Author: ecogrow

If the Q1 and Q3 are quite tight, then the quantification results are quite reliable. If the reciprocal labeling gives similar results, such as SR45, then the quantification should be reliable, even if the total peptides from this protein are only a few. We recommend at least three to four biological experiments be done for quantification, including at least one reciprocal labeling experiment . After the quantification is done, the users can evaluate the quality of the quantification of each protein and peptide of interest. Protein Prospector provides interactive feedback during the quantification process to allow for manual validation of the quantification results or visual assessment of what went wrong in case the ratio is incorrect. For protein quantification, a tight range between Q1 and Q3 often indicates the quantification is reliable. In cases where the range is big and the protein itself is of interest, then users can use the Cosine Similarity score to determine the quality of the matches or manually check them.Once the peptide sequence is identified, the elemental composition of the peptide is generated based on the peptide sequence. The CS score, similar to the Isotope Dot Product “idotp” product used in Skyline , automatically measures the similarity between the experimentally measured isotope pattern and the calculated pattern using the M, M+1, M+2 peaks, thus reducing manual checking time by auto-flagging the contaminated peaks .

The CS score ranges between 1.0 and 0.0 and can be determined by measured peak intensity or area. Figure 6A shows the pair have both good CS scores and the L/H ratio of this peptide is close to the median number of the protein. Figure 6B shows one peptide in the pair of another peptide from the same protein gives a lower CS score ,grow bucket and thus the L/H ratio of this peptide will produce an outlier ratio. Importantly, Protein Prospector takes account of labeling efficiency when calculating the CS score, as low labeling efficiency changes the isotope pattern quite dramatically. It should be noted that the CS score will be less accurate when the peak intensity of the peptide is very low. Users can create a cache file when submitting the quantification in Search Compare. This stores the data required to regenerate the Search Compare report in a JSON file. The cache function is quite useful for various reasons: when the user needs to retrieve the data or manually check the data, there is no need to re-calculate the quantification, which can take many hours for a large dataset. With the cache file, the reports come up quickly for a few seconds rather than hours; Often it is hard to display an HTML peptide report when many proteins or peptides are quantified. The cache function can allow visualization of such reports easily. This workflow allows users to report quantification of thousands of proteins and is applicable to the quantification of the total proteomes, sub-proteomes, and immuno precipitated samples . During the extraction of elution profile of every peptide identified, Protein Prospector averages together scans over a time window but doesn’t fit the peak shape to a Gaussian function. Therefore, each identified peptide/protein will be quantified and none gets discarded due to failing the scoring threshold for fitting the Gaussian function. To get high quality data, we recommend to get 97% or above labeling efficiency to achieve higher ID rates in 15N samples, so more proteins will be reproducibly identified and quantified between different replicates. Data acquired on high resolution and high accuracy instruments will also improve the quality of the dataset. A systematic normalization is normally required before comparing results between different experiments , as the samples are rarely mixed at exactly 1:1.

One choice is to use the median number of all the quantified proteins, or median number of top one hundred abundant proteins . Alternatively, users can use housekeeping proteins that are assumed to not change for normalization. Statistical analysis of quantification data on three or more replicates is advised. Users need to determine how to apply statistical analysis on the data using a separate tool. Benjamini-Hochberg multiple hypothesis test has been used to determine significant regulated PTM peptide groups in 15N metabolic labeled samples . A standardized statistic pipeline for protein quantification is still lacking, particularly a pipeline that can leverage quantification ratios of each peptide from a protein. Our current workflow uses a median value, which takes advantage of the quantification ratios of each peptide but is less affected by outliers than using the mean. However, the statistical power utilizing quantifications from these multiple peptides from single protein has not been explored and awaits development in the future. A targeted quantification strategy is recommended for further analysis of proteins of interest because this provides more accurate quantification and is less likely to have missing values, particularly in the 15N labeled samples . In addition to targeted analysis, data-independent acquisition can also be utilized, which can be done in label free samples or combined with 15N metabolic labeling in the future. DIA benefits from having few missing values, but more efforts will be needed to deconvolve the mixed MS2 spectra in DIA datasets. 15N metabolic labeling has been utilized in studies of analyzing protein synthesis and degradation . These studies are based on incomplete and often low incorporation rates which result in very broad satellite peak distributions and cause 15N labeled peptide isotope clusters to overlap with 14N labeled peptide clusters. As Protein Prospector doesn’t deconvolve the 15N distribution from the 14N distributions , the presented workflow will not provide accurate quantification in this type of study. On the other hand, for chase studies that analyze the assembly kinetics in vitro , the presented workflow can be applied because the proteins involved have a very high labeling efficiency.

This workflow can be also applied to the quantification of post-translational modification with a slight modification. The users will include related PTM search parameters into data search. Instead of reporting median number at protein level, ratios from each peptide are reported and then compared across different replicates. Trait analysis, especially genome-wide trait analysis, is centered on how genetic variation gives rise to phenotypic variation. This type of analysis relies on statistical methods and tools to perform association mapping between causal genetic variants and resulting phenotypes, which can determine the heritability of a trait at a subset of genetic variants and delineate regions of the genome that control the trait, thereby providing markers that can be utilized to accelerate breeding by marker-assisted selection. Because of the great success of genome-wide association studies , hundreds of SNPs conferring genetic variation of complex traits have been identified and reported. However, the genetic structures of most traits remain unexplained, as associated SNPs detected from GWAS explain only a small fraction of heritability and a much smaller percentage of the total phenotypic variance. This is mainly because a number of these studies employed only additive models that fail to account for epistasis,blueberries container or the interaction between multiple loci and the environment. Xu et al. proposed a new linear mixed model for mapping quantitative loci by incorporating multiple polygenic covariance structures. Based on this model, a pipeline for estimating epistatic effects was developed to com prehensively estimate additive effects, dominance effects, and interaction effects between multiple genetic loci. PEPIS allows analysis of genome-wide genetic architectures, including genotype interaction effects , and can thereby explain more than 80% of phenotypic variance. Compared with standard GWAS tools that consider only additive effects, the PEPIS pipeline is equipped with a more complex polygenic linear model that can explain more phenotypic variance. However, neither of these methods can explain nearly 100% of phenotypic variance, as neither considers the interaction between genotypes and environments . Today, the predominant thinking in biology is that the orchestrated expression of many genes in different environmental conditions affects the transcriptome, proteome, and metabolome to produce a final observable phenotype. Recent work in Saccharomyces cerevisiae suggests that GxE can occur at the individual locus level and the group level for multiple loci, leading to environment dependent epistatic interactions. Although Muir et al.conceptualized the partitioning of GxE into two possible inter action types, our mathematical understanding of the genetic and molecular mechanisms by which GxE collectively gives rise to phenotypes is still incomplete. The central dogma of biology is that the genome, tran scriptome, proteome, and metabolome are cascading and connected to the end phenome. The development of life science technologies enables transcriptomic, proteomic, and metabolomic events to be analyzed in detail within the same biological system, allowing the systematic study of a complete biological system.

Out of all the omic data from the same biological system, genomic data generally remain constant across environments, although the same genotype subjected to different environments can produce a wide range of phenotypes by triggering the expressions of different genes, downstream enzymes, and metabolites. Most current association methods and analysis tools perform associa tion mapping based on fundamental relationships between DNA sequence variation and phenotypic variation without addressing environmental variation. GxE can be understood by observing and measuring the expression of genes or metabolites. Harper et al.developed an associative transcriptomic approach to study complex traits in the polyploidy crop species Brassica napus by correlating trait variation with the quantitative expression of genes and sequence variation of transcripts, with the consistent physical positions of the two kinds of associative markers allowing the identification of high-confidence transcription factor candidates. However, their method is based on a pure additive model only, and they make no mention of interaction effects between biomarkers or their contribution to phenotypic variation. To overcome the limitation of standard GWAS that fails to consider the GxG and GxE effects, we extend associative geno mics and transcriptomics into a broader associative omics by systematically integrating all available omic data into one analytical model. Here we propose a new LMM and describe the development of a pipeline for analyzing traits through ome-wide association studies to implement the model. The proposed LMM considers not only the additive effects of each biological marker but also the interaction effect of each marker pair. The marker pairs’ interaction effect introduced here corresponds to two-dimensional association mapping, which is complementary to one-dimensional association mapping in regular GWAS. Consequently, the proposed model and PATO WAS pipeline are not limited to GWAS for genotype-to phenotype mapping ; instead, they are capable of per forming multiple types of ome-wide association studies, such as transcriptome-wide association studies for transcript-to phenotype mapping and metabolome-wide association studies for metabolite-to-phenotype mapping . We submit a rice recombinant inbred line dataset with three omics markers and two agronomic traits to PATOWAS for comprehensive analyses of associative omics. The results demonstrate that our proposed LMM and the pipeline PATO WAS can effectively address the GxG effect and the GxE effect, perform multiple-level associative omics in one platform, and innovatively provide a systems biology view into the traits analyzed.We aimed to systematically integrate multiple associative omic results to provide more biological insights into the phenotypic traits to be analyzed. We first collected a dataset of 210 rice RILs geno typed with 1619 marker bins, profiled with 22,584 transcripts and 1000 metabolites, and phenotyped with two agronomic traits . The phenotypic traits were yield and thousand grain weight , and the omic quantitative markers were bin based genotype data, Affymetrix RNA microarray-based gene expression data, and mass spectrometry-based profiling of metabolite abundance data. We presumed that expressed tran scripts, proteins, and metabolites are prone to vary when sub jected to the environments, while the genetic variants are considerably stable. Therefore, compared with genome-wide genotypic data, we further presumed that measured gene expression and metabolite abundance contain both gene and environment information and expect that associative transcriptomics or metabolomics could explain more phenotypic variance . Motivated by our consideration of genetic epistasis and our desire to explain more phenotypic variance, we next proposed astatistical LMM that considers not only the additive effects of the two components, the lower the residual component and the each marker variant but also the interaction effects of each more phenotypic variance can be explained by the model.

Crucially, fast initial fern growth in the medium-textured soil, likely due to higher nutrient content and/or lower arsenic phytoavailability, led to a decrease in effluent flow and therefore arsenic leaching, as transpiration exceeded water application. In this soil, the fern required less energy to acquire nutrients, released less arsenic from soil via nutrient scavenging, and produced greater biomass such that arsenic concentrations in biomass were lower. The increase in mass of arsenic accumulated from 11 to 21 weeks, coupled with lower final biomass of mature and young compared to senescent fronds, shows that arsenic accumulation continued even as growth slowed. This arsenic accumulation could be due to increased nutrient scavenging associated with drought stress.The increase in arsenic concentrations in leachate in the presence of ferns growing in the medium-textured soil reveals the importance of rhizosphere processes to arsenic release for uptake and leaching. We found that arsenic depletion from the medium-textured soil was the greatest in surface soil where P. vittata roots are primarily located . Moreover, in both soils we calculated that arsenic concentrations in rhizosphere pore water must be greater than those in bulk soil pore water,square planter pot because assuming arsenic concentrations to be the same in rhizosphere and bulk pore water indicated a discrepancy between arsenic intake through transpiration flux, and fern arse nic content. Processes other than mass flow of soluble arsenic from bulk soil to roots must be important for arsenic uptake .

If nutrients availability in soil is lower than P. vittata demand, P. vittata could employ nutrient-scavenging processes that release iron, phosphorus, and therefore arsenic from soil into pore water in the rhizosphere , increasing pore water arsenic concentrations locally to potentially very high concentrations. We suggest that the majority of the arsenic taken up into P. vittata was mobilized directly in the rhizosphere, similarly to others who found greater desorption of cadmium in cadmium hyper accumulator rhizospheric compared to bulk soils . We hypothesize that higher diffusivity due to greater connected pore space in the medium-textured soil could lead to transport of the arsenic re leased in the rhizosphere to the bulk soil, where it is then available for leaching. Similarly, rhizosphere DOC could be transported to the bulk soil and promote release of arsenic. However, in the coarse-textured soil characterized by lower porosity, larger pores, lower saturated fraction, and pre dominantly advective flow, arsenic and DOC released in the rhizosphere did not contribute to bulk leachate arsenic concentrations and, conversely, arsenic in the bulk pore water was not as accessible to the plants.We suggest rhizosphere arsenic mobilization is a byproduct of nutrient scavenging processes, particularly iron-scavenging in the medium-textured soil, where we found higher iron concentrations in ferns and in root zone pore water. Specifically, arsenic release from soil could be coupled to phosphorus and iron release from soil iron oxide minerals . Release processes could include ion exchange, ligand-enhanced dissolution, and reductive dissolution , likely tied to release of root exudates from P. vittata roots . We found primarily oxidized arsenic in our well-drained rhizosphere soil, suggesting processes including ion exchange and ligand-enhanced dissolution, likely coupled to rhizosphere DOC, are more important than reductive dissolution, similarly to in the Pine rhizosphere . Alternately, the predominance of oxidized species could indicate P. vittata preferentially took up reduced species, leaving oxidized species behind. We found evidence of reductive processes in the rhizosphere, with up to 41% of the arsenic present as arsenic in rhizosphere soil, up to 100% of the arsenic present as arsenic on and within roots, and iron phases in rhizosphere soil, suggesting reduced arsenic and iron could play a secondary role in arsenic release and uptake.

A high fraction of surficial arsenic could indicate transport of arsenic toward the root and accumulation in the rhizoplane, with slower uptake of arsenic enriching arsenic relative to arsenic on the root surface. The presence of arsenic on the root surface could also indicate efflux of arsenic from roots, which has been proposed to be a secondary tolerance mechanism in P. vittata and other plants under arsenic stress . In bulk pore water, bulk soils, and soil aggregates, the predominance of arsenic indicates arsenic can leach under oxic conditions. Arsenic availability for leaching, whether due to soil characteristics or influence of plant growth, is not dependent on reducing conditions. Indeed, arsenic mobility in soil increases at the circumneutral to alkaline pore water pH we observed . Arsenic mobilized as arsenic could be oxidized, perhaps coupled to reduction of the moderately-available soil manganese. Leaching of root derived dissolved organic carbon could also increase arsenic release from bulk soil for leaching.Rhizosphere nutrient acquisition processes have a specific significance in the case of hyper accumulators. Infertile soils could characterize the hyper accumulator ecological niche , such that P. vittata employs scavenging techniques and associates with indigenous AMF to acquire necessary phosphorus and other nutrients. We found Glomus spp. including F. mosseae were present across all treatments whether due to colonization by indigenous mycorrhiza or due to inoculation. In the very low nutrient coarse-textured soil, we hypothesize that extensive use of these scavenging processes cost metabolic energy, locally in creased already high arsenic availability, led to high uptake of arsenic and consequently even more energy expenditure to sequester this arsenic, and ultimately resulted in low biomass containing arsenic at high concentrations. The lack of effect of supplemental phosphorus in the coarse textured soil suggests it is a balance of phosphorus and other nutrients which are required to meet P. vittata nutritional needs. In contrast, in the medium-textured soil, we hypothesize the ferns used less energy to acquire nutrients.

Iron scavenging here was successful, apparently meeting fern nutrient needs and therefore limiting “byproduct” arse nicreleased from soil. Hence, P. vittata growing in the medium-textured soil experienced lower metabolic costs and consequently higher biomass until drought stress limited biomass production. In keeping with evolution under phosphorus starvation conditions , our results suggest P. vittata is less tolerant to extractable phosphorus concentrations greater than that of the medium-textured soil . Fronds of P. vittata growing in its native habitat in China were only 0.08% phosphorus, and ferns including P. vittata had the lowest phosphorus content of any flora group in China . We found phosphorus application delayed fern growth in both medium- and coarse-textured soils,hydroponic nft channel as has been shown for tropical forest ferns , leading to smaller senescent fronds containing lower amounts of arsenic.Our findings suggest that P. vittata is a good choice for remediation at the mesoscale, because arsenic uptake in ferns exceeded cumulative loss by leaching by an order of magnitude, and transpiration limited leaching compared to the absence of ferns. Decreased effluent volumes and cumulative arsenic leaching in both soils in the presence of ferns confirms the critical role transpiration plays in limiting water percolation and leaching of soluble, plant available constituents . The leaching to uptake ratio measured in this mesocosm system is not directly scalable to field conditions. We demonstrate that arsenic leaching during phytoextraction depends on soil characteristics, fern growth, and water input/evapotranspiration ratios, and therefore must be measured at the field scale. The constant water application required in our column study design could have increased leaching of arsenic relative to field applications. On the other hand, our experimental design could have limited plant growth and therefore nutrient scavenging activities, which we showed can increase arsenic release from soil. Larger biomass under field conditions could increase the influence of the nutrient scavenging geo chemical processes observed here and lead to increases in arsenic mobilization for both uptake and potential leaching, explaining the excess loss of arsenic from soil observed under field conditions . Counter intuitively, because we showed that P. vittata continued to phytoextract arsenic under drought conditions from the medium-textured soil to effectively limit arsenic leaching, phytoextraction could be best suited for dry soils with lower arsenic availability. Here, even though frond arsenic concentrations were an order of magnitude greater in coarse-textured soil ferns, mass of accumulated arsenic in coarse-textured soil ferns was only 1.2 to 2.4 times that of medium-textured soil ferns, while leached arsenic was also greater in coarse-textured soil, due to the lower biomass and lower transpiration. Alternatively, phytostabilization with species with high transpiration rates but lower iron demand could limit biotic and abiotic arsenic leaching.Such substrates typically have an inorganic and organic component . The organic component provides high porosity, low bulk density, and nutrient retention , which makes Sphagnum peat moss a strongly suitable option with widespread use .

However, increasing expense and competing uses for peat , impacts of its harvest on wetland ecosystems , including loss of peat bogs as a key global C sink , and its perception as unsustainable have spurred recent investigations of substitutes for peat in soil-free substrates, including biomass waste products such as compost and sawdust . Biochar has been recently proposed as a strong candidate to substitute for peat because of its high porosity, low density and high cation-exchange capacity. Biochar is a carbon -rich material produced by pyrolysis of biomass and has been a major subject of study as a soil amendment in the last decade . In addition to providing high nutrient and water retention, replacing peat with BC could offset or reverse the C footprint of soil-free substrates into a net C sink . Evidence to-date suggests neutral or positive effects of BC use in substrates on nutrient availability and plant growth , though many studies examine additions of BC to peat-based substrates, rather than replacing a substrate component such as peat .Evaluating effects of high BC substitution rates on substrate properties and plant growth is necessary to understand the extent to which BC can replace peat. At low amendment or substitution rates, BC has been found to maintain or improve plant growth as a result of increased nutrient availability , reduced nutrient and water loss , and amelioration of peat acidity , though these effects may be BC-specific due to feed stock and pyrolysis influences on BC properties . However, at high substitution rates, substrate properties conducive to plant growth may be compromised. In particular, the high pH of many BCs could result in BC-substituted substrates with pH values unfavorable to plant growth. For example, pelleted wood BC substitution for peat required adjustment of pH due to the liming effect of the BC . The neutral to alkaline pH of BCs and their liming potential means that BC substitution for peat can increase pH beyond optimum for plant growth in potting media . Explicit eva luation of BC effects on substrate pH and plant performance provides a basis to improve design of BC-based substrates and inform trade-offs in this application of BC . The objective of this study was to determine the effects of BC substitution for peat and substrate pH on greenhouse production, using marigold as a model crop. In the United States, the wholesale value of marigolds plants was 30.3 million USD in 2015 . Softwood BC was substituted for peat in a typical 70:30 peat:perlite mixture at 10%v increments. Since many BCs are alkaline and will increase pH of substrates in proportion to the degree of substitution, the effect of adjusting pH of substrates to typical soil free substrate values was also evaluated. Marigold germination and growth were measured over 9 weeks. We hypothesized that under greenhouse conditions , marigold germination and growth would be more sensitive to BC substitution at higher rates and that this would be due to elevated substrate pH. Additionally, we hypothesized that pH adjustment of BC substrates would increase the extent to which this softwood BC could be substituted for peat without compromising plant growth.Marigold var. ‘Crackerjack’ seeds were sown directly in 0.7 L of substrate pre-fertigated to 100% WHC using 0.5% Hoagland solution in 1.2 L polypropylene pots in a greenhouse at the UC Davis Plant Growth Facility. Pots were arranged 18 cm apart in a completely randomized block design with four replicates per substrate-pH treatment . Pots were drip fertigated with 0.5% Hoagland solution at 66 mL d−1 for weeks 1–6 and 99 mL d−1 for weeks 7–9.

For many important horticultural crops, ex ports constitute a large share of output, so FTO under IP must include freedom in foreign markets. Since the various IP rights important for plants are administered nationally, an exporter must check FTO separately in each foreign market. In general, the tools of biotechnology are more likely to be patented in just the major markets — such as the United States, Europe and Japan — and less likely to be patented in countries with smaller markets. Uses of bio-technologies specifically for minor crops are less likely to be widely patented in multiple countries than are uses in important field crops. However, as a result of the International Union for the Protection of New Varieties of Plants agreement first established in 1961, PVP systems are widely available overseas for the protection of clonally propagated varieties, and such varieties do tend to be widely registered in multiple countries. Still, not all types of bio technologies, genes or plant germplasm can be protected in all countries. For example, utility patenting of plants is allowed in only a few countries . Beyond these trends, however, there are no hard-and-fast rules as to which technology will be protected in which country, as each inventor decides where to seek protection . As a result, those seeking FTO are confronted by an often bewildering international patchwork of IP rights, where the negotiations needed for a particular transgenic variety can differ significantly each time it crosses a national border.Unless a new transgenic variety is developed by an integrated effort at a large company backed by a broad IP portfolio, a number of different owners — including companies, individuals,ebb and flow trays universities and even governments — will have valid IP claims over the technologies and genetic contents that end up being included in it.

That means there are numerous owners to track down, negotiations to conduct, billable legal time to hire, and multiple royalty payments to administer. The costs and headaches involved in working out “who owns what” and “who owes what to whom” can balloon into what economists call the “tragedy of the anti commons” and render the development process unfeasible.Given the smaller markets involved, there is less incentive in industry to consolidate IP portfolios around horticultural crops. Also, not one of the public-sector organizations or their typically smaller commercial partners in horticultural crop development has a complete IP portfolio in plant biotechnology.When technologies are patented, it is often not clear who currently owns particular aspects of each technology. This uncertainty is cleared up in the courts through patent interference cases, where attorneys and scientists under take intensive “surveying” of the “property lines” between the patents and technologies in question. Some times these cases drag on for years, keeping key technologies in legal limbo and the R&D community guessing as to who is the rightful owner. Yet, for most registered patents there is no such scrutiny. As a result, the boundaries for a considerable expanse of technological territory are not clearly demarcated, creating considerable uncertainty as to when a new application could be considered to be infringing or “trespassing.” In horticultural crops, the lack of clarity about the scope and validity of patent claims is especially important. Because the markets are smaller, fewer products have been developed and fewer contests have been fought to establish legal precedents. Furthermore, just the anticipation of possible legal costs can shut a project down before it ever gets off the ground.IP covering a crop variety may be sold, licensed or transferred to another organization at any time. The transfer of rights can occur either in part or in whole .

The transfer can happen in just one territory where it is protected or in multiple territories. The transfer of rights for a biotechnology tool or gene could be specified for use in just one crop , in several crops , or in any and all crops. Finally, to make matters worse, the fact that the IP rights have been transferred may be considered commercially sensitive information and not be made public.Any organization managing the release of a new crop variety faces uncertainty about which IP rights actually cover what technologies, who holds those rights in which countries, and to what degree a specific new transgenic variety infringes on those rights. Resolution of such uncertainty is not less costly for crops with smaller market values. Even after reliable information is obtained, uncertainty remains about negotiating the permissions. IP owners are not required to negotiate licenses, and they may feel there is not enough potential revenue in minor crops to make their licensing efforts worthwhile. They may also be concerned about technology stewardship, given the nervousness among consumers about food biotechnology and its status as a hot media topic. They may worry that the mishandling of their technology by a small and relatively inexperienced horticultural player could lead to stronger regulations, potentially eroding that technology’s value in its major crops, or jeopardize public perceptions about biotechnology overall.In response to IP congestion and continuing uncertainties, several leading U.S. public-sector agricultural re search organizations have come together to create the Public Intellectual Property Resource for Agriculture , an organization providing col laborative IP management solutions to public-sector and smaller private-sector players in horticulture . While individual universities and even the USDA have small and uncoordinated IP port folios in plant genetics, together they hold a fairly comprehensive set of technologies that could be useful for developing transgenic varieties .

PIPRA seeks to coordinate the disparate portfolios of its member organizations to support specialty crop applications. With the offices of technology transfer of its member organizations, PIPRA is pursuing several cooperative strategies.First, PIPRA seeks to develop and adopt more precisely focused terms of licensing, with specific distinctions for the “fields of use” to which a technology is licensed. A company that licenses a technology in vented at a university can still get the full benefit of using the technology in those major row crops in their line of business,grow strawberries container even if the license clearly defines and grants exclusive use of the technology in just those crops. Such a license effectively “reserves” the rights to use the technology in any other crops. Horticultural firms could then make separate agreements with the university to use the technology in only their defined specialty crops. An ad vantage of this strategy is that it can also apply to other minor uses, including “alternative” crops and humanitarian applications in staple crops for developing countries . By discriminating be tween big markets and multiple smaller markets — including those with limited commercial value but important social benefits — public-sector scientists could see their inventions earn royalties in the big markets of ma jor row crops while still helping to improve smaller crops or increase food security in world’s poorest regions.A database will, for the first time, list in one place current information about all of the patents of PIPRA’s members and their availability for licensing alongside information about technologies published in the scientific literature , in sufficient detail to identify which technologies can be accessed for which uses. The database will offer a clear, complete and certain “universal listing” of technologies available from PIPRA’s member organizations and the public domain. Commercial patent databases and professional legal staff are available to researchers in large private companies for searching through the “prior art” to make FTO analyses of a new product’s IP position. Such resources are seldom available to academic and government researchers.

The PIPRA database will decrease uncertainty about what cannot be used by showing what can be used.PIPRA is investigating the creation of patent-pooling mechanisms, which would collect IP submitted from its member organizations, package the technologies together and offer unified licenses for the “bundled” IP in a field of use, such as a specific crop, or in a particular state or country. This process mimics, in a virtual way, how large commercial firms have assembled their IP portfolios to provide FTO in major field crops. Its feasibility will depend — at least at the outset — on the extent to which public sector organizations are able and willing to provide access to patents covering key enabling biotechnology tools already licensed to the corporate sector. Even if used to access technologies on just a patent-by-patent basis, coordinated information and streamlined access to academic and government owned IP could help decrease trans action costs and improve efficiency in technology-transfer markets. There is ample room for improvement here, as some have complained that negotiating licenses from universities and government agencies is often less efficient than negotiating licenses from firms. PIPRA can improve public-sector technology transfer for agriculture by providing information, tools and precedents for efficient licensing. Greater opportunities lie in the The costs and headaches involved in working out “who owns what” and “who owes what to whom” can balloon into what economists call the “tragedy of the anti-commons” and render the development process unfeasible. steps being taken to coordinate access, package IP bundles and target uses in lower-value markets such as horticultural crops and traits important for food security in developing countries. These are, generally speaking, areas that commercial firms are not interested or capable of serving. Such collaboration is not surprising, given the history and ethos of cooperation among agricultural experiment stations within the land-grant system. Public-sector institutions also have greater legal flexibility to enter into collective IP management arrangements, given historical antitrust concerns about abuses of patent-coordination efforts in industry. Even more important will be the establishment of ongoing precedents and mechanisms for the treatment of future IP. Academic and government re searchers will go on making important discoveries and inventing new technologies for agriculture. Those future inventions will, from their inception, be handled in ways — such as being listed in the universal database, licensed for targeted “fields of use” and included in IP pools — that will make them accessible in a carefully proscribed manner, not just to top commercial bidders, but to anyone else in the broader agricultural community who can make good use of the technology, including horticultural researchers and growers.Current practices in patenting and intellectual property protection have created barriers to the use of biotechnology and advanced agricultural technologies for the creation and commercialization of new crop varieties. The complex and cumulative nature of biological innovation requires access to multiple technologies that are often exclusively owned or licensed. For example, commercializing a single variety of transgenic tomato could involve obtaining the rights to use a variety of technologies and genes from numerous life-sciences companies, government agencies and universities. Obtaining “freedom to operate” for transgenic crop varieties is difficult. There is considerable uncertainty as to who holds what rights to particular technologies, and negotiating access to those rights is time-consuming and costly. This is a problem for the major international agricultural companies that focus primarily on high-volume crops such as corn, soybeans and cot ton; for research institutions that work on specialty crops grown on much smaller acreages, such as tomatoes, strawberries, apples and cabbage; and for public institutions that work on staple crops for humanitarian use in developing countries. The international agricultural companies have taken steps to solve their FTO problems through mergers and cross-licensing agreements that bring together essential IP components within one company. However, public-sector institutions — such as universities, government agencies, international agricultural research centers and others working on specialty and staple crops — are still struggling to find ways to gain FTO. In addition, donor agencies such as the Rockefeller and McKnight Foundations, which have a long history of investing in agricultural research that benefits subsistence farmers in developing countries, have also found that IP constraints are reducing the flow of technology.Universities and other nonprofit institutions have generated many key patents related to agricultural bio technology and they will most likely remain an important source of innovation. However, no single institution has the complete package of technologies required for commercialization of a biotech variety. Although some institutions are developing ways to deal with these problems, there are still many examples of public-sector inventions that have been licensed exclusively to private-sector partners. In late 2002, representatives of more than a dozen U.S. public-sector agricultural re search institutions joined with the U.S. Department of Agriculture and the Rockefeller and McKnight Foundations to discuss access to patented agricultural technologies for the development and distribution of improved specialty crops and those targeted for the developing world.

A stainless steel hand auger with a Teflon® coated-core sampler was used to collect the soil samples. To minimize cross contamination, a polyethylene core liner was utilized. Soil samples were obtained from the topsoil and composited. The forage soil samples were obtained by utilizing a topographic soil zone sampling pattern using a random zig-zag pattern. Soil samples were weighed at 100 g. Physicochemical properties such as temperature, pH, Munsell color, depth, and moisture were obtained. The sheep tissue samples were paired with the forage, soil, and water samples. The mean age of adult sheep harvesters was 58.67 ± 2.89 years; two of three participants were male. All sheep parts were consumed by the participants for a mean of 52.33 ± 10.78 years. The sheep harvesters reported that 35% of their overall meat intake came from sheep they raised and harvested locally. On average, all the participants reported consuming locally raised sheep once a week. The local harvesters reported other important non-food uses for sheep. All participants reported selling wool, and two reported using the locally harvested wool to create textiles to sell for income. One of three harvesters reported selling live sheep to market, and two reported selling sheep or lamb cuts to market. All the participants reported sharing sheep meat for free with others. On average, each sheep harvester distributed free meat to two households. Multiple sheep parts were reportedly used for various ceremonial or cultural purposes by all harvesters.The existing literature reports HM levels in kidney tissue,ebb flow table but typically there is no comparison between the kidney medulla and the renal cortex. In this study, the kidney medulla rather than the kidney cortex showed an increased uptake of U, Se, Mo, and As.

The renal proximal tubule epithelia arechemically damaged by high acute levels or prolonged low doses of U; the proximal tubules are housed in the renal cortex. The administration of toxic doses of Se demonstrated histopathological changes in the proximal tubules of the sheep. The kidneys maintain Se homeostasis. Renal compromise may cause dysregulation of Se. Our study indicates there may be a difference between HM accumulation in the medulla and the cortex. The renal toxic effects of U and Cd are well supported in the literature. The effects of associated heavy metals on the sheep kidney need further exploration. Meat protein is richer in Se than plants. The literature supports that Se commonly concentrates in the liver and kidneys of animals. Of all sheep organs, elevated Se levels were found in the liver and kidney medulla . In a lamb tissue study, it was reported that Se concentrations in the kidneys were seven to 44 times higher than in other tissue and organs. Similarly, in our study, the medullary levels contained the higher concentrations of Se . The above lamb study reported that leg muscle contained the lowest Se concentrations of the tissues sampled. We also found that leg muscle contained the lowest Se concentrations in our examination . There is a narrow margin between Se requirement and toxicity. Therefore, taking an accurate measure of food intake containing Se, particularly meat protein, is important. Food processing such as cooking via baking, boiling, and grilling may alter the amount of Se in food. Whether food processing has an additive or minimizing effect on Se concentrations in food is to be determined by research. Adjusting food intake and cooking habits based on various HM measurements and bio-availability may be a plausible intervention once it is informed by research. Elevated levels of Se and Pb were found in sheep wool in the current study. Further, though Th was negligible in all other sheep tissue, it was detected in sheep wool .

This finding may indicate that Th may be accumulating across time in sheep wool. Direct dirt and dust aerosol capture and the effects of lanolin may be contributing exposure factors. We did not measure the effects of lanolin in this study. The current study community relies on wool to create textiles. It is common practice to place local plants in hot water to pigment the wool. The wool is handled often by weavers once the wool is removed from the animal, hand-carding the wool, hand-spinning, dyeing, and weaving the textile. The entire process often takes weeks to months, suggesting a potential lengthy human exposure to heavy metals. A considerable amount of time is spent outdoors for such activities, and exposure to various sources of contaminants such as soil, water, and air is a concern. Although this study of three sheep provided interesting insight, future studies should focus on determining the speciation of heavy metals and evaluate which metals have a greater affinity to wool. The majority of the time for this study, heavy metals were found in the greatest amounts in soil > forage roots > above-ground forage parts, respectively. The current study mean Se soil concentrations were equivalent or exceeded the exposed soil and were greater than the control concentrations reported by Dreesen and Cokal. The above-ground forage parts contained the least amount of heavy metals, except for Mo and Cd. The bio-accumulation ratio can partially demonstrate the ability of particular plants to absorb soil heavy metals and transport them to the above ground portions of a plant. The data shows that the uptake of Cd, Mo, and Se by most plants sampled were high under current soil conditions. The highest BF ratios were seen in most forage for Mo , Cd , and Se , which needs further exploration. The high BF ratios seen may indicate a low tolerance of various plants to high concentrations of Mo and Cd. In particular, B. gracilis the most abundant plant, was associated with elevated BF ratios for Cd, Mo, and Se.

Generally, in the biota samples there were greater heavy metal concentrations in the plant roots than the above-ground portions, which is consistent with several other plant studies that found that U translocates in greater amounts to the roots than the shoots. Similar to the current study, Soudek et al. reported that U was more localized in the root system. Uranium accumulation was less in grasses than root crops and Brassica spp.. Uranium uptake was found to be 3.9 or 4.5higher in the presence of phosphate deficiency. The micro and macro-nutrients available in soil affected the uptake of Cd in one source. Geochemical characteristics have an important influence on HM plant uptake. Future investigations can focus on the interactions between trace elements or other factors that may demonstrate an influence on HM plant uptake. Forage and water intake are important considerations in livestock,air pruning pots and soil ingestion must also be considered. In the current examination, the soil samples showed greater HM concentration than sheep tissue and forage samples. It has been demonstrated that sheep, a ground feeding animal, eat one to two percent of soil when good forage is available and about 18% when low quality forage is available. It has been shown that sheep intake and digestibility of more mature plant material decreases with advancing maturity due to greater effort and time in chewing by the animal; sheep selectively graze in high-quality forage areas when they are available. The forage environment of the sheep in the current study area exhibited high stocking rates, sparse vegetation, and mature forage samples, which may have contributed to higher HM concentrations in forage. Dung analysis can evaluate the amount of inorganic material in sheep diets and may be useful in future studies in the current study area. Previous studies have reported the concentrations of HMs in sheep tissues, plants, and soil in the target study area . In most categories, our study results were comparable to or less than what was previously found.

The current study tissue measurements were below the exposure and control concentrations reported by Millard et al. and Ruttenber et al. in the 1980s. No excess cancer risk was calculated to be attributed to eating sheep meat, liver, kidney, and soup bone by humans; researchers recommended continued monitoring at that time. The highest HM metal concentration in the diet was used for each animal to calculate and compare to the maximum tolerable concentrations, and the lowest concentration for each HM was used to compare to the requirements for Mo and Se. The calculations are based only on the forage samples collected and are not representative of the complete sheep intake. Maximum tolerable concentrations are established for sheep intake for As Cd, Pb, Mo, and Se. All study animals did not exceed the calculated maximum tolerable concentrations for As, Cd, Mo, and Pb. All study sheep met the Mo and Se dietary requirements. Liver is the organ of choice to diagnose Se deficiency, and concentrations less than 0.21 mg/kg in sheep liver are considered deficient. All study sheepliver concentrations did not indicate deficiency. In sheep, Se toxicity was reported at 0.25 mg/kg of body weight chronically. However, the National Research Council set the maximum tolerable concentrations for Se at 5.0 mg/kg of DM. Of the donated sheep, the shepherds did not report indicators of acute or chronic Se poisoning . Other supplementary sources of forage were not reported at the time of sampling; sheep harvesters reported relying on alternative fodder sources for their sheep in the late winter months only . New Mexico is one state that was reported to have high Se concentrations in soils and those in areas with low annual rainfall or alkaline soil. The mean study soil pH was 7.31 ± 0.51. Primarily, most of the Se is absorbed in the small intestines of ruminants and less absorption is seen in forage based diets versus diets based on concentrate. Plants that accumulate Se may be unpalatable to grazing animals, but if there is lack of more palatable forage, animals may develop signs of toxicity from ingestion. According to one study calculation, selenosis can occur in lambs ingesting 0.2% BW of Se accumulating plants. Soil ingestion during foraging, seasonal soil forage adhesion , pulling up of roots while foraging, and licking snouts by livestock may also contribute to higher HM concentrations. The forage plants that we sampled were not known Se obligate or secondary accumulator plants. Aside from Se, whether study plants accumulate Mo and Cd needs further evaluation. Selenosis diagnosis is primarily based on Se measurements , anemia and the presence of physical examination findings identifying toxic levels.One source reported that plant forage containing >3–5 mg/kg induced toxicity in sheep. In our study, several plant roots exceeded 3 mg/kg, which is a concern with the pulling up of roots when sheep forage. The amount of root consumption in relation to the total sheep forage intake is important to determine. Further work examining these factors is an area of future research. Based on drinking water standards for livestock, none of the heavy metal concentrations were above maximum tolerable concentrations . Heavy metal water measurements collected by the DiNEH study from two of the water sources identified for Sheep 3 contained lower concentrations of Pb in comparison to our data ; the remaining HM data were less than what the DiNEH researchers found. Most of the shepherds obtained public water for sheep consumption, which was reflected in the concentration levels found in sheep water. Harvesters in the study reported a history of consuming unregulated water intended for livestock. However, the As, Cd, Pb, Se, and U concentrations did not exceed the maximum contaminant levels set for human consumption. The implementation of water use maps may have contributed to the use of safer alternative water sources for these shepherds. Continued emphasis on the use of safe alternatives for water use in sheep and human consumption put forth by deLemos et al. is essential. Harvested food selling and sharing was common among the participants in the study. Emphasis should be placed on determining the incidence and frequency of food selling and sharing when assessing food chain contamination. Harvesting locations and activities can overlap in mining impacted areas. A few important factors to consider include the availability of harvest items based on seasonal variation and peak consumption periods . It is important to consider the consumption of contaminated food not only by individuals and their families but potentially the whole community and beyond.

It could hardly be otherwise, given that it is such a highly profitable business. When coca paste leaves Peru, it is worth US$400 per kilo; it then reaches Colombia, where it is processed and becomes cocaine, valued at US$1,200 per kilo; in Miami, the same amount is sold for US$20,000, and is transported to Chicago, where it fetches US$30,000 wholesale and is sold to individuals for approximately US$140,000 per kilo. Figures for heroin are even more fabulous; its sale is four times more profitable. This being so, one might spray Colombia all over, from the Amazon to the Andes, with every kind of chemical or fungus available, and the effect would be precisely the same: the drug phenomenon will continue to thrive. Meanwhile, this phenomenon is rapidly becoming a tremendous catalyst for a kind of rebellion, one which is brewing amongst those who have traditionally been excluded from Colombia’s society. It may not constitute a genuine revolution, but could well explode in an amorphous, uncontrollable uprising by the dispossessed. Since its launch in 2009, Evergreen Co-operative Corporation, a network of worker-owned co-operatives in Cleveland Ohio, has magnetized media, political elite, and academic attention. Evergreen has garnered supportive coverage in the Economist, Harper’s, The Nation, The New York Times, Fast Company, Time, and Business Week. Sarah Raskin lauded the initiative in 2013 while she was serving on the Board of Governors for the Federal Reserve System .Ron Sims, then Deputy Secretary for the U.S. Department of Housing and Urban Development, referred to the Evergreen network as “brilliant” during a 2011 interview .

Intellectuals on the Left have also been attracted to the initiative: Noam Chomsky has celebrated Evergreen in interviews and public talks,ebb and flow bench and the initiative has been cited by numerous academics as a hopeful alternative to the capitalist firm and its social and environmental externalities . Evergreen is currently comprised of three worker-owned co-operative enterprises: Evergreen Laundry , Evergreen Energy Solutions , and Green City Growers . Evergreen was designed to capture procurement flows from area “anchor” institutions: large hospitals and universities that are unlikely to leave the community, have a general commitment to improving it, and can do so by leveraging their purchasing power in support of local economic development . While Evergreen currently employs approximately 120 people, the vision is that it will become a large network of worker co-operatives that can rejuvenate the depressed regional economy in Cuyahoga County and inspire replications in other regions across the United States. Evergreen’s key features are ensuring worker ownership, harnessing the local wealth of anchor institutions, and prioritizing sustainable service delivery.Supporters refer to Evergreen as the “Cleveland Model,” an approach that can be pursued in communities across the country . According to leaders with Evergreen, “What’s especially promising about the Cleveland model is that it could be applied in hard-hit industries and working-class communities around the nation” . Despite all of the attention, the Evergreen case has not yet been studied in a sustained way. Furthermore, there is a dearth of literature on co-operative development in general . With this article we aim to contribute to the collective learning that can happen from successful and failed co-op development experiments. Building this knowledge is especially important at a time when heightened contestation over neoliberal capitalism has intensified interest in the co-operative model . Our primary finding is that Evergreen’s development depended on contextual factors that might not be replicable: a supportive and wealthy community foundation and champions within local government.

The post-2008 period of contested neoliberalism in which Evergreen emerged created opportunities for new alliances, as diverse actors were willing to consider alternative economic models. These alliances were critical to Evergreen’s emergence, but similar connections might not be available elsewhere. The fact that Evergreen’s start-up relied so heavily on context-specific private and ad hoc arrangements suggests that moresystematic, government-supported programs of financing and technical support are needed if worker co-operatives are to thrive in North America. We conclude that bottom up, movement-driven action often precedes – and creates a climate for – policy change. Our analysis therefore falls within the social movement approach to co-operative development, which argues that robust popular movements are integral to successful development of co-operatives and often predicate policy breakthroughs . While Evergreen’s replicability may be limited, its social movement orientation and ambition to scale up the co-operative alternative to neoliberal capitalism position it as a contributor to the important project of movement building that can facilitate the policy change needed to grow the co-operative economy. To contextualize our case study we conducted an extensive literature review on co operative policy, focused specifically on co-op dense regions . Our research team then visited Cleveland in May 2013. We did site visits to Green City Growers and Evergreen Laundry, interviewing management and speaking with employees at each location. We also conducted semi-structured interviews with key actors involved in the conception and implementation of the project. We sought from the outset to make our findings relevant not only to academics, but also to practitioners in the co-operative movement.We thus undertook this project as a form of “movement-relevant” research . According to Bevington and Dixon, movement-relevant research “emerges out of a dynamic and reciprocal engagement with the movements themselves.

This engagement not only informs the scholarship but also provides … accountability” . Our research team would like to see Evergreen and the co-operative movement in general thrive, and this article is an effort to understand the conditions that might enable this success. We believe, following Bevington and Dixon, that this commitment to the co-operative movement does not lead to bias, but instead adds incentive to provide the “best possible information” to movement participants and supporters. Interest in the “Cleveland Model” has cut across the political spectrum, coming not only from progressive media and academics on the Left, but also from conservative venues like the Economist and the Federal Reserve Board. Evergreen emerged one yearafter the 2008 financial crisis, during heightened contestation over the philosophy and policy mix that has guided political economic affairs for the past forty years: neoliberalism. Neoliberalism involves a significant reduction in the state’s social and environmental welfare role coupled with an expansion in the state’s facilitation of private capital accumulation . While neoliberalism has been consistently challenged since it arose in the late 1970s,strawberry pots contestation became mainstream after 2008, as the financial crisis raised questions about the viability of under-regulated financial markets and the growing inequality that helped fuel increased consumer reliance on credit . During this period, too, climate change moved into the mainstream of political debate: in 2007 Al Gore’s film An Inconvenient Truth won an Academy Award, and Gore shared the Nobel Peace Prize with the Intergovernmental Panel on Climate Change. As Gore himself articulates, neoliberal philosophy and policy has been a significant impediment to strong government action on climate change . This more mainstream contestation of neoliberalism – fuelled by economic crisis, rising inequality, and climate change – has not facilitated the emergence of broadly accepted alternatives, leading some critics to worry about a “zombie neoliberalism” that will not die . While the post-2008 period of contestation has not enabled a consensus solution to neoliberal capitalism’s contradictions, it has powered the search for alternatives . Ideological perspective necessarily conditions the kinds of solutions different actors seek and support. American political elites like Sarah Raskin and Ron Sims, for example, are interested in innovative ways of addressing inequality and ecological strain that leave in place the fundamentals of capitalism . Radical critics like Chomsky are interested in systemic alternatives to not only neoliberalism but also capitalism itself . Co-operatives, Evergreen founders note, provide alternative economic models that “cut across ideological lines – especially at the local level, where practicality, not rhetoric, is what counts in distressed communities” . Evergreen, then, is an ideologically flexible initiative: an innovative market-based poverty alleviation strategy or the germ of capitalism’s successor, depending on one’s point of view. While Evergreen has benefitted from the surge of interest in economic alternatives post-2008, the whole co-operative movement is experiencing resurgence. The General Assembly of the United Nations declared 2012 the International Year of Co-operatives. The International Co-operative Alliance , an organization representing the global co-operative movement, recently reflected that “rarely has the argument in favor of co operatives looked stronger”.

Co-ops can be read as either an ethical supplement to neoliberal capitalism, one that evens out its contradictions in distressed communities, or they can be read as the basis for a systemic alternative. Leaders of the Cleveland Model explicitly subscribe to the latter, more radical view, even as they strategically benefit from the former. Evergreen is modeled after the Mondragon Cooperative Corporation in Spain, which has long been a model for large-scale co-operative development worldwide. Founded in 1956, MC is now a conglomerate including 110 worker co-operatives, and employing more than 80,000 workers . Mondragon does business in manufacturing, retail, finance, and knowledge . As a worker owned co-operative system, Mondragon has several features that distinguish it from traditional capitalist firms: for example, a pay cap specifies that top earners with MC can only earn six times the pay of those in the bottom bracket . By comparison, CEOs for US corporations regularly make 400 times an average worker’s salary – a rate that has increased twenty fold since 1965 . Mondragon is one of the largest employers in the Basque region of Spain where it is centered . The Mondragon model is not without its challenges, including the recent bankruptcy of Fagor, one of its larger companies , but it remains an example of how co-operatives can operate on a large scale, produce considerable wealth, share it equitably, and promote relative worker satisfaction. As such, Mondragon is a longstanding inspiration for movements and intellectuals interested in alternatives to the capitalist firm and economy . Replicating Mondragon’s successes, however, is no easy task. The region’s political culture is an enabling factor: Basque country is home to a robust nationalist and separatist movement, and considerable associational energy is generated from feelings of marginalization at the hands of a dominant majority .Political culture supportive of co-operative development is not easily replicated, a fact that limits the ability of the co-operative movement to transplant successes from one region to another . Evergreen has generated popular and movement excitement partly because it appears to have successfully adapted the Mondragon model for North America. Northeast Ohio is not home to a political culture distinctively supportive of co-operatives. As Ted Howard, one of Evergreen’s leaders, told us, “Some people think there must have been something about the Cleveland community that would welcome this co-operative development, but it was a foreign concept” . As we learned, however, key supports were available in Cleveland – mainly a wealthy community foundation and champions in local government – that might not exist in other North American communities.Cleveland is still struggling to recover from the economic decline that began in the late 1960s. Once the fifth-largest city in the US and a center for manufacturing, Cleveland was hit hard by forces of economic globalization and the deindustrialization they brought. Plant closures, unemployment, and out-migration contributed to a depressed urban economy. Between 1970 and 1980, the city lost 24 percent of its population, one of the steepest drops in US urban history . Those who left generally had the means to do so, “with the poor, elderly, structurally unemployed, or marginally unemployed remaining behind” . White flight, the large-scale migration of whites from racially mixed urban neighborhoods to more suburban regions, was also a factor in the hollowing out of Cleveland . In the 1990s Cleveland began to slowly recover economically, and the city is currently a hub for health care services, biotech, and polymer manufacturing . The two largest employers in the region are the Cleveland Clinic and University Hospitals, together employing 46,000 people . Both institutions are located in the Greater University Circle , an area four miles east of downtown that is also home to Case Western University, the Cleveland Museum of Art, and the Museum of Contemporary Art.

Wang et al. investigated the role of light quality, specifically, low red to far-red ratios , on photo protection during cold stress in tomato. They showed that L-R/FR activated two pathways associated with cyclic electron flow : the PGR5/PGRL1A- and NDH dependent complexes, respectively. These CEF complexes help to reduce cold-induced photo damage of the photosynthetic machinery by accelerating the thermal dissipation of excess energy, enhancing ROS scavenging, and reducing the hyper reduction of the electron transport chain. This work therefore provides a better understanding of the mechanistic relationship between varying light quality and low temperature in plant photosynthetic performance in temperate climates when seasonal variation induces these conditions. Spring frosts cause important economic losses in many fruit-producing areas of the world, and there is interest in identifying feasible approaches to mitigate these risks. Ethylene controls fruit ripening in climacteric species but it also plays an important role in plant stress responses . Published literature on the use of ethylene or ethylene-based compounds for protecting temperate fruit orchards against frost damage was reviewed . Experimental evidence of ethylene modulation of bud dormancy and blooming were presented and discussed. It was suggested that ethylene-delayed bloom and the associated frost protection may result from either the slowing down of floral bud responsiveness to seasonal temperature changes, an antagonistic interaction with other hormones such as abscisic acid or gibberellins, plant sensing of exogenous ethylene as a stress signal leading to longer dormancy, or ethylene-enhanced ROS accumulation resulting in extended bus dormancy.

Because chilling stress in plants often leads to ROS accumulation, the questions arises whether improving the antioxidant capacity of tissues by the exogenous application of antioxidant treatments may help improve tolerance to cold as well as to other types of abiotic stress. To this purpose, Tang et al. treated low bush blueberry seedlings with hydrogen sulfide,vertical farming racks and found that treated plantlets performed better under low temperatures than the untreated controls, as shown by the alleviation of membrane peroxidation, the reduction of chlorophyll and carotenoid degradation, and the lessening of photo system I and II photo inhibition. Conversely, the application of hypotaurine, a H2S scavenger, aggravated the oxidative symptoms under cold stress. Brassinolide is an important plant stress hormone shown to promote plant resistance to low-temperature environments. Zhang et al. investigated the effects of exogenous BR on the photosynthetic characteristics, leaf anatomical structure, and chloroplast ultra structure of two species of tung tree seedlings under different temperature conditions. The results suggested that long-term low temperatures significantly reduced the photosynthetic efficiency of tung tree seedlings, affecting the formation of the internal structure of plant leaves and destroying the integrity and function of the chloroplast. To prevent this, external application of BR to tung tree seedlings could enhance the photosynthetic potential of tung trees by maintaining the stability of the leaf structure and morphology and alleviating the damage caused by cold injury. In summary, the papers in this collection illustrated the breadth of research aimed at understanding chilling responses in horticultural crops, but more importantly provided new insights that will further our future basic and applied research in this area.

Agriculture has been an important engine of economic development, and the mainspring of economic progress in agriculture has been productivity improvements driven by technological change that is fueled by re search and development . Since World War II, agricultural productivity has more than doubled in the United States, as in many other countries. California agriculture today produces more than twice the output of 1950, using roughly the same total input — although with less labor and land, and more capital and purchased inputs. These gains can be attributed to new biological, mechanical and chemical technologies, including improved genetic material, machines, fertilizers and pesticides, and knowledge. The current wave of technological progress continues this pattern, while emphasizing information technologies and biotechnology — in particular genetically modified crops. For many, GM crops represent the hope for a future with less hunger and malnutrition, and for a more sustainable agriculture with more varied, cheaper and safer food. For others they are cause for serious concern about the environment and food safety. Regardless of how we may feel about it, the juggernaut of technological change continues and the biotechnology revolution is well under way in the United States and other countries. The challenge for public policy is to determine what regulations should be applied to govern the development and use of these technologies, and what other types of intervention may be necessary, such as public investments in research to correct for private-sector under investment. In the case of horticulture — the cultivation of fruits and vegetables, tree fruits and nuts, turf grass, flowers and ornamental crops — these is sues are sharply drawn because the private sector has not found it profitable to develop or commercialize many GM crops in the current political, legal and market environment.

What will happen in biotechnology applied to horticultural crops is up to the government, for a variety of economic reasons. Some of these aspects may be unique to GM horticultural crops but many are common to GM crops generally, and similar issues arise with some new non-GM technologies.Without government intervention, the rate of innovation will be too slow, reflecting both under investment in research and under adoption of some research results. Both problems are related to the nature of property rights for re search results. “Free-rider problems” occur when property rights are incomplete, and privateinves tors can capture only part of the re turns to their investments in certain types of research ; as a result, their incentives to invest are reduced. On the other hand, when the rights to research results are protected, such as by patents or trade secrets, the owner of a new variety can charge monopoly prices,maceta cuadrada 25 x 25 unduly limiting the use of that variety. Intellectual property rights are a double-edged sword: to the extent that they pro vide a greater incentive for investing in research they are also likely to result in lower adoption rates. Governments have addressed the incentive problems in agricultural research in several ways. Federal and state governments have funded agricultural research at public institutions such as the U.S. Department of Agriculture and state agricultural experiment stations associated with land-grant colleges. This approach allows an increase in total research with out the problems associated with monopoly pricing of inventions. How ever, even though the investment has paid handsome dividends, it is increasingly difficult to sustain the past levels of funding for public agricultural R&D, in the face of general budget problems and declining political sup port for public science funding, including agricultural science . Governments have also acted to strengthen IPRs applied to plants and animals as well as mechanical technologies; and changes in IPRs, especially in the 1980s, were crucial for the agricultural bio-technol ogy development that followed. Partly as a reflection of enhanced IPRs, in the United States, private-sector funding of agricultural research has been growing faster than public-sector funding and now exceeds it. The balance in agricultural R&D be tween the private and public sectors varies among types of research. For in stance, until recently the private sector emphasized agricultural R&D pertaining to mechanical and chemical technologies, especially pesticides, where IPRs are effective; and the government was more important in other areas such as improving crop varieties. Private involvement was dominant in crop variety research only in hybrid corn, where the returns were well protected by technical restrictions on copying or reusing saved seed, trade secrets and other legal rights. Changes in the institutional environment and the form of new IPRs, combined with new scientific possibilities associated with modern biotechnology, resulted in a shift in the private public balance in research to improve crop varieties.

As the balance shifts toward private re search, new attention must be paid to old questions about whether the private investment in crop research will be sufficient, whether the allocation of those resources will be optimal, whether the results will be adopted rap idly and widely, and what role the government should play.The development of new technologies through R&D is only one element of the picture. The technologies must also be approved for commercial application and economically attractive enough to be adopted by farmers. Biotech crops have been a commercial reality only for a few years but they have made very rapid inroads in some parts of the market. In particular, pest resistant and herbicide-tolerant corn, soybeans, canola and cotton were rap idly developed and adopted in the United States and to a lesser extent in other countries . To date, the successful GM crop varieties have emphasized “input traits,” related to reducing the use of chemical pesticides or making them more effective, rather than “output traits,” related to product quality. Why has there been rapid development and adoption of GM crop ping technologies for these crops and not other important crops, such as wheat and rice? The likely reasons re late to the nature of supply and demand for new technology, and the economics of adoption.The total benefits from farmers adopting any new cropping technology are approximately equal to the benefits per acre times the number of acres affected. With pest-resistant crop varieties, these benefits come primarily from reduced costs for applying chemical pesticides and increased yields, after an allowance for regulatory requirements for refugia to manage resistance. The distribution of these total benefits between farmers on the one hand, and the technology suppliers on the other, is determined by the size of the premium charged for the use of the new technology, but this premium also affects the incentives for farmers to adopt the technology. Economic studies suggest that farm ers and biotech companies have shared in the benefits of biotech crops and that the net benefits have been large. Gianessi et al. conducted 40 detailed U.S. case studies of biotech cultivars. They estimated that in 2001, eight biotech cultivars adopted by U.S. growers provided a net value of $1.5 billion to growers, reflecting increased crop values and cost savings. They further estimated that the 32 other case-study cultivars would have generated an additional $1 billion in benefits to growers if they had been adopted, bringing the total potential benefit in 2001 to $2.5 billion. Of this annual total, the lion’s share was for herbicide-tolerant crops , followed by insect-resistant crops . These estimates do not represent the total economic impact because the geographic analysis was limited in scope, and they do not include any benefits to the seed companies and biotech firms that produced the technology. Environmental concerns. Private benefits and costs from biotech crops accrue to growers and consumers of the products, along with seed companies and biotech firms. If the new technology involves environmental risks or replaces technology that involves environmental risks, there will be additional environmental costs and benefits to take into account as an element of national costs and benefits. For instance, pest-resistant crops can reduce the application of broad-spectrum chemical pesticides, which are hazardous to farm workers, compromise food safety and impose a burden on the environment. The economic studies to date have not assessed these environmental costs and benefits. However, Gianessi et al. estimated that adoption of the eight current cultivars allowed a redution in pesticide use of 46 million pounds in 2001, and the 32 potential cultivars could have allowed a further reduction of 117 million pounds. The relevant comparison then is between the environmental risks associated with these biotech crops and those associated with the annual burden on the environment of 163 million pounds of chemical pesticides that could be avoided by growing biotech crops instead – 66 million pounds in California alone, where 185.5 million pounds of pesticides were used in 1999 Market acceptance. On the demand side, farmers will adopt biotech varieties if the perceived net benefits to them are large enough, and this depends on the perceived market acceptance of GM crops. Concerns have been raised about the possibility that GM crops may be unsafe for consumers because of allergens or other, as yet unidentified risk factors, about risks to the environment and to the economy from uncontrolled genetic drift, and about the moral ethics of tampering with nature.

In both cases, strong competition occurs between plants and microbes so that actual nutrient uptake by individual consumers is often less than their demand due to limited supply and uptake of a nutrient by one consumer suppresses the functioning of other consumers . Furthermore, as CO2 concentrations increase, nutrient competition between plants and microbes is expected to intensify. Because elevated CO2 concentrations fertilize plant carbon productivity, plants will require more soil nutrients to facilitate enhanced photosynthesis and for tissue construction . On the other hand, enhanced carbon assimilation dilutes tissue nutrient concentrations and lowers litter quality . Decomposing lower quality litter implies that soil microbes may need to immobilize nutrients to maintain their stoichiometric balance . In addition, under elevated CO2 conditions, available nutrients will progressively move from fast cycling tissues to slow cycling tissues , which induces progressive nutrient limitation that further exacerbates nutrient limitations. Although increased external nutrient inputs and accelerated nutrient mineralization rates under warming soil conditions may enhance soil nutrient availability and partly ease plant– microbe nutrient competition, these additional nutrients may be insufficient to satisfy the enhanced plant nutrient demands . To investigate nutrient competition and its effects on the terrestrial carbon cycle,maceta 15 litros different theories of plant-soil nutrient competition have been developed and implemented in Earth System Models . However, the oretical justification and observational support for these theories are rarely discussed, which may have resulted in large biases in modeled nutrient and carbon cycling.

To reconcile this inconsistency between theory, observations, and models, we focus on one overarching question in this study: Is there an observationally consistent, theoretically supported, and mathematically robust theory that is simple enough to implement in ESMs while accurately representing plant–microbe competition for nutrients? To answer this question, we first survey four existing nutrient competition theories and their implementation in ESMs . In Results, we discuss in detail these four competition theories: CT1, no direct competition; CT2, microbial decomposers out compete plants; CT3, competition depends on pore-scale soil fertility heterogeneity; and CT4, plant–microbe relative demand controls competition. Then we describe a new theory of nutrient competition based on Equilibrium Chemistry Approximation kinetics . We test our new theory together with other existing competition theories against a unique observational data set of N competition in a grassland ecosystem.To inform the development of ESM land models, observations have to satisfy two criteria. First, observations should capture plant and microbe competition at the whole-soil level, because the significance of microsite heterogeneity diminishes at this spatial scale. Second, measurements should target short-term nutrient uptake, thus enabling relatively clear separation of the instantaneous competitive interactions from other ecosystem dynamics that occur over longer time scales . Among the four existing theories surveyed, the traditional Nutrient Competition Theory assumes that plants and microbes do not compete for nutrients. This theory presumes that plants can assimilate carbon directly from the atmosphere but rely on nutrients released from soil microbial activity, so plants are carbon rich but nutrient limited . Conversely, because soil microbes decompose soil organic matter to obtain carbon and nutrients , they are relatively nutrient abundant but carbon limited.

A second reason ecologists hypothesize that plants and microbes do not compete is that microbes can directly use organic N during decomposition , while plants primarily use inorganic N . How ever, depending on their carbon use efficiency and biomass stoichiometric imbalances against substrates , microbes do immobilize inorganic nutrients and thus directly compete with plants, creating the first contradiction against the CT1 theory. Further, plants may also utilize some low molecular weight amino acids through mycorrhizal fungi associations or direct root uptake , which creates a second contradiction to the theory. However, no existing ESMs apply CT1 to represent nutrient com petition . The second theory posits that microbial decomposers out-compete plants in nutrient acquisition. This theory assumes that microbial nutrient uptake is extremely efficient , and microbes assimilate as much nutrients as they can during decomposition, provided they are not carbon limited. When carbon is limited, mineral nutrients are released as a “waste product” . This concept leads to the classic idea that plants can only use “leftover” nutrients after microbial demands are satisfied , which is why measured net mineralization rates are commonly used as a proxy for plant-available nutrients . However, no evidence exists to support its validity at the whole-soil or ecosystem level. In contrast, 15N labeling studies have demonstrated that plants can continuously acquire inorganic nutrients, even when both plants and microbes are nutrient limited . Other observations indicate that plants may even suppress microbial nutrient uptake . CT2 has been applied in several ESMs. HadGEM2 and GFDL assume that soil microbial decomposers always outcompete plants and have priority for available nutrients . IPSL and BNU-ESM also assume that microbial immobilization has priority, but apply this priority to the estimated gross mineralization flux in the current model time step, as opposed to the nutrient pool. The third competition theory applies the emerging perspective that plant–microbe nutrient competition depends on the spatial heterogeneity of soil nutrient fertility, and therefore plants do not completely lose the competition at the whole-soil or ecosystem level.

In a heterogeneous soil medium, inorganic nutrients move from nutrient-rich microsites toward nutrient-limited microsites , with roots potentially intercepting the nutrients . CT3 has been integrated into very fine-spatial scale models that explicitly consider the role of microsite soil nutrient heterogeneity, nutrient diffusion, root–microbe interactions , and microbe–microbe competition . In these models, plants do not completely lose the competition with microbes because they can take advantage of fine-scale spatial gradients between immobilizing and mineralizing microbes. The emergent responses from these models indicate that nutrient diffusion rates, sink strength ,indoor garden and competitor spatial distributions are the most important factors affecting plant competitiveness. However, these models’ fine spatial resolution is not directly applicable to ESMs. In ESMs, each soil column is assumed to be a well-mixed environment of nutrients and competitors. Such an assumption is currently necessitated, at least, by limited computational power and observations. Although ESM spatial resolutions likely will become finer, simulating microsite-level soil heterogeneity will remain impractical in the near future. In addition, a model based on CT3 may have high explanatory value but low predictive value, because it requires fine resolution observations of soil heterogeneity.In these ESMs, plant nutrient demand is simulated based on potential Net Primary Production in the absence of nutrient constraints and the plant C to N ratio ; an analogous approach is taken for microbial nutrient demand. When soil nutrient supply is insufficient to satisfy these demands, both plant and microbial demands are reduced in proportion to their respective demands . The actual NPP is then calculated by rescaling NPP demand with the reduction factor. This “relative demand” theory implicitly assumes that the consumer with higher demand will be relatively more competitive. While being simple, the CT4 predicted plant nutrient uptake is mechanistically inconsistent with measurements , although Goll et al. argued that the “demand-driven” approach requires fewer model parameters. The ESMs that apply CT4 include CLM-CN and NorESM , CLM-CNP , and JSBACH-CNP .We compared observations from the 15N tracer study with three model structures for competition: CT2 , CT4 , and CT5 . We were unable to build a model based on CT3 for the study site due to a lack of detailed information about soil N heterogeneity, root architecture, and N diffusion and mass flow rates. Further, such a complex model structure would currently be computationally intractable for ESM applications, although below we discuss a possible intermediate-complexity approach based on CT3 concepts that could be integrated with CT5 in an ESM land model. The CT2 model predicts that topsoil plant 15N uptake is very small due to large microbial nutrient demand . In contrast, because of lower microbial nutrient uptake at depth, there are more “left-over” nutrients and plant 15N uptake is relatively higher, although root biomass density decreases with depth.

Therefore, there is an increasing microbial to plant 15N uptake ratio with increasing root biomass for the CT2 model . For relative-demand-based competition , the predicted microbial nutrient uptake declines with depth, because topsoil litter substrates are nutrient depleted and microbial biomass declines sharply with depth . However, in this calculation, the whole plant nutrient demand is fixed. This constraint implies that microbial decomposers are more competitive in the topsoil than they are in subsoil, while plant competitiveness remains constant across the soil profile. Therefore, the predicted ratio of microbial to plant 15N uptake increases with increasing root biomass . The CT2 and CT4 models were unable to match the observed nitrogen partitioning between microbes and plants. Comparing CT2 and CT4 in the topsoil, CT2 predicted a much higher ratio of the microbe to plant 15N uptake, because plants do not completely lose the competition in the relative demand approach . Importantly, in our evaluation, both CT2 and CT4 resulted in nutrient competition profiles qualitatively opposite to those observed. We also confirmed that no combination of parameters for either CT2 or CT4 could reproduce the qualitative shape of the observed competitive relationship because, for both CT2 and CT4 models, the target variable UPmic/UP plant is proportional to microbial biomass . Shaping parameters only affect the steepness of UPmic/UPplant, but not the general trend. The ECA approach explicitly considers the substrates and enzymes competitive interactions throughout the profile. It captures the general competition pattern using literature-derived parameters from other ecosystems , and qualitatively and quantitatively captures the competition pattern using parameters derived for this site .The ECA representation of nutrient competition provides a theoretical and modeling construct that resulted in very good comparison with the nitrogen uptake partitioning. These predictions demonstrate that integrated across the soil profile, plants were less competitive than microbial decomposers; plant competitiveness against microbes is a spatially distinct property and there is no simple coefficient that can scale their “competitiveness”; the ECA framework offers a theoretically consistent approach to continuously update individual competitiveness; plant competitiveness is controlled by functional and structural traits ; and in the topsoil, plants might out-compete microbes and consequently suppress microbial nutrient uptake. Of course, applying the ECA competition to ESMs comes at the cost of introducing new parameters and additional uncertainty associated with those parameters. However, the ECA approach does not necessarily increase overall model uncertainty . In fact, ECA competition largely reduced the uncertainty in global-scale predictions by considering essential processes that govern system dynamics . We argue that an analogous result occurred in this analysis, i.e., that the uncertainty reduction in model structure overwhelmed uncertainty associated with new model parameters. In addition, most of the ECA parameters are kinetic parameters, which can be directly measured or optimized , implying that targeted experiments and model calibration could further reduce parameter uncertainty.Nutrient competition constantly occurs between plants and microbes in natural terrestrial ecosystems and it will likely intensify under climate change . Therefore, two fundamental questions arise: what controls the partitioning of limited nutrient resources between plants and microbes and how should short-term competition be modeled? Regarding the first question, we highlight the very few observations available to quantitatively partition nutrient acquisition by plants and microbes, and contend that such observations are critical to improve carbon-climate feedback predictions. As we showed here, the detailed 15N tracer experiment used in this study allowed us to evaluate the existing and newly developed plant–microbe N competition hypotheses, because the experiment was conducted at the plot scale and 15N was directly injected in the rooting zone . Thus, most of the observed plant N uptake pattern reflected the direct competition between roots and microbes, via nutrient carrier enzymes quantity and quality. Regarding the second question, we show here that plant and microbial nutrient uptake can be mechanistically explained as different nutrient transporter enzymes reacting with soil nutrients in a competitive manner. By linking plant root and microbial biomass density to nutrient transporter enzyme abundances, our new competition theory produces qualitatively correct competition patterns with literature-derived parameters from other ecosystems, and is easy to calibrate for specific ecosystems. Further, the linkage of nutrient competition with plant and microbial traits will allow a model to represent the competitors’ dynamic allocation of resources to acquire necessary nutrients.

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,growing lettuce hydroponically 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 plant cell 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, 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,maceta de 30 litros 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 host genome, but these will take months or even years to develop, optimize, and scale-up. Among the available transient expression systems, only plants can be scaled-up to meet the demand for COVID-19 countermeasures without the need for extensive supply chains and/or complex and expensive infrastructure, thus ensuring low production costs . These manufacturing processes typically use Nicotiana benthamiana as the production host and each plant can be regarded as a biodegradable, single-use bioreactor . The plants are grown either in greenhouses or indoors, either hydroponically or in a growth substrate, often in multiple layers to minimize the facility footprint, and under artificial lighting such as LEDs. In North America, large-scale commercial PMP facilities have been built in Bryan, TX , Owensboro, KY , Durham, NC , and Quebec, Canada . The plants are grown from seed until they reach 4–6 weeks of age before transient expression, which is typically achieved by infiltration using recombinant A. tumefaciens carrying the expression cassette or by the introduction of a viral expression vector such as tobacco mosaic virus , for example, the GENEWARE platform . For transient expression by infiltration with A. tumefaciens, the plants are turned upside down and the aerial portions are submerged in the bacterial suspension.

A moderate vacuum is applied for a few minutes, and when it is released, the bacteria are drawn into the interstitial spaces within the leaves. The plants are removed from the suspension and moved to an incubation room/chamber for 5–7 days for recombinant protein production. A recent adaptation of this process replaces vacuum infiltration with the aerial application of the A. tumefaciens suspension mixed with a surfactant. The reduced surface tension of the carrier solution allows the bacteria to enter the stomata, achieving a similar effect to agroinfiltration . This agrospray strategy can be applied anywhere, thus removing the need for vacuum infiltrators and associated equipment . For transient expression using viral vectors, the viral suspension is mixed with an abrasive for application to the leaves using a pressurized spray, and the plants are incubated for 6–12 days as the recombinant protein is produced.Depending on the batch size , the vacuum infiltration throughput, and the target protein production kinetics, the infiltration/ incubation process time is 5–8 days. The inoculation/incubation process is slightly longer at 6–13 days. The overall batch time from seeding to harvest is 33–55 days depending on the optimal plant age, transient expression method, and target protein production kinetics . Importantly, plant growth can be de-coupled from infiltration, so that the plants are kept at the ready for instant use, which reduces the effective first-reaction batch time from gene to product to ~10–15 days if a platform downstream process is available . The time between batches can be reduced even further to match the longest unit operation in the upstream or downstream process. The number of plants available under normal operational scenarios is limited to avoid expenditure, but more plants can be seeded and made available in the event of a pandemic emergency. This would allow various urgent manufacturing scenarios to be realized, for example, the provision of a vaccine candidate or other prophylactic to first-line response staff.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 biofacilities of the future . Accordingly, continuous harvesting and extraction can be carried out using appropriate equipment such as screw presses , whereas continuous filtration and chromatography can take advantage of the same equipment successfully used with microbial and mammalian cell cultures . Therefore, plant-based production platforms can benefit from the same >4-fold increase in space-time yield that can be achieved by continuous processing with conventional cell-based systems .

Several growers noted the long pipeline to the development of good varietals, because of the lengthy time needed for testing and propagation. Some were also aware of the difficulty in breeding for multiple diseases and were skeptical that a truly disease-resistant variety could be developed. Some growers even suggested that industry and ecological conditions might be too dynamic for cultivars bred for specific conditions to be of use by the time they are developed. The last survey question asked about the policies or practices that would encourage planting of a disease resistant cultivar instead of fumigating and asked respondents to choose all answers that applied. Here again it appeared that additional regulatory restrictions would increase interest in disease-resistant cultivars , although it was clear that few growers would wish for such a situation. Were it to come about, sup port from UC Cooperative Extension could help aid the transition, as could financial support in terms of higher prices or subsidies. As of now, however, with fumigation still allowed, albeit restricted, most growers were concerned with other challenges: “To be honest, right now our focus is definitely in other factors. If the economics don’t work, we can have the disease-resistant variety but we’re not going to be able to farm it.”Overall, while there was still keen interest in seeing disease-resistant cultivars developed, disease resistance has become less of a priority for growers, mainly because other pressures have overtaken concerns with disease. It is also clear that disease-resistant varieties alone are unlikely to replace fumigation or, more to the point,hydroponics growing system convince growers to take the risks of reducing or forgoing fumigation.

As emphasized in a report issued by the California Department of Pesticide Regulation encouraging research into fumigation alternatives, with out the magic bullet of chemical fumigation, disease management is more complex, and strawberry growers would need to incorporate a combination of complementary methods and technologies to address the changing economic, ecological and regulatory environment of strawberry production . These complementary methods need continuing research support and testing in combination with each other. Consideration should also be given to ways of mitigating the costs of growing berries in this ever more challenging economic environment. Does that mean breeders should turn to other priorities than disease resistance? Not at all. Regulation is unlikely to become less restrictive or pathogens less virulent, and at some point disease resistance will become imperative. Given the difficulty of breeding effectively for all desirable traits, it is arguable breeders should even double down on disease resistance and lighten up on yield. Although growers want yield, breeders responding to that are perpetuating the technology treadmill that contributes to low prices. Indeed, it is important that super industry forces, those whose interests surpass those of individual growers, including university scientists, shippers and policy makers, aim to curb this prioritizing of yield by attending to the economic exigencies that make yield so important for growers.Chlamydomonas reinhardtii is a single-celled green alga that has been determined to be able to grow in the absence of light, and therefore does not require photosynthesis, by utilizing carbon containing substrates such as acetate. This is known as heterotrophic growth, where growth and propagation occur under dark conditions with metabolism of external carbon sources. This growth cannot be considered entirely decoupled from photosynthesis, however, because essentially all of these carbon substrates are derived from photosynthesis, including petroleum products which are the result of ancient photosynthesis.

A new, carbon fixing electrocatalytic process developed at the University of Delaware has been shown to be able to fix CO2 and CO into acetate rich product streams. This technology utilizes a copper nanosheet cathode and an IrO2 anode to catalyze the reduction of these single carbon substrates, demonstrating a relatively high efficiency of approximately 54% conversion into acetate. Due to the high acetate content of the product stream, when incorporated into a common algal growth media, Tris-Acetate-Phosphate , a new media can be produced that can possibly harbor algal growth. This process can be powered entirely by electricity and thus, photovoltaic technology can be employed and direct comparisons regarding efficiency given a fixed solar footprint can be made. By combining these two processes, there lies potential for developing an artificial photosynthetic system that can possibly match or exceed the efficiencies of conventional plant or algal growth, offering unforeseen advantages. Without the reliance on light, inconsistencies of sunlight due to climate variations can be remedied; this is a commonly discussed advantage of hydroponic agriculture. This project has significant implications for introducing alternative methods of agriculture that can aid in the battle against food shortage without further expanding agricultural lands. The first experiment conducted was testing the growth of the algae on a modified version of TAP media, where the acetate was exchanged with the acetate contained within a simulated, chemically identical effluent, as the effluent produced by the University of Delaware, was not yet accessible. The purpose of this experiment was to observe if media produced utilizing the effluent can harbor algal growth in heterotrophic conditions. The amount of effluent added to supplement acetate was enough to replicate the typical acetate concentration used to grow algae . This also came with the potentially cytotoxic chemicals included with the effluent and was labeled as TP-Effluent media. The positive controls for this experiment were TAP-dark, and TAP-light, where the cultures were grown on TAP media in the absence and presence of light, respectively.

TAP media is typically sterilized using an autoclave, but it was discovered that the effluent contained heat or pressure sensitive chemicals that would drastically increase the pH of solution and develop white, crystalline precipitate following auto-claving. Due to this issue,macetas de cultivo after adjusting media pH utilizing 5.0M HCl and 5.0M NaOH to approximately 7.23, the media was instead vacuum filtered for both the positive controls and experimental cultures. Inside of a bio hood, the corresponding media solutions were placed into 125 mL, pre-autoclave sterilized Erlenmeyer flasks, topped with pink caps for sterile air flow and wrapped in aluminum foil for the dark cultures. The flasks were then inoculated with 21gr- Chlamydomonas reinhardtii strain from an agar-plate preculture, placed under a light source and left to grow for 1 week.Growth parameters were measured by aliquoting 1.0 mL of culture for cell count and OD750 analysis. For cell culture analysis, a Biorad TC20TM automated cell counter was used, where 10 µL of culture was placed into both A/B sides of the slides, measured, and averaged. These parameters were measured at time 0, and at 1 week. The second experiment aimed to determine cytotoxic chemicals in the effluent and TP Effluent media, in order to develop potential treatment methods to improve growth. This was done by omitting chemical constituents from the simulated effluent and examining if growth improved. Because there were four chemical candidates for growth inhibition , each of the experimental effluent solutions omitted one candidate, labeled -KHCO3, – Ethanol, -1-Propanol, and -Propionic Acid, respectively. Preparation of the media was identical to that of the first growth experiment. The inoculation protocol for this experiment was slightly different than the first, instead using liquid pre-cultures of 21gr+ Chlamydomonas reinhardtii grown under light for 7-days. The inoculation of the control and experimental cultures was to achieve 5.0✕105 cells/mL cell density. The cell density of the pre-cultures was determined using the Biorad TC20TM automated cell counter, diluted to the target density and inoculated into pre-autoclaved flasks similar to that of the first experiment, but instead with 50 mL of media. All of the samples were grown in triplicate, with the positive controls; TP-effluent, TAP, and the experimental flasks; KHCO3, – Ethanol, -1-Propanol, and -Propionic Acid. The growth parameters used for this experiment differed slightly from the previous experiment, with the cell counting method being the same as before, but now with the inclusion of optical density measurement at 750 nm with a Molecular Devices QuickDropTM Spectrophotometer. This is a common method of quantifying algal growth. This measurement was blanked with a small aliquot of TAP media with no algae. Microscopic analysis was also performed using an Olympus BX51 Fluorescence Microscope. For each of these methods, 1.0 mL of culture was taken from each flask at time 0, and every 48 hours subsequently until 16 days total growth. On day 16, the dry biomass of the cultures by separating the cells from the media, baking overnight, and weighing the cell mass.

The third experiment was the Media Optimization Experiment, where effluent and thus acetate and cytotoxic chemical concentrations were altered to investigate the thresholds of algal growth on TP-effluent media. The percentages of acetate used in this experiment were 25%, 50%, 75% and 100% . TAP and TP were included as positive and negative controls. This experiment sought to determine how algal growth can be affected with lower concentrations of effluent, predicted to worsen growth due to less acetate but also improve growth due to lower concentrations of cytotoxic chemicals. The same effluent recipe from the drop out experiment was used in this experiment. Inoculation and culturing protocols were very similar to that of the “Drop-Out” experiment, with the same growth parameter measurement methods, consisting of cell counts and OD750 every 48 hours for a 16-day period and dry biomass measurements at the very end of the experiment. The same strain of 21gr+ Chlamydomonas reinhardtii was used. For this experiment, the algae were grown in the dark, by wrapping the flasks with aluminum foil.The fourth and final experiment was the growth experiment where the actual effluent derived from the electrocatalytic reduction process was tested. The inoculation protocol was similar to previous experiments, but cell counts for this experiment were done manually with a hemocytometer for higher fidelity. The other growth parameters measured were the OD750 and dry biomass. To optimize growth for this experiment, the culture flasks were placed on a shaker with a controlled temperature of 30°C. Effluents of various compositions were tested in this experiment, as the collaborating laboratory was able to produce various kinds of different compositions, some containing entirely different chemicals. The same strain of 21gr+ Chlamydomonas reinhardtii was grown in the dark using aluminum foil. Although the different effluents contain different acetate concentrations, the effluent was added to the media so that the final medias had the same acetate concentration . The first growth of the algae with effluent proved to be unconvincing, with poor growth even in traditional photosynthetic conditions. There was also noticeable clumping of the cells inside the effluent flasks, suggesting cell stress. This indicated that there was a strong cytotoxic component in the media, and therefore in the simulated effluent as well. In the drop-out experiment, after 16 days of growth, the results strongly suggested that the cytotoxic component in the effluent was potassium bicarbonate, or KHCO3. In Figure 2A, the appearance of the culture grown in media with KHCO3 omitted was similar to the positive control, TAP. This growth appeared to be lush and dark green, while the other flasks had relatively patchy growth. This could be, however, the result of inadequate mixing. The OD750 and cell count assays demonstrated similar findings, with the TAP and -KHCO3, cultures growing considerably better than the other experimental cultures. For cell density, the results even suggested that the -KHCO3 cultures grew better than standard TAP, although the errors for this proved to be very large. Dry biomass collected at the end of the growth period showed that the TAP, -KHCO3 and -propanoic acid cultures grew best. This was unexpected considering the OD and cell count measurements. By analyzing microscopic images, the TAP and -KHCO3 cells were dispersed and not clumped. All KHCO3 containing cultures had both lower cell density and cell clusters. This reflected what was observed in the initial growth experiment. For the media optimization experiment, it was found that for medias containing TP Effluent, 75% TP-Effluent and 50% TP-Effluent, there were not significant differences in cell viability over the course of a 16-week growth period, shown in Figures 3B and 3C. The 25% TP Effluent showed weaker growth, possibly because the cells were too quickly depleted of a carbon source, limiting their growth even when the presence of cytotoxic chemicals was reduced.

Microbial food safety issues are rare events and tracking the source of disease outbreaks is extremely complex, making it difficult to predict or determine their cause . Thus, the best way to minimize these events is to perform risk assessment analyses . As discussed above, it has become evident that the plant is not a passive vehicle for microbial food hazards, hence providing opportunities to breed crops for enhanced food safety. The challenge remains to identify effective traits and genetic variability useful for breeding. It has long been possible to breed plant germplasm that is resistant to plant pathogens. For example, the Fusarium pathogen synthesizes toxic DON and/or fumonisins and reduces seed set and fill in wheat; Aspergillus flavus can cause ear rots of maize in environmental conditions suitable for fungal growth. In both cases, these fungi can reduce plant yield and germplasm resistant to these pathogens is available . However, in cases where the fitness of the plant is not as directly reduced by the presence of the pathogen, traits that could potentially increase food safety may be harder to find and may require indirect or more creative solutions. They also compete with priorities for crop production and quality in breeding programs. Edible plants carrying human pathogens generally do not show visual symptoms as they would when infected with plant pathogens, particularly when they occur at low levels. This creates a challenge in developing screening assays to identify phenotypes with useful variation to support breeding efforts. Unlike the challenges associated with microbial hazards,maceta 7 litros detection of elements such as nitrates or heavy metals is relatively easy with standard tissue analysis.

Allergens can often be detected by routine assays . However, for human pathogens, rapid and cost-effective assays still need to be developed for routine screening of breeding populations, although some efforts have been made in this direction 9 . These assays will allow large scale assessment of germplasm to find the best expression of useful traits and their introgression into cultivated varieties. Despite the challenges, variations in human pathogen colonization of lettuce, tomato, and spinach genotypes have already been determined. An additional hurdle comes from the fact that microbial colonization is a complex behavior influenced by the plant host– pathogen combination and crop management practice such as irrigation type and crop fertilization . Human pathogen–plant models should be developed for the purpose of breeding efforts to enhance food safety based on enteric pathogen strain– plant commodity variety pairs identified from prominent or recurring foodborne illness outbreaks. At the same time, plant genetic resources that may facilitate genome-wide association studies should not be excluded. Furthermore, the use of human pathogens in routine assays requires highly trained personnel and laboratory/greenhouse bio-safety conditions according to NIH guidelines, in addition to considerable costs associated with the handling of microbial hazards in contained facilities. These approaches will require collaborative efforts among food safety experts, plant–microbe interaction biologists, microbiologists, and crop breeders for successful advancements in the field. to enhance food safety based on enteric pathogen strain– plant commodity variety pairs identified from prominent or recurring foodborne illness outbreaks.

At the same time, plant genetic resources that may facilitate genome-wide association studies should not be excluded. Furthermore, the use of human pathogens in routine assays requires highly trained personnel and laboratory/greenhouse bio-safety conditions according to NIH guidelines, in addition to considerable costs associated with the handling of microbial hazards in contained facilities. These approaches will require collaborative efforts among food safety experts, plant–microbe interaction biologists, microbiologists, and crop breeders for successful advancements in the field. to enhance food safety based on enteric pathogen strain– plant commodity variety pairs identified from prominent or recurring foodborne illness outbreaks. At the same time, plant genetic resources that may facilitate genome-wide association studies should not be excluded. Furthermore, the use of human pathogens in routine assays requires highly trained personnel and laboratory/greenhouse bio-safety conditions according to NIH guidelines, in addition to considerable costs associated with the handling of microbial hazards in contained facilities. These approaches will require collaborative efforts among food safety experts, plant–microbe interaction biologists, microbiologists, and crop breeders for successful advancements in the field. The growth of the human population places an ever-increasing demand on freshwater resources and food supply. The nexus of water and food is now well recognized. One promising strategy to sustain food production in the face of competing water demands is to increase the reuse of treated human wastewater. Municipal wastewater reuse for food production has been successfully adopted in some regions of the world. For example, Israel uses ~84% treated wastewater in agriculture production . However, Southern California, a region that suffers from a similar degree of water shortage, currently uses less than ~3% of municipal wastewater in agriculture, while discharging ~1.5 million acre-feet effluent per year into the Pacific Ocean . Secondary municipal waste water effluent for ocean discharge is often sufficient to support both the nutrient and water needs for food production.

Water reuse in agriculture can bring municipal water reclamation effluent to nearby farms within the city limit,vertical grow rack thus promoting local agriculture and also reducing the rate of farmland loss to urban development. While the use of reclaimed water in agriculture offers a multitude of societal and agronomical benefits, broader adoption faces great challenges. One of the important challenges is ensuring the safety of food products in light of a plethora of human pathogens that may be present in recycled wastewater. Past studies have identified risks associated with irrigating food with recycled wastewater through the retention of the irrigation water on edible plant surfaces during overhead irrigation . With the emphasis on water conservation and reduction of evapotranspiration, subsurface drip irrigation is gaining popularity . Since there is lesser contact between water and the plant surface, the chance of surface contamination of pathogens is reduced. However, this new practice presents risk of uptake of microbial pathogens into plants. Such internalized pathogens are of greater concerns as washing, even with disinfectants, may not affect pathogens sheltered in the vasculature. Although pathogen transport through root uptake and subsequent internalization into the plant has been a growing research area, results vary due to differences in experimental design, systems tested, and pathogens and crops examined . Among the array of pathogens causing foodborne illness that may be carried by treated wastewater, viruses are of the greatest concern but least studied. According to the CDC, 60% of U.S. foodborne outbreaks associated with eating leafy greens were caused by noroviruses , while Salmonella and E. coli only accounted for 10% of the outbreaks . Estimates of global foodborne illness prevalence associated with NoV sur pass all other pathogens considered . Viruses are also of concern because they persist in secondary wastewater effluents in high concentrations . They do not settle well in sedimentation basins and are also more resistant to degradation than bacteria . Therefore, in the absence of solid scientific understanding of the risks involved, the public are likely less receptive to adopting treated wastewater for agricultural irrigation. NoV internalization in hydroponic systems has been quantified by DiCaprio et al. . Internalization in crops grown in soil is considered lesser but nevertheless occurs. However, the only risk assessment that considered the possibility of NoV internalization in plants assumed a simple ratio of viruses in the feed water over viruses in produce at harvest to account for internalization. The time dependence of viral loads in lettuce was not explored and such an approach did not permit insights into the key factors influencing viral uptake in plants. In this study, we introduce a viral transport model to predict the viral load in crisp head lettuce at harvest given the viral load in the feed water. It is parameterized for both hydroponic and soil systems. We demonstrate its utility by performing a quantitative microbial risk assessment . Strategies to reduce risk enabled by such a model are explored, and a sensitivity analysis highlights possible factors affecting risk.

Some parameters to complete the conceptual viral transport model were obtained from the literature. Others were estimated by fitting the model to published data from experiments using NoV seeded feed water to grow crisp head lettuces in a hydroponic system . The initial volume of 800 mL for the hydroponic growth medium was adopted based on these experiments.For the soil system, the volume of the growth medium equals the volume of water contained in the soil interstitial spaces in an envelope around the roots. This envelope is a region around the roots that the plant is assumed to interact with. Vg, s is given by Eq. 17, where θ is the volumetric water content obtained from Clapp and Hornberger . Estimates for Ve spanned a large range and a middle value of Ve = 80000 cm3 was adopted and assumed to be constant over the lettuce growth period. This assumed value was also verified to have minimal impact on the model outcome .The plant transpiration rate was adopted as the viral transport rate ) based on: 1) previous reports of passive bacterial trans port in plants , 2) the significantly smaller size of viruses compared tobacte ria, and 3) the lack of known specific interactions between human vi ruses and plant hosts . Accordingly, viral transport rate in hydroponically grown lettuce was deter mined from the previously reported transpiration model , in which the transpiration rate is proportional to the lettuce growth rate and is influenced by cultivar specific factors . These cultivar specific factors used in our model were predicted using the hydroponic crisp head lettuce growth experiment carried out by DiCaprio et al. described in Section 2.3 . Since the transpiration rate in soil grown lettuce is significantly higher than that in the hydroponic system, viral trans port rate in soil grown lettuce was obtained directly from the graphs published by Gallardo et al. using WebPlotDigitizer .In the absence of a published root growth model for lettuce in soil, a fixed root volume of 100 cm3 was used. In the viral transport model, viral transfer efficiency was used to account for the potential “barrier” between each compartment . The existence of such a “barrier” is evident from field experiments where some microbial pathogens were inter nalized in the root but not in the shoot of plants . In addition, viral transfer efficiencies also account for differing observations in pathogen internalization due to the type of pathogen or lettuce. For example, DiCaprio et al. reported the internalization of NoV into lettuce, while Urbanucci et al. did not detect any NoV in another type of lettuce grown in feed water seeded with viruses. The values of ηgr and ηrs were deter mined by fitting the model to experimental data reported by DiCaprio et al. and is detailed in Section 2.3. The values of ηgr and ηrs predicted for the hydroponic lettuce were assumed in the soil case. The viral removal in the growth medium includes both die-off and AD, while only natural die-off was considered in the lettuce root and shoot. AD kinetic constants as well as the growth medium viral decay constant in the hydroponic case were obtained by fitting the model to the data from DiCaprio et al. . Viral AD in soil has been investigated in both lab scale soil columns and field studies . In our model, viral AD constants in soil were obtained from the experiments of Schijven et al. , who investigated MS2 phage kinetics in sandy soil in field experiments. As the MS2 phage was transported with the water in soil, the AD rates changed with the distance from the source of vi ruses. To capture the range of AD rates, two scenarios of viral behavior in soils were investigated. Scenario 1 used the AD rates estimated at the site closest to the viral source , while scenario 2 used data from the farthest site . In contrast to lab scale soil column studies, field studies provided more realistic viral removal rates . Using surrogate MS2 phage for NoV provided conservative risk estimates since MS2 attached to a lesser extent than NoV in several soil types .