Month: December 2024

Infection and dieback severity varied considerably across the landscape, however, there is some evidence to suggest that populations at lower elevations may be at highest risk for severe dieback, either due to increased water stress, close proximity to fungal inoculum sources, or both. Additionally, shrubs located on southwest-facing slopes may also be more vulnerable due to increased sun exposure and thus environmental stress. Management efforts may want to focus on these areas when this region experiences future drought. Finally, although extreme dieback was recorded throughout the study, none of the observed shrubs succumbed to mortality. This may be the result of overall physiological resiliency, and the ability of adult shrubs to allocate resources to keep portions of the canopy alive. It could also be that the region’s slightly more mesic climate offers a climatic buffer that prevented shrubs from reaching their mortality thresholds. More research is needed to identify these exact mechanisms and thresholds in A. glauca. Collectively, the results of this dissertation work provide valuable knowledge on the severe dieback of an important chaparral shrub during an historic drought, with the potential for ecologically and economically costly consequences. Additionally, the data I present provide insight into the scale and progression of A. glauca dieback in a chaparral system, and potential patterns of future dieback in the face of predicted climate change. Future research that seeks to further resolve landscape and environmental variables contributing to plant stress would help in identifying these patterns.

Heterogeneity and rugged topography across the landscape, round garden pot while likely beneficial for the resilience of regional A. glauca populations during drought, presents significant challenges for on-the-ground monitoring. Out of necessity for safe access , many of the plants surveyed were located on the outer boundaries of stands, where edge effects may have been a factor. Monitoring intact, undisturbed stands using drones would yield valuable additional insight into the extent of disease deeper into stands and in stands on steep terrain or that are outside of normal visual range. The challenges of working in rugged landscapes covered in impenetrable vegetation highlight the need for using and refining remote sensing technologies, such as drone imaging, Light Detection and Ranging , and hyperspectral imaging as monitoring tools. Large-scale, long-term monitoring using these tools would allow researchers to retrieve data in areas that have previously been inaccessible, while also gaining a larger scale understanding of drought impacts. They ultimately will enable future studies to reveal more nuanced patterns across the landscape and between years of varying climatic conditions.Plant pest and disease outbreaks play a major role in shaping ecosystems around the world. Outbreaks can alter ecosystem structure and function, often with substantial consequences . Over the past 200 years, pest/disease outbreaks have increased due to mass exchange of biological materials from global trade and a rise in unusual climate events resulting from global climate change .

Prolonged climate irregularities can subject plants to environmental stress outside of their normal resistance thresholds and make them susceptible to pests and pathogens . For example, the increase in extreme droughts, defined here as greater in intensity and duration than historical drought regimes, has been directly linked to enhanced mortality in woody plant systems worldwide, often in association with pest/pathogen outbreaks . Plant disease outbreaks are often economically costly , and can result in loss of ecosystem services in natural ecosystems. With global trade continuing to spread pests and pathogens, and global change-type drought events predicted to increase , incidences of plant disease outbreaks are expected to increase. Understanding the role of drought and pathogens in plant dieback and mortality is therefore of critical importance. Latent fungal pathogens are of particular concern for natural ecosystems yet their ecological roles remain poorly understood. These pathogens can live as asymptomatic endophytes within their hosts and remain undetected for long periods of time . The Botryosphaeriaceae fungi, a groupthat causes considerable damage to hundreds of agricultural, ornamental, and naturally occurring host species around the world , includes many latent fungal pathogens that are difficult to detect in wild plant populations. Members of this diverse family can occur as endophytes, pathogens, and saprophytes on diverse woody hosts . They are best known as pathogens that cause leaf spots, cankers, severe branch dieback, and death in economically important hosts such as grapevines , avocado , and eucalyptus . While Bot. fungi are rapidly becoming one of the most important agents of disease in agricultural plant hosts , relatively few studies have been conducted on these pathogens in natural systems .

The Bot. fungi have a long history of taxonomic confusion, in part due to indistinctive morphological characteristics among species and from other fungal taxa, as well as historically poor and inconsistent descriptions early on in their discovery . Furthermore, Bot. host specificity and pathogenicity can vary widely among species and across geographical regions, complicating our understanding of their influence in various host species and across systems . While advances in molecular sequencing and data basing have added clarity in this area , challenges remain in understanding the diversity and pathogenicity of Bot. species among hosts and across regions. As a result, there is a dearth of knowledge on their ecological roles, particularly in native ecosystems.One consistent finding is that disease outbreaks from Bot. fungi in agriculture are often associated with environmental stress, such as extreme heat fluctuations and drought . Furthermore, studies have shown latent pathogens like Bots cause more damage to water-stressed hosts , and some Bot. species have been shown to grow well in water potentials much lower than what their plant hosts can tolerate , suggesting drought conditions increase virulence by these pathogens. Therefore, regions that have historically dry climates or experience periodic extreme drought may be especially vulnerable to disease outbreaks from latent pathogens as they are predicted to experience an increase in drought events due to climate change . Mediterranean-type climate areas are projected to be global change “hot spots” , and dry shrublands are predicted to experience some of the most rapid increases in mean temperatures . Indeed, recent drought-related morality in California’s semi-arid Mediterranean climate shrublands has provided support for these predictions . Furthermore, the combination of dense human settlement and agricultural lands in close proximity to many natural shrubland habitats in southern California creates a likely pathway for exotic pathogen introductions and movement of pathogens from agricultural settings into wildland species. Not surprisingly, Bot. species have been retrieved on a variety of native chaparral shrub species in California, including Ceanothus spp. , Malosma laurina , and other species of Arctostaphylos . Understanding the response of native species and these pathogens to extreme weather conditions will help to predict future vegetation change and potential species losses . From 2011-2018, southern California experienced one of the most severe droughts in recorded history, with 2014 being the driest in the past 1,200 years . Field observations in winter 2014 identified high levels of branch dieback, and in some cases mortality, large round plant pots in a common ecologically important shrub, Arctostaphylos glauca in coastal California. Two well-known Bot. species were isolated from the symptomatic shrubs . Like other members of the Bot. family, both N. australe and B. dothidea infect a broad range of hosts, and are known to be responsible for disease outbreaks associated environmental stress in agricultural species . While B. dothidea is well established in California, with over 35 different host species having been identified , phylogenic evidence suggests N. australe may be more recently introduced . Its impact on shrublands of California has not been quantified.

Preliminary observations suggested high levels of branch dieback, and in some cases mortality, at lower elevation sites and along exposed ridges compared to higher elevations in coastal montane settings. We hypothesized that identifiable patterns would exist in the distribution of B. dothidea and N. australe across these landscapes that correlate with branch dieback and environmental variables associated with drought stress. Manzanita dieback has previously been causally associated with Bot. infection . A greenhouse experiment by Drake-Schultheis et al. , revealed that drought enhances onset of stress symptoms and mortality in young A. glauca inoculated with N. australe compared to shrubs subjected to drought or inoculation alone. However, to the authors’ knowledge no previous quantitative studies exist on the distribution of Bot. species in California shrubland environments with Mediterranean climates. To better understand the occurrence, distribution, and severity of Bot. infections in chaparral shrublands, we surveyed infection in A. glauca between April and September 2019. We also collected data on site elevation, aspect, and average percent canopy dieback at each site sampled for infection. While a variety of landscape variables are likely to influence plant stress at any given site , we focused on elevation because A. glauca already tends to occur mostly on xeric and rocky soils of exposed slopes, and therefore elevation was presumed to be the most significant factor influencing precipitation and water availability in this setting. Also, other studies have used elevation as a proxy for climate variation . We also recorded aspect of each sampled shrub since it influences sun exposure, temperature, and water stress. To test our hypothesis that Bot. fungi and level of stress each played a role in extensive canopy dieback in A. glauca, the following questions were addressed: What is the distribution of Bot. infection in A. glauca stands across the chaparral landscape in coastal Santa Barbara County? How do levels of infection by the two Bot. fungi, N. australe and B. dothidea, compare across elevation? and How do stand-level infection and elevation correlate with dieback severity? We predicted N. australe and B. dothidea to be presentacross all sites and elevations, but also that N. australe, having been previously isolated with high frequency in the area , would likewise have the greatest incidence in this study. Furthermore, we expected levels of Bot. infection and dieback severity to be greater at lower elevations compared to higher elevations, because lower sites typically receive less annual rainfall, thus exacerbating drought stress. This study presents the first quantitative survey summarizing the severity and distribution of Bot. fungi in natural shrublands, and seeks to identify important patterns of infection and dieback in A. glauca to predict future vulnerabilities across the landscape.The study sites were located on the generally south-facing coastal slopes of the Santa Ynez Mountains in Santa Barbara, California, USA . The sites range from a lower elevation of ~550m to an upper elevation of ~1145m, and cover an area of ~47km2 . This region is characterized by a Mediterranean climate, with wet winters and hot, dry summers. Mean annual precipitation ranges from 68.4cm at lower elevations to 90.6cm at upper elevations . During the 2013-2014 wet season, which was two years into a multiyear drought and one of the driest years on record in California , these areas received only 24.8cm and 31.6cm precipitation, respectively .Sites were initially randomly generated from polygons drawn in the field around relatively pure stands of A. glauca , and Drake-Schultheis, unpublished data. Polygons were then categorized according to elevation , and numbered within their respective elevation categories. Ten sites per elevation zone were randomly selected using random number generator for a total of 30 sites. When necessary, some randomly generated sites were substituted with nearby stands that were more accessible. Furthermore, any randomly selected sites that were discovered to be in recent fire scars were exchanged for nearby stands that contained intact, mature A. glauca.Elevation data were collected in situ using Altimeter GPS Pro and corroborated using Google Earth . Aspect was recorded in situ in degrees, then converted to radians and transformed to linear data for analysis of “southwestness” using cos according to Beers et al. . This yielded aspect values ranging from -1 to 1 , which were then used for modeling the effects of aspect on shrub dieback and Bot. infection. Sites were demarcated by >50% A. glauca cover within a stand, as determined by visual on the-ground assessments where the tops of the canopies could be viewed. Stand dieback was then visually estimated by two-to-three people as the percent of “non-green” vegetation compared to live, green vegetation within the defined boundaries of a site . Categories of NGV included yellow, brown, and black leaves, and bare/defoliated canopy, and percentages were summed to reflect total NGV within a site.

As wind turbines account for a major proportion of the wind farm initial investment, previous researches mainly focused on optimizing the number and locations of wind turbines. In [6] and [7], the genetic algorithm was proposed to iteratively optimize gridded turbine layouts on flat terrains. In [8] and [9], the greedy algorithm was introduced to search for the optimal gridded turbine layouts on hilly wind farms. The modified particle swarm optimization algorithm was applied to solve the turbine layout optimization problem in a continuous solution space. The civil works include the construction of access roads, turbine foundations, crane hard-standings, cable-trenching, and substation buildings. Their cost can be substantial. Access roads are greatly influenced by topographic characteristics, and their cost is much higher for hilly or mountainous wind farms than plain ones. A comparative strategy and the minimum spanning tree were proposed to search for the shortest road network for wind farms in flat areas. In [11], the Euclidean Steiner tree was introduced to further reduce the length of the road network. However, these methods are not directly applicable to hilly or mountainous wind farms where the road gradient must be considered to build winding roads capable of accommodating significant weight. Wind farms are generally constructed in rural areas with challenging topography. With the rapid expansion of wind farms, black plastic planting pots new farms have to be built in hills or mountains, where designing suitable access roads for construction, turbine erection and maintenance is not straight forward.

It is necessary to know roughly which route should be chosen to connect turbines and how much they will cost, while placing turbines at the peaks for high energy production. The selection of appropriate access roads is essential for the overall planning of the wind farm construction. A well-chosen road network can also reduce the wind farm construction period and lower the environmental impact. In this paper, the constraints on the maximal gradient of access roads are guaranteed when developing an automatic contour-based road-network design model. The design of access roads and the evaluation of their cost are simultaneously considered in the process of optimizing turbine layouts, which results in a more technically feasible and economically beneficial micrositing of wind farms. The remainder of this paper is organized as follows. Section II proposes an automatic model for road network design. The problem of wind farm micrositing is formulated in Section III. Section IV gives the simulation results. Finally, Section V makes some concluding remarks.In civil engineering, a feasible route between the starting and end points is selected by establishing a series of control points on the terrain. Using these control points as an initial alignment, the horizontal and vertical curves are then located, subject to the road design constraints including geometric specifications and environmental requirements. Heuristic optimization algorithms such as the genetic algorithm, the Tabu search, and the simulated annealing algorithm have been applied to optimize horizontal and vertical alignments of roads. These algorithms depend on a high-resolution digital elevation model to support the analysis of road design features such as ground slopes and other landform characteristics.

The calculation of an optimal route between two given locations is time consuming and could even take several hours. The micrositing optimization process usually involves hundreds or thousands of iterations to evaluate the wake effects between turbines and to update the turbine layout. The process without road design is already intensive. If a complex road network for the whole farm needs to be designed in each iteration by the aforementioned heuristic optimization algorithms, the computation burden is prohibitive and even impossible. In this paper, only the control points of access roads are selected during the micrositing optimization process. Such a relatively simple and functional road design is suitable for low-volume access roads, which are mainly for wind farm construction and maintenance. It is also adequate for the design at the strategic and tactical level. Based on the above assumption, a fast automatic method for road-network design is developed to estimate the cost of access roads during the optimization of wind farm micrositing.Forest engineers usually use large-scale contour maps to select preliminary routes with dividers to connect two locations, a process known as route projection. Control points of a road can be selected during the route projection process. In this paper, contour lines as a group of nested closed curves are chosen to store topographic information of wind farms. A distinct advantage of the contour-line data over a digital elevation model is that the vector representation lends itself to the object-oriented modeling of terrains, which provides a natural mechanism for sorting terrains and facilitates the search through a contour map.

Given a technically feasible gradient, a route is projected from the starting point toward the end across adjacent contour lines. This process can be efficiently automated with a mathematical model. The basic idea of the route projection process is to determine the projected route segment between two neighboring contour lines, as the elevation of the terrain rises continuously. Every projected segment of the route must begin from a point on a contour line and end on another one.Two comparative cases are studied to investigate the effectiveness of the proposed road network design model and the significance of road network optimization for farms in hills or mountains. Due to the randomness of genetic algorithms, ten independent runs are performed for each case. Table IV summarizes the results, including the best, average, and worst values of the best solutions achieved in each independent run. In Case 1, the micrositing problem is solved in a two-stage manner, i.e., we first search for an optimized turbine layout without considering access roads and then design the road network for the given layout. The best configuration of the wind farm is shown in Fig. 7, where the blue triangle represents the entrance of the wind farm, red points mean the locations of wind turbines, and black polylines are the designed road network. In Case 2, the turbine layout and its corresponding road network are optimized simultaneously, i.e., in each optimization iteration, the road network design model is performed for every individual turbine layout generated by the genetic algorithm. The best configuration of the wind farm is shown in Fig. 8. The simulation results of both cases are summarized in Table IV. Note that, the simulations for both cases are time consuming. The simulations were carried out in an IBM Blade Center HS22 with 12 Intel Xeon X5650 CPUs at a frequency 2.66 GHz. Parallel computing was employed to speed up the simulation progress. In each run of the 10 independent simulations, 12 CPUs were utilized simultaneously and each CPU was responsible for each wind direction. Even with the parallel computing Fig. 7. Best wind farm configuration of Case 1. technique, plant plastic pots the running time for each simulation is still around 7–10 h. For both turbine layouts, reasonable road networks with feasible winding roads are obtained, which demonstrates the effectiveness of the proposed road network design model. Comparing Figs. 7 and 8, it is clear that the configuration of the road network depends on the turbine layout and the farm topology considerably. Therefore, to create a cost-effective road network, it is necessary to consider these two parts simultaneously during the microsting optimization process. Indeed, since the road network is simultaneously considered in Case 2, while the cost of the road network is ignored in Case 1, Case 2 consistently has a higher net present value than Case 1 in all independent runs. In the simulations, the net present value is determined by the annual energy production and the length of the road network.Methane released into the atmosphere as a result of agricultural activity, such as enteric fermentation and anaerobic digestion, significantly contributes to overall greenhouse gas emissions in the United States . The California Air Resources Board attributes approximately 60 % of recent anthropogenic CH4 emissions in California to agriculture, with 45 % of CH4 emissions directly related to dairy farm activity for 2013 . Reduction strategies proposed by CARB seek to lower California’s CH4 emissions to 40 % below 2013 rates by 2030 , thereby emphasizing the need for accurate methods to directly quantify the contribution of different CH4 sources within agricultural operations. Estimates of CH4 emissions due to dairy livestock can be calculated using inventory emission factors combined with activity data on animal populations, animal types, and details about feed intake in a particular country . Other methods to estimate CH4 emissions from ruminants involve direct atmospheric measurements.

Emissions from dairy farms have been estimated in the Los Angeles Basin, California, using downwind airborne flux measurements . Farmscale measurements of CH4 have been made using a variety of techniques and instruments, such as open-path infrared spectrometers , tunable-infrared direct absorption spectroscopy , and column measurements employing solar absorption spectrometers with comparisons to cavity ring-down spectrometers . Several studies of various CH4 sources assert that inventory-based calculations tend to underestimate emissions compared to atmospheric observations and modeling . Atmospheric studies have often used specific gases as tracers to distinguish a sample of interest from background conditions or interferences. Tracer gases released at known rates have been employed in experiments looking at chemical transport , dispersion , source allocation , and model verification using mobile laboratories , radiosondes, sampling towers, and ground-based equipment. Application of tracer gases in agricultural studies have involved insertion of a sulfur hexafluoride permeation tube into the rumen of a cow with subsequent collection of time-integrated breath samples . Inverse-dispersion techniques have employed line-source releases of SF6 within a dairy farm combined with open-path measurements to understand whole-site emissions . Release of a tracer gas directly into the atmosphere, 2–3 m above ground level, can be used to determine and distinguish CH4 emissions from various sources within a site . This study quantifies CH4 emissions using the well-established tracer flux ratio method at two dairy farms over the course of 8 summer days . Controlled releases of tracer gas from various areas on each farm mixed with site-derived emissions were observed by an instrumented aircraft and mobile laboratory . Using this technique provided the flexibility to estimate entire dairy farm emissions and apportion emissions among sources on multiple scales. Uncertainty in measurements from low-flying airborne studies has been attributed to the need to extrapolate results below the minimum safe flight heights as regulated by the Federal Aviation Administration . Prior to this study, Aerodyne Research, Inc. performed controlled ground releases of ethane in Colorado and Arkansas, while Scientific Aviation made measurements in a similar aircraft to the one used in this study . The original release rate of C2H6 was estimated via a refined mass balance technique, with a +2 % difference observed during tests in Colorado and +24 % difference in Arkansas as described in Conley et al. . These releases did not correspond to any CH4 source but demonstrated the feasibility of using a low-flying aircraft to successfully quantify flow rates from controlled tracer gas releases. Using tracer flux ratio in this study, we again utilized the aircraft to detect emitted tracer gas and then compared with dairy farm emissions to evaluate CH4 emission rates. This field study was originally focused on estimating CH4 emissions from dairy farms and distinguishing on-site sources using established techniques . An intentional effort was made to align measurement time windows of the mobile laboratory and aircraft for the purpose of inter-comparison between the tracer flux ratio and mass balance methods. As a result, the aircraft was exposed to several hours of ground-released tracer gas. Due to this overlap in time, we were able to further assess the viability of observing enhanced concentrations of a ground-released tracer gas from an aircraft at low flow rates, compare CH4 and C2H6 enhancements emitted from within dairy farms via tracer flux ratio to determine emission rates, and directly compare the application of tracer flux ratio methodology to simultaneous ground and airborne measurements of the same air mass.In a collaborative effort, SA and ARI attempted a flight-based tracer release experiment to quantify CH4 emissions from two dairy farms in central California. This study reanalyzes data collected as part of an Environmental Defense Fund coordinated project that occurred in June 2016 . ARI employed tracer flux ratio methodology with two tracer gases and a mobile laboratory, while SA conducted a mass balance experiment from a light aircraft.

While surveys often provide a way to overcome time and budget constraints to learn about farmer knowledge, this study suggests that to achieve specificity and depth in analysis of farmer knowledge requires an interactive approach that includes – at a minimum – relationship building, multiple field visits, and in-depth, multi-hour interviews. Accessing farmer knowledge necessitates locally interactive research; this knowledge may or may not be immediately generalizable or scalable without further locally interactive assessment in other farming regions.The impact of the COVID-19 pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 was foreshadowed by earlier epidemics of new or re-emerging diseases such as SARS , influenza , Middle East Respiratory Syndrome , Ebola , and Zika affecting localized regions . These events showed that novel and well-known viral diseases alike can pose a threat to global health. In 2014, an article published in Nature Medicine stated that the Ebola outbreak should have been “a wake-up call to the research and pharmaceutical communities, and to federal governments, of the continuing need to invest resources in the study and cure of emerging infectious diseases” . Recommendations and even new regulations have been implemented to reduce the risk of zoonotic viral infections , but the extent to which these recommendations are applied and enforced on a regional and, more importantly, local level remains unclear. Furthermore, large plastic gardening pots most vaccine programs for SARS, MERS, and Zika are still awaiting the fulfillment of clinical trials, sometimes more than 5 years after their initiation, due to the lack of patients .

In light of this situation, and despite the call to action, the SARS-CoV-2 pandemic has resulted in nearly 20 million infections and more than 700,000 deaths at the time of writing based on the Johns Hopkins University Hospital global database. The economic impact of the pandemic is difficult to assess, but support programs are likely to cost more than €4 trillion in the United States and EU alone. Given the immense impact at both the personal and economic levels, this review considers how the plant-based production of recombinant proteins can contribute to a global response in such an emergency scenario. Several recent publications describe in broad terms how plant-made countermeasures against SARSCoV-2 can contribute to the global COVID-19 response . This review will focus primarily on process development, manufacturing considerations, and evolving regulations to identify gaps and research needs, as well as regulatory processes and/or infrastructure investments that can help to build a more resilient pandemic response system. We first highlight the technical capabilities of plants, such as the speed of transient expression, making them attractive as a first-line response to counter pandemics, and then we discuss the regulatory pathway for plant-made pharmaceuticals in more detail. Next, we briefly present the types of plant-derived proteins that are relevant for the prevention, treatment, or diagnosis of disease. This sets the stage for our assessment of the requirements in terms of production costs and capacity to mount a coherent response to a pandemic, given currently available infrastructure and the intellectual property landscape. We conclude by comparing plant-based expression with conventional cell culture and highlight where investments are needed to adequately respond to pandemic diseases in the future.

Due to the quickly evolving information about the pandemic, our statements are supported in some instances by data obtained from web sites . Accordingly, the scientific reliability has to be treated with caution in these cases.A major advantage of plants in this respect is the ability to test multiple product candidates and expression cassettes in parallel by the simple injection or infiltration of leaves or leaf sections with a panel of Agrobacterium tumefaciens clones carrying each variant cassette as part of the transferred DNA in a binary transformation vector . This procedure does not require sterile conditions, transfection reagents, or skilled staff, and can, therefore, be conducted in standard bio-safety level 1 laboratories all over the world. The method can produce samples of even complex proteins such as glycosylated monoclonal antibodies for analysis ~14 days after the protein sequence is available. With product accumulation in the range of 0.1–4.0 g kg−1 biomass , larger-scale quantities can be supplied after 4–8 weeks , making this approach ideal for emergency responses to sudden disease outbreaks. Potential bottlenecks include the preparation of sufficiently large candidate libraries, ideally in an automated manner as described for conventional expression systems, and the infiltration of plants with a large number of candidates. Also, leaf-based expression can result in a coefficient of variation >20% in terms of recombinant protein accumulation, which reduces the reliability of expression data . The variability issue has been addressed to some extent by a parallelized leaf-disc assay at the cost of a further reduction in sample throughput .

The reproducibility of screening was improved in 2018 by the development of plant cell pack technology, in which plant cell suspension cultures deprived of medium are used to form a plant tissue surrogate that can be infiltrated with A. tumefaciens in a 96-well microtiter plate format to produce milligram quantities of protein in an automated, high-throughput manner. The costs can be as low as €0.50 per 60-mg sample with a product accumulation of ~100 mg kg−1 and can typically result in a CV of <5% . These costs include the fermenter-based upstream production of plant cells as well as all materials and labor. The system can be integrated with the cloning of large candidate libraries, allowing a throughput of >1,000 samples per week, and protein is produced 3 days after infiltration. The translatability of cell pack data to intact plants was successfully demonstrated for three mAbs and several other proteins, including a toxin . Therefore, cell packs allow the rapid and automated screening of product candidates such as vaccines and diagnostic reagents. In addition to recombinant proteins, the technology can, in principle, also be used to produce virus-like particles based on plant viruses, which further broadens its applicability for screening and product evaluation but, to our knowledge, according results had not been published as of September 2020. In the future, plant cell packs could be combined with a recently developed method for rapid gene transfer to plant cells using carbon nanotubes . Such a combination would not be dependent on bacteria for cloning or gene transfer to plant cells , thereby reducing the overall duration of the process by an additional 2–3 days . For the rapid screening of even larger numbers of candidates, cost-efficient cell-free lysates based on plant cells have been developed and are commercially available in a ready-to-use kit format. Proteins can be synthesized in ~24 h, potentially in 384-well plates, and the yields expressed as recombinant protein mass per volume of cell lysate can reach 3 mg ml−1 . Given costs of ~€1,160  ml−1 according to the manufacturer LenioBio , this translates to ~€400 mg−1 protein, an order of magnitude less expensive than the SP6 system , which achieves 0.1 mg ml−1 at a cost of ~€360  ml−1 based on the company’s claims. Protocol duration and necessary labor are comparable between the two systems and so are the proteins used to demonstrate high expression, e.g., luciferase. However, the scalability of the plantcell lysates is currently limited to several hundred milliliters, and transferability to intact plants has yet to be demonstrated, i.e., information about how well product accumulation in lysates correlates with that in plant tissues. Such correlations can then form the basis to scale-up lysate-based production to good manufacturing practice -compliant manufacturing in plants using existing facilities. Therefore, the cell packs are currently the most appealing screening system due to their favorable balance of speed, throughput, large plastic growing pots and translatability to whole plants for large-scale production. In any pandemic, the pathogen genome has to be sequenced, made publically available, and freely disseminated in the global scientific community to accelerate therapeutic and vaccine development. Once sequence information is available, a high priority is the rapid development, synthesis, and distribution of DNA sequences coding for individual viral open reading frames. These reagents are not only important for screening subunit vaccine targets but also as enabling tools for research into the structure, function, stability, and detection of the virus .

Because many viral pathogens mutate over time, the sequencing of clinical virus samples is equally important to enable the development of countermeasures to keep pace with virus evolution . To ensure the broadest impact, the gene constructs must be codon optimized for expression in a variety of hosts ; cloned into plasmids with appropriate promoters, purification tags, and watermark sequences to identify them as synthetic and so that their origin can be verified ; and made widely available at minimal cost to researchers around the world. Not-for-profit plasmid repositories, such as Addgene and DNASU, in cooperation with global academic and industry contributors, play an important role in providing and sharing these reagents. However, the availability of codon-optimized genes for plants and the corresponding expression systems is often limited . For example, there were 41,247 mammalian, 16,560 bacterial, and 4,721 yeast expression vectors in the Addgene collection as of August 2020, but only 1,821 for plants, none of which contained SARS-CoV-2 proteins. Sharing plant-optimized SARS-CoV-2 synthetic biology resources among the academic and industry research community working on PMPs would further accelerate the response to this pandemic disease. Screening and process development can also be expedited by using modeling tools to identify relevant parameter combinations for experimental testing. For example, initial attempts have been made to establish correlations between genetic elements or protein structures and product accumulation in plants . Similarly, heuristic and model-based predictions can be used to optimize downstream processing unit operations including chromatography . Because protein accumulation often depends on multiple parameters, it is typically more challenging to model than chromatography and probably needs to rely on data-driven rather than mechanistic models. Based on results obtained for antibody production, a combination of descriptive and mechanistic models can reduce the number of experiments and thus the development time by 75% , which is a substantial gain when trying to counteract a global pandemic such as COVID-19. These models are particularly useful if combined with the high-throughput experiments described above. Techno-economic assessment computer aided design tools, based on engineering process models, can be used to design and size process equipment, solve material and energy balances, generate process flow sheets, establish scheduling, and identify process bottlenecks. TEA models have been developed and are publicly available for a variety of plant-based bio-manufacturing facilities, including whole plant and plant cell bioreactor processes for production of mAbs , antiviral lectins , therapeutics , and antimicrobial peptides . These tools are particularly useful for the development of new processes because they can indicate which areas would benefit most from focused research and development efforts to increase throughput, reduce process mass intensity, and minimize overall production costs.The rapid production of protein-based countermeasures for SARS-CoV-2 will most likely, at least initially, require bio-manufacturing processes based on transient expression rather than stable transgenic lines. Options include the transient transfection of mammalian cells , baculovirus-infected insect cell expression systems , cell-free expression systems for in vitro transcription and translation , and transient expression in plants . The longer term production of these countermeasures may rely on mammalian or plant cell lines and/or transgenic plants, in which the expression cassette has been stably integrated into. The speed of transient expression in plants allows the rapid adaptation of a product even when the process has already reached manufacturing scale. For example, decisions about the nature of the recombinant protein product can be made as little as 2 weeks before harvest because the cultivation of bacteria takes less than 7 days and the post-infiltration incubation of plants takes ~5–7 days. By using large-scale cryo-stocks of ready-to-use A. tumefaciens, the decision can be delayed until the day of infiltration and thus 5–7 days before harvesting the biomass . This flexibility is desirable in an early pandemic scenario because the latest information on improved drug properties can be channeled directly into production, for example, to produce gram quantities of protein that are required for safety assessment, pre-clinical and clinical testing, or even compassionate use if the fatality rate of a disease is high . Although infiltration is typically a discontinuous process requiring stainless-steel equipment due to the vacuum that must be applied to plants submerged in the bacterial suspension, most other steps in the production of PMPs can be designed for continuous operation, incorporating single-use equipment and thus complying with the proposed concept for bio-facilities of the future .

For Wichman and Norman et al. , the detour apparatus consisted of an arena with a starting compartment in the center, across from a holding compartment. The starting compartment consisted of three walls, two opaque and one of wire mesh, so that the holding compartment was visible through the wire mesh. Opposite the wire mesh screen was an opening in the back of the compartment allowing access to the rest of the arena. The holding compartment consisting of a screen so that its contents could be seen by the individual in the starting compartment. The test chick was placed in the central starting compartment and two of its companions from it home pen were placed in the holding compartment. To solve this task, the chicks had to walk away from the familiar chicks, out of the central staring compartment, and around this compartment to reach their companions. Wichman tested ninety 3 day old chicks on a detour test to evaluate their spatial ability. The young chicks had not yet developed perching behavior at the time they were tested. As with the radial arm experiment, all birds were raised in three different rearing environments: control, floor enrichment, and hanging enrichment. The time it took chicks to reach their companions was recorded or, if the chick was unable to successfully complete the task, square pot the trial elapsed at 10 minutes.Forty out of the ninety chicks successfully solved the detour task, however, no relationship was found between onset of perching behavior and the performance of the chicks on the detour test.

This result suggests that the detour test is not a good predictor of early perching behavior in pullets. Additionally, there was no significant effect of rearing environment complexity on the success of chicks in the detour task. Norman et al. also employed a detour task in order to assess if early access to elevated structures affects spatial working memory and route planning in laying hens. They raised chicks from one day of age in either a control or enriched treatment. The control treatment had no elevated structures and the enriched treatment had eight wooden perches arranged in an A-frame structure. In addition to the perches the enriched treatment also had a ramp leading up a platform placed on the second rung of perches. Norman et al tested 48 chicks from both rearing treatments on a detour task at either 14-15 days of age or 28-29 days of age. The test chick was placed in the starting compartment and two of its companions from their home pen were placed in the holding compartment. If the chick left the start box and began walking around the compartment, but then reentered the center of the arena, this was considered an orientation error. Chicks were given a maximum of 5 minutes to complete the task. Out of the 96 birds tested, 67 completed the detour task. There was no significant difference between treatment groups for the number of orientation errors, however the latency to complete the task was significantly shorter for the enriched as compared to the control chicks.

Morris also used a detour task to assess birds from two different rearing treatments on their spatial reasoning . For this study, the unenriched condition included shavings, a hanging feeder, and a nipple line drinker. The enriched treatment included two perches, three live plants for cover, a dust bathing box, two hanging party decorations, and hidden meal worms. At 10 weeks of age 42 birds were tested on a detour task. The target consisted of three familiar birds from their home pen. A barrier was located at the other end of the arena and was made of a mesh screen with an opening on the far-left side, across from the target. The test bird was placed in a starting location behind the barrier and was given 3 to 5 minutes to acclimate in a mesh enclosure. Once the enclosure was lifted, the birds were given 5 minutes to reach their companions. The latency of each test bird to navigate around the barrier and come within 0.5 m of the target was recorded. No significant differences between treatment groups in time taken to reach companions were found. These results contradict the finding from Norman et al. that birds with accesses to vertical structures at a young age did have a shorter latency to complete the detour task than birds without access to vertical structures. It is possible that the difference in these results could be due to the different rearing environments and detour design of these two experiments. The design of the detour task in Morris differed from the design of Norman et al. and Wichman . Instead of the chick walking away from the companion chicks to reach the goal, the Morris design required the chicks to walk towards the companions with them in view at all times through a mesh screen.

The birds were unable to take the most direct route, a diagonal path across the arena, but were not required to walk away from the target at any point to successfully solve the task. Due to this, the Morris detour task does differ from the classic detour task used by Wichman and Norman et al. . However, the inconsistent results between Wichman and Norman et al. should be taken into account when interpreting the impact of rearing environment on performance in a detour task. Due to the varying results across these studies, it is difficult to make definitive conclusions. However, the lack of relationship found by Wichman between early success on the detour task and onset of perching behavior suggests this task may not be relevant to use of vertical space.There is growing evidence in the literature that vertical complexity of the rearing environment of pullets can impact spatial abilities of domestic laying hens. However, tasks such as the hole board test, radial maze, and detour task only test spatial navigation and memory and this is done on a single geometric plane, the ground. Adult hens utilize three dimensional space and therefore their spatial cognitive abilities should also be investigated in relation to depth. While these tasks evaluate spatial memory in hens, a useful skill for finding resources in the aviary, they are not appropriate for assessing visual perception as it relates to navigating around structures in the aviary. In contrast, the jump test does use three-dimensional space and does not directly test spatial memory, making it more relevant to the use of vertical space in commercial aviaries. However, due to the increasing physical difficulty of this test, visual perception and physical skill cannot be separated. There is a great need to study the depth perception of laying hens in relation to complexity of rearing treatment by using both a floor test and a test that utilizes three dimensional space. This methodology would allow for the comparison of performance on both two-dimensional and a three-dimensional test, square plastic plant pots providing greater evidence for the translation of floor tests of spatial cognition to the use of vertical space. It is also important to investigate depth perception as this aspect of spatial cognition has not been specifically evaluated in relation to rearing environment. Adequate perception of depth seems to be vital for gauging the distance to fly or jump between perches and platforms in an aviary. Without proper depth perception, collisions and falls would be likely to occur due to misjudgment of distances.The aim of this study is to gain a better understanding of how rearing environment impacts the cognitive and spatial development of laying hens by examining the development of depth perception. Laying hens reared in three environments of differing vertical complexity were tested on a Y-maze and visual cliff task to evaluate depth perception acuity. Both tasks utilize hens’ motivation to escape after being caught and handled, by offering them the option to exit the experimental apparatuses. Previous studies have used food or social rewards in spatial cognition tasks. In this study, variance in responses due to appetite or sociality was removed by utilizing the hens’ motivation to escape. The Y-maze uses arms of varying lengths to determine if the bird can assess distance and choose the shortest path to escape. The visual cliff was used to evaluate perception of depth as well as method of crossing the visual cliff. In order to account for the disadvantages associated with the traditional visual cliff test, the plexiglass was illuminated and the table was covered with a canopy to reduce reflection.

Additionally, a perch was added in the center of the table, 15 cm above the plexiglass. This prevented tactile information from pecking the glass and the birds’ feet, further concealing the plexiglass. These tests offer greater insight into the topic of spatial cognitive development by examining a previously overlooked, yet relevant aspect of spatial cognition: depth perception.Data Collection At each of three time points a sample of 150 Dekalb White hens were tested . Due to camera malfunctions, video data from three birds were excluded from the visual cliff data set. An equal number of birds were randomly selected from each of three rearing treatments. All birds were tested on the Y-maze task the first week and the visual cliff the second week of testing. Birds were caught from their home pen just prior to testing by corralling them with a metal catch pen. All trials were recorded using a Sony Action Camera and specific behaviors were coded from the video data using Noldus Observer XT . Y-Maze The Y-maze was constructed out of 1 m high wooden boards and included a starting chamber and two arms . The starting chamber was 40 cm long and 60 cm wide, with a guillotine door. The two arms of the maze were 60 cm wide and were joined at a 50o vertex. The arms were adjustable in length so that each arm could be either 30 cm or 90 cm long. The maze was covered by soft, plastic black mesh, preventing the birds from flying out. Both arms were open at the end, allowing the bird the choice to escape into an arena via one of the arms. Chalk lines were drawn where the entrance met each arm of the maze to indicate that the bird had entered an arm of the maze. Additional lines were also drawn at 30 cm from the entrance to mark the point the bird had chosen that side of the maze. Birds were tested with two consecutive trials where the length of each arm varied so that the birds are presented with a 1:3 ratio and a 1:1 ratio .The Y-maze was randomly configured into one of three different orientations: equal length arms or unequal with either the right or left arm being shorter in length. The equal configuration had a 1:1 ratio , while the unequal configuration consisted of a 1:3 ratio . A bird was randomly selected, caught using a catch pen, and assigned an ID number. The bird was then placed into the entrance of the maze facing the vertex of the two arms of the maze. When the bird was placed, a timer was started and the guillotine door of the entrance was closed behind them. If the subject had not exited the maze after two minutes had elapsed, the guillotine door was lifted and then immediately closed to encourage movement of the bird out of the maze. Trials ended after the bird had successfully crossed one of the 30 cm lines or after 2.5 minutes had elapsed. Trials were then repeated once more with the same individual so that each bird was presented with both the equal and the unequal configuration. Time spent in each arm, exit choice, and frequency of flying within or out of maze was recorded . The exit choice was considered correct if, when presented with arms of different lengths, the bird chose the shortest path to the arena . Visual Cliff Two visual cliff tables were used. A small table was used for the 8-week-old pullets while a larger table was used for the 16 and 30-week-old birds . This allowed for the proportions of the table to remain consistent as the birds grew.

The price premium is significant for almond and alfalfa . However, organic almonds suffer from an average 20% of yield loss, which hinders the transition . For alfalfa, the price depends on the organic status as well as quality, which is hard to control for organic growers due to weed and pest pressures . The z-test results in Table 1.3 and Table 1.4 show that the coefficients of Organicare similar to or larger, in absolute value, than those in the full sample estimation, which implies that the difference between two production systems are larger in the sub-sample than full sample.Differences in environmental impacts between organic and conventional production vary across crops. The full-sample regression is estimated for selected crops individually, except for lettuce where an additional time dummy is added to split the sample in half, to highlight important patterns of pesticide use in conventional and organic production. The specifications without grower or field fixed effects provide similar results and therefore results are not presented here for individual crops. The PURE index values are plotted for conventional and organic lettuce fields in Figure 1.4. The risk index from pesticides used in conventional lettuce fields decreased since growers have gradually transitioned from organophosphates to pyrethroid and neonicotinoid insecticides over the past twenty years and organophosphate insecticides are more toxic than their pyrethroid and neonicotinoid alternatives . Prior to 2005, diazinon was the most used insecticide in conventional lettuce production while the usage of lambda-cyhalothrin , was limited in lettuce.

However, by 2015, blueberry grow bag size lambda-cyhalothrin was the most used insecticide in conventional lettuce fields while fewer than 30 acres of lettuce were treated with diazinon. Consistent with these changes, in Table 1.5, the coefficients for Organic × 06_15 are significant and positive showing that the difference in the environmental impacts from pes-ticides use between conventional and organic lettuce production decreased in the second half of the study period. In Table 1.6, differences in environmental impacts between conventional and organic strawberries are largely driven by the environmental impacts of pre-plant soil fumigation, which is used by conventional but not organic strawberry growers. Soil fumigation is a common practice for managing pathogens, nematodes, and weeds in conventional strawberry fields. While soil fumigants are most commonly regulated because of their negative effects on human health via the impact on air quality and ozone layer, most soil fumigants are also highly toxic to earthworms . Accordingly, the PUREindex for soil is large. Consequently organic strawberry production achieves a 78% reduction in the environmental impact on soil. Conventional strawberry production also poses higher impacts on surface water because several AIs are highly toxic to fish and aquatic invertebrates , including abamectin for controlling spider mites , malathion for white flies , and pyraclostrobin for gray mold . As a result, the coefficient of Organic for surface water is larger than average. The difference in the PURE index for air is smaller because azadirachtin and clarified neem oil, two primary AIs contributing to VOC emissions in the nonattainment area of Ventura , a major strawberry producing county, together accounted for 18% of treated acreage for organic strawberries.

Comparing the results in Table 1.7 with other tables in this section, organic processing tomato production reduces the environmental impact on air by a larger percentage than all organic production on average. The key difference between processing tomatoes and other crops is that processing tomatoes are more threatened by diseases than by insects or nematodes . The two most common diseases are powdery mildew and bacterial speck, which are treated with sulfur and copper hydroxide respectively in organic production . In 2015, the acreage treated with these two AIs accounted for 42% of total acreage treated for organic processing tomatoes. In comparison, the share of sulfur-and copper hydroxide-treated acreage is below 10% for production of lettuce and strawberries and 25% for all organic crops. These two AIs have lower VOC emissions than other AIs used in organic production such as pyrethrins, azadirachtin, and clarified neem oil, which together accounted for nearly 30% of treated acreage for organic lettuce and strawberries, 18% for organic processing tomatoes, and 18% for all crops. However, the impact is increasing as indicated by the positive coefficient for the variable Organic × Year. Wine grape production occurs in many regions in California, and pest and disease pressures vary across production regions due to different climate and soil conditions. In the North Coast production region, which includes Napa and Sonoma counties among others, powdery mildew is a common disease because the fungus prefers a cooler temperatures, ideally around 21◦C, to grow . Measured by treated acreage, 9 out of the 10 most used AIs are fungicides targeting powdery mildew in this area. In the San Joaquin Valley, in contrast, powdery mildew is rarely seen because of high temperatures. Due in part to the large number of frost-free days per growing season, insects are the primary concern .

For wine grapes, the most used AIs beside sulfur are abamectin targeting spider mites, imidacloprid targeting vine mealybugs, and methoxyfenozide targeting lepidoptera . These insecticide AIs aremore toxic for humans, earthworms, and honeybees and have larger VOC emissions than the fungicides used for powdery mildew , so the estimated intercept in Table 1.8 is larger in the San Joaquin Valley than in Napa and Sonoma counties and the state as a whole for groundwater, soil air, and pollinators. Powdery mildews in grapes are often treated with sulfur . In 2015, table, wine, and raisin grapes accounted for 77% of acreage treated with sulfur among all crops. To control powdery mildew, organic growers also rely on bio-ingredients such as Bacillus pumilus and Bacillus subtilis, which have larger VOC emissions than sulfur and mineral oils. Thus, organic wine grapes growers in Napa and Sonoma counties only achieve a 38% reduction in the PURE index for air while the reduction in the San Joaquin Valley is 45%.Using a consistent index, this essay quantifies the environmental impacts of pesticide use in conventional and organic fields and how they have changed over time. Information from this analysis could benefit organic crop production worldwide because California is an important production region with a diverse set of crops and environmental conditions. Previous studies rarely focused on the use of specific AIs or the change in the structure of pesticide use when evaluating the environmental impact of organic agriculture. To the best of my knowledge, the PUR database has never been used to compare pesticide use for conventional and organic production. The U.S. organic agriculture sector has grown significantly over the past two decades, after the launch of the NOP in 2002. Organic farming has the potential to continue to ex-pand in the future. Pesticides are essential for both conventional and organic crop production. However, pesticide use is not static. The pesticide portfolio changed dramatically for both farming systems in the study period. Based on field-level pesticide application information, this essay shows that the environmental impact of pesticide use on air increased in organic fields due to the adoption of new chemicals and the reduction in the use of sulfur, which has zero VOC emissions. Pesticides used in organic agriculture had lower environmental impacts per acre on surface water, groundwater, soil, air, blueberry box and pollinators depending on the pesticide portfolios for conventional and organic growers. However, the difference between two systems is decreasing over time for all five dimensions. Notably, they had almost the same level of VOC emissions in 2015. In both production systems, increases in growers’ total acreage were associated with increases in the environmental impacts of pesticide use in all dimensions. Increases in grower experience were associated with increases in the environmental impacts of pesticide use to surface water and groundwater, and decreases in the impacts on soil, air, and pollinators. The magnitude of effects of these two variables is smaller than the effect of the organic status of the field. Pesticide use in organic agriculture has evolved to have greater environmental impacts over time. This is consistent with findings in Läpple and Van Rensburg , who showed that late adopters, those who adopted organic farming after the launch of government supporting program, are more likely to be profit-driven and less likely to be environmentally concerned than early adopters. New policy instruments could alter the current situation.

When reviewing pesticide and fertilizer AIs used in organic agriculture, the NOSB could focus on environmental criteria such as VOC emissions, which has not been considered previously. Such policy instruments could partially offset the negative environmental impacts of pesticide used in organic fields. Whether organic farming is the most cost-effective way to reduce the environmental impacts of agriculture remains unclear because the changes in PURE index values does not directly translate to a one-dimensional environmental or food safety benefit that is comparable across commodities or farming methods. An alternative approach to reducing environmental impacts is to regulate pesticide use directly, which could have a significant cost. For example, the ban of methyl bromide was estimated to result in an annual revenue loss of $234 million and a 10% revenue loss for the strawberry industry in California . However, as the result shows, the PURE air index for strawberry did not decrease in conventional production after the ban. In addition, the groundwater index value increased because alternatives to methyl bromide have a greater impact on groundwater. In previous studies, demographic variables, such as gender and education, were shown to be determinants of the adoption of organic farming . Here, these characteristics are addressed by using time-invariant grower fixed effects. More information regarding the determinants of pesticide use decisions might be revealed if those characteristics data were available. Future research could focus on impacts on human health rather then the environment and cal-culate the monetary value of reduced mortality and morbidity of converting to organic production. And, estimating the value of improved environmental quality associated with organic agriculture, identified in this essay, is another research direction. While pesticide use remains important for both farming systems, another caveat is that this essay does not investigate the environmental impacts of non-chemical pest management practices, such as biological, cultural, and mechanical/physical controls. However, if one were to pursue that direction by collecting data on non-chemical practices, the analysis would necessarily be done on a relatively small scale, unlike the comprehensive data used here.Organic agriculture has been proposed as an essential part of sustainable food systems . In 2016, over 5 million acres of land were certified organic in the United States, which generated over $7.5 billion worth of agricultural products. California is the leading state as a producer of organic crops in the United States, accounting for 12% of organic cropland and 51% of crop sales value in 2016 . According to Willer and Lernoud , the United States is the largest market for organic products and accounted for 43% of global organic retail sales in 2017. Organic land use data for California have been collected for a limited number of years by two government agencies, the United States Department of Agriculture and the California Department of Food and Agriculture . Farm-level acreage and location information are not publicly available from either source. Detailed crop acreage data would facilitate further investigation of key topics such as the spatial distribution of organic fields, which previously could be studied only at a very small geographic scale using other data sources . In this context, California’s unique Pesticide Use Report database serves as an alternative source of very detailed and long-term data, which allows the identification of individual organic fields based on their historical pesticide use records. The PUR database contains information on all commercial agricultural pesticide use in California since 1990, including information on the chemicals used, crops and acreages for millions of individual applications. Pesticide use patterns for organic fields and their environmental impacts have not been studied previously. Existing studies often evaluate the environmental performance of organic agriculture as a system, rather than focusing on specific farming practices . To the best of my knowledge, no study has quantitatively described pesticide use in organic agriculture or assessed its environmental impacts for ecosystems on a large scale across numerous crops and over a long time period .