimens for taxonomic research, to record the flora of a region, to document their economic uses, and as a social hobby. Traditionally specimens were not collected with the specific intent to study phenology per se. As plant collection became more widespread among professional botanists in the 18th and 19th centuries, however, the ancillary information recorded and retained with each specimen became more detailed and standardized, and thus more amenable to phenological research. Most specimen labels created during the past 150 years provide information on locality, date of collection, and habitat. In addition to label data, physical specimens are rich with information regarding plant health, morphology, and phenological status. From these data researchers can derive descriptive estimates of a species’ reproductive season for inclusion in published floras, species identification, and application in horticulture. The use of such data for more detailed studies of ecological and evolutionary processes, such as phenological sensitivity to temperature, has been limited . Phenology as a field of study dates to the 18th century in Europe and even earlier in Japan and China, where observers recorded the flowering dates of culturally significant plants such as cherry trees. Careful observations of plant phenology and its relationship to meteorological records became common in many European countries, the USA, Japan, South Korea, and China during the 19th century; these observations have a rich tradition in horticulture and agriculture and natural history and in the past couple of decades have been used for climate change and ecological research. It is only relatively recently that researchers have begun to use herbarium specimens for plant phenological research.

The composition of ESMs varies widely, from simple mixtures of stones and native soil to patented commercial products. Highly porous ESM mixes provide ample infiltration and pore space for temporary storage of surface runoff. Also, they support tree growth by providing more water and aeration to roots than compacted native soil alone. ESMs can reduce conflicts between surface roots and sidewalks by promoting deeper rooting systems. In California alone, over $70 million is spent annually to remediate damage by shallow tree roots to sidewalks, curbs and gutters, and street pavement. In Davis, California, a bioswale installed next to a parking lot reduced runoff from the parking lot by 88.8% and the total pollutant loading by 95.4% during the nearly two year monitoring period. Furthermore, a bioswale installed next to a turf grass patch at the University of California-Davis campus eliminated dry weather runoff from an irrigated urban landscape. The ESM used in these studies offered several advantages over other ESMs because the main structural element was locally quarried and relatively inexpensive lava rock . This ESM had a high porosity, high infiltration rate, and a high water storage capacity . It effectively fostered the growth of biofilms that retain nutrients and degrade organic pollutants. Because vegetated bio-swale research is in its infancy, very few studies have monitored vegetation growth and its impacts on bioswale performance. Moreover, plant pot with drainage evaluation of system performance is generally conducted before vegetation is fully established.

In contrast, this study evaluated the effectiveness of two bioswales on surface runoff reduction, pollutant reduction, and tree growth eight years after construction. The control bioswale contained native soil and the treatment contained an ESM. At the time of this study, the trees in the control and treatment bioswales were fully established and approaching mature size. Measurements recorded the differences in surface runoff dynamics and pollutant reduction rates, as well as tree and shrub growth. This study provides new information on the long-term effectiveness of engineered bioswales in a region with a Mediterranean climate.The water collection system was installed in 2007 to collect composited samples from natural runoff . In this study, surface runoff samples from the control site were collected at a high frequency using grab samples to better observe pollutant concentration dynamics for each experiment throughout a storm hydrograph. A test run was conducted on 10 October 2013 to determine the optimal runoff sampling time intervals and the number of samples needed to capture the peak and total loadings. For the test run, the water soluble fertilizer was applied to both of the sites at a rate of 2.24 g m−2 . Grab samples were collected at a 10-min frequency over a two hour irrigation period from the control site. Water samples were collected immediately before the runoff was directed to the underground tank. The water samples were coarse filtered during sample collection with coffee filters to remove large tree leaves, grass clippings and large soil particles. Based on the results of the test run, the water sampling frequency for subsequent trials was extended to six hours with a variable sampling interval to better characterize the runoff pollutant pattern. The composite water sample from the treatment site was used for calculating the total loading of the treatment site where little surface runoff occurred in this study.

A 5.1 cm diameter and 0.9 m long PVC drainage pipe was vertically installed into the middle of the treatment bioswale to collect a representative water sample for monitoring pollutants concentration dynamics in the bioswale. The treatment site water samples were not affected by successive flow from the control site because the treatment site was located upslope of the control site. The water sample collected from the control site was surface runoff, which was not affected by subsurface flow of the treatment site because of the site’s relatively flat surface.This treatment bioswale had slightly higher pollutant reduction rates as compared to the bioswale with ESM installed adjacent to a parking lot in a previous study. The parking lot bioswale reduced the nutrients by 95.3% and organic carbon by 95.5%. The peak pollutant concentration reduction rates found in this study were a minimum of 53% higher than those reported in the parking lot study. One possible explanation for this difference is that the trees and shrubs in the bioswale were more extensive and older than the tree in the parking lot site. Tree and shrub roots can function as a biofilter, where pollutants are immobilized, transformed, or degraded. Although data were not available for below ground biomass, a more extensive rooting system and associated microorganisms in this study’s bioswale could be partially responsible for its improved performance. Another possible explanation is the difference in pollutant inflows. The primary pollutant source in the parking lot study was from atmospheric deposition, with lower concentrations when compared to the fertilizer rates applied in this study. Stormwater BMPs, such as bioswales, are reported to have higher removal rates when treating stormwater with high inflow concentrations. Concentration of Zn, Cd, Ni, Cu, and Pb are key water quality concern parameters.

They were excluded from this analysis because their concentrations were below detection levels in the irrigation source waters and these metals are not identified as impairments in the study area.The interpretation of results from this study is subject to some limitations. Pollutants can leave the system via infiltration deeper into the soil and potentially enter the groundwater. Deep leaching can be an important flow path affecting the fate of pollutants, and was not included in the scope of this study. Caution should be taken regarding the potential for groundwater contamination when considering the use of ESM in bioswale projects. In this experiment, the trees were eight years old and their root systems were well established. Trees received excess surface irrigation runoff during the hot/dry summer. Because the ESM in this study was 75% lava rock it may not retain enough moisture for tree roots during long dry periods. Trees in bioswales with ESM may require more irrigation than trees in native soils, especially for establishment. In this study, the pollutants were artificially added to the system by using dissolved fertilizer. Actual storm runoff includes pollutants from atmospheric deposition and has a more complex mixture of pollutants. These factors introduce uncertainty in extrapolating the pollutant reduction efficiency of the bioswale to other sites. It is unclear whether all of the pollutants retained by the bioswale were fully retained by the vegetation and soil, or if a portion of these pollutants were only temporarily immobilized in the system by the soil-tree root system. The bioswale system tested in this study was eight years old, relatively young when compared to its 20 to 30 year life expectancy. Long-term monitoring of system performance is needed to document bioswale performance over longer time periods typical of urban green infrastructure. Additional research is needed that follows the fate and transport of pollutants after infiltration. In particular, chemical analyses of soil and tree samples are needed to understand the fate and transport of the pollutants in the bioswale system.The oomycete Phytophthora cinnamomi Rands is the causal agent of Phytophthora root rot of avocado, the most destructive disease in the avocado industry worldwide . PRR affects approximately 75% of California avocado growers, historically causing losses of $40 million annually . The pathogen mostly infects the feeder roots, causing the roots to be blackened, brittle, or necrotic. Infected trees usually develop symptoms from pale green to yellow leaves, wilting, to heavy leaf fall and dieback, greatly reducing fruit yield. Trees eventually become leafless and die . P. cinnamomi is considered one of the most invasive pathogens in the world, because the pathogen has a wide host range, infecting over 3,000 hosts, including forest trees, such as eucalyptus, pine, and oak ; ornamental plants, such as camellia, Rhododendron, and Azalea; and fruit crops, such as pineapple, peach, and highbush blueberry . P. cinnamomi is a hemibiotrophic pathogen feeding initially from living host cells and then switching to necrotrophy by killing the host cells and feeding from the nutrients released by them . The entry into the plant is achieved by the adhesion of the motile zoospores to the host tissue, encystment, and germ tube formation. The germ tubes usually grow and penetrate the root surface via appressorium-like swelling structures and then plant tissue is rapidly colonized .

During its biotrophic stage, P.cinnamomi projects haustoria into the plant cells for the acquisition of nutrients and release of pathogen proteins to aid the infection process in the host . This is followed by a necrotrophic stage characterized by host cell death, hyphal proliferation, pots with drainage holes and production of numerous sporangia . P. cinnamomi is heterothallic and both pathogen mating types are pathogenic, however, the A2 mating type is more invasive and is generally recognized as being the more aggressive . P. cinnamomi isolated from infected avocado trees almost exclusively consist of an A2 clonal population with no sexual reproduction evident. Even when both mating types are present in the population, diversity develops asexually in the form of clonal populations. Only one A1 P. cinnamomi isolate has been discovered on avocado in California, which appears to infect mainly alternate hosts such as camellia . Once P. cinnamomi is introduced in a new avocado area, it cannot be eradicated, and for this reason, the ability to rapidly and accurately detect this pathogen, monitor its population, and differentiate their variants becomes more urgent to apply appropriate management strategies to reduce crop yield loss and pathogen spread. Despite the economic and ecological importance of P. cinnamomi worldwide, there are limited studies regarding: the genetic diversity of the pathogen population , the efficacy of novel Oomycota fungicides to manage avocado PRR, and the molecular and genetic basis of host-P. cinnamomi interactions .The first main objective of this dissertation was to assess the phenotype of avocado P. cinnamomi isolates representing the current clonal populations recovered in California. Recently, two different A2 clonal P. cinnamomi populations were found in California : the prevalent A2 clade I population and the more geographically specific A2 clade II population. The phenotypes of several avocado P. cinnamomi isolates corresponding to these A2 clades regarding in vitro mycelial growth rate, optimal growth temperature, sensitivity to registered Oomycota fungicides mefenoxam, potassium phosphite, and new compounds fluopicolide, and oxthiapiprolin, and virulence were evaluated. Finally, a detached leaf assay P. cinnamomi inoculation method using Nicotiana benthamiana was developed and validated to circumvent the difficulties associated with the avocado root inoculation method to assess the virulence of P. cinnamomi isolates. Control strategies of avocado PRR are limited, and include: the use of resistant rootstocks, cultural practices, and chemical treatments. Commercially available root rot resistant rootstocks include Dusaâ, Steddom, Thomas, Uzi, and Zentmyer . Among them, Dusaâ is the industry standard currently in California, which enables growers to cultivate avocado in P. cinnamomi infested soil and maintain avocado production. Cultural practices include using certified disease-free nursery stock, keeping well-drained soil, cleaning tools and equipment, and applications of gypsum and mulch . Resistant rootstocks and cultural practices are effective ways to manage PRR, however, losses of avocado production to PRR are still substantial. Resistant rootstocks may also be overcome by aggressive P. cinnamomi populations . Until rootstocks with strong levels of quantitative resistance are developed, there will be a need for chemical treatments to mitigate losses caused by PRR in avocado production. At present, the only fungicides available to control PRR of avocado are phosphonate fungicides, e.g. potassium phosphite, and phenylamide fungicides, e.g. mefenoxam. Potassium phosphite injection is the preferred treatment by avocado growers due to the lack of reported resistance as well as the ability to apply this chemical as a fertilizer. Mefenoxam is used to a much less extent because of resistance development and the relatively higher cost. Both chemicals have been used for decades against Phytophthora spp. including P. cinnamomi. Aluminum tris-O-ethyl phosphonate was first introduced by Rhone-Poulenc Agrochimie Laboratories in 1977 with the product name Fosetyl-Al . It was later discovered that potassium phosphite was the active ingredient and that alkyl phosphonates were degraded in the plant .

Counts, binned data, or other data representations can be mapped to the PPO and by so doing, assure interoperability with field monitoring data. There are some taxonomic groups that have very small floral structures that either require an onerous amount of time to score or require expertise to determine what kinds of organs are actually present on the sheet. Members of Poaceae, Cyperaceae, and Juncaceae, as well as certain Asteraceae, are a few examples for which second-order scoring may be more challenging. However, it should be relatively easy to apply first-order scorings to these groups, thereby greatly increasing the utility of these specimens for phenological research. Our protocol does not address the presence/ absence or abundance of male vs. female flowers, or distinguish between perfect and imperfect flowers in gynodioecious, gynomonoecious, or and romonoecious species, largely due to the fact that these categories have seldom been included in phenological research. The timing of reproduction is not the only important phenological event of interest to be tracked in plants. Leaf bud break and leaf-out are important phenomena for deciduous forests, as is autumn senescence. These vegetative characters are often tracked via satellite imagery and in situ monitoring efforts. Scoring phenological leaf traits on herbarium specimens is rare , vertical aeroponic tower garden but it provides valuable insights into the effects of climate change .

A similar scoring protocol is recommended for foliar structures, although we do not specify a protocol here. Online documentation, including definitions and examples, is provided for each term used in the Darwin Core . Any attempts to share phenological or other trait data from specimens should utilize Darwin Core fields to assure that the basic specimen occurrence information is standardized. However, phenological trait descriptions are not part of the Darwin Core and therefore other mechanisms are needed to support narrower or broader data-sharing approaches. We are proposing to share phenological data using the Darwin Core Extended MeasurementOrFact extension . This extension provides a mechanism whereby many measurements or facts can be shared for each specimen record in a Darwin Core Archive. This extension allows for sharing of metadata associated with each phenological scoring. For example, when evaluating data quality, it can be useful to know when, how, and by whom scorings were recorded. Accuracy may be affected by whether the specimen was scored from a web-based image or the physical specimen. An eMoF record can contain a definition of the type of measurement, the value and units of the measurement, the method of measurement, and by whom and when it was measured. Table 5 shows an example Darwin Core Archive file of the eMoF extension of two herbarium sheets that were scored using our protocol . The first record was scored with a measurement Value = ‘Reproductive.’ Additionally that same record is scored as measurement Value = ‘Open flowers.’ The second record, united by catalog number, is scored ‘Reproductive,’ ‘Open flowers,’ and ‘Fruiting.’ Using the eMoF extension has a potential disadvantage, namely that it does not allow the measurement to be rigorously tied to a particular aspect of the core record.

This means that any user can define a new and non-standard ‘measurement Type’ and ‘measurement Value’ , which could lead to difficulty compiling data. Unless various measurement Types and measurement Values are rigorously defined, an excessive number of unique text strings could be generated. To address this, we are working toward defining these terms within Apple Core. Apple Core is a set of best practice guidelines for publishing botanical specimen information for herbaria. A goal of the guidelines is to mitigate the generality of Darwin Core by providing detailed guidelines for publishing botanical specimen information in Darwin Core. These guidelines will include recommended terms, specific definitions, multiple examples, common issues, and controlled vocabularies where appropriate that are specific to herbarium specimens. Apple Core is a community-curated resource that is still being refined, and interaction with phenological researchers will help to strengthen this resource. Finally, use of this approach is complementary with broader sharing initiatives that utilize ontologies, such as the Plant Phenology Ontology. In the near future, using the eMoF extension will allow for phenological scorings to be published in iDigBio, the Global Biodiversity Information Facility , and other public repositories. Darwin Core Archive publishing services are available within all Symbiota portals and form the basis from which iDigBio harvests specimen data from these portals . Adherence to our protocol at local institutions will facilitate the search functions provided and developed by large aggregators such as iDigBio and GBIF.The questions presented here provide important data for researchers while also requiring minimal effort from herbarium curators. Phenological questions are easily integrated into standard label digitization workflows or could be subsequently scored from images.

Due to the nested nature of the questions, a third-order question can be scored initially, with the appropriate second- and first-order questions automatically populated. For example, a report of “fruits present” on a specimen would automatically score a “yes” for the first-order question, indicating that reproductive structures are present. To answer first-order questions, the person who is performing the initial data entry for a specimen need only look at the sheet and check a box indicating whether reproductive structures of any kind are present. For databases that do not have the infrastructure to accommodate this type of scoring, a few alternatives are presented below.Phenological scores can be recorded at a number of steps in a digitization workflow. In the case of an object to-data workflow, scores could be made directly from the sheet as label data are being captured. With an image-based workflow, the scoring of specimens can be achieved by visual inspection of their images. The latter approach provides the option of making the image available online where the public can record phenological observations. Machine learning approaches are likely to facilitate our ability to score images at scale in the near future. Database fields in local databases need to be modified to accommodate the proposed structure. Implementation of controlled vocabularies can be facilitated with drop-down menus or pick-lists ; however, providing such functionality might require changes to database management software. Fortunately, a number of tools have been developed for scoring the phenological status of specimens. For curators who do not have a database with Symbiota-type functionality that provides phenological check boxes corresponding to our proposed protocol, we suggest that users enter phenological information into an appropriate text field within their existing database with the expectation that new tools will enable users to search these text fields and score the specimens appropriately . Ideally, every institution’s home database will include a text field dedicated exclusively to information pertaining to phenology. However, including phenological information as text anywhere within a given specimen’s label data is better than not capturing any phenological traits. To choose the best text field within a local database, it is important to know how the specimen data appear when shared using a Darwin Core Archive. If, for example, one’s local database conforms to Darwin Core, reproductive traits should be included in the ‘reproductive Condition’ field. The words entered into the text field should be unambiguous and should correspond to the protocol above . This is an action that all curators can immediately integrate into their current digitization workflows.Those managing or implementing digitization workflows should consider incorporating the scoring of phenological data into their workflows. At the very least, vertical gardening in greenhouse first- or second-order phenological data should be considered for capture. Doing so will facilitate future scoring of the specimens. If time does not permit training herbarium personnel to record challenging second-order scorings, then simply adding the word “reproductive” somewhere in a relevant database field will aid future work and research use.Part of the NEVP project was the development of a tool to score phenological traits using digitized label text . This tool allows a user to search for specific words in database fields and map these to the proposed vocabulary. For example, using this tool to search the field ‘reproductive Condition’ within SEINet resulted in over 4000 unique text strings . The Attribute Mining Tool allows one to select all records containing text that refer solely to a single scoring category. For example, if a user were scoring “open flowers present” only, the user could select all the highlighted rows in Table 1 and click “Open flowers present.” In the example from SEINet presented in Table 1, this single scoring event would result in the selection and scoring of 1,031,786 records.

In a separate scoring event, the user could select all records that make reference to both open flowers and fruits and then select “open flowers present” and “fruits present.” Because a curator is responsible for mapping free text strings from the database to a controlled vocabulary, this method does not rely on computerized inference. The ability to apply phenological scoring to any specimen within a Symbiota portal is highly efficient, and these types of tools should be developed within other database platforms.Many platforms have been developed for remotely scoring images of specimens, and we review them below. It is vital that future scoring platforms conform as closely as possible to the proposed protocol to facilitate data integration. Furthermore, it is vital that specimen trait data, even when scored outside of the local database, remain associated with the original specimen record. This will allow trait data and occurrence data to travel together through the data aggregation process, preventing duplicated scoring efforts.The new Image Scoring Tool, developed as part of the NEVP project, allows Symbiota network users to filter images and apply a phenological score to them . This approach has facilitated the scoring of over 240,000 images of New England specimens to date. Phenological scorings are being shared with end users through the Consortium of Northeastern Herbaria portal via the Darwin Core Extended MeasurementOrFact extension and Darwin Core Archives, as outlined above. This functionality will soon be available to all Symbiota-based databases.Notes from Nature is an online citizen science platform originally developed to support the transcription of specimen labels, but it has expanded to include phenological classifications. Notes from Nature extends the Zooniverse model by providing a simple way for curators or researchers to bundle and upload images, set targets for transcriptions or scoring, launch new expeditions, and engage volunteers . Notes from Nature addresses data quality by requiring a minimum of three replicated classifications for each imaged specimen. When expeditions are completed, a suite of tools are available for reporting outcomes of efforts, and automated reconciliation occurs to produce a “best classification.” This includes data for phenological categories and for counts of reproductive structures. Notes from Nature phenology expeditions have so far solicited reports of flowering and fruiting as well as counts of reproductive structures for Quercus L., Coreopsis L., and Cakile Mill. Notes from Nature has launched expeditions asking for simple annotations of open flowers or fruits present, to more complex expeditions where users are asked to count numbers of unopened flowers, open flowers, and fruits. Asking users to report first- and second-order scorings generated large volumes of accurate phenological data, whereas expeditions asking for more complex scorings, such as counts, had lower participation from the community of citizen science annotators and took much longer.CrowdCurio is a new online platform designed to give researchers the ability to design and implement crowd sourcing projects tailored to their specific interests and data sources . Most recently, a CrowdCurio project, titled “Thoreau’s Field Notes,” demonstrated that the platform was an effective tool for crowd sourcing the collection of phenological data from digitized herbarium specimens . Participants are presented with an image of a herbarium specimen and asked to annotate the image by clicking on each visible unopened flower, open flower, and fruit. These annotations are then transformed into counts that can be used to approximate the phenological stage of a given individual specimen. In a preliminary study of the efficiency and quality of CrowdCurio data collection, Willis et al. compared data collected by expert and non-expert participants for two common New England species: greater celandine and lowbush blueberry . They found that non-expert counts were similar to expert counts, but that non-experts were able to record nearly twice as much data at less cost over the same amount of time. Data collected via crowd sourcing, however, are not without limitations.

Tasks such as picking, pruning, or planting can be easily measured and tracked, making piece rates feasible. In these cases, productive workers can earn considerably higher incomes under a piece rate scheme than under an hourly wage scheme: Moretti and Perloff find that US agricultural workers paid by piece rate earn 26% more than their hourly counterparts. This number is slightly misleading, and certainly not causal, considering that workers select into particular work in part based on the compensation scheme. Rubin and Perloff note that piece rate workers tend to be disproportionately young or old: “[a]pparently, prime-age workers find that higher earnings in piece-rate jobs do not compensate for the difficulty of more intensive effort, more variable incomes, and possible greater injury risk or shortened farm-work career” . However,these selection issues are irrelevant if the goal is to understand how piece rates affect the productivity of workers who select into such work in the first place.Harvesting fresh blueberries is a labor intensive process. Berries grow in small bunches and ripen at differing times. This means that a single blueberry bush can be harvested multiple times each season. However, since each berry-bunch contains both ripe and unripe berries, pickers must harvest fruit carefully by hand. Mechanized blueberry harvesters exist, but they are imprecise and are used primarily for harvesting berries destined for the processing market.16 Berry-pickers collect fruit in small buckets fastened on the front of their bodies. Once the buckets are full, the workers carry their harvest to a weigh-station at the end of a field row. Workers pour their berries into standardized bins which are then weighed, packed into trucks, square plastic pot and driven to a refrigerated packing plant. Because blueberries are delicate and perishable, they must be refrigerated quickly after being picked.

When workers bring their berries to be weighed, a foreman closely watches the process to ensure quality control. If a picker’s fruit is intermingled with too many twigs, leaves, or unripe berries, the foreman will warn the picker that their quality must improve to keep their job. The farms I study both utilize an automated system to track workers’ productivity and calculate payroll. Each picker is given a unique barcode that they wear as a badge, and each fruit tray is assigned its own barcode as well. When a picker brings their fruit to be weighed, the weigher scans both the picker’s barcode and the tray’s barcode to record the tray weight. The picker then receives a receipt of their weigh-in. The farmer likes the barcode system because it is quick, automatic, reliable, provides real-time data, and replaces a cumbersome paper-and-pencil system. Pickers like the barcode system because they are able to witness the fruit-weighing and are thus confident that the farmer is paying them honestly for the fruit they pick. At the beginning of each work day, around 6:00 or 6:30 a.m., the farmer sets the day’s piece rate wage and posts the wage in a public spot for all workers to see. Workers are paid the piece rate for each pound of berries they harvest, and the rate does not change throughout the day. The piece rate does, however, change over the course of the season . As fruit becomes more abundant on the bushes through May and June, picker productivity rises. Farmers therefore generally lower the piece rate wage throughout the season as more and more berries ripen. Anecdotally, farmers say they lower their piece rates “when there’s a lot of fruit in the field” with the goal of maintaining a relatively stable effective hourly wage for the average berry picker. If any one worker picks a small enough quantity of fruit that their effective hourly wage for the day falls below the legal minimum wage, the farmer pays them according to the hourly minimum wage. In these cases, the farmer often then gives the picker in question additional training and a warning that they may be fired if they do not quickly improve.

Anecdotally, the hourly minimum wage is most likely to bind during a new employee’s first few days on the job as they develop their skills as a fruit picker. If a worker consistently falls below the minimum wage cutoff, they frequently quit on their own accord or are effectively fired and asked not to return the next day. Because blueberries are delicate and highly perishable, they are not bought and sold in a central commodity market. Instead, individual producers set short-term contracts with different marketers or buyers to provide a certain quantity of berries in particular packaging at a particular time. These contracts are set on a near-daily basis, and prices can change quickly throughout the season. While there is certainly some quality differentiation within the blueberry market, buyers and marketers view different producers as close substitutes. This means that individual producers have relatively little, if any, market power. I thus take California blueberry contract prices as an accurate reflection of a competitive market price for blueberries in the state. Blueberry prices in California are highly seasonal: prices are quite high at the beginning of the season in April, and much lower near the end of the season in June. This seasonality in price is largely explained by variation in aggregate production throughout California, and variation in the availability of blueberries from other global producers. In the early spring, the United States imports fresh blueberries at high prices from Mexico or other countries since domestic production is agronomically infeasible. By mid-to-late-June, farms in northern states such as Washington, Oregon, and Michigan begin to produce berries in large quantities, driving down the market price. California blueberry farmers therefore face a relatively short season when it is profitable to harvest and sell their fruit. While blueberry bushes continue to yield berries through June and into July, labor costs are too high relative to market prices at that time for California farmers to justify continued production. To summarize, the California blueberry season begins agronomically, but ends economically.

While the harvesting process is identical for conventional and organic berries, organic bushes produce fewer berries per bunch. Thus, pickers of organic berries spend more time finding and harvesting berries than do their conventional counterparts. Additionally, fruit quality is more variable in organic blueberries. This leads to a smaller proportion of berries ultimately reaching market.As described in the previous section, the farms I study use a digital fruit weigh-in system to track worker productivity and generate payroll data. I utilize data from these weigh-ins to conduct my analyses. In particular, I observe the weigh-in time, the berry picker’s unique employee identifier, the field where the berries were picked, and the weight of the picker’s harvest. I divide the harvest’s weight by the time elapsed since the picker’s previous weigh-in to obtain a weight-per-hour measure of worker productivity. For the first weigh-in of the day, I use time elapsed since morning check-in to calculate this measure. As reported in table 1.1, average productivity pooled across both farms is just over nineteen pounds picked per hour. This number, however, masks significant heterogeneity across farm, day, and worker. At the San Diego farm, which grows organic berries, average productivity is slightly under fourteen pounds per hour, while at the Bakersfield farm, which grows conventional berries, average productivity is over twenty-two pounds per hour. Figure 1.3 plots the distribution of workers’ average productivities, while figure 1.4 plots the distribution of each day’s average productivity, in both cases separated by farm. These two figures highlight substantial variation in picker skill, as well as in daily productivity. In southern California and the central valley, where the farms I study are located, temperatures peak in the mid-to-late afternoon. To avoid the hottest part of the day, most pickers begin work as early as 6:00 a.m. and end around 3:00 p.m. This pattern is reflected in figure 1.5: most fruit picking ends by mid-afternoon. The average picker works around eight hours each day, as shown in figure 1.6. Under California law in my sample period , 25 liter pot agricultural workers do not earn overtime pay until after working ten hours in a single day. In my data, only the San Diego farm ever lets pickers work more than ten hours in any given day. Farms employ pickers on a day-to-day basis, either directly or through a labor contractor. Some pickers only work for a day or two, but others work continuously for several weeks or months as shown in figure 1.7. A handful of pickers return to their respective farm each year. Indeed, several employees in my data work for a farm in two or all three of the years I study. Unfortunately, I do not observe each worker’s initial date of hire, so I am unable to confidently measure lifetime worker tenure on either farm.I utilize high-frequency temperature data sourced from the MesoWest database maintained by the University of Utah. 

In particular, for each year, I find the temperature monitor closest to each farm with hourly or finer temperature readings. In order to protect the identity of each farm, I cannot share the precise locations of these monitors, but they are all between 1.2 and 14.8 miles away from their respective farm. Temperature readings are available at least hourly, with some available at fifteen-minute intervals. I match temperature observations to each “picking period” – the span between two of a worker’s sequential weigh ins – using a time-weighted average of observed temperature. Figure 1.8 describes in detail how I calculate this time-weighted average. By using individual picking periods as my unit of observation, and matching these periods to time-weighted temperature measurements, I am able to exploit variation in temperature throughout each work day that accurately captures the heat exposure faced by outdoor laborers at different points of their shift. Figure 1.9 displays the distribution of time-weighted average temperatures within my data. There are few observations with extremely high temperatures, largely due to the fact that berry pickers usually end work in the mid-afternoon, before the hottest part of the day.While I know each farm’s daily piece rate wage from the its payroll data, I obtain information on market prices for California blueberries from the Blueberry Marketing Research Information Center of the California Blueberry Commission . As an official agricultural commission, the CBC legally requires all blueberry producers in the state to report daily production and sales figures. The CBC then publishes daily summary statistics of these data through BMRIC. Individual blueberry producers are able to access a daily BMRIC report online that summarizes the high, low, and weighted average prices received by blueberry producers throughout the state on the previous day. Separate statistics are provided for conventional and organic blueberries. In order to capture the information a farmer could have accessed on any particular day, I use each day’s most recent previous BMRIC report as the relevant measure of market prices. Because BMRIC publishes a daily report each weekday except for holidays, the relevant market price data for harvest data collected on a Thursday is from the Wednesday prior. Similarly, the relevant market price data for harvest data collected on a Monday is from the Friday prior. Based on personal conversations, the blueberry farmers I study track these BMRIC reports quite closely throughout the season. From April to June each year, both market prices and piece rate wages fall as the California blueberry season progresses. Figure 1.10 documents this relationship across the three years and two farms in my dataset. Recall that the San Diego farm grows organic blueberries while the Bakersfield farm grows conventional berries. This distinction accounts for why the two farms face differing market prices in the same year. Market prices and piece rate wages are highly correlated over time, due in large part to seasonality in blueberry production. Figure 1.11 plots each farm’s daily total production over time for each season. At times of high production, blueberry bushes are likely to be full of easily-pickable ripe berries. This abundance of fruit leads farmers to cut the piece rate as described in the previous section. In order to disentangle the various factors that affect farms’ piece rate wages in my empirical exercises, I control both for seasonality in production as well as the field where berries are harvested.In my subsequent econometric analyses, I estimate the causal effects of piece rate wages and temperature on picker productivity.

One of the challenges of similar foods being studied in differing formats and by various research groups is the utility of the data as a combined set. Differences in test materials and experimental designs make integration of data difficult. The proper curation of combined data, whether physiologic, metabolomic, or genomic, is critical to ensure that combined datasets provide synergy, statistical power, and enhanced usefulness.The cardiometabolic benefits from regular consumption of nuts or berries are widely reported and include improved vascular function, reduction of cardiovascular disease risk factors, improved insulin sensitivity, and reduced risk of type 2 diabetes mellitus. Metabolic outcomes may be context-specific and related to the physiologic state of the individual and host microbiome composition, among other factors. Examples include findings of ellagitannin and ellagic acid rich foods resulting in differential responses in healthy individuals compared to those with prediabetes, who are dependent on gut microbial-derived metabolite profiles. Many factors contribute to interindividual variability in response to diet that can extend to context-specific aspects influencing the magnitude of health benefits and reinforces the importance for further research aimed at advancing discoveries in precision nutrition. Additional health outcomes related to nut or berry intake are outlined below.Adding nuts or berries to the daily diet may be advantageous for weight management for several physiological reasons. One is that these foods produce feelings of satiety, helping to reduce the desire to consume calorie-rich snacks that are low in vitamins, minerals, and fibers, dutch buckets ultimately improving body composition over time. A second possibility is due to urolithins, secondary metabolites produced from ellagitannins in nuts and berries.

Urolithins increase the activation of the adenosine monophosphate-activated protein kinase pathway, resulting in anti-obesogenic properties in vitro and in animal models. AMPK increases fatty acid oxidation and decreases triglyceride accumulation. Phosphorylation of AMPK may also decrease cholesterol synthesis and lipogenesis by downregulating 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and sterol regulatory-element binding protein expression. In clinical studies exploring the relationship between food and body composition, the incorporation of nuts and berries into the diet was associated with weight loss or maintenance.Regular consumption of nuts or berries has been reported to support brain health and cognitive function, motor control, mood, and executive function at physiologically relevant intakes. Middle-aged and older adults experienced improvements in balance, gait, and memory, and children experienced higher executive function and positive affect after acute and regular intake of both strawberries and blueberries. These beneficial effects may be the result of direct effects on brain signaling or indirect effects through oxidant defense and anti-inflammatory properties of polyphenols and other bio-active compounds in nuts and berry foods. The gut-brain axis is an emerging area of research. Most studies are preclinical in nature using animal models but are suggestive of a significant role of gut microbial-derived ellagitannin metabolites on brain health and neuroprotection.The influence of nuts and berries on skin health and appearance is an emerging area of research. Regular intake of almonds, a good source of fatty acids and polyphenols, has been associated with a significant decrease in facial hyperpigmentation and wrinkle severity. A walnut protein hydrolysate administered to rats exposed to ultraviolet radiation significantly reduced skin photoaging and enhanced skin elasticity. Supplementation with ellagic acid, a compound found in many berries, prevented ultraviolet B -related inflammation and collagen degradation related to skin wrinkling and aging in a murine model.

More human studies, using objective measures of skin wrinkles, skin elasticity and response to low-dose UVB radiation exposure are warranted. Monitoring skin responses to a UVB radiation challenge has been used as a marker of whole-body antioxidant status in response to almond consumption. The response to a UVB challenge has also been used to monitor oxidant defenses and changes in skin microbiome following the intake of pomegranate juice.Age-related macular degeneration is the third leading cause of vision loss worldwide. Anthocyanins, carotenoids, flavonoids, and vitamins C and E, found in many berries, have been shown to reduce risk of eye-related diseases. Goji berries, containing the highest amount of zeaxanthin of any known food, hold particular promise since this compound binds to receptors in the macula to offer protection from blue and ultraviolet light. Regular supplementation with 28 g/d of goji berries for 3 mo increased macular pigment optical density, a biomarker for AMD, as well as the skin carotenoid index. Nuts may also be protective against AMD since they are a rich source of vitamin E and essential fatty acids. Regular intake of nuts has been associated with a reduced risk and slower progression of AMD in 2 epidemiological studies, thought to be due to the beneficial role of polyunsaturated fatty acids.Identification of new cultivars with traits desirable for growers, processors, and consumers is a continuous effort. As researchers continue to produce new varieties by both conventional and molecular-driven approaches, assessing these varieties for nutritional value is a challenge. A combination of broad targeted and untargeted metabolomic approaches, along with defined functional phenotyping could be used for rapid screening and defining of mechanistic pathways associated with health. However, consumer preferences for new cultivars are often driven by size and appearance of the berry or nut and flavor, rather than its nutritional value. This would further confirm the need to balance improvements to nutritional profiles with enhancement of consumer-driven traits, maintaining the marketable nature of the berries and nuts.Biomedical research, particularly for clinical studies, is expensive and resource intensive. Although the USDA competitive grants program offers funding for outstanding research projects, budget limitations favor animal or in vitro study proposals. Compelling pilot data is needed to be competitive for clinical studies funded by the USDA or NIH, so many researchers submit their initial ideas to commodity groups representing specific nuts or berries. Commodity groups represent farmers, processors, and distributors and have been instrumental in supporting fundamental and applied research focused on their specific berry or nut. The perception that studies funded by nut and berry commodity groups are inherently biased in favor of the test food is an issue sometimes raised by critics, journalists, and the general public. As in all nutrition research, ethical considerations regarding the structure of research questions, hypotheses, study design, outcome measures, interpretation of data, and conclusions must be rigorously considered. The food and beverage industries have played a key role in providing funds and supporting nutrition research on individual foods and beverages, including berries and nuts. Although this draws scrutiny regarding scientific integrity and data reporting, collaboration between academia and industry compared to exclusive corporate funding may help offset some of these concerns. For example, in multiple reported studies, matching funds were also provided by nonindustry sources, including institutional and federal agencies. In other cases, while the food industry provided the test agents, key research personnel and staff were not supported by the same funding source.

The academia-industry collaboration has also led to the formation of scientific advisory committees that evaluate and recommend proposals for funding, a peer review process that helps ensure rigorous study designs, data reporting, and dissemination of results. Human studies of sufficient statistical power are expensive, labor-intensive efforts requiring sophisticated and costly laboratory equipment and supplies. In order for research proposals to be competitive for funding from the USDA or NIH, pilot data is required, grow bucket and for nuts and berries, the only realistic source of funding for these exploratory trials is from industry sources. Critics of industry support for nutrition research have yet to propose realistic alternatives for funding needed to generate initial data. Further, ongoing industry funding of nuts and berries research has yielded important insights into the molecular and physiological understanding of mechanisms of action. Without industry support, provided in an ethical and transparent manner, advances in our understanding of the role of nuts and berries in a healthy dietary pattern would be limited. A risk-of-bias study of 5675 journal articles used in systematic reviews published between 1930 and 2015, representing a wide variety of nutrition topics, concluded that ROB domains started to significantly decrease after 1990, and particularly after 2000. Another study examined the incidence of favorable outcomes reported in studies funded by the food industry in the 10 most-cited nutrition and dietetics journals in 2018. Of the 1461 articles included in the analysis, 196 reported industry support, with processed food and dietary supplement manufacturers supporting 68% of the studies included. Studies supported by any nut or berry commodity group were not considered due to an incidence lower than 3% of qualifying articles. Studies with food industry support reported favorable results in 56% of their articles, compared to 10% of articles with no industry involvement. The authors offer a number of suggestions to help minimize real or perceived bias, calling on research institutions to enforce strict, regularly updated, and transparent oversight of all research projects involving industry. Suggestions in support of research transparency and integrity have also been advanced from guidelines adapted from the International Life Sciences Institute North America. This served as the basis for the development of consensus guiding principles for public-private partnerships developed by a group of representatives from academia, scientific societies and organizations, industry scientists, and the USDA, NIH, US Centers for Disease Control, and the US Food and Drug Administration. These provisions include full disclosure of funding and confirmation of no direct industry involvement in the study design, data and statistical analyses, and interpretation of the results and only minimal, if any, involvement of industry coauthor, often given as a courtesy to acknowledge funding and logistical support by the investigators with no intellectual involvement by the study sponsor. This is in contrast to industry-initiated research, where the industry office or commodity group sets predetermined research objectives, provides intellectual collaboration, and often has input on the study design, interpretation of results, and decisions regarding publication. Although some critics may argue that repeated industry funding in support of research groups that report favorable results on a particular nut or berry shows a bias toward positive outcomes, other interpretations are also possible. First, few labs have the infrastructure, detailed methodology and analytical equipment, and trained personnel to conduct clinical studies in an efficient and timely manner. Industry funded studies conducted at major universities have layers of review and accountability within their organizations to guard against malfeasance, and while these layers may not focus directly on precise elements of research design and interpretation of results, faculty members at such institutions generally have a level of integrity and accountability, knowing that administrative review exists. Calls for industry-funded research are often broad in scope, which allows researchers to generate proposals, research questions, and hypotheses that do not have preconceived outcomes. A third consideration is that the nuts or berries under study may simply have sufficient bio-activity to produce favorable outcomes, independent of potential researcher bias.With the depletion of natural resources and growing populations, there is a great motivation for recovering valuable compounds from fruit and vegetable wastes and converting them into functional ingredients as a part of sustainable agricultural practices. Pomegranate is an important California commodity and pomegranate by-products are a large portion of the process. These by-products could be repurposed into valuable compounds, but they are not unutilized or underutilized, ending up in landfills or animal feeds. In this dissertation, physiochemical properties and extraction methods of bio-active compounds in pomegranate peel were comprehensively studied to determine the feasibility of converting these pomaces into healthful food ingredients. The research objectives were to identify the physiochemical characteristics of pomegranate peel and assess different size reduction methods to obtain suitable peel particles for effective extraction by using water as a solvent; evaluate the effects of extraction temperature, time, and water usage on the phenolic yield, phenolic composition, and antioxidant activity to optimize the extraction conditions; study the bio-active properties and nutritional benefits of pomegranate peel powder and extract through animal models; investigate the characteristics of food products developed with pomegranate peel powder and extract; and study the co-extraction mechanism of pectin and polyphenol to improve extract yield and phenolic stability. There are 6 chapters in this dissertation. Chapter 1 includes an introduction, research objectives, and literature review on the processing and waste utilization of pomegranate fruits, with a focus on the current waste management practices and effects of punicalagin, which is the most unique polyphenol in pomegranate.In Chapter 2, the physicochemical properties of pomegranate fruits were investigated to explore the potential of recovering bio-active food ingredients from the inedible fruit peel.

There is also evidence that CRISPR-Cas9 modifications to upstream open reading frames can impact metabolite regulation across species, as vitamin C concentrations increased by 150% in Arabidopsis and lettuce by removing the regulatory effects of the associated uORF . While altering the levels of endogenously produced chemicals is effective, this approach is limited in the diversity of nutrients it can deliver. However, plant synthetic biology offers a means to bypass this limitation through the introduction of heterologous biosynthetic pathways. One of the first successes in this came with the production of golden rice, a ß-carotene enhanced rice grain that was generated through the addition of three genes . Since then, replacement of the daffodil phytoene synthase with a maize phytoene synthase has increased the titers of ß- carotene in golden rice 2 , providing greater nutritional benefits . Furthermore, the authors were able to not only add ß-carotene to rice grains but also increase the levels of iron and zinc with a single genetic insertion. This shows the potential of a biofortified staple crop to have an enhanced nutritive capacity suitable to meet multiple minimal daily requirements. Additional work has shown that the added ß- carotene in the rice grain can be further modified into astaxanthin and canthaxanthin, two antioxidants of interest, with one or two additional genes . Iron deficiency is one of the world’s leading micronutrient deficiencies and is responsible for around one million deaths annually. As such, hydroponic nft channel iron has been a target of biofortification efforts over the last few decades.

Early work showed that the overexpression of a root zinc transporter from Arabidopsis could not only increase the zinc concentration in grains, another significant deficiency, but it also increased the iron content for Hordeumvulgare . The addition of genes increasing nicotianamine, the nicotianamine-iron transporter and the iron storage protein ferritin was shown to have a concerted effect on rice seed iron concentration of more than fourfold . Perhaps, with the combination of nutrient transport and sequestration mechanisms in the grain or fruit, levels of many micronutrients could be enhanced. Other vitamin deficiencies also pose major concerns to human health and plant synthetic biology efforts can alleviate some of these nutrient shortcomings. Inadequate folate consumption is linked to cardiovascular disease, Alzheimer’s disease, and cancer, but it is especially important for pregnant women, as deficiencies can cause neural tube defects or megaloblastic anemia . Folate content in rice has been improved by 150-fold through the expression of a folate binding protein and a folylpolyglutamate synthase , offering a means to increase folate consumption without dietary supplementation. This work also showed that the binding protein provided stability to folate so that it was maintained at a similar level even after months of storage, which is a concern for any crop that does not get eaten immediately after harvest.

Vitamin B6 deficiency has been shown to be linked to cardiovascular disease, diabetes, neurological diseases, as well as nodding syndrome . Efforts to biofortify cassava, a staple vegetable crop in many parts of Africa, have been successful with the expression of the Arabidopsis thaliana genes PDX1.1 and PDX2, which are involved in de novo production of vitamin B6. Root-specific expression of PDX1.1 and PDX2 increased vitamin B6 and B6-glycoside concentrations by 4- and 14-fold, respectively, even after cooking . While biofortification of crops has taken great strides forward, there remain several considerations for effective implementation of this synthetic biology strategy. As more compounds are engineered into crops, it will be important that metabolic flux and regulation are not drastically altered, as this can have detrimental pleiotropic effects on the plant’s health. For example, the overproduction of γ-aminobutyric acid in tomatoes causes gross developmental defects . Moreover, there will be clear challenges in transferring metabolic engineering strategies from one crop to another. There has been moderate success in this, such as with enhancing ß-carotene in golden bananas , but each crop is unique and requires experimentation to optimize production. As we continue to understand the intricacies of plant metabolism, these issues will be more easily addressed.Nutraceuticals are defined as consumed bio-active compounds that are not essential for nutrition but offer other health benefits. These molecules are distinct from pharmacological compounds—though in some cases they do overlap—and do not require extraction and purification or semi-synthetic modifications before final dosage and distribution, as the source of nutraceuticals are the foods themselves.

While these compounds are not essential for growth and development and many of their health benefits are still under investigation, their presence in our diet has the potential to improve human health. One nutraceutical compound that has been of great interest is resveratrol. This naturally occurring compound, found in wine and chocolate among other plants, has been studied since the 1990s for its health benefits due to its association with the “French paradox,” the quandary of a low incidence of heart disease in a population that eats a high-fat diet. Many health benefits, including prevention of cardiovascular disease and treatment of diabetes, have been attributed to resveratrol and validated clinically, but its low concentration in native plants poses a challenge to extraction for medicinal use . Additionally, consumption of purified resveratrol is mostly metabolized, greatly reducing its beneficial effect, but ingestion alongside other food-based phenolic compounds increases resveratrol levels found in blood serum. Therefore, the addition of resveratrol to foods could provide a more efficient means for delivery than purified supplements. Since all plants contain the precursor molecules for resveratrol, 4-coumaroyl-CoA and malonyl-CoA, only a single gene, stilbene synthase, is required to produce minor amounts of the metabolite in any plant. Optimization of precursor pathways would also be necessary to enhance nutraceutical levels in any given food . Glucosinolates, and their hydrolysis products, isothiocyanates, are another family of compounds with great health potential that have been studied thoroughly . A recent review examined the vast diversity of these compounds, totaling 156 unique entries, emphasizing their identification and synthesis in plants . This diversity can be broken down into three types of glucosinolates based on the amino acid precursor: aliphatic, indolic, or benzenic, with the vast diversity derived from secondary modifications to these basal categories. Over decades of study, biosynthetic pathways for each type have been elucidated . Glucoraphanin is a glucosinolate found in high concentration in broccoli and has been a subject of intensive study due to the health benefits of it and its isothiocyanate, sulforaphane . Various studies have displayed the promise of sulforaphane in treating diseases such as neurological disorders, diabetes, cancer, and cardiovascular disease with variable efficacy observed over a vast range of dosages and sources of the metabolite; however, further clinical validation is needed to verify these health benefits . Medicinal interest in glucoraphanin led to its successful production in Nicotiana benthamiana through transient expression, though only small amounts were produced . This was one of the first examples of a complex pathway being heterologously expressed in plants, and with some optimization, nft growing system it could be stably introduced into a crop species for consumption. The use of breeding practices that select plants for favorable agricultural traits can remove important nutrients from cultivated crops. This can also reduce some of the important nutraceutical compounds found in the foods that we eat. For example, there is a complete lack of iridoids, a class of monoterpenes suggested to benefit human health, in most cultivated varieties of blueberry on the market, while every wild species tested was abundant with them . While this was an inadvertent consequence of breeding, it illustrates the need to investigate wild relatives of food crops to identify nutraceuticals lost during domestication.

Using the tools of plant synthetic biology, we should be able to quickly and easily reintegrate the pathways lost into commercial cultivars without the need for the lengthy breeding process. As we move forward in our efforts to produce advanced nutritive foods, it is important to examine the past uses of nutraceuticals in the prevention and treatment of disease. Traditional medicinal plants have seen renewed interest as sources for undiscovered bio-active compounds . This highlights the need for additional compound and pathway discovery efforts. There are many other reviews that discuss the different types of specialized metabolites found in medicinal plant species . Historically, medicinal plant extracts have been used as food additives, but introduction of their biosynthetic pathways into common food crops offers a means of increasing the availability of nutraceuticals to a broader market. In some cases, the biosynthetic pathways are known and efforts are being made to sequence more medicinal plant genomes in order to accelerate enzyme discovery in candidate organisms . Nevertheless, the major limiting step hampering efforts to engineer plant nutraceuticals is still the discovery of the enzymes involved in the biosynthesis of target plant natural products. Thus, new tools to streamline such research is of great interest to the larger plant synthetic biology field.Plants have many attributes that make them an ideal platform for the production of plant natural products. They are autotrophic, can be grown at a large scale, contain various intracellular compartments and tissue types, are used as a nutrient delivery system, and retain similar cellular features to the productproducing plant that may be required for efficient biosynthesis of compounds. Together, this enables countless strategies for the production and administration of plant natural products . However, the state of plant biotechnology has imposed limitations on the utilization of plants. In this section, we focus on the strengths of plants as a production platform and the technologies needed to improve their efficacy.Perhaps one of the greatest strengths of plants as a synthetic biology platform is their scalability. Large-scale plant production has been a focus of human society and technology since the first crops were cultivated. In addition to a long history of scalable plant cultivation for consumption, agriculture also offers a unique platform for the production and extraction of valuable therapeutic plant natural products. Opioids from Papaver somniferum, taxol from Taxus brevifolia, and QS-21 adjuvants from Quillaja saponaria, are several notable examples of therapeutic molecules that are produced and extracted from their native plant host . While many therapeutic chemicals can be extracted from their native producer, many species are difficult to cultivate or suffer from limited yields . Past efforts have focused on the production of nutrients and phytochemicals in microbes or with the use of chemical synthesis, but plants are an ideal platform for the production of small molecules at a large scale, as they are autotrophic and can be grown in non-axenic conditions like an open field. There are two main approaches for the heterologous expression of a specialized metabolic pathway in planta: transient expression or expression in stable plant lines. Transient expression typically utilizes Agrobacterium tumefaciens to insert genes of interest into a host plant. This process allows for the expression and characterization of genes in 3–6 days as opposed to the months required to make stable plant lines . Additionally, multiple strains of Agrobacterium harboring different genes can be co-infiltrated into host tissue, limiting the need for creating multi-gene constructs . These attributes have made it a valuable tool for in planta pathway discovery and protein production. Transient expression is usually conducted in a species of the genus Nicotiana but is typically only applied at small scales, limiting its viability as a production platform. Recent improvements by Reed and Osbourn have demonstrated the scalability and viability of transient expression for the production of phytochemicals. Through the use of full plant transient expression via vacuum infiltration, gram-scale quantities of various triterpenes were able to be produced and purified from Nicotiana benthamiana . As well as being a production platform, transient expression permits the testing of gene construct efficacy prior to stable line generation; however, the efficiency of Agrobacterium-mediated transient expression is variable in different hosts . This poses a limitation of Agrobacterium as a tool to screen genes in host plants. Additionally, transiently expressed pathways in Nicotiana benthamiana do not always function as expected in the desired host, limiting its use for screening constructs before generating stable lines . One method to circumvent these issues is the use of protoplasts, calli, and cell suspension cultures. These cell types are suitable for the use of direct transformation methods such as electroporation, gene gun bombardment, and microinjection. However, generation of these cell lines is often inefficient depending on the species used, produces fragile cells, and may not behave as an intact plant would .

Counts of uniquely mapping reads were generated through HTSeq for all 35 RNA-seq datasets . Multimapping reads were excluded from the analysis except for the tandem gene expression analysis. Differential gene expression analysis was performed using the DESeq2 pipeline across fruit developmental stages with three biological replicates per developmental stage . Gene expression values were derived by calculating the fragments per kilobase per million reads mapped values using the standard formula for FPKM /gene length in kilobases. To construct the gene co-expression network, genes that were not expressed or very weakly expressed in 30 or more conditions were first excluded from the analysis. The count data was then transformed into variance stabilized values using the variance stabilizing transformation function in DEseq. Pairwise correlations of gene expression were calculated using Pearson correlation coefficient and mutual rank using scripts available for download from the project’s data repository. MR scores were transformed to network edge weights using geometric decay function e−; five different co-expression networks were constructed with x set to 5, 10, 25, 50, and 100, respectively. Edges with PCC <0.6 or edge weight <0.01 were excluded. For each network, modules of coexpressed genes were detected using ClusterONE v1.0 using default parameters, blueberry grow pot and modules with P value > 0.1 or quality score <0.2 were excluded. The results from all co-expression networks were then combined by collapsing modules into meta modules of non-overlapping gene sets.

Total antioxidant capacity of tissues from the fruit developmental panel was analyzed using the ORAC assay. Briefly, ∼20–30 mg of frozen ground fruit tissue was measured for tissue samples prior to extraction. Sample extractions were performed on ground tissue using 1.8 mL of ice cold 50% acetone. Samples were vortexed and then put on a shaker for 5 minutes at room temperature. Samples were then centrifuged at 4◦C for 15 minutes . The ORAC assay was performed in a 96-well black microplate using the FLUOstar OPTIMA microplate reader . Each reaction well contained 150 μL of 0.08 μM fluorescein and 25 μL of 75 mM phosphate buffer , Trolox standards , or diluted sample extracts. For blueberry tissue samples, 1:80–1:20 dilutions were used. Upon loading all appropriate wells, the 96-well microplate was put into the microplate reader and incubated for 10 minutes at 37◦C. Following incubation, 25 μL of 150 mM AAPH was added to each well, and fluorescence measurements began immediately. Fluorescence measurements were taken for 90 seconds per cycle for 70 cycles until the fluorescent probe signal was completely quenched. The area under the fluorescence decay curve was calculated for each well. The total antioxidant capacity of a sample was calculated by subtracting the AUC from the blank curve from the AUC of the sample curve to obtain the net AUC. Using Trolox of a known concentration, a standard curve was generated , and the total antioxidant capacity of each sample was calculated as Trolox equivalents. Each sample was run twice for two technical replicates. The coefficient of variation between technical replicates was required to be less than 0.20. Biological replicates were run for all tissues in the fruit developmental series.Berries from ”Draper” were collected as described above.

Approximately 100 mg of each frozen ground sample was resuspended in extraction solvent in a 2 mL tube . Ground tissue was immediately mixed thoroughly to prevent thawing during extraction and to prevent metabolism of analytes by enzymes in the samples. All tubes were spun down for 10 minutes at 13,000 × g to pellet protein and other insoluble material. Then, 1 mL of supernatant was transferred to an autosampler vial. Anthocyanin content was evaluated by LC-MS as follows: 5 uL of sample extract were separated using a 10 minute gradient on a Waters Acquity HSS-T3 UPLC column on a Waters Acquity UPLC system interfaced with a Waters Xevo G2-XS quadrupole time-of-flight mass spectrometer . Column temperature was maintained at 40◦C, and the flow rate was 0.3 mL/min with starting conditions of 100% solvent A and 0% solvent B . The gradient was as follows: hold at 100% A for 0.5 minutes, ramp to 50% B at 6 minutes, then ramp to 99% B at 6.5 minutes, hold at 99% B to 8.5 minutes, return to 100% A at 8.51 minutes, and hold at 100% A until 10 minutes. Mass spectra were acquired in positive ion mode electrospray ionization over m/z 50–1500 in continuum mode using a data-independent MSE method that acquires data under both low and high collision energy conditions with the high collision energy setting using a ramp from 20–80 V. Capillary voltage was 3 kV, desolvation temperature was 350◦C, source temperature was 100◦C, cone gas flow was 25 L/hr, and desolvation gas flow was 600 L/hr. Correction for mass drift was performed using continuous infusion of the lock mass compound leucine encephalin. Anthocyanins and other related flavonoids were identified based on accurate mass and fragmentation pattern. Peak areas were determined using Quanlynx within the Masslynx software package . Relative anthocyanin content was calculated for each sample using the formula: reported peak area of the compound/peak area of internal standard/weight of extracted tissue .Insect repellents have been used since antiquity to fend off disease-transmitting mosquitoes and other arthropods. They developed gradually from smoke generated by burning plants and topical applications of essential oils into repellent substances, including those isolated from plants and a broad-spectrum synthetic repellent DEET , which was discovered in the early 1940s from a screening of more than 7000 compounds . Thereafter, other synthetic repellents have been developed, including IR3535, aminopropionate and picaridin piperidine-1-carboxylate, but DEET remains the most widely used repellent substance worldwide , particularly in the United States of America. Repellents work primarily as spatial and contact repellents. Mosquitoes attracted to and flying towards vertebrate hosts may make oriented movements away from the source upon approaching chemically treated skin surfaces. In this case, the chemical is a repellent sensu stricto . Because the repellent is acting from a distance, it may be referred to as a spatial repellent . When mosquitoes land on a chemically treated skin thus making contact before starting increasing locomotion activity or taking off, the chemical is called a contact repellent, which is sometimes referred to as excitorepellent, irritant, or contact irritant . From a strict mechanistic viewpoint, these two groups of compounds should be named non-contact and contact disengagent for spatial and contact repellents, respectively. It is now known that at least Culex and Aedes mosquitoes smell DEET . More importantly, it has been demonstrated that an odorant receptor from the Southern house mosquito, Cx. quinquefasciatus, CquiOR136, is essential for reception of DEET as a noncontact disengagent . Recently, it has been demonstrated that as a contact disengagent, DEET is detected by sensilla on the tarsal segments of the legs of the yellow fever mosquito Aedes aegypti , but the receptors remain elusive. Lastly, it has been suggested that DEET merely masks the reception of human emanations by Anopheles coluzzii , thus reducing the attractiveness of the host. Although its modes of action remain a matter of considerable debate, DEET is a gold standard repellent. It also has many “off-label” properties that do not directly affect human mosquito interactions. For example, DEET is a feeding deterrent , hydroponic bucket but if this were the primary mode of action, it would have little value in epidemiology.

The great value of repellents is that they reduce biting rates, which represents a second order parameter in vector capacity . Another property that may have a value in epidemiology, albeit not by decreasing vector capacity, is the deterrent effect of DEET on oviposition, as first observed for Ae. aegypti . While using the Xenopus oocyte recording system to deorphanize odorant receptors involved in the reception of oviposition attractants, we observed that DEET elicited outward currents in our preparations, in contrast to oviposition attractants and other compounds that generated inward currents. We have now systematically investigated this phenomenon using different ORs from three different species of mosquitoes. Here, we report that DEET, IR3535, and picaridin elicit outward currents on OR involved in the reception of mosquito oviposition attractants in the Southern house mosquito, Cx. quinquefasciatus, and orthologues from the yellow fever and malaria mosquitoes. Dose dependent outward currents were also observed with compounds in a panel that included plant-derived and plant-inspired repellents. Like DEET, IR3535 and picaridin , plant-inspired compounds, elicited robust inward current in the DEET receptor, CquiOR136, and showed repellency activity.In vitro transcription, oocytes microinjection, and electrophysiology were performed as previously described . Briefly, in vitro transcription of cRNAs was performed by using an mMESSAGE mMACHINE T7 kit , according to the manufacturer’s protocol. Plasmids were linearized with NheI, SphI, or PstI, and capped cRNAs were transcribed using T7 or SP6 RNA polymerase. cRNA samples were purified with LiCl precipitation solution and resuspended in nuclease-free water at a concentration of 200 μg/mL and stored at −80°C in aliquots. RNA concentrations were determined by UV spectrophotometry. cRNA samples were microinjected into stage V or VI Xenopus laevis oocytes . A two-electrode voltage clamp was used to detect currents. Oocytes were placed in a perfusion chamber and challenged with test compounds. Odorant-induced currents were amplified with an OC-725C amplifier , the voltage held at −80 mV, low-pass filtered at 50 Hz and digitized at 1 kHz. Data acquisition and analysis were carried out with Digidata 1440A and pClamp10 software .In short, two 50-mL Dudley bubbling tubes painted internally with a black hobby and craft enamel were held in a wooden board , 17 cm apart from each end and 15 cm from the bottom. The board was attached to the frame of an aluminum collapsible field cage . Two small openings were made 1 cm above each Dudley tube to hold two syringe needles to deliver CO2. To minimize handling of mosquitoes, test females had been kept inside collapsible field cages since the latest pupal stage. These female cages had their cover premodified for behavioral studies. A red cardstock was placed internally at one face of the cage, openings were made in the cardboard and in the cage cover so the cage could be attached to the wooden board with the two Dudley tubes and CO2 needles projecting inside the mosquito cage 6 and 3 cm, respectively. Additionally, windows were made on the top and the opposite end of the red cardstock for manipulations during the assays and a video camera connection, respectively. The two cages were connected at least 2 h prior to bioassays. At least 10 min before the assays, water at 28°C started to be circulated with a Lauda’s Ecoline water bath, and CO2 at 50 mL/min was delivered from a gas tank just at the time of the behavioral observations. Sample rings were prepared from strips of filter papers 25 cm-long and 4-cm wide and hung on the cardstock wall by insect pins to make a circle around the Dudley tubes. Cotton rolls were loaded with 100 μl of defibrillated sheep blood purchased from UC Davis VetMed shop and placed between a Dudley tube and CO2 needle. For each run, one paper ring was loaded with 200 μL of hexane and the other with 200 μL of test repellent in hexane. The solvent was evaporated for 1-2 min, blood-impregnated cotton plugs and filter paper rings were placed on the arena, CO2 was started, and the assays recorded with an infrared camera . During the assay, the arena was inspected with a flashlight with a red filter. After 5 min, the number of females that have landed and continued to feed on each side of the arena was recorded. Insects were gently removed from the cotton rolls and the assays re-initiated after rotation of sample and control. Thus, repellence for each set of test mosquitoes was measured with the filter paper impregnated with the same sample at least once on the left and once on the right side of the arena.To revisit our earlier observation of repellent-induced outward currents on OR sensitive to oviposition attractants, we challenged CquiOR21/CquiOrco-expressing oocytes with DEET and then skatole. CquiOR21, formerly known as CquiOR10 , is narrowly tuned to the oviposition attractant skatole . CquiOR21/CquiOrcoexpressing oocytes generated robust inward currents when stimulated with 10 μM skatole, whereas 1 mM DEET elicited outward currents . These outward currents were dose-dependent and were not observed when oocytes were injected only with CquiOrco cRNA or CquiOR21 cRNA .

Briefly, for PD3 and PW with and without BSA, media plates solidified with agar or Gelrite were divided into two halves, 10 to 12 spots of each mutant strain were made using a sterile toothpick, and plates were incubated at 28°C for 4 to 5 days. For XFM with and without BSA, plates solidified with agar were used and incubated for 10 to 12 days before measurements were recorded. Colony peripheral fringes were observed under 10 magnification using a Nikon Eclipse Ti inverted microscope , and fringe widths were measured for six colonies per plate per strain, with at least seven measurements per colony using a Nikon DS-Q1 digital camera connected to a Nikon Eclipse Ti inverted microscope and controlled by NIS-Elements imaging software version 3.0. Twitching experiments were performed at least three times independently for PD3 and PW with and without BSA and once for XFM with and without BSA. Three growth conditions were used: solid agar plates , liquid culture tubes , and continuous liquid flow . PD3 without antibiotics was the medium used, and the initial inocula of the NS1-CmR and pglA-KmR mutants were prepared as described above. Competence in tubes. Twenty-five-milliliter glass test tubes containing 3 ml of PD3 were inoculated with 100 l of each of the OD adjusted strain suspensions as donor and recipient cells . Tubes containing single strain inoculations were included as control treatments.

Tubes were then incubated with shaking . After 3 days, the tubes were vortexed well to mix the biofilm formed on the air-liquid interface with the rest of the suspension and serially diluted and plated as described above. Three independent experiments were performed, nft hydroponic system and three replications were included in each experiment . Competence in MCs. MCs were prepared as previously described . Briefly, two parallel channels with separate inlets for bacterial cells and growing media were etched on a silicon wafer. The channels were modeled into polydimethylsiloxane and sandwiched between the PDMS layer and a glass cover slide. The inlets and outlets were then connected to tubings that were connected to syringes . The syringes were connected to pumps which control the media flow rate in the MC. The MC was mounted onto a Nikon Eclipse Ti inverted microscope to observe cell attachment and biofilm formation using phase-contrast and Nomarski differential interference contrast optics. Time-lapse video was taken using a Nikon DS-Q1 digital camera connected to the microscope and controlled by NIS-Elements imaging software version 3.0. For preparing the inocula for MCs, equal volumes of the strain pairs were mixed and inoculated into the cell inlet syringes, and growing medium was injected in the media syringes. MCs were run for 5 to 7 days with a media flow rate of 0.25 l min 1 until abundant growth of biofilm was observed. At the end of the experiment, the fraction of cells collected in the outlet syringe was harvested, and the fraction formed inside the channels was detached and pushed to the outlet collection syringe by increasing the flow rate to 30 to 40 l min 1 . Serial dilution, plating, CFU counts, and the frequency of recombination calculations were done as described above. Four independent experiments were performed with seven replicates in total . 

Competence in MCs with grapevine sap. Grapevine sap was collected from a X. fastidiosa-susceptible variety in Dahlonega, GA, and a tolerant variety in Tallahassee, FL, at the end of the dormant season . A new season cane was pruned, and sap was collected in a 50-ml conical tube, which was stored in ice until it was brought back to the lab. Xylem sap was sterilized by filtering with a 0.22- m vacuum filter and stored at 80°C until used. Sap experiments were performed in the MCs with both pure sap and 50% sap mixed in PD3 . Natural competence assays were same as those for the MC experiment with PD3. Experiments were repeated at least three times for both sap types. Natural competence with heat-killed donor cells and confirmation of homologous recombination. Confirmation of homologous recombination occurring via natural competence was performed by using heat killed donor cells in the solid agar plates. Suspensions of the donor cells were incubated at 90°C for 15 min for heat killing. Complete killing was confirmed by plating an aliquot onto PW plates. The heat-killed donor and live recipients were then spotted on PD3 plates as described above. For confirmation of homologous recombination at the desired genome region, randomly selected recombinant CFU were restreaked onto new double-antibiotic PW plates, and colony PCR was performed using the primers targeting the flanking region of the construct used to generate the mutants according to Kung et al. . Sequences of the flanking regions of antibiotic cassette insertion sites between the parent strains were compared using the muscle pairwise alignment algorithm within the Geneious 9.0.3 platform . Statistical analysis. The number of recombinants, total CFU, and recombination frequency data were analyzed in PROC GLIMMIX , which fits statistical models to data with nonnormal distribution and nonconstant variability.

For the analysis of frequency, the response distribution was used as the binomial distribution of number of recombinants/ total cells. Least-squares differences of means among the treatments were separated by Tukey’s honestly significance difference test at the significance level P 0.05. For the repeated experiments, time factor was used as a random variable. The fringe widths of bacterial colonies among different media also were compared using PROC GLIMMIX in SAS. Accession number. Sequences of the flanking regions of antibiotic cassette insertion sites between the parent strains were deposited in NCBI under accession numbers KU873007 to KU873014.Several hypotheses have been proposed to explain the existence of natural competence in bacteria. One explanation is that starvation signals induce competence, and the incoming DNA serves as a nutrient source under poor nutrient conditions as demonstrated in H. influenzae , Pseudomonas stutzeri , and R. solanacearum . Based on the results with a minimal medium and a rich undefined medium , a previous study speculated that growth in a low-nutrient medium favors natural competence inX. fastidiosa. However, the results of this study with these two media and PD3, another undefined rich medium, demonstrated that growth in PD3 significantly increases the recombination frequency. This suggests that starvation is not necessary to induce competence in X. fastidiosa. Further investigations of the differences between PD3 and PW were performed by either removing or adding these components to/from one another. Initial screening with the components showed a pronounced effect of BSA on the number of recombinants recovered. Additional experiments confirmed that BSA significantly reduces the recombination frequency when present in PD3, PW, and XFM. Since both XFM and PW contain BSA, this may explain the lower recombination frequencies in these media. In a previous study, BSA had been found to reduce the surface attachment and twitching motility of X. fastidiosa . In fact, natural competence and twitching motility are dependent on the activity of type IV pili in X. fastidiosa . Therefore, in this study the correlation between twitching movement and natural competence in different media was investigated. Interestingly, PD3 allowed the highest fringe width, and the presence of BSA significantly reduced twitching motility in all three media. Twitching motility in XFM was lower than in either PD3 or PW as poor growth in XFM resulted in smaller colony sizes. Still the fringe widths of colonies in XFM without BSA were bigger than in XFM with BSA.

Most of the colonies spotted in XFM and XFM-BSA showed very little or no visible growth. This can be expected as XFM is a nutrient-limited minimal medium. Moreover, the pglA-KmR mutant that did not show twitching movement was not competent when tested with heat-killed NS1-CmR mutant and plasmid DNA as the donor. These results of concomitant decreases in natural competence and twitching motility in BSA-supplemented media and non-competency of twitch minus strain suggest that twitching motility is correlated with natural competence in X. fastidiosa. Natural competence in other Gramnegative bacteria is mediated by type IV pili-like structures . In light of the effect of BSA on twitching, hydroponic nft system it remains to be determined if BSA only alters movement or bio-genesis of type IV pili. Our results with different growth settings showed that the recombination frequency is significantly higher in the MC_in fraction than in the MC_out fraction. The MC_in environment closely mimics xylem vessels and the insect foregut with respect to continuous liquid flow, adhesion of cells on channel walls in a fashion similar to adhesion of cells on xylem vessels and the insect foregut, and formation of biofilms. This environment is conducive for both biofilm formation and twitching motility as demonstrated in previous studies . Moreover, expression levels of some of the type IV pili genes were shown to be increased in the MC_in environment compared to those under the other growth conditions , implying that activity of type IV pili is increased in this system, which may explain the higher rates of recombination in the MC_in fraction. The MC_out environment, on the other hand, consists mostly of planktonic cells and some detached biofilm fraction from MC_in, which is washed away with the liquid flow. The differences in recombination frequencies in these two environments suggest that the continuous media flow condition of the xylem vessels and growth in biofilm may increase the chances of recombination. Batch cultures in tubes also allowed recombination but at a lower rate than the continuous flow environment of MC_in and surface-attached condition of solid agar plates. A previous study also showed that growth in solid plates increases recombination compared to the growth in the liquid culture tubes .Recombinantsin theMC_outfraction were recovered when profuse biofilm growth was observed in the MC_in fraction with many recombinants formed. It is possible that the recombinants recovered in the MC_out fraction are due to detachment and washing away of portions of biofilms from the MC_in fractions, supporting the proposition that biofilm formation induces competence. Biofilm formation and quorum sensing signals have been shown to induce natural competence in other naturally competent bacteria such as Vibrio cholerae, Acinetobacter sp. , and Streptococcus mutans . Biofilms, in addition to having dense populations of cells, contain elevated amounts of extracellular DNA , which can be used for transformation by competent cells. Extracellular DNA has been shown to enhance biofilm formation in X. fastidiosa . Moreover, Kung et al. also showed that a knockout mutant on a biosynthetic gene for diffusible signaling factor , a cell-cell communication signal in X. fastidiosa, had a reduced rate of recombination, implying that a cell-cell communication signal also may be involved in regulating natural competence in X. fastidiosa. MC experiments with grapevine sap provide a closer resemblance to the natural habitat than MCs with the artificial culture medium. Previously, we have shown that the biofilm structure in grapevine sap is more similar to the natural biofilm than are the aggregates observed in synthetic medium inside MCs . The experiments with amendments of sap in the MCs detected natural competence, providing an indication that natural competence occurs in the xylem vessels of host plants and possibly in the insect vectors. Although the results with pure sap experiments were not reproducible due to inconsistent growth of one of the strains used, recombinants were recovered once with pure Chardonnay sap as the medium. Recombinants were readily recovered with the 50% sap in PD3 for both tolerant and susceptible varieties. Maintenance of competence with the addition of xylem sap indicates that sap components support DNA acquisition and transformation. Natural competence occurring in environments resembling natural habitats also have been demonstrated in other naturally competent bacteria such as P. stutzeri and V.cholerae, in which artificial medium resembling natural soil extract and natural growth substrate , respectively, induced competence. In R. solanacearum, another xylem-colonizing plant pathogen, natural competence has been demonstrated in planta , and the recipient strains were shown to have increased virulence, acquiring DNA regions as long as 40 kb from donor strains. Findings from competence experiments with grapevine sap and the MCs suggest that when two different strains are established together in the xylem vessels or in the vector foregut, recombination is possible. Noteworthy is the fact that in the experiments reported here, recombination was higher with sap from a tolerant grapevine variety, where infection by X. fastidiosa is symptomless.

Patients with inflammatory bowel disease have a significantly increased risk of developing CRC, while long-term aspirin treatment is associated with a significantly decreased risk of CRC. The mechanisms by which chronic inflammation promotes tumor development often involve the immune system. For example, the IL6/STAT pathway discussed earlier is also implicated in cancer formation. Overexpression of IL6 leads to excess STAT3 transcription, causing unwanted cell proliferation not only in T cells but also in the intestinal epithelium. Another inflammatory cytokine of note is TNF α. While the intestinal bacteria can promote inflammation, they may also affect the likelihood of CRC more directly. Once the intestinal mucus layer is thinned, and direct bacterial-epithelial cell interactions occur, certain bacterial strains promote tumor development. E. coli strains bearing the pks island are of particular interest. This genetic locus codes for the secondary metabolite colibactin, along with the enzymes necessary for its production. Colibactin has been shown to crosslink with DNA, producing double-stranded breaks. Furthermore, pks+ E. coli strains have been shown to be prevalent in CRC patients. In one study, big round plant pot nearly two-thirds of CRC patients had pks+ E. coli strains in their intestinal bacteria. In the same study, pks+ E. coli also existed in about 20 percent of healthy individuals.

Colibactin, however, is a reactive and short-lived protein, requiringclose contact with epithelial cells to cause DNA damage. A healthy mucosal barrier keeps colibactin at a distance and reduces the chance of affecting the intestinal epithelium. Evidence for the pathogenic relationship between diets, Fusobacterium nucleatum, and CRC has been emerging. The F. nucleatum levels have been shown to be higher in CRC than in adjacent normal mucosa. Utilizing the molecular pathological epidemiology paradigm and methods, a recent study has shown the association of fiber-rich diets with decreased risk of F. nucleatum-detectable CRC, but not that of F. nucleatum-undetectable CRC. Experimental evidence supports a carcinogenic role of F. nucleatum, as well as its role in modifying therapeutic outcomes. The amount of F. nucleatum in CRC tissue has been associated with proximal tumor location, CpG island methylator phenotype , microsatellite instability , low-level CD3+ T cell infiltrate, high-level macrophage infiltration, and unfavorable patient survival. The amount of F. nucleatum in average increased in CRC from rectum to cecum, supporting the colorectal continuum model. Future studies should examine the role of diets, microbiota, and CRC in detailed tumor locations. Dietary prevention of CRC, then, has two intertwined aims: to reduce inflammation and to promote a healthy intestinal microbiota. As already discussed, preclinical evidence implies that dietary bio-active compounds, particularly anthocyanins, can reduce symptoms of lowgrade chronic inflammation as well as oxidative stress. It can also aid in balancing the intestinal microbiota by promoting the growth of beneficial bacteria and by reducing the populations of pro-inflammatory bacteria.

Clinical trials have had mixed results, but anthocyanins and some polyphenols have shown to counteract against CRC actively. More research, however, is necessary for conclusive results. How, then, are individuals to consume enough bio-active compounds to have an effect on health? Some answers may be found in the food consumption practices of cultures with historically low CRC incidence. Parts of India, for example, have had some of the lowest CRC incidence rates in the world; however, this status has been changing. In recent decades, increasing urbanization and similar factors have led to progressively Westernized diet patterns and lifestyle. CRC incidence rates are similarly rising, lending weight to the hypothesis that the traditional Indian diet may help prevent CRC. Furthermore, Indian immigrants to Western countries have a much higher incidence of CRC compared to Indians in India. Typical components of traditional Indian meals include a broad variety of flavors, as promoted in Ayurvedic medicine, and a variety of other foods. Both are facilitated by using a thali platter to serve the meal. The traditional American main meal includes an entree , one or more carbohydrates , and one or more vegetables. This basic structure can potentially be adapted with inspiration from thali meals by reducing the size of the main dish and serving more vegetables, legumes, pulses, herbs, and spices to accompany it. The inclusion of multiple colors in a meal is desirable, because certain bio-active compounds, particularly anthocyanins are also pigments. Blue, purple, and red-purple colors in plant foods indicate high anthocyanin content. Purple-pigmented potatoes can be prepared in the same way as traditional white potatoes, but the anthocyanin content is significantly higher in the pigmented varieties.

Purple sweet potatoes also contain more anthocyanins than the more common orange varieties and can be easily substituted for them. Other vegetables with red or purple cultivars include carrots, cauliflower, and cabbage. Different colors can indicate the presence of other bio-active compounds, such as orange , yellow , and red/pink . Thus, healthy bio-active compound consumption may be increased by selecting colorful vegetables. Another way to increase consumption of bio-active compounds is to increase their presence in available foods. The content of bio-active compounds in plant foods is highly influenced by genetics. The agricultural industry could greatly impact health by adopting food plant cultivars that produce bio-active compounds in larger amounts than is currently common. New cultivars may need to be developed that retain desirable characteristics such as large size, pest resistance, reduced spoilage, etc., but also have high bio-active content at the time of consumption. bio-active compounds, with some exceptions, tend to deteriorate during storage. Even when compounds have not deteriorated, storage may reduce the anti-inflammatory/antioxidant activity of bio-active compounds to affect health. A second systemic change that would promote increased bio-active compound consumption involves reworking how fruits and vegetables are currently stored and processed, as well as reducing the average storage time and adapting processing to optimize the amount of bio-active compounds. Presently, “nutritional adequacy” does not consider many of the bio-active compounds discussed in this paper. Further clinical studies are needed to support and elucidate the role of bio-active compounds in the prevention and treatment of disease.Recent research provides preclinical evidence that phytochemicals, especially anthocyanins, promote gut microbial health, reduce inflammation, and lower the risk of colorectal cancer. Clinical evidence is sparse but indicates that anthocyanins and other bio-active compounds do have an effect on colon cancer. Both are consistent with low cancer rates in India, where both traditional diet and Ayurvedic medicine promote consumption of many classes of phytochemicals. Long-term, diet-based randomized clinical trials are both difficult to conduct and prohibitively expensive. However, given the strong evidence from basic studies, observational data, and randomized clinical studies with short-term surrogate outcomes, steps should still be taken to improve the consumption of bio-active compounds, particularly in countries which contain a large proportion of CRC patients. Eating a wide variety of plant foods has no ill effects, and is indeed a commonly recommended part of a healthy lifestyle. Increasing bio-active intake among Westerners will require modifications in both individual eating habits and food system practices.Survival of humans will depend on increased agricultural productivity. Agricultural productivity is not only more yield per area, but also higher nutritional content, round plant pot less dependence on fertilizers, and more resilience against environmental hazards. All of these traits impinge upon plant metabolism. Plants carry out a myriad of metabolic reactions that are intricately connected into complex networks. To understand and engineer plant metabolism, it is important that metabolic complements of plant genomes are accurately and consistently annotated across species. To provide the research community with comprehensive information about plant small-molecule metabolism, we previously introduced the Plant Metabolic Network , a plant-specific online resource of metabolic databases .

PMN consists of PlantCyc, a database of all experimentally-supported information found in the literature from any plant species, as well as 22 single-species databases with a mix of experimentally-supported and computationally-predicted information, which allow researchers to explore each species’ unique metabolism. Here we describe the substantial expansion of PMN in both quantity and quality, which includes 126 single-species databases. We demonstrate the utility of the PMN resource by applying recently published omics data to gain insights into plant physiology and cellular level metabolism. Additionally, we systematically compare 126 species in the context of metabolism to identify metabolic domains and pathways that distinguish plant families. Finally, we present new website tools for viewing and analyzing metabolic data including a CoExpression Viewer and subcellular boundaries for metabolic pathways.PMN is a compendium of databases for plant metabolism with a substantial amount of experimentally supported information. The latest release contains 126 databases of organism-specific genome-scale information of small-molecule metabolism alongside the pan-plant reference database PlantCyc . Together, these databases include 1,280 pathways, of which 1,163 have direct experimental evidence of presence in at least one plant species. In addition, PMN 15 includes 1,167,691 proteins encoding metabolic enzymes and transporters where 3,436 have direct experimental evidence for at least one assigned enzymatic function. There are 9,129 reactions , and 7,316 compounds. Compared to the PMN 10 release described in Schläpfer et al. , PMN 15 increases the number of species 4.7-fold and proteins 8-fold, and adds 2,929 more reactions, 2,178 more compounds, 66 more pathways, and 3,229 more references . Data in the PMN databases are represented using structured ontologies consisting of hierarchical classes to which pathways and compounds are assigned by PMN curators, which makes statistical enrichment analyses possible. The pathway and compound ontology classes, alongside the phylogeny of the included species, illustrate the breadth of metabolic information and species included in the database . Prominent specialized metabolism classes such as terpenoids and phenylpropanoids are highly represented in the databases. This large volume of metabolic information makes PMN a unique resource for plant metabolism. The reference database, PlantCyc, is a comprehensive plant metabolic pathway database. PlantCyc 15.0.1 contains experimentally supported metabolic information from 515 species. Most of the data come from a few model and crop species . For example, Arabidopsis thaliana contributes to 43.4% of experimentally supported enzyme information in PlantCyc, followed by 7.46% from Chlamydomonas reinhardtii and 3.37% from Zea mays. Compared to other metabolic pathway databases such as KEGG and Plant Reactome , PlantCychas substantially higher numbers of experimentally supported reaction and pathway data . PlantCyc 15 includes 3,077 experimentally validated reactions with at least one curated enzyme and 1,163 curated pathways . Plant Reactome includes 1,887 and 320 curated reactions and pathways, with 677 reactions and 266 pathways predicted to occur in A. thaliana , while KEGG includes 543 experimentallysupported pathways as of February, 2021, with 136 occurring in A. thaliana. Data on the number of reactions in KEGG that were experimentally validated were not available at the time of publication. The reference information in PlantCyc is incorporated into MetaCyc, which also includes experimentally supported metabolic information from non-plant organisms and is used to predict species-specific pathway databases . In addition to the reference database PlantCyc, PMN 15 contains 126 organism-specific metabolism databases . These databases range widely in the plant lineage including several green algae and nonvascular plants. The majority of the plants are angiosperms with the Poaceae family most highly represented with 25 organisms. There are also 8 pairs of wild and domesticated plants, including rice, wheat, tomato, switchgrass, millet, rose, cabbage, and banana, alongside their wild relatives . Finally, PMN 15 includes 6 medicinal plants : Camptotheca acuminata, Cannabis sativa, Catharanthus roseus, Ginkgo biloba, Salvia miltiorrhiza, and Senna tora. The newest addition to the list of the medicinal plants is Senna tora, which is a rich source for anthraquinones and whose recent genome sequencing and metabolic complement annotation helped discover the first plant gene encoding a type III polyketide synthase catalyzing the first committed step in anthraquinone biosynthesis . This rich collection of species-specific metabolic pathway databases enables a wide range of analyses and comparisons. To promote interoperability and cross-referencing with other databases, PMN databases contain links to several compound databases such as ChEBI , PubChem , and KNApSAcK . PubChem containins over 270 millionchemical entries as of March 2021, and 95% of PMN compounds link to it. ChEBI release 197 has 58,829 entries and serves as a primary source of compound structural information during curation into PMN databases. Within PMN, 65% of compounds link to ChEBI. Examining 50 randomly chosen compounds that are not mapped to ChEBI suggest that the majority of the remaining 35% compounds do not yet exist in ChEBI . KNApSAcK links are comparatively rare, as only 1.7% of compounds have had a KNApSAcK link added by curators.

This modeling tool may improve current management practices by predicting pest pressure independent of trap catches or samples of infested fruit. We also see potential applications of this model for research in other fields of study and for broadening the understanding of how pests interact with the environment.The population projection model was written in the open source statistical environment R version 3.0.2. The model calculated the matrix based on mean temperature input. Briefly, the matrix calculations were based on age-specific regressions of temperature-dependent population parameters as highlighted by Tochen et al.. Whereas immature life stages of D. suzukii may experience different environmental conditions than adults because these life stages are completed within the fruit, in this study, ambient air temperatures were used to predict population dynamics for all life stages. To return age-specific population vectors for 50 age-classes of D. suzukii for each test case, a vector of mean daily temperatures for each site was input into the R statistical interface. The biofix, square pot plastic or the point where the model began in the spring, was determined using methods described in Tochen et al.. Biofix essentially described the earliest point in the season when the temperature allows the population to increase. Calculations for population estimates were initiated on the biofix date of 2 February in Parlier and 1 April in Wilmington and Salem . In Pergine and Sant’Orsola, estimates were initiated on 6 April .

The population matrices were initiated with 100 flies in the population vector for 41–50 day-old females based on the assumption that females of this age group represent flies that would be emerging from diapause in spring. The logtransformed sum of D. suzukii from all life stages for each day represented the total population estimate except where age distributions are considered. For daily age distribution of D. suzukii from Parlier, Salem and Wilmington during 2013, 1–3 day-old D. suzukii were classified as eggs, 4–7 day-olds were larvae, 8–9 day-olds were pupae, and 10–50 day-olds were classified as adults. Among the most important assumptions of the model are that populations of D. suzukii would not be limited by host availability, are not density dependent, do not exhibit Allee effects, and that response to current temperature is not dependent on previous temperature exposure.Seasonal weekly trap catches of D. suzukii were recorded in all study sites, except Riva del Garda, but model estimations for this location was included because the climate here is much different from the other locations studied in Italy. Trap counts were pooled data from commercial blueberry fields in Wilmington ; unsprayed apricot, blackberry, blueberry, cherry, peach, and citrus orchards in Parlier ; commercial blueberry fields and surrounding blackberry vegetation in Salem ; strawberry, blackberry, cherry and blueberry fields in Pergine ; and unsprayed strawberry and raspberry fields in Sant’Orsola . In Wilmington and Salem, traps were made of clear plastic cups, ca. 1 liter in volume each. Each trap had 6–15 entrance holes 4.5– 9 mm in diameter.

Trap baits in Wilmington consisted of a yeast and sugar water mixture containing 6 g yeast and 40 g sugar dissolved in 710 ml water. In Salem, traps were baited with 100– 200 ml natural apple cider vinegar and 1–3 ml unscented liquid soap to break water surface tension. In Parlier the traps were made to the specifications of the ‘‘Haviland Trap’’ design for D. suzukii monitoring. A 750-ml plastic container served as the basin for each trap. A 7.5-cm diameter hole was cut in the lid, over which a piece of 0.6-cm wire mesh was attached. Each trap was covered with a Pherocon trap cover , which had a built-in wire hanger. Each trap was filled with 250– 300 ml of apple cider vinegar with 15 ml of unscented soap added as a surfactant to each container of vinegar. In Trentino the containers were 1000-ml graduated white polyethylene bottles filled with 200 ml apple cider vinegar . All traps were placed near the fruiting level of host plants or on stable surfaces in shaded areas and were checked weekly. The contents of each trap were collected into a separate container that was taken to the laboratory for processing, and at the same time, the traps were refilled with fresh apple cider vinegar and unscented soap, as described above, in the field. The liquid and contents from each trap sample were strained in the laboratory and the numbers of adult SWD collected were recorded by gender. All data from traps were analyzed to display mean weekly D. suzukii per trap for each of the regions. In Wilmington, winter and late dormant mean temperatures were never below 0uC and reached 20uC multiple times prior to 31 April .

At this site, mean high summer temperatures exceeded 25uC multiple times during July, and mean low temperatures were below 10uC during November. In Parlier, winter and late dormant temperatures were never below 0uC and were higher than observed in both Salem and Wilmington during this period . Here temperatures were above 25uC multiple times prior to 31 April. Mean daily temperatures were consistently above 25uC during June–September, and daily means in Parlier sometimes exceeded 33uC. In Parlier, temperatures dropped to below 10uC during November. In Salem, early-season temperatures were warmer during January and February of 2012, compared to 2013 . Temperatures observed from March through May were slightly higher during 2013 compared to 2012. In Salem, winter and late dormant temperatures were never below 0uC or above 15.3uC until 31 April 2013 . Daily mean temperatures gradually increased to 25uC during July, after which daily mean temperatures dropped to below 10uC during November. For the Italian sites Riva del Garda had higher late dormant and fall temperatures than Pergine and then by Sant’Orsola . Summer temperatures were similarly ranked higher in Riva del Garda, followed by Pergine and then Sant’Orsola during 2013. In Pergine during winter 2012 , late dormant temperatures were frequently below 0uC and were as high as 20uC before 31 April. Fluctuations of temperature were more pronounced in 2012 in Pergine, compared to 2013 . Very low temperatures were recorded in Pergine from 3–12 February 2012, followed by relatively warm temperatures from 24 February to 13 March. Temperatures were also comparatively low from 9–11 April 2012. In Pergine, daily mean temperatures increased to 25uC during July, after which they dropped to below 10uC during November. Mean temperatures were well below 0uC during December.Population estimates using temperature data indicate that D. suzukii populations are able to increase to high levels in all of the studied locations . The population estimates in all regions broadly tracked demographic trends of D. suzukii caught in traps . When comparing early-season population estimates between Wilmington, Parlier, and Salem , the population estimates were highest in 2013 in Wilmington followed by Parlier and then Salem. However, the population estimate for Salem surpassed Wilmington by 15 June and surpassed Parlier on 16 July, as Salem population estimates continued to climb while the latter sites experienced declining populations after reaching the first peak of their bimodal distributions. In Parlier, the early-season population peaked on 16 June, subsequently decreasing to a low on 10 September before increasing to a second population peak on 9 November, large plastic planting pots then decreasing again as winter progressed. In Wilmington, the population curve peaked on 21 June, then the population curve declined slightly for an extended period, followed by a second period of population increase beginning on 19 September to a population peak in November. In Salem, populations consistently increased from 25 April to a peak on 22 October, followed by a steep decrease. When comparing population estimates between seasons for the initial harvest period of early- to mid-season blueberries in Salem , the majority of model outputs for this period estimated greater populations for 2013.

When comparing populations along the elevation gradient of the three Italian sites, higher early-season populations were predicted at the lowest elevation Riva del Garda, followed by Pergine and then Sant’Orsola . In Pergine, greater population numbers were estimated for the majority of the growing season during 2013 compared to 2012 . In all model predictions, immature life stages comprised by far the majority of the population, except at the beginning or end of the season when adults tended to dominate . One exception was Wilmington, where temperatures remained favorable for reproduction into the late fall so that immature stages remained a majority of the population . In Salem, fall temperatures initially caused cessation of reproduction, leaving a majority of adults, but December temperatures allowed for some reproductive activity to occur . In the early spring, a higher relative percentage of adults occurred due to the overwintering adults that were initiating their first reproduction. In part, this was an artifact of initiating the model with only older adult females. In the fall, environmental conditions became unfavorable for reproduction but may not have had strong effects on adult survival. Overall, no populations reached a completely stable age structure, but the highest relative stability for each site occurred in the middle of the season. Stability of age structure was the highest in Wilmington, followed by Parlier and finally Salem, which had a high degree of instability . Demarcation of distinct generations was very clear for the first part of the season in Parlier and Salem , but during the mid season at these sites and in Wilmington , it was very difficult to distinguish individual generations to distinguish complete generations from partial generations.In Wilmington, D. suzukii counts were first recorded on 5 May 2013 at one fly per trap with an erratic increase to a peak in numbers at 26 flies per trap on 26 July . After this period, the trap numbers gradually decreased to six flies per trap until 4 December, at which point D. suzukii trapping was discontinued. In Parlier, two population peaks were found during the crop season, one during the early part of the season, followed by a long mid-summer period without fly captures, and a second peak during the latter portion of the season . Adult D. suzukii were first caught on 19 March 2013 at one fly per trap and increased to a high of six flies per trap on 16 May, after which they decreased to zero on 27 July. The trap numbers remained at this level until 19 September, after which numbers continued to increase into December. In Salem and Wilmington only one population peak was observed during the summer period . During 2012 in Salem , D. suzukii trap counts consistently increased starting on 5 July from one fly per trap per week to a maximum average of 17 flies per trap on 6September. During 2013 in Salem , the first D. suzukii trap counts were observed on 30 May at an average of three flies per trap per week and gradually increased until 10 September, when a maximum of 27 flies per trap was observed. The first trap counts during 2013 were therefore consistently recorded four weeks before those found in 2012 and higher levels of flies were found in traps during 2013 in Salem. In Pergine and Sant’Orsola, fly counts were first observed 23 June 2013 , and on 7 July 2013 in respectively. In Italy, one population peak was visible each year for Pergine and Sant’Orsola. In 2012 in Pergine, the first flies were trapped on 25 July, approximately four weeks before those caught during 2013. The mean number of flies per trap per week increased to a peak of 4.6 on 10 September 2012 to a peak of 11.3 on 29 August 2013. Infested fruit was first found in 2013 on 24 June in Pergine , and on 19 July in Sant’Orsola . First fruit infestation in Pergine in 2012 was determined on 24 June , compared to 28 July 2013 .In order to compare environmental differences between all of the regions in this study, we illustrate degree-day accumulation for 2013 . When comparing differences between seasons, we found basic differences in accumulation for Salem and Pergine during 2012 and 2013. Of the three US locations, accumulation was initially the greatest in 2013 in Wilmington, followed by Parlier and then Salem . However, accumulation of degree-days increased at a higher rate in Parlier and exceeded the accumulation in Wilmington by 3 March.