Horticulture

The homolog of Arabidopsis TCP4, AqTCP4, was expressed at fairly consistent levels across all stages in the spurred taxa, but showed a substantial increase in expression in A. ecalcarata at DS3 and DS4, while AqTCP5 showed a gradual decrease in expression across all taxa, but a slightly higher level of expression in A. ecalcarata.Aquilegia has two BB homologs, one that is expressed at a low level in all taxa and shows a pattern of decreasing expression over development , and one that has fairly steady expression in the spurred taxa across phase I, but starts with a higher level of expression in A. ecalcarata .Variation in the shape of plant organs is determined by combinatorial differences in cell number and cell shape. The Aquilegia petal has evolved substantial morphological variation, particularly in several aspects of nectar spur shape, including length, width, and curvature. Our current understanding of the development of the Aquilegia nectar spur involves two phases, an early mitotic phase and a later cell expansion phase . Despite differences in adult morphology, detailed ontogenetic studies using SEM have shown that from petal initiation through the earliest stages of Phase I, petals of a spurred species, A. olympica, and those of the spurless species, A. ecalcarata, are quite similar in shape. In other species comparisons,vertical hydroponic garden differences in spur length at maturity have been largely attributed to differential cell elongation during phase II of development, however, the developmental basis of other aspects of spur shape have not been studied in detail.

Based on the sampling conducted here, discernible differences in several axes of shape, particularly width and curvature, are apparent quite early in Phase I of petal development, suggesting that cell division plays a role in determining their differences. By examining gene expression across early petal development in several species with variable morphologies, this study allows us to identify gene expression modules that appear to be conserved across petals with diverse morphologies as well as modules that differ in correlation with variation in spur morphology. Further, this comparative approach may provide insight into the developmental processes that underlie the morphologies.Over the course of petal development, GO enrichment analysis of genes commonly DE across all four taxa detected a pattern of declining expression of genes involved in mitotic activity. This pattern was also supported by correlations between WGCNA modules and developmental stages. Modules with eigengenes highly positively correlated with DS1 are enriched for GO terms related to mitosis. This finding of a decrease in mitotic activity throughout development is consistent both with petal developmental patterns in other model systems and with previous studies in Aquilegia. In contrast to this pattern, an enrichment of genes involved in oxidation reduction processes were found to be up-regulated at later developmental stages in all four taxa.

This is supported by both the DE analyses between DS1 and DS5 and by the WGCNA analyses where several modules with eigengenes highly positively correlated with DS5 are enriched for loci involved in oxidation-reduction processes. Exploring the types of genes that are differentially expressed between the spurred taxa and A. ecalcarata detected up-regulation of loci with GO categories related to mitosis in the spurred taxa, but only at DS5 . As Aquilegia petals develop, they begin to transition from cell division to expansion and differentiation, starting at the distal margin of the blade. In spurred taxa, this transition progresses from the petal margins toward the nascent nectary, with cell division persisting longest in the spur itself. Considering the pattern seen in the developmental comparisons and what is known about cellular processes during spur development, the increased expression of genes related to mitosis in the spurred taxa relative to A. ecalcarata suggests that the entire A. ecalcarata petal shifts into the differentiation phase at an earlier time point than in spurred taxa. Another pattern that supports this assertion is that there is an enrichment of loci involved with oxidation reduction processes expressed more highly late in development when considering all taxa, while when considering the loci DE between the spurred taxa and A. ecalcarata across development, there is an enrichment for loci with these processes at early stages in A. ecalcarata . A closer examination of genes with oxidation-reduction GO categorization in the developmental and spurred/spurlesss comparisons revealed a number of cytochrome P450 monooxygenases that at a molecular level function through heme/iron binding and oxidation. Although CYPs have similar molecular functions, they comprise the largest enzymatic gene family in plants and have evolved to play diverse roles in an array of cellular, developmental, and metabolic functions from hormone synthesis to pigment production. Many of these CYP functions appear to be important in differentiated cell types, rather than undifferentiated mitotically active cells. Thus, the upregulation of CYPs earlier in A. ecalcarata development may be consistent with our hypothesis that these petals are accelerated in their differentiation relative to those in spurred species.

Although the WGCNA identified a module that is highly correlated with the presence or absence of spurs , this module showed no GO enrichment. A curious result that emerged when examining the genes that are commonly up-regulated across development in only spurred taxa was the enrichment of genes involved in photosynthetic processes. This was also seen in WGCNA module 20, which contains genes expressed more highly in spurred taxa at DS5 and is enriched for GO terms related to photosynthesis. While this enrichment of photosynthetic genes seems perplexing given that petals are generally not considered photosynthetic organs, we hypothesize that this result is likely an indirect consequence of spur development, rather than a cause. During phase I of A. ecalcarata development, the entire petal is shaded from light by the enclosing sepals. In the spurred taxa, however, elongation of the petal spurs causes them to emerge from the bud and become exposed to direct light, likely inducing baseline expression of photosynthetic loci . Thus, this appears to be a background temporal component of spur development rather than a controlling factor.Although one might predict that more genes would be required for the development of the nectar spur, given its more complex three-dimensional structure compared to laminar petals and blades, a number of lines of evidence indicate that the number of loci required for early spur development may actually be relatively small. Considering the PCA conducted across the set of genes DE between DS1 and DS5 in any taxon , one might have expected that the presence or absence of spurs would be a major PC resulting in the grouping of samples from the spurred taxa apart from A. ecalcarata. However, none of the first 10 principle components examined capture such variation. The first principal component clustered samples by developmental stage,vertical home farming which is not surprising given how genes were selected for inclusion in the analysis. The second PC in this data set groups samples by geographic origin, with the Eurasian taxa clustering together and the North American taxa clustering together. This suggests that phylogenetic relatedness explains more shared developmental differences in gene expression in our data set than whether or not a nectar spur is produced. Several scenarios may contribute to this phenomenon. This pattern may indicate that relatively few genes are necessary to make a nectar spur and, thus, loci that consistently vary between the spurred taxa and A. ecalcarata do not explain a significant proportion of variation in this data set. Not mutually exclusive to this possibility, it may be that the genes important for spur production do not have variable expression levels across phase I of development and therefore were not captured in this set of analyses. Regardless, this result underscores the importance of sampling more than two species across divergent lineages for this type of study since, for instance, a pairwise comparison of A. ecalcarata and any single spurred species might have primarily identified loci that differ due to phylogenetic divergence rather than their morphological differences.

Given that crucial loci in nectar spur development may not be differentially expressed between DS1 and DS5, we also compared expression differences between the spurred taxa and A. ecalcarata at each of our developmental stages. These comparisons showed that during the earliest stages of spur development , there are fewer genes up-regulated in the spurred species than in A. ecalcarata. One possible explanation may be related to the proportion of differentiating cells in the petals of A. ecalcarata versus the spurred species. Given that the A. ecalcarata petal does not produce a spur and is entirely composed of blade tissue, a greater proportion of the A. ecalcarata petal may be in the differentiation phase relative to an equivalent sized petal that is producing a spur. Although a large set of genes are necessary for mitosis, the differentiation of cells into many specialized types may require the up-regulation of an even greater number of loci. In support of this hypothesis, several comparisons from the A. coerulea ‘Origami’ expression data set show a similar pattern. Comparing expression between the blade and the spur showed more loci are up-regulated in the blade and developmental comparisons between the 1mm and 3mm blade and spur cup tissue samples demonstrated that a greater number of genes are up-regulated in both tissues at the later developmental stage . Another consistent data point is seen in the expression of AqTCP4 . Previous studies have found that AqTCP4 is critical for the cell division to cell expansion/differentiation transition in Aquilegia and is expressed in a wave that starts at the blade margin and progresses towards the spur tip. However, in the comparison between the four sampled taxa, AqTCP4 is observed to peak during DS3-4 in A. ecalcarata while it is present at lower levels in the three spurred taxa. This is likely due to the absence of the prolonged proliferation needed for spur development, but may also reflect a developmental acceleration in A. ecalcarata relative to the spurred taxa, representing a heterochronic shift. Therefore, we infer that the greater up-regulation of genes in A. ecalcarata petals suggests that, across the organ, a larger proportion of cells have blade identity and have transitioned to expansion and differentiation. This pattern is likely to change over development as a greater proportion of the spurred petal begins to differentiate, including into cell types that are not present in the blade, such as trichomes and cells associated with the complex nectary. Along these lines, it was surprising to find that STY homologs were not strongly differentially expressed between A. ecalcarata and the spurred taxa, given the absence of nectaries in A. ecalcarata. This may reflect the fact that in situ expression studies have revealed early expression of STY homologs at the distal tip of developing petals, which is consistent with the more deeply conserved role for STY homologs in controlling auxin homeostasis in lateral organs. It is likely that this distal expression domain is also present in A. ecalcarata during the early stages sampled here, making the detection of differential expression in whole petals more difficult than in the previous study where dissected blade and spur tissues were compared at later stages. Even so, there is a discernible trend of increasing AqSTY expression across time in the spurred species relative to A. ecalcarata. While a relatively small number of genes were found to be consistently differentially expressed in entire early petals between the three spurred species and A. ecalcarata, an even smaller number of these genes were also found to be differentially expressed between the blade and the spur tissue of A. coerulea ‘Origami’. Only 35 genes showed consistent differential expression in what we term the ‘blade’ and ‘spur’ comparison classes between these studies, with 27 genes falling into the ‘blade’ class and 8 genes in the ‘spur’ class. Key loci in the development of nectar spurs are likely to act by prolonging mitosis in the spur cup, but none of these 35 loci are homologs of genes known to regulate the transition from cell division to differentiation in Arabidopsis by promoting or repressing mitosis. This list of 35 loci also does not contain several genes that are known to be necessary for proper spur formation. For example, the Aquilegia homolog of JAG has been shown to promote cell proliferation in petals as well as other organs. In the current dataset, AqJAG is expressed at somewhat lower levels in A. ecalcarata versus the spurred taxa, but the temporal expression dynamics across the five stages are quite similar .

As more sampling is completed, e.g. baseline surveys and during pilot mining programs, there will be an opportunity to build a database for genetic information that can be linked to specific species. Accomplishing this aim may require a combined approach.Benthic organisms can be classified by size: megafauna , macrofauna , meiofauna , and microbial communities. Megafauna are the only group that can be readily identified with imagery generated by photo or video methods, which can also contribute to our understanding of organism behavior and interaction . On the other end of the spectrum, microbial biodiversity is identified via molecular techniques such as sequencing . Macro- and meiofauna fall somewhere in the middle, amenable to both approaches but perhaps both are not always simultaneously appropriate. Macrofauna are often used as environmental indicators , and there is work to develop similar deep-sea measures using meiofauna where they dominate the biological community . It has been shown that MBT and metabarcoding can perform equally as well for biomonitoring of freshwater systems and assessment of marine aquaculture impacts .

In the deep sea,vertical aeroponic tower garden the limited taxonomic expertise on deep-sea meiofauna , may make the latter approach more appropriate for rapid assessment of biodiversity and environmental impacts.Molecular methods are better suited for identifying rare and cryptic species than MBT. Because of their low abundance and patchy distribution, missing rare species because of under sampling is often a problem, especially in the abyssal deep sea. Additionally, small, soft-bodied protists often disappear in fixed sediment and only show up in eDNA . Although rare species do not have significant biomass, they can contribute unique functions , and new species are discovered frequently with few representative individuals . Choosing clustering cutoffs for OTUs can affect results, losing rare species if too conservative and retaining technical artefacts if too lenient . Cryptic species usually cannot be identified via morphology and, therefore, require molecular methods to distinguish them. They have been documented in deep-sea taxa, such as polychaetes and gastropods , and can significantly impact estimates of species richness .MBT can provide demographic information and material that allow for examination of life histories, lifestyles, and functional attributes. On a practical level, sequencing technology cannot yet reveal these kinds of data. Whether demographic information is relevant to mining environmental requirements depends on the goals and objectives set by the ISA . Metrics such as density, dispersal, size structure, sex ratio, and physiological responses to environmental changes are often used to assess ecosystem health .

Metabarcoding is sufficient to estimate relative abundance, dispersal, and community changes , but additional techniques are required for the remaining metrics . However, capacity to interpret molecular data continues to increase, such as experimental calibration to calculate abundance data , or computational techniques to identify differentially abundant taxa and community shifts .In order to establish identification of deep-sea species and their ecological roles, it is likely MBT and molecular approaches are needed in combination. Metabarcoding yields sequences of genes that are clustered into OTUs which can serve as a proxy for species. However, in order to assign an OTU to a species, that species must have been morphologically identified and sequenced previously, or belong to a taxonomic clade well-supported in a robust phylogeny . Once described morphologically and genetically, metabarcoding alone can then be used to identify it. However, a molecular approach may greatly benefit from the improvement of MBT-based analyses, such as collating phylogenetic histories with environmental data . The former is sufficient if species diversity is the only interest. However, ISA draft exploitation regulation calls for the application of “an ecosystem approach” which implies information on functional diversity and ecological interactions are also necessary .There could be scenarios in which rapid results are required in order to respond appropriately to environmental impacts, such as when an environmental threshold is surpassed or a trigger is set off indicating serious harm to the marine environment . An ecological threshold is a “tipping point” at which the system undergoes rapid and irreversible change that triggers an action .

As an example, a possible threshold to cessation of mining may be the loss of a specified amount of local biodiversity , implied by current mining guidelines for environmental management and monitoring plans , ISA, 2017; Annex VII , ISA, 2019. In a scenario using MBT, results can be obtained only after months of work, whereas metabarcoding results can be obtained within days after return to shore. This difference in response time may have significant impacts on the amount of environmental damage that ensues and, ultimately, the ability for an ecosystem to recover. The amount of time required to develop the expertise necessary for either approach differs greatly. Depending on taxon, it can take multiple years for a taxonomist to become accurate and efficient at species identification using MBT. In the deep sea, this expertise is especially lacking, particularly for small taxa, which can overwhelm experts’ workloads and increase turnaround time. Parataxonomy, which is the delegation of taks in an MBT workflow to non-experts, can alleviate some pressure on taxonomic experts. Although parataxonomy does not yield reproducible results, it can be useful when rapid biodiversity assessments are necessary . In contrast, popularity of molecular approaches has caused the proliferation of sequencing facilities, both academic and commercial, for turnaround time of approximately 2- 3 weeks from sample submission to data return. The more limited training required to process sequencing samples makes molecular approaches more accessible, and the automation that is actively being developed will further reduce processing times. Additionally, loss of taxonomic expertise over time can be mediated by genetic information which can be used as an immutable characteristic for taxonomic identification.One of the biggest challenges facing both MBT and molecular approaches is developing an adequate sampling design. There are still many gaps in our knowledge of deep-sea biodiversity , but it is unclear at what spatial and temporal scales scientists need to sample in order to accurately characterize it . Spatial heterogeneity is poorly defined and likely differs among habitat and location. In the CCZ, there can be unexpectedly high spatial heterogeneity . Additionally, adequate temporal resolution of sampling for accurate characterization of deep-sea habitats is unknown . MBT and metabarcoding often provide one snapshot in time and, given time constraints associated with MBT, a comprehensive monitoring program may incorporate both methodologies by employing metabarcoding more frequently and MBT at longer intervals. The study of deep-sea habitats is plagued by a lack of data, creating challenges that are exacerbated by the sheer vastness of the deep sea. The CCZ claim areas span almost 4500 km wide,vertical gardening in greenhouse larger than the continental U.S., which leads to high levels of uncertainty because of data limitation. A precautionary approach, wherein adequate measures are taken in order to manage risk, should be invoked . Therefore, collection of robust baseline data is critical for protection of the marine environment , as well as exhaustive monitoring, especially at the early stages of commercial exploitation . MBT and molecular approaches can be utilized together in these programs to facilitate future environmental assessment and monitoring.There are direct and indirect costs involved in collecting and processing sediment samples for MBT and metabarcoding. Here, they are separated into costs associated with consumables and labor . Other costs, such as laboratory equipment and bio-informatics pipelines, are held fixed. Choices within the decision networks are discussed in terms of how they affect the short-run economic cost. We assume that laboratories have basic amenities and access to people who are trained to do these tasks, i.e. identifying target taxa, preparing sediment samples for sequencing. Deep-sea samples, whether sediment, water, or individual organisms, are relatively expensive to collect because they often require research expeditions on global-class vessels equipped with specialized instruments.

One ship-day on a U.S. vessel can range from $42,000- 48,000 USD, which includes food, fuel, crew, and two technicians . The amount of time it would take to sail from the Scripps Institution of Oceanography pier to the eastern edge of the CCZ is approximately six days at full speed without stopping, resulting in over a quarter million dollars in transit time one-way. This cost estimate does not include the use of autonomous underwater vehicles , remotely-operated vehicles , or human-occupied vehicles , commonly used on deep-sea expeditions. In addition to collecting sediment samples for both MBT and sequencing, deep submergence vehicles can conduct visual surveys. The U.S. National Deep Submergence Facility operates their instruments at day-rates of $14,000 for AUV Sentry, $23,000 for ROV Jason, and $45,000 for HOV Alvin . Other sampling equipment includes box corers, multi-corers, epibenthic sleds, and CTD rosettes which can be deployed without special vehicles. Relevant results and sampling regimes from studies included in this paper are summarized in Table 3.2. Sediment samples were taken with multi-corers, box corers, or an epibenthic sled in one case, and the same sampling devices can be used for both approaches. The total number of samples used for analysis ranged from 5-41 and 15-42 per cruise for MBT and metabarcoding, respectively. Here, we assume number of samples equates to number of vertical fractions . Sampling costs are likely the biggest element but should not contribute significantly to cost differences between the two analytical approaches. However, the different methods used to process deep-sea sediment samples are a major factor.Consumables required for MBT are common to most labs and are readily available .Formalin is cheaper, easier to ship , and fixation results in little distortion of morphology. However, specimens fixed in ethanol can later be used for molecular studies which is advantageous and cannot be done with specimens fixed in formalin. The total cost of lab consumables for MBT ranges from $13.10-16.29 USD. Labor and expert time are the dominant costs of the MBT workflow . Based on a graduate student stipend in the U.S. of $29,500 USD annually, time alone ranges from $325-2,875 per sediment core slice. Additionally, the estimates shown here are only for the taxa listed for one vertical fraction of a sediment core although the top several centimeters are likely necessary for biodiversity characterization. The extensive time needed to manually sort and identify organisms, especially when using smaller sieve sizes that retain more sediment, is a large cost and a disadvantage of using MBT. Ludoxâ flotation, a method of concentrating animals in a liquid medium, can reduce picking and sorting times but adds additional consumable costs. The estimates we have provided are based on hand-sorting. There are many options when processing marine sediment for metabarcoding , influencing scientific outcomes and economic costs. A metabarcoding approach can also be used in parallel with other omics methods if relevant to objectives . The number of replicates in a study can refer to either sample replicates or PCR replicates. If there is enough sediment material in each sample, then pseudo-replicates can be added. PCR replicates are more often used and can help minimize errors due to PCR, such as unequal amplification and chimeras . Additional replicates yield more robust results and higher statistical power, but increase total cost . Sampling replicates and pseudoreplicates can also apply to MBT. Metabarcoding utilizes highly conserved fragments of DNA that all organisms have and within these fragments are markers, which are highly variable regions that allow for higher resolution taxonomic classification. Choosing markers, how many and which ones, can affect results and will depend on scientific objectives. 16S rRNA can be used to assess microbial diversity whereas eukaryote diversity is typically assessed with markers within the 18S rRNA or cytochrome oxidase I genes . More comprehensive diversity assessments may require multiple markers in order to completely capture the community. Advantages to working with widely-used markers include a more optimized protocol, the ability to compare to other studies, and a larger database. However, there is no truly universal marker that allows the retrieval of all taxa without bias. Additionally, a universal marker must be very conserved and therefore less useful for species-level taxonomic assignments. Development of markers targeting indicator taxa allows for better detection of them . These bioindicators can then be used for characterizing specific habitats, such as sediment plumes, and early detection of mining impacts .

Assessing the central nutritional role of indigenous agriculture alongside that of hunting and gathering might well suggest that malnutrition, rather than simply the spread of diseases through concentrated settlement, made communities less likely to recover from infectious epidemics. According to Snow, had “European expansion been less rapid, and had lethal epidemics not swept the landscape clear of Indian resistance as effectively as they did, the dynamics of historic cultural adaption” on the Great Plains and at the previous sites of European contact might have been different. But it is worth students and researchers asking a slightly different set of questions: Did the inability to reproduce horticultural and hunter-gathering methods actively contribute to Native American demographic decline following epidemics, rather than simply demonstrating another unfortunate result of the Biological Exchange? Is it possible to draw any stronger conclusions in regard to direct causation, rather than more general association, in assessing the dual disruption to hunter-gathering and horticultural practices? Did the change in Native American diets following European contact directly impact the attendant increase in mortality rates – as distinct from epidemics elsewhere in the world where demography was able to re-stabilize after around a century?

In order to consider such a possibility,plastic pots 30 liters students and researchers should turn to the literature on medical anthropology, paleo-archaeology, and modern experimental data on the link between health, immunity, and the consumption of vitamins and important minerals. Disrupted access to macro-nutrients and micro-nutrients – whether derived from hunted and gathered animals and plants, from indigenous agricultural practices, or a combination of both – might be defined as a co-factor alongside specifically pre-determined genetic loci and/or the biological exchange of diseases. Infectious diseases began to ravage Native American populations from the moment of first contact. But an inability to recover immunity and/or fertility, according to a working hypothesis, might have been exacerbated by declining access to ancestral sources of food. To assess these possibilities in greater detail, and how they might be understood by researchers and students, let us turn to more specific examples that relate to the hunting of animal products and the gathering and/or managed cultivation of vegetables, fruits, tubers, and seeds. They allow a potentially fruitful synthesis between early-contact history and modern research in evolutionary medicine and nutrition. Emphasizing the role of colonial human intervention rather than an amorphous biological exchange, modern historical scholarship provides further evidence of the problematic impact of European domestic agriculture on Native American health and fertility after contact.

Aside from its greater propensity to affect the spread of infectious diseases, we now know that the proliferation of small enclosures of cattle and grain physically disrupted hunting and gathering practices, as well as pre-contact forms of plant horticulture, and reduced their attendant nutritional gains. According to biological anthropologist Clark Spencer Larsen, the emphasis on disease in the biological exchange thesis “has overshadowed a host of other important consequences of contact such as population relocation, forced labor, dietary change, and other areas.”Meister similarly notes that “later population decline resulting from disease was made possible because Indians had been driven from their land and robbed of their other resources [including hunted animals and cultivated crops].”According to Anderson, “before long, the expansion of livestock-based agriculture ceased being a model for Indian improvement and instead served almost exclusively as a pretext for conquest, a very different expression of the cultural impact of distinct farming practices” among Europeans and Native Americans in eastern North America from the 1600s.As Kunitz has pointed out in a discussion of the paleoepidemiology of southwestern Native American communities, and their malnutrition following subsequent European colonization, “one does not need to invoke large-scale dramatic epidemics; prosaic entities like malnutrition… are more than sufficient to do the job [in demographic collapse].”Though it remains difficult to address the direct triggers for final mortality, Thornton has assessed much evidence on the history of Plains Native Americans in the two centuries after contact and concluded that their “mortality and fertility” was severely impacted “when the great herds of buffalo were destroyed” by European agricultural patterns, Native American over-hunting in response to curtailed nutritional sources elsewhere, and open warfare.

Other scholars concur that an association can be drawn between worsening health, the declining ability to hunt animals and/or cultivate plants, and a new reliance on European agricultural production as animals on the Great Plains came to be over-hunted.Before turning to cultivated plants, let us now consider how students and future researchers might understand the ways in which declining access to hunted animal products reduced the consumption of important minerals and fat-soluble vitamins, and affected overall health, immunity, and fertility. We would begin with a historical assessment of the role of hunted animals in Native American history. Particularly during the winter, many Native American communities relied on hunting and gathering fatty cuts of meat for optimal nutritional health, long after Neolithic-era Europeans had moved towards domesticated animal husbandry and grain production. Frison’s classic work on the pre-historic practices of the High Plains, Great Plains, and Rocky Mountain regions, for example, suggests that nutritious organs and fats from buffalo meat were favored over and above other portions.Having considered the importance of hunting in ancient populations, students might then turn to seventeenth-century and eighteenth-century European observations of indigenous communities in the northernmost parts of continental North America. Hunting and gathering patterns in those regions were less altered by colonization, and so provide a more accurate picture of their ancestral nutritional profile within their regional context. Their populations relied on fats from fish and land animals as a greater proportion of their diet due to their climate and ecology.Students could then move on to observational studies from pioneering ancestral health theorists such as Weston A. Price, as well as those from more recent anthropologists who have worked in regions such as British Columbia and sub-Arctic Canada. As Hearne noted elsewhere, community members tended to select only the fattiest parts of the animal, or nutrient-dense organ meats, throwing the rest away: “On the twenty-second of July we met several strangers,round plastic pots whom we joined in pursuit of the caribou, which were at this time so plentiful that we got everyday a sufficient number for our support, and indeed too frequently killed several merely for the tongues, marrow and fat.”Students might then examine oral histories, archival collections, archeological records, interviews, and participant observation of contemporary practices, using methods and materials from ethnographers such as Richard Daly, who has noted the historical preference for fish fats among Delgamuukw indigenous peoples in British Columbia, going back several centuries in communal memory. According to Daly, fat “rendered from salmon heads was prepared in summer, hung in bladder pouches in the rodent-resistant family meat caches, and saved for winter use.” Oils were “prepared from fatty fish and meat such as oolichan, salmon and beaver. Special processes were involved in preparing the heads- drying or boiling them for oil-as well as the eyes, bellies and eggs”. Moreover the “arrival of the oolichan. . . was traditionally announced with the cry, ‘Hlaa aat’ixshi halimootxw!’ or, ‘Our Saviour has just arrived!’” Ooligan grease was thus a prized gift in feasts and between neighbors.Surveying modern communities of Native Americans, Weston A. Price’s 1939 Nutrition and Physical Degeneration noted a similar preference for animal and fish fats, and organ meats, and suggested its provenance in ancestral food patterns that dated back centuries and even millennia.

The indigenous communities Price encountered were seen to prize the fattiest parts of meat and fish, including organ meats, rather than muscle-cuts. As Fallon Morell and Enig have summarized, Price linked a diet high in fats from mammals and fish to “an almost complete absence of tooth decay and dental deformities among native Americans who lived as their ancestors did… [including among] the nomadic tribes living in the far northern territories of British Columbia and the Yukon, as well as the wary inhabitants of the Florida Everglades, who were finally coaxed into allowing him to take photographs… Skeletal remains of the Indians of Vancouver that Price studied were similar, showing a virtual absence of tooth decay, arthritis and any other kind of bone deformity…”Ironically, in order to move beyond conjecture when discussing declining health markers after European contact, paleo-anthropological and paleo-archaeological evidence for the precontact Native American consumption of maize provides students and researchers with a helpful framework. Evidence for diminishing health following the introduction of maize raises the hypothesis that consumption of the grain came at the expense of more nutrient-dense calorie sources from animals and other plant species. Familiarity with such a hypothesis should help students and researchers to examine a similar – albeit greatly increased – association between the intervention of colonial European agriculture and the failure of Native Americans to recover demographically from infectious diseases in the three centuries after contact . In eastern North America from A.D. 800 to 1100, a shift towards maize-centered agriculture took place in regions as far afield as the Southwest and the Eastern seaboard. Hunted and gathered meats, as well as nutrient-dense crops such as gourds, seeds, and tubers, declined as a proportion of overall calorific consumption. Across the South Atlantic and Gulf Coastal plains, the shift towards maize cultivation as a vital source of calories was associated with the development of “socially ranked societies” and “fortified civic ceremonial centers” placed near maize storage centers. Corn came to be central to the activities of Iroquois confederations stretching from the east coast to the Ohio River valley, as well as in the Mississippian chiefdoms that grew along the river-ways of the Southeast and Midwest.

On the one hand, the cost-to-yield ratio of pre-contact maize cultivation may have become seemingly more attractive because of an increased demographic pressure on both wild and domesticated resources. That is to say, increased mortality due to food scarcity from hunted and gathered sources might have been diminished by the calories provided by maize. Yet on the other hand, there is a growing scholarly consensus that health and immunity decreased following the indigenous production of maize among North American communities around 2000 years ago, particularly in the American southwest. It may well have provided a source of energy to keep Native American communities alive. But those communities, when compared to populations who had preceded the demographic pressure placed on hunter-gathering, seemed to register declining health markers. That which saved them from death through famine was not necessarily nutritionally optimal as a dominant calorie source. To be sure, we can find historical records from the contact and pre-contact era that suggest several indigenous Native American methods to increase the nutritional profile of corn, some of which may have been introduced in pre-historic communities as they shifted towards the grain. These include soaking it in lye made from wood ashes, in order to make it more easily ground. Doing do also likely made the protein and niacin in corn more bio-available, due to the alkalinizing effect of the wood ashes/slaked lime solution. Greater absorption of Niacin is associated with greater bio-availability of important minerals such as Calcium and Potassium. Nonetheless, the production of “hominy” from corn in this way may not always have been enough to counteract potential nutritional deficiencies associated with the shift toward the grain.In order to understand the distinction between famine-prevention and optimal nutritional health, students and researchers would be able to consider a number of different case studies. From the Great Lakes region to the southern plains, and from eastern coastal to Pacific populations, for example, the regular consumption of bison, deer, and fish likely prevented protein and iron deficiencies that later accompanied the reliance on maize for a greater proportion of the daily calorie output of Native American communities.Analysis of bones from prehistoric North America reveals that, long before European contact, potentially problematic indicators followed the move towards maize consumption. Several studies have focused on growth retardation and suggested its negative association with health markers more generally. Declining stature has been correlated with the onset of agricultural production of maize during the first centuries of the Common Era. Archaeological evidence from late prehistoric Dickson Mounds populations in west-central Illinois show that agricultural intensification led to a decline in skeletal weight and height. In the Lower Illinois Valley children under six in nascent maize producing societies have been found to have suffered growth retardation compared to hunter-gathering communities from nearby excavations.

Soil pathogen control with 1,3-D followed by metam sodium and 1,3-D with intermittent water seals was inconsistent between the two experiments, which suggests that specific micro- and macro-level differences in environmental and field conditions may contribute to greater treatment variability and risk to growers.When 1,3-D was sealed with HDPE and VIF, broad leaf weed density was reduced to less than 6 weeds per square meter, which was comparable to methyl bromide . These results are similar to a previous nursery study that indicated 1,3-D or 1,3-D plus chloropicrin sealed with HDPE or VIF resulted in weed seed viability and hand-weeding time comparable to methyl bromide . Generally, intermittent water seals after a 1,3-D application resulted in broad leaf weed density similar to the untreated control. Most weeds germinate near the soil surface,microgreen fodder system thus techniques such as intermittent water seals that limit upward fumigant movement into surface soils can adversely affect weed control.

The other surface treatments 1,3-D dual application and 1,3-D followed by metam sodium had intermediate broad leaf weed densities compared to untreated plots and methyl bromide. All fumigation treatments reduced grass weed populations compared to the control plots; however, the greatest reductions were observed in plots treated with methyl bromide, 1,3-D sealed with HDPE or VIF, and 1,3-D followed by metam sodium. It was clear in this study that effective surface treatments can greatly increase weed control with 1,3-D; however, even the best treatments will likely require supplemental weed control to meet grower expectations.Effects of surface seal treatments and 1,3-D soil fumigation on nursery stock vigor and performance in two nursery trials were evaluated in 2007 to 2010 . In the rose nursery trial, all treatments had similar root stock vigor and number of marketable plants except when 1,3-D was followed by metam sodium. During the 2008 growing season, roses grown in plots treated with 1,3-D followed by metam sodium had lower vigor than the other treatments; however, by harvest at the end of the second year, no differences in marketable plants were observed. In the tree nursery trial, tree root stock vigor was reduced in plots treated with 1,3-D followed by metam sodium and1,3-D with intermittent water seals compared with the other fumigation treatments, but root stock caliper at the end of the first growing season did not differ among treatments.

Compared with some other fumigation-dependent industries, perennial fruit and nut nursery stock production systems face a more difficult transition to methyl bromide alternatives . Despite several years of research, the following significant challenges to widespread adoption of alternatives in the perennial crop nursery industry remain: National and international market expectations for nematode-free nursery stock limit nursery stock producers to alternatives with very high nematode efficacy at significant depths in the soil. To meet California nursery certification requirements, producers are required to use approved fumigant treatments or conduct a post production inspection. A failed inspection may result in an essentially nonsalable crop. Most alternative treatment schedules are based on the use of 1,3-D , a fumigant that faces its own serious and evolving regulatory issues in California. No currently available alternative fumigant can be used in California to meet certification requirements in nurseries with fine-textured soil at registered rates. Methyl iodide, the alternative fumigant with performance most similar to methyl bromide, is not currently registered in the United States due to a voluntary withdrawal by the manufacturer. Concerns over control of weeds and fungal and bacterial pathogens in the short and long term may further limit adoption of alternatives with a narrower pest control spectrum. 

Containerized nursery stock production systems are being used in some parts of the industry, but the production costs, market acceptance and long-term viability of this system have not been addressed at the required scale.Adoption of methyl bromide alternatives, where they exist, in the perennial crop nursery industry will ultimately be driven by state and federal regulations and economics. Although it’s heavily regulated, 1,3-D is a viable alternative for growers with coarse-textured soil, but if 1,3-D becomes more difficult to use due to shortages or increasingly stringent regulations, it may be only a short-term solution. No viable fumigant alternatives exist for California nurseries with fine-textured soil, and some of them may be unable to produce certified nursery stock in the absence of methyl bromide. The cost of producing perennial nursery stock using more expensive, laborious or economically risky production methods will ultimately be passed on to customers and could have long-term impacts on the nursery, orchard, vineyard and ornamental industries.Citrus fruit is very important to the Indonesian farmer, since it can give him more income than other crops. According to some surveys , he can get at least five to six times as much income from a 5-6- year-old citrus planting as from groundnut and four to five times as much as from rice cultivated on equal acreages. Because of good yields and prices for certain mandarin varieties, more and more acreage is being planted. Although no exact statistics are available, it is estimated that in West Java alone, at least 10,000 ha are planted with citrus . Except in some places, i.e., the southern part of West Java and the northern part of West Borneo, mandarin trees are not planted as special citrus orchards, but are usually mixed together with other crops. Citrus plantings are scattered throughout the larger islands of Sumatra, Java, Madura, Borneo, Sulawesi, Bali, Lombok, etc. There are varieties that can be grown well at lower altitudes, like the Siam, but there are also good varieties that can grow and give good quality fruit at altitudes of 700 to 900 m like the well-known Garut variety. Therefore,barley fodder system the farmer has the opportunity to choose varieties suited for his situation. The gradual, but steady extension of citrus plantings carries with it some problems that are becoming more and more serious. Nearly every citrus grower knows that in Some places the original good quality mandarin trees which were many years old declined and were replaced by small, chlorotic citrus trees with marble-sized fruits. The farmer could not realize that introductions of new stock material from certain other areas could endanger the already established citrus trees, and instead, soil and climatic factors were blamed for the disease situation. The farmers tried to eliminate the symptoms by putting on more manure and fertilizers, and even by spraying the trees with pesticides. Presently, most of the citrus growers know that these efforts are useless. The magnitude of destruction is not small. In West Java alone, it was estimated that from 1960 onward, not less than 3 million trees were destroyed , and we know that this destruction is still taking place. Research on this problem was begun in 1954 and research workers of the Horticultural Research Institute, the Bogor Institute of Agriculture, and the Padjadjaran University have made contributions. Since deficiency symptoms of some macro- and microelements were found on leaves of declining trees nearly everywhere, much was done to correct these symptoms by applying fertilizers containing these elements to the soil or by foliar sprays. Reitsma et al.were optimistic that some carbamates such as Zn-dithiocarbamate, Fe-dithiocarbamate, and Mn-dithiocarbamate could remedy these respective deficiencies.

Soil applications of NPK fertilization trials together with microelements were made by some other workers at the Bogor Institute of Agriculture. Some of these trials yielded a more-or-less positive result, but generally it was temporary. After the applications had ceased for some months, the deficiency symptoms reappeared as before. The idea that there was not only a shortage of some elements, but that there could be other factors causing the deficiency symptoms was suggested by some workers. Reitsma and Hadiwidjaja stated, “Though as a rule poor drainage and hardpan are the main fac- tors which lower the vitality of the trees, giving rise to injury of the rootlets by secondary parasites, malnutrition, on the other hand, resulting from insufficient or inadequate fertilization may indirectly intensify the severity of the disease.” Indeed. this statement was not totally incorrect, i.e., for trees not affected by the disease which will be discussed later. Tylenchulus semipenetrans. Cobb, a citrus nematode that causes some problems in other citrus regions of the world, was found by Thrower on citrus roots in several places in Java. Also he found Xiphinema sp. He applied Nemagon to correct the general vigor of the trees, but no positive reAsults were obtained. Inoculation trials with T. semipenetrans also failed to produce chlorotic symptoms on the inoculated seedlings, although the nematode was established on the roots . It was Terra who first expressed the opinion that the probable cause of the bad vigor of citrus trees in general, and the decline of some varieties on sour orange, in particular, was tristeza virus. Inoculation trials done by Thrower on lime seedlings indeed proved that tristeza virus was present in the declining mandarin trees tested. For the moment, it seemed certain that tristeza was to blame for the destruction of so many trees. However, if one considers the problem more closely, symptoms manifested on indicator plants such as vein clearing, vein flecking, and stem pitting merely indicate the presence of tristeza, and these symptoms are by no means generally found on declining mandarin or other citrus trees, except perhaps on limes. So these results did not yet prove that the decline was caused bv tristeza. Proof that the decline was caused by a virus or a complex of viruses required that certain symptoms generally present on declining trees could be reproduced constantly on inoculated seedlings of the same variety. Tirtawidjaja showed that certain symptoms could be reproduced consistently on graft-inoculated seedlings of several species and varieties, including the variety of mandarin declining in the field. At that time, there was no knowledge about mycoplasmas causing plant disease, so it was concluded that the cause of the constant and persistent symptoms was a virus or a complex of viruses . Owing to the absence of similarities with symptoms and other properties of known virus diseases at that time, the authors concluded that this was a new virus disease. It was named “Citrus vein phloem degeneration disease,” for the most specific symptom, the collapse of certain cells of the phloem in leaf veins. The phloem of the mature, yellow or chlorotic leaves is much thicker than normal . Sieve tubes and companion cells are collapsed, and form white bands extending from the sclerenchyma to the xylem. Ray cells remain intact, but are filled with abnormal quantities of starch granules.The external and the internal symptoms were used for diagnosis of CVPD,first in Java and later in other islands of western and central Indonesia. In 1964, the disease was found widespread in several places in west Java and was also found in central and southern parts of central Java and in certain areas of east Java . Further surveys in recent years revealed that CVPD is not confined to the island of Java, but is also present in several places in Sumatra. Nearly all provinces of the island have citrus areas with declining trees which show the characteristic symptoms. On the other hand, no indication of the disease was encountered in the citrus areas of west Borneo, south Borneo, Sulawesi , Madura, Lombok, and others, except on limited numbers of trees in some yards of Pontianak and Ujungpandang . So it is Sumatra and Java that suffer the most from CVPD. This is probably due to the fact that Pasarminggu was and still is functioning as a center of distribution for citrus nursery stock, and Pasarminggu is blamed for the distribution of CVPD in Java and Sumatra. A certain mandarin variety was once known only in Pasarminggu, but now it is widely grown throughout Java and Sumatra. The spread of CVPD was further enhanced by the acquired ability of nurserymen to produce budlings. In certain places, which are CVPDendemic areas, hundreds of thousands of mandarin budlings are being produced, sold and distributed to new and old citrus centers. The Garut area is the most important source of CVPD-affected nursery stock. With past experience that one could obtain more income from citrus than from other crops, but without the new knowledge that CVPD can wipe out whole areas of citrus, this nursery material is being used extensively, especially in Java and Sumatra.

Jasmonic acid and ET are known to be involved in defense responses against necrotrophs, such as mediating the host’s responses against them, but ET is also required for fruit ripening and senescence processes, which are conducive to disease susceptibility. Jasmonic acid can also mediate the disease resistance of fruit by increasing the fruit antioxidant capacity, but some fungi are able to hijack the JA signaling pathway to cause disease. Although the early steps of JA biosynthesis were highly induced upon M. laxa inoculation, downregulation of receptor genes was observed in mature fruit inoculated with M. laxa when compared to controls. These findings suggest that M. laxa could be somehow blocking the JA signaling pathway, although the mechanisms involved are unknown. Ethylene biosynthesis increases during ripening of climacteric fruit, such as nectarines. In our study, the control immature fruit produced basal ethylene levels, whereas ethylene production in control mature fruit increased through time after harvest. In inoculated immature fruit, there was a significant peak of ET production as compared to the control at 24 hpi. This discrete induction of ET can be part of the fruit defense responses against M. laxa.

Alternatively,dutch bucket for tomatoes the pathogen could be inducing fruit ethylene biosynthesis in immature fruit to accelerate ripening, in an attempt to promote fruit physicochemical changes that are conducive to disease. Along this line, ACS2 and ACO1, involved in system 2 ET production, were over expressed in inoculated immature tissues. Previous studies have reported on a similar modulation of ET biosynthesis by the pathogen. However, after 24 hpi, ethylene levels in inoculated immature fruit fell to control levels, and the fruit remained resistant. This may be in part due to the upregulation of the ethylene signaling inhibitors EBF1/2, which could mitigate the ethylene-induced ripening processes that contribute to susceptibility. In contrast, in inoculated mature fruit, ET production and signal transduction were lower at 6 hpi in inoculated fruit but grater from 24 hpi onward, following the autocatalytic system 2 ethylene biosynthesis. Overall, the results indicate the ability of M. laxa to differentially alter ET production to promote susceptibility and, in turn, the ability for immature fruit, but not mature fruit, to mitigate the consequences of this induction. The above observations indicate that although M. laxa was deploying some strategies to infect the immature tissues, it was not able to overcome either the surface or the active defense responses deployed by the immature fruit. Monilinia laxa remained on the immature tissue, increasing its biomass and multiplying on the surface, until 14 hpi when it ceased to grow.

It is known that Monilinia spp. can remain quiescent on fruit surfaces and that they can employ appressoria as resting structures on immature nectarines. After 14 hpi, M. laxa biomass and reads started to decrease, switching its transcriptional machinery by employing different sets of genes in order to deploy different strategies to survive on the fruit’s surface. Some results point out that M. laxa could either be starting a quiescence period or moving toward an autolysis process, breaking cells to feed on its remains. Another possibility is that the remaining M. laxa cells on immature fruit were being attacked by the host defenses. This is supported by the expression of M. laxa genes associated with response to oxidative stress at late time points, such as catalases, previously reported in detoxification during infection of tomato leaves by B. cinerea. Thus, it is likely that immature fruit was generating reactive oxygen species during the interaction through an oxidative burst to kill the pathogen. Monilinia laxa could also be producing ROS for its development and as a pathogenicity mechanism to damage the host tissue. Particularly, the NADPH oxidase complex is involved in both fungal ROS production and its use in sclerotia development and virulence. Some genes encoding the Nox regulator R  were found to be upregulated at 24 hpi in both mature and immature tissue. At later stages, a highly induced alcohol oxidase expressed in immature tissue at 48 hpi could be another ROS producer, previously described as an alternative ROS production system. Lin et al. demonstrated that AOX1 was involved in pathogenicity and oxygen stress responses in B. cinerea.

Concomitantly, nectarine counteracted the pathogen oxidative burst by expressing genes of antioxidant metabolism compounds such as glutathione and redox-related amino acids . Plant secondary metabolites such as terpenoids have been described to protect the fruit under biotic and abiotic stresses, although their role can be tissue dependent. Overall, the enrichment of genes involved in secondary metabolite biosynthesis was higher in resistant immature than susceptible mature tissue, which suggests that either the host was producing terpenoids in the resistant immature tissue to prevent the attack or that M. laxa was inhibiting its biosynthesis on mature tissue. Monilinia laxa could also be able to degrade and transform terpenoids as described for B. cinerea. The phenylpropanoid metabolism is also triggered in response to brown rot. In both immature and mature fruit, from 14 hpi to 48 hpi, phenylpropanoid-related pathways were highly induced. While on the immature tissue, these pathways could be involved in reinforcing the cell wall through lignin production, the role in the mature fruit could be more focused on the detoxification of fungal ROS production. Nevertheless, these hypotheses need to be further tested. On mature nectarines, M. laxa deployed other virulence factors in addition to ROS production and scavenging. The pathogen expressed upregulated DEGs related to proteolytic activity, containing domains such as the Pro −kumamolisin domain . The list of genes summarized in Table 1 could be putative pathogen target genes as they were expressed only when M. laxa infected the mature tissues, as none of the top five upregulated genes in mature tissue was found in the immature fruit. For instance, the highest expressed protease at in all time points is a homolog of a nonaspartyl protease found during pathogenesis in Sclerotinia sclerotiorum. Cell wall-degrading enzymes are commonly produced by necrotrophic fungi as virulence factors and their secretion by Monilinia spp. on culture media has been previously reported. A rhamnogalacturonan hydrolase ,blueberry grow pot which was highly expressed at both 24 and 48 hpi, was already described as a putative virulence factor in M. laxa infecting peaches. Current information regarding the strategies utilized by either Monilinia spp. or stone fruit or during their interaction is mainly focused on specific metabolic pathways or actions developed by one of the two players. As a novel feature of the present research, we demonstrated the synchronized responses from nectarine and M. laxa, by utilizing a resistant immature and susceptible mature fruit throughout a course of infection. Future research studies should be focused on delving into the host defense system for the ongoing development of nectarine cultivars with increased resistance to brown rot, as well as conducting in-depth fungal studies to alter the ability of M. laxa to cause disease.“Venus” nectarines Schneider were obtained from an organic orchard located in Raïmat . Fruit was bagged 6 weeks before the last harvest and then harvested at two different fruit developmental stages, “mature” and “immature” , and used immediately after harvest.

Injured or deformed fruit was discarded, and fruit for analysis was further homogenized by using a portable DA-Meter , based on the single index of absorbance difference . Other assessments of quality parameters were performed on 20 randomly selected fruit , according to the method of Baró-Montel et al..The M. laxa single-spore strain 8L was used for all experiments. Fungal conidial suspensions were maintained and prepared, as described by Baró-Montel et al..Each fruit was inoculated with the application of six 30- μL drops of a conidial suspension at a concentration of 106 conidia mL−1 on the fruit surface. Mock-inoculated fruits were equally treated with sterile water containing 0.01% Tween-80. Fruit were placed in closed containers with a relative humidity of 97 ± 3% at 20 ± 1 °C.Six cylinders of peel and pulp tissue encompassing the inoculation sites were sampled from each fruit and pooled for each replicate. Samples were immediately flash-frozen in liquid nitrogen and stored at −80 °C until extraction. For symptom analysis, inoculated fruit was imaged at the set time points. Ethylene production of both mock and M. laxa inoculated immature and mature fruit was determined, as described by Baró-Montel.In the high input year round strawberry and vegetable cropping systems of coastal California, organic growers have to balance their desire to reduce environmental impacts with the multiple challenges of maintaining economically sustainable yields. These challenges include providing adequate plant nutrients while minimizing losses to protect water quality, maintaining low levels of disease, and building effective system-based pest management strategies. In comparison with conventionally managed systems, little research on organic systems has been available for growers to help meet these challenges and so in 2004 we formed a research-extension-grower network to develop and implement a collaborative organic research program . The California Collaborative Research and Extension network which we report on here grew out of this initial program. The first 4-5 years of research, known as the Organic Research Network Project, was funded by the USDA-Organic Agriculture Research and Extension Initiative program in 2004 , with the goals of building an organic research and extension network to support organic vegetable and strawberry producers in the region and to develop integrated fertility and pest management strategies to minimize negative impacts of agriculture on surrounding natural ecosystems and improve the economic viability of organic farming. Multiple field experiments were carried out including cover crop/fertility trials , a strawberry rotation trial , studies of the role of trap crops and hedgerows in arthropod biological control , and development of anaerobic soil disinfestation as a technique for controlling soil borne diseases in strawberry production . The current CAL-CORE program represents an extension and expansion of this earlier effort, bringing additional researchers, farmers, regions, and organizations into the network. Our goal is to provide a dynamic platform for network members to collaboratively research and evaluate integrated systems approaches to improve environmental sustainability and economic viability in a comprehensive manner. Using the information generated previously, a series of field experiments have been undertaken to examine the effect of crop rotations and specific management practices on productivity and economics as well as ecosystems services such as nutrient cycling; pest, disease, and weed suppression; soil carbon sequestration; and greenhouse gas emissions. A combination of direct measurement, modeling and Life Cycle Analysis are being used to describe the environmental imprint of the management systems being tested. The centerpiece of the project is a replicated rotation study being conducted at the University of California, Santa Cruz, Center for Agroecology and Sustainable Food Systems farm site which has been under organic management for 40 years. In addition a subset of the treatments in this study were chosen by six farmer collaborators to test on their fields. A number of additional satellite studies are also underway, but here we report on some of the preliminary findings from the main rotation study which is now in its 4th year and reflect on the functioning of the network as a mechanism for collaborative research and outreach. The goals of this study are to compare rotation length and crop composition on soil fertility and soil borne disease dynamics when used alone or in conjunction with soil amendments or anaerobic soil disinfestation. The CASFS field is an Elkhorn sandy loam , and the experiment is a split-split plot design with four replicates. It compares two and four year vegetable/strawberry rotations using combinations of crops believed to be either suppressive of a major soil borne disease , or more profitable but more conducive to disease. Superimposed on the rotations are fertility treatments : legume/cereal cover crop only, legume/cereal cover crop + compost + additional fertility amendments, cereal cover crop + mustard seed meal, or untreated control. In the two legume/cereal cover cropped treatments anaerobic soil disinfestation is used for disease management prior to planting strawberries. The study is in year 4 and all treatments were planted to strawberries in 2014. Winter cover crops in treatments 1a, 2a, 5a, 6a, 1b, 2b, 5b, and 6b were a mixture of bell bean 45%, purple vetch 45%, and cereal rye 10% and were planted at a rate of 367 kg ha-1 in November each fall and incorporated with a spader in April to May. In treatments 2a, 6a, 2b and 6b, compost and organic fertilizer were added pre-plant for each crop.

Recent years have seen rapid growth in the number of published studies, increased numbers of scientific conferences and development of legal framework and legislation. These trends will inevitably improve the image of this industry and the efficacy of products. Two significant problems still exist within the industry broadly: preparations of products with highly complex multi-component and incompletely identified composition make the identification of a primary mode of action extremely difficult and the current classification and legislation/legal framework for regulation of bio-stimulants is based primarily on source material and not on biological mode of action. Hence there is insufficient capacity to differentiate products, and there is the potential for the successful demonstration of a single product within a bio-stimulant category, to falsely indicate the efficacy of the group as whole. Modern bio-stimulants are complex mixtures derived from raw materials of highly diverse origin utilizing highly diverse manufacturing processes and as such can be expected to have a broad spectrum of possible biological activity and safety. To distinguish bio-stimulants from the existing legislative product categories including essential nutrients, pesticides, or plant hormones a bio-stimulant should not solely function by virtue of the presence of elements or compounds of known function.

We propose, therefore,vertical hydroponic nft system a definition of a bio-stimulant as “a formulated product of biological origin that improves plant productivity as a consequence of the novel or emergent properties of the complex of constituents and not as a sole consequence of the presence of known essential plant nutrients, plant growth regulators, or plant protective compounds.” Consistent with this definition, the ultimate identification of a novel molecule within a bio-stimulant that is found to be wholly responsible for the biological function of that bio-stimulant, would necessitate the classification of the bio-stimulant according to the discovered function. This novel definition is inspired by three observations: that the development of the bio-stimulant industry will inevitably result in the discovery of novel biologically active molecules and that the identification and classification of these molecules will benefit biological discovery more greatly if these molecules are explicitly described than if they were merely labeled as “bio-stimulants,” that there is a need for the nascent bio-stimulant industry to explicitly discourage the inclusion of nutrient elements and known biologically active molecules under the guise of a “bio-stimulant” and that there is a need to recognize that classic reductionist biology/chemistry may indeed be insufficient to explain biological complexity . The definition provided here is important as it emphasizes the principle that biological function can be modulated through application of complex mixtures of molecules for which an explicit mode of action has not been defined. The definition also requires a demonstration of beneficial impacts of the bio-stimulant on plant productivity.

Given the difficulty in determining a “mode of action” for a bio-stimulant, and recognizing the need for the market in bio-stimulants to attain legitimacy, we suggest that the focus of bio-stimulant research and validation should be upon determining the mechanism of action, without a requirement for the determination of a mode of action. This can be achieved through careful agronomic experimentation, molecular or biochemical demonstration of positive impact on biological processes or the use of advanced analytical equipment to identify functional constituents. Given the prerequisite multi-component and emergent characteristics of bio-stimulants, the discovery of the mode of action is likely to require application of new techniques in bio-informatics and systems biology. While the definition proposed here suggests that the development and marketing of a bio-stimulant does not require a demonstration of the mode of action, it is still in the interest of the commercial producers of these products to pursue an understanding of these products so that the product can be improved and optimized for use in various environments and cropping systems. While there is a clear commercial imperative to rationalize bio-stimulants as a discrete class of products, there is also a compelling biological case for the science-based development of the bio-stimulant science that is grounded in the observation that the application of biological materials derived from various organisms, including plants, that have been exposed to stressors can affect metabolic and energetic processes in humans, animals, and plants . This hypothesis is based upon the premise that functional chemical communication occurs between individuals or organs that favorably modulate metabolic pathways and networks at different plant hierarchical levels.

Inter and intra organism communication and consequent molecular and metabolic regulation are at the heart of the science of systems biology and the tools of systems biology will inevitably be critical to the realization of mode of action of many bio-stimulants. Continued investments by commercial entities in bio-stimulant research and product development will serve as a critical driver of discovery in this realm and will inevitably lead to the identification of novel biological phenomenon, pathways and processes that would not have been discovered if the category of bio-stimulants did not exist, or was not considered legitimate.Obesity is a multi-factorial disease that is challenging to treat, requiring several considerations and components encouraging behavioral modifications. Adolescent obesity can have lasting consequences on health as it is associated with adulthood obesity and thus may affect long-term quality of life and lead to development of chronic diseases. Prevalence of adolescent obesity has progressively increased over the last several years and adolescents consistently have the highest rates of obesity among youth. In 2016, 1 in 5 adolescents were classified as obese, with prevalence of obesity highest in Hispanic and non-Hispanic Black adolescents with 25.9% and 25%, respectively. These values were above the 20.6% average for adolescents aged 12–19 years and higher than that of non-Hispanic White youth within the same age group.Youth who reduce incidence of obesity mitigate the associated increased risk of adulthood chronic disease, instead exhibiting comparable risk to that of youth who were never obese. This supports an urgent need to educate adolescents as they transition to experiencing more autonomy in food choices and other lifestyle behaviors that arise with emerging adulthood. Poor diet quality may contribute to the high prevalence of adolescent obesity. Over 50% of youth had poor diet quality in 2016 . Youth are well below meeting dietary recommendations despite having quite high nutritional requirements to support a period of immense growth. Diet quality progressively decreases as youth advance in age, with high school-aged adolescents having lower diet quality compared to youth of elementary school age. In particular, adolescents aged 14–18 years do not meet recommendations for consumption of fruits, vegetables, and whole grains. Adolescents in the lowest quartiles of intake for each food group tend to continue having low levels of intake into adulthood. Consistently and of particular concern, youth from low-income communities tend to have the poorest diet quality. While not the only consideration, poor diet quality of adolescents may be attenuated with advancement of food literacy. Beyond the focus of traditional nutrition education,nft hydroponic system food literacy requires understanding of food procurement and preparation. Food literacy involves having the knowledge and skills necessary to make healthy dietary choices and comprises 11 components within 4 domains and 15 attributes within 5 categories. Many nutrition education programs utilize some of these elements, however few incorporate all. Components of food literacy were extrapolated from surveying experts and young adults and attributes were identified through a scoping review of the literature. Food literacy components are specific while attributes are more thematic. For example, the component “determine what is in a food product, where it came from, how to store it and use it” encompasses several attributes related to food selection and preparation. These elements include both critical knowledge, such as understanding nutrition-related information, and functional knowledge, wherein application of knowledge through skills and choices is essential, that intersect to aid in developing and maintaining healthy food behaviors. Education in one domain or category is not sufficient for achieving food literacy, instead scaffolding of knowledge and skills from the various topic areas is required. A systematic and narrative review of food literacy programs for high school-aged adolescents found that interventions at least 4 weeks in length that included opportunities for advancement in knowledge and self-efficacy were most likely to affect short-term dietary behavior. Additionally, several recommendations for implementing food literacy interventions have been identified. Such recommendations include utilizing settings where adolescents normally congregate and engaging in weekly experiential activities that provide opportunities for application of food-related knowledge and skills.

Furthermore, it is recommended to tailor the program approach to the specific age group being targeted and to provide opportunities that support positive youth development. Despite the need, especially considering the high rates of obesity, food literacy programs targeting older adolescents are limited. This dearth in food literacy programming prevents adolescents from gaining knowledge and skills needed to make healthy food choices as young adults and perpetuates unhealthy food practices observed during childhood. Previous findings from a study conducted within the 4-H Youth Development Program found that adolescents did not have foundational knowledge to effectively lead garden-enhanced nutrition and cooking lessons. Focus groups completed in Australia found that adolescents had some prior food-related knowledge from participation in yearlong required courses, but had limited opportunities to apply that knowledge through food preparation. Participants in the focus groups expressed an interest in increasing food literacy through home economics courses. Home economics courses are increasingly rare in the United States and topics relevant to food literacy are often categorized into health courses. However, national Health Education Content Standards include a plethora of topics that must be covered in one semester and thus completing food literacy education outside of the typical school day may be more feasible. Informal settings, such as after school programs, encourage the acquisition of knowledge through lifelong, life-wide, and life-deep learning, which incorporate the people, places, and culture that every individual brings to a learning environment, whether in or outside of a formal classroom. This is especially helpful for learning concepts that directly impact learners’ everyday lives and require synthesis of various prior experiences in conjunction with newly acquired information. Unlike traditional classroom learning, which mostly applies to meeting objectives of school, such as completing exams and assignments, informal learning objectives can be directly applicable to knowledge needed for daily life activities. With this, the objective of this project was to develop a comprehensive food literacy curriculum for high school-aged adolescents to be implemented through after school programs. Developing curricula based on theories and recognizing needs of the target population are recommended for maximum efficacy. Furthermore, curricula that focus on behavior change and skill development in addition to knowledge attainment tend to be more successful . Therefore, Social Cognitive Theory and Constructivism were selected as theoretical frameworks while also considering the Social Ecological Model.Social Cognitive Theory is widely utilized in nutrition interventions and conceptualizes dietary change with consideration for the intersection of personal, environmental, and behavioral factors. Constructivism functions through a community of learners engaged in active discourse, allowing for creating knowledge together with the goal of deep and sustained learning. The Social Ecological Model provided context for factors that affect food choices of adolescents at various levels including local access, peer influence, and preparation skills, among others. The food literacy curriculum was developed following systematic procedures previously utilized to design a garden-enhanced nutrition curriculum for a multi-component school-based nutrition intervention called the Shaping Healthy Choices Program. The process began with assembling a development team including three experts in the overarching topic areas, agriculture, nutrition, and cooking, which were deemed necessary for development of food literacy through consolidation of the components and attributes, and 13 undergraduate interns. The experts collectively had extensive knowledge in curriculum development, nutrition, sustainable agriculture, food systems, garden-based education, recipe development, and cooking techniques. To develop the curriculum with intention, Backward Design was employed. The first step of Backward Design is to identify desired results, which was implemented through determining concepts that youth should learn after participating in the curriculum lessons. Interns were instructed to independently search for learning concepts by reviewing reputable resources, including peer-reviewed literature, government reports, and educational standards. Under supervision of the relevant content expert, learning concepts were grouped and consolidated into the three topic areas in addition to being reviewed for alignment with aspects of food literacy. This was proceeded by the second step of Backward Design, determine acceptable evidence, which was employed to develop learning objectives guided by authentic assessment.

However, more work is required to determine how to better represent coupled microbe–plant nutrient competition and transport limitations. For example, we have previously shown that one can apply a homogenous soil environment assumption and include the substrate diffusivity constraint in the ECA competition parameters. In this approach, the diffusivity constraint can be directly integrated into the substrate affinity parameter. The “effective” KM would be higher than the affinity measured, e.g., in a hydroponic chamber . We hypothesize that our calibrated KM value, which led to an excellent match with the observations , effectively accounted for this extra diffusive constraint on nutrient uptake. A second approach would be to explicitly consider fine-scale soil fertility heterogeneity, explicitly represent nutrient movement , and apply the ECA framework at high resolution throughout the rhizosphere and bulk soil. However, to test, develop, and apply such a model requires fine-scale measurements of soil nutrient concentrations,ebb flow tray microbial activity, and rhizosphere properties and dynamics; model representation of horizontal and vertical root architecture and microbial activity; effective nutrient diffusivities; and potentially, computational resources beyond what is practical in current ESMs.

Yet, there is potential value in this approach if we can produce a reduced order version of the fine-scale model that is reasonable and applicable to ESMs. A third approach, of intermediate complexity, would be to simplify the spatial heterogeneity of root architecture, soil nutrient distributions, and nutrient transport. Roots could be conceptually clustered in the center of the soil column, where nutrients would become depleted and competition between microbes, roots, and abiotic processes would occur. The “radius of influence” concept that defines a root influencing zone could be used to simplify heterogeneity, with CT5 competition applied to this root influencing zone. More model development, large-scale application, and model-data comparisons are needed to justify such an approach. As we argued above, the choice of nutrient competition theory used by ESMs faces a dilemma between necessary model simplification and accurate process representation. Our goal is to rigorously represent nutrient competition in ESMs with a simple framework that is consistent with theory and appropriate observational constraints while not unduly sacrificing accuracy. We conclude that our ECA nutrient competition approach meets this goal, because it is simple enough to apply to climate-scale prediction and is based on reasonable simplifications to the complex nutrient competition mechanisms occurring in terrestrial ecosystems.Over the past two decades, the ecological significance of anadromous Pacific salmon has been well documented in aquatic ecosystems throughout the Pacific coastal ecoregion.

The annual return of salmon to fresh waters and the associated decomposition of post-reproductive carcasses result in the transfer of marine-derived nutrients and biomass to largely oligotrophic receiving ecosystems. Such inputs have been shown to increase primary production , invertebrate diversity and biomass , and fish growth rates . Since rates of primary production are typically low in many coastal salmon-bearing streams, even small nutrient inputs from anadromous salmon may stimulate autotrophic and heterotrophic production and produce cascading effects through the entire aquatic food web. In addition to subsidizing riverine biota, salmon-borne MDN also benefit vegetation within the riparian corridor . Marine nutrients are delivered to the terrestrial environment via deposition of carcasses during flood events, absorption and uptake of dissolved nutrients by riparian vegetation and removal of carcasses from the stream by piscivorous predators and scavengers . Empirical studies have shown that as much as 30% of the foliar nitrogen in terrestrial plants growing adjacent to salmon streams is of marine origin and that growth rates of riparian trees may be significantly enhanced as a result of salmon-derived subsidies . Nitrogen availability, in particular, is often a growth-limiting factor in many temperate forests and annual inputs of marine-derived nitrogen may be critical to the maintenance of riparian health and productivity in Pacific coastal watersheds. Our understanding of the ecological importance of MDN subsidies has been greatly advanced by the application of natural abundance stable isotope analyses.

A biogenic consequence of feeding in the marine environment is that adult anadromous salmon are uniquely enriched with the heavier isotopic forms of many elements relative to terrestrial or freshwater sources of these same elements. When fish senesce and die after spawning, these isotopically heavy nutrients are liberated and ultimately incorporated into aquatic and terrestrial food webs. Our research has determined that the stable nitrogen isotope “fingerprint” of adult anadromous salmon returning to coastal California basins is 15.46 ± 0.27‰ , a value markedly higher than most other natural N sources available to biota in coastal watersheds. This salient isotopic signal allows the application of stable isotope analyses to trace how salmon-borne nutrients are incorporated and cycled by organisms in the receiving watersheds. Researchers interested in the utilization of MDN by riparian trees have generally inferred sequestration and incorporation from foliar δ15N values. Nitrogen is a very minor constituent of wood cellulose and natural abundance levels of δ15N in tree rings have rarely been determined. Poulson et al. were among the first to successfully analyzed δ15N from trees , but analysis required combustion of prohibitively large quantities of material per sample. Since that time, advancements in stable isotope analytical techniques have made it possible to detect δ15N from small samples of material . This permits for non-destructive sampling of live trees via increment cores and provides a novel opportunity to assess the transfer of salmonderived nitrogen into the riparian zones of salmon-bearing watersheds. Reimchen et al. recently reported that wood samples extracted from western hemlock trees in British Columbia yielded clear evidence of SD-nitrogen incorporation with reproducible δ15N values. Intuitively, if spawning salmon represent a significant source of nitrogen to riparian trees in salmon-bearing watersheds,flood and drain tray information on salmon abundance may be recorded in the growth and chemical composition of annual tree rings. By quantifying the nitrogen stable isotope composition of tree xylem it would be possible to explore changes in SD-nitrogen over decadal or sub-decadal time increments and determine whether the nutrient capital of riparian biota has been affected by diminished salmon returns. Moreover, if δ15N can be quantified from annual growth rings, it may be possible to infer changes in salmon abundance over time and reconstruct historical salmon returns for periods and locations where no such information presently exists. Nearly all salmon recovery programs are built upon very uncertain estimates of population sizes prior to European settlement. The development of robust paleoecological methods to determine historical salmon abundance and variability would greatly assist resource managers in identifying and establishing appropriate restoration targets. Our study was carried out in two geographically disparate coastal basins that are widely regarded as important salmonid spawning and rearing habitat in California. The initial method development phase of our research was conducted in the West Branch Mill Creek watershed, a fourth-order tributary of the Smith River in northern California . The WB Mill Creek drains an area of 23.6 km2 and supports five species of anadromous salmonids: coho salmon , fall chinook salmon , chum salmon , steel head trout , and coastal cutthroat trout . The WB Mill Creek watershed has an extensive history of timber harvest dating back to the mid 1800s, with concentrated commercial harvest of coast redwood and Douglas-fir occurring between 1950 and 2000. In 2002, a major portion of the Mill Creek watershed was acquired by Save-the-Redwoods League and subsequently incorporated into the California State Parks system.

Coast redwood trees presently dominate the upland forest while the riparian zone is comprised mainly of second-growth redwood and Douglas-fir, Bigleaf maple , willow , red alder , and California-laurel . The WB Mill Creek was selected for study because standardized annual salmon escapement surveys have been conducted since 1980 providing 23 years of continuous abundance data. The stream at this location is low gradient and the channel is largely unconfined with an extensive floodplain. Additional reach characteristics are described in Waldvogel . Our second study site was Waddell Creek, a small coastal watershed in Santa Cruz County, California . The headwaters of Waddell Creek originate in the Santa Cruz Mountains at an elevation of approximately 600 m and the creek drains to the Pacific Ocean ~27 km north of MonTherey Bay, California. Waddell Creek has two major forks, East and West Waddell, which join to form the main stem approximately 4.8 river km from the ocean. Approximately 84% of the Waddell Creek watershed is located within the boundaries of Big Basin Redwoods State Park. Waddell Creek supports steel head trout and one of the southernmost extant populations of coho salmon . Both anadromous species have access to the main stem of Waddell Creek, the lowermost 2.4 m of the East Fork Waddell and up to 10 km of the West Fork Waddell. High stream gradient and natural barriers restrict coho distribution in the West Fork, however, and they are scarcely reported upstream of the confluence of Henry Creek . For our initial pilot study we collected increment cores from 10 extant riparian Douglasfir trees growing adjacent to WB Mill Creek. All trees were located within 10.0 m of the active stream channel. Core samples were collected on 17 January 2004 from a 250 m section of riparian zone located immediately downstream of the 2.7 km index stream reach used by Waldvogel to derive minimum annual escapement estimates. Small diameter increment core samples were extracted from each tree and prepared for dendrological analysis using standard methods . We concurrently collected a second, large diameter , increment core from four trees for determination of annual nitrogen content and natural abundance stable nitrogen isotope ratios . Diameter at breast height , distance from the active stream channel, and general site characteristics were also recorded for each tree sampled. Increment core samples from Waddell Creek were collected on 16-18 October 2005. Collections were made from two distinct areas within the watershed: a ~750 m length of riparian zone adjacent to the creek where salmon spawning is known to occur , and a ~500 m section of riparian zone located above a natural barrier to salmon migration . We collected paired increment cores from a total of 10 Douglas-fir and 16 coast redwood trees in the Waddell Creek watershed . Distances from the stream channel, DBH, and site characteristics were also determined for each tree sampled. Two coast redwood cores from the upstream control site were later determined to be damaged or unreliable and excluded from our analyses. Small-diameter increment core samples were air dried, mounted, and sanded for analysis of annual growth rings. Prepared cores were converted to digital images and ring widths were measured to the nearest 0.001 mm using an OPTIMAS image analysis system. Each increment core was measured in triplicate and mean ring widths values were used to generate a time series for each tree. Each time series was then detrended using the tree-ring program ARSTAN to remove trends in ring-width due to non-environmental factors such as increasing age and tree size. Detrending was accomplished using a cubic smoothing spline function that preserved ~50% of the variance contained in the measurement series at a wavelength of 32 years. Individual growth index values were derived by dividing the actual ring-width value by the value predicted from ARSTAN regression equations. Chronologies using the growth index values were subsequently combined into a robust growth index series for each sample site. Cross-dating of coast redwood trees from Waddell Creek was not successful due to the presence of anomalous rings, a high degree of ring complacency in some cores, and small sample sizes. Previous dendrochemical research has found than nitrogen may be highly mobile in the xylem of some tree species . Although the degree to which coast redwood and Douglas-fir trees exhibit radial translocation of nitrogen is largely unknown, such mobility could potentially obscure interpretation of nitrogen availability at the time of ring formation . To minimize potentially confounding effects associated with the translocation of nitrogenous products across ring boundaries, increment core samples from Waddell Creek were pretreated to remove soluble forms of nitrogen following the “short-duration” protocol outlined in Sheppard and Thompson . Briefly, tree-ring samples were sequentially Soxhlet extracted for 4 h in a mixture of ethyl alcohol and toluene , 4 h in 100% ethyl alcohol, and 1 h in distilled water .

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 . As a consequence, a larger amount of product can be delivered earlier, which can help to prevent the disease from spreading once a vaccine becomes available. In addition to conventional chromatography, several generic purification strategies have been developed to rapidly isolate products from crude plant extracts in a cost-effective manner . Due to their generic nature, these strategies typically require little optimization and can immediately be applied to products meeting the necessary requirements, which reduces the time needed to respond to a new disease. For example, purification by ultrafiltration/diafiltration is attractive for both small and large molecules because they can be separated from plant host cell proteins , which are typically 100–450 kDa in size, under gentle conditions such as neutral pH to ensure efficient recovery . This technique can also be used for simultaneous volume reduction and optional buffer exchange,ebb and flow bench reducing the overall process time and ensuring compatibility with subsequent chromatography steps.

HCP removal triggered by increasing the temperature and/ or reducing the pH is mostly limited to stable proteins such as antibodies, and especially, the former method may require extended product characterization to ensure the function of products, such as vaccine candidates, is not compromised . The fusion of purification tags to a protein product can be tempting to accelerate process development when time is pressing during an ongoing pandemic. These tags can stabilize target proteins in planta while also facilitating purification by affinity chromatography or non-chromatographic methods such as aqueous two-phase systems . On the downside, such tags may trigger unwanted aggregation or immune responses that can reduce product activity or even safety . Some tags may be approved in certain circumstances , but their immunogenicity may depend on the context of the fusion protein. The substantial toolkit available for rapid plant biomass processing and the adaptation of even large-scale plant-based production processes to new protein products ensure that plants can be used to respond to pandemic diseases with at least an equivalent development time and, in most cases, a much shorter one than conventional cell-based platforms. Although genetic vaccines for SARS-CoV-2 have been produced quickly , they have never been manufactured at the scale needed to address a pandemic and their stability during transport and deployment to developing world regions remains to be shown.

Regulatory oversight is a major and time-consuming component of any drug development program, and regulatory agencies have needed to revise internal and external procedures in order to adapt normal schedules for the rapid decision-making necessary during emergency situations. Just as important as rapid methods to express, prototype, optimize, produce, and scale new products are the streamlining of regulatory procedures to maximize the technical advantages offered by the speed and flexibility of plants and other high-performance manufacturing systems. Guidelines issued by regulatory agencies for the development of new products, or the repurposing of existing products for new indications, include criteria for product manufacturing and characterization, containment and mitigation of environmental risks, stage-wise safety determination, clinical demonstration of safety and efficacy, and various mechanisms for product licensure or approval to deploy the products and achieve the desired public health benefit. Regardless of which manufacturing platform is employed, the complexity of product development requires that continuous scrutiny is applied from preclinical research to drug approval and post-market surveillance, thus ensuring that the public does not incur an undue safety risk and that products ultimately reaching the market consistently conform to their label claims.

These goals are common to regulatory agencies worldwide, and higher convergence exists in regions that have adopted the harmonization of standards as defined by the International Council for Harmonization ,2 in key product areas including quality, safety, and efficacy.Both the United States and the EU have stringent pharmaceutical product quality and clinical development requirements, as well as regulatory mechanisms to ensure product quality and public safety. Differences and similarities between regional systems have been discussed elsewhere and are only summarized here. Stated simply, the United States, EU, and other jurisdictions follow generally a two-stage regulatory process, comprising clinical research authorization and monitoring and result’s review and marketing approval. The first stage involves the initiation of clinical research via submission of an Investigational New Drug application in the United States or its analogous Clinical Trial Application in Europe. At the preclinicalclinical translational interphase of product development, a sponsor must formally inform a regulatory agency of its intention to develop a new product and the methods and endpoints it will use to assess clinical safety and preliminary pharmacologic activity . Because the EU is a collective of independent Member States, the CTA can be submitted to a country-specific regulatory agency that will oversee development of the new product. The regulatory systems of the EU and the United States both allow pre-submission consultation on the proposed development programs via discussions with regulatory agencies or expert national bodies. These are known as pre-IND meetings in the United States and Investigational Medicinal Product Dossier 3 discussions in the EU. These meetings serve to guide the structure of theclinical programs and can substantially reduce the risk of regulatory delays as the programs begin. PIND meetings are common albeit not required, whereas IMPD discussions are often necessary prior to CTA submission.

At intermediate stages of clinical development , pauses for regulatory review must be added between clinical study phases. Such End of Phase review times may range from one to several months depending on the technology and disease indication. In advanced stages of product development after pivotal, placebo-controlled randomized Phase III studies are complete, drug approval requests that typically require extensive time for review and decision-making on the part of the regulatory agencies. In the United States, the Food and Drug Administration controls the centralized marketing approval/authorization/ licensing of a new product, a process that requires in-depth review and acceptance of a New Drug Application for chemical entities, or a Biologics License Application for biologics, the latter including PMP proteins.The Committee for Medicinal Products for Human Use , part of the European Medicines Agency , has responsibilities similar to those of the FDA and plays a key role in the provision of scientific advice, evaluation of medicines at the national level for conformance with harmonized positions across the EU,4x8ft rolling benches and the centralized approval of new products for market entry in all Member States.The statute-conformance review procedures practiced by the regulatory agencies require considerable time because the laws were established to focus on patient safety, product quality, verification of efficacy, and truth in labeling. The median times required by the FDA, EMA, and Health Canada for full review of NDA applications were reported to be 322, 366, and 352 days, respectively . Collectively, typical interactions with regulatory agencies will add more than 1 year to a drug development program. Although these regulatory timelines are the status quo during normal times, they are clearly incongruous with the needs for rapid review, approval, and deployment of new products in emergency use scenarios, such as emerging pandemics.Plant-made intermediates, including reagents for diagnostics, antigens for vaccines, and bioactive proteins for prophylactic and therapeutic medical interventions, as well as the final products containing them, are subject to the same regulatory oversight and marketing approval pathways as other pharmaceutical products. However, the manufacturing environment as well as the peculiarities of the plant-made active pharmaceutical ingredient can affect the nature and extent of requirements for compliance with various statutes, which in turn will influence the speed of development and approval. In general, the more contained the manufacturing process and the higher the quality and safety of the API, the easier it has been to move products along the development pipeline. Guidance documents on quality requirements for plant-made biomedical products exist and have provided a framework for development and marketing approval . Upstream processes that use whole plants grown indoors under controlled conditions, including plant cell culture methods, followed by controlled and contained downstream purification, have fared best under regulatory scrutiny. This is especially true for processes that use non-food plants such as Nicotiana species as expression hosts. The backlash over the Prodigene incident of 2002 in the United States has refocused subsequent development efforts on contained environments . In the United States, field-based production is possible and even practiced, but such processes require additional permits and scrutiny by the United States Department of Agriculture .

In May 2020, to encourage innovation and reduce the regulatory burden on the industry, the USDA’s Agricultural Plant Health Inspection Service revised legislation covering the interstate movement or release of genetically modified organisms into the environment in an effort to regulate such practices with higher precision [SECURE Rule revision of 7 Code of Federal Regulations 340].4 The revision will be implemented in steps and could facilitate the field based production of PMPs. In contrast, the production of PMPs using GMOs or transient expression in the field comes under heavy regulatory scrutiny in the EU, and several statutes have been developed to minimize environmental, food, and public risk. Many of these regulations focus on the use of food species as hosts. The major perceived risks of open-field cultivation are the contamination of the food/feed chain, and gene transfer between GM and non-GM plants. This is true today even though containment and mitigation technologies have evolved substantially since those statutes were first conceived, with the advent and implementation of transient and selective expression methods; new plant breeding technologies; use of non-food species; and physical, spatial, and temporal confinement . The United States and the EU differ in their philosophy and practice for the regulation of PMP products. In the United States, regulatory scrutiny is at the product level, with less focus on how the product is manufactured. In the EU, much more focus is placed on assessing how well a manufacturing process conforms to existing statutes. Therefore, in the United States, PMP products and reagents are regulated under pre-existing sections of the United States CFR, principally under various parts of Title 21 , which also apply to conventionally sourced products. These include current good manufacturing practice covered by 21 CFR Parts 210 and 211, good laboratory practice toxicology , and a collection of good clinical practice requirements specified by the ICH and accepted by the FDA . In the United States, upstream plant cultivation in containment can be practiced using qualified methods to ensure consistency of vector, raw materials, and cultivation procedures and/or, depending on the product, under good agricultural and collection practices . For PMP products, cGMP requirements do not come into play until the biomass is disrupted in a fluid vehicle to create a process stream. All process operations from that point forward, from crude hydrolysate to bulk drug substance and final drug product, are guided by 21 CFR 210/211 . In Europe, bio-pharmaceuticals regardless of manufacturing platform are regulated by the EMA, and the Medicines and Healthcare products Regulatory Agency in the United Kingdom. Pharmaceuticals from GM plants must adhere to the same regulations as all other biotechnology-derived drugs. These guidelines are largely specified by the European Commission in Directive 2001/83/EC and Regulation No 726/2004. However, upstream production in plants must also comply with additional statutes. Cultivation of GM plants in the field constitutes an environmental release and has been regulated by the EC under Directive 2001/18/EC and 1829/2003/EC if the crop can be used as food/feed . The production of PMPs using whole plants in greenhouses or cell cultures in bioreactors is regulated by the “Contained Use” Directive 2009/41/EC, which are far less stringent than an environmental release and do not necessitate a fully-fledged environmental risk assessment. Essentially, the manufacturing site is licensed for contained use and production proceeds in a similar manner as a conventional facility using microbial or mammalian cells as the production platform.

Reports from all over the world show that several destructive isolates of CTV, not dependent on sensitive rootstocks, may suddenly appear as a result of rearrangements or mutations of the genome. Also, it appears that bio-indexing on indicator plants or other official equivalent methods cannot help to limit the introduction of exotic strains, given that biological indexing is time consuming and molecular methods have a limited range of discrimination. With this in view, and thanks to the recent progress in CTV genome sequencing, we developed a fast diagnostic assay in which multiplex RT-PCR combined with a sequential hybridization step on the InCheck Platform allows the genotyping of CTV isolates. The 44 probes were designed on the complete genome of 38 CTV representative isolates of six phylogenetic clades . Tests carried out with isolates from different countries were used to validate the diagnostic procedure: single or mixed MMM genotypes inducing seedling yellows and stem pitting on sour orange and grapefruit; VT+T3 and VT genotypes inducing SY/SP on sweet orange; and mild isolates, with T30+T3 or T30 genotypes. Quadruplex primers Qua1 and Qua2 targeting eight genes, and, coupled with the panel of specific probes, after the hybridization step,mobile grow system resulted in signals of VT-like group probes for SY isolates and RB probes for isolates inducing SY and SP.

T30-like isolates from asymptomatic as well as combined infections were also detected. According to the results the detection and hybridization process is easy, rapid and accurate and can also be run by someone with no background in biology. With such potential it could dramatically increase the capability of diagnostic laboratories and contribute to minimizing the impact of new emerging CTV strains.Unlike most citrus infecting viruses, Citrus tristeza viruspossesses a number of distinct strains that produce a range of disease syndromes on different host species. In addition, these strains are frequently found in mixed populations within a single host. While we have some knowledge of the symptoms produced by a single strain, there is little understanding of how combinations of strains affect symptom expression and disease severity. To test the effect of population structure on symptom expression, we inoculated Citrus paradisi and C. aurantii with a range of single strains and mixed field populations of CTV. Seedling yellows symptom development was assessed six months post-inoculation. Real-time qRT-PCR was used to assess relative population titre, and these data were compared with symptom severity. We found that there was no obvious correlation between the dominance or presence of specific isolates in the population and the incidence or severity of seedling yellows; there was also no correlation between total viral load within infected tissues and symptom severity. These data further suggest that symptom severity is not due to quantitative differences between strains in a population, but indicates that qualitative differences within and between each population influence symptom development.

Citrus tristeza virusis the cause of an important and devastating citrus disease with great economic loss to some citrus species in certain regions of the world. In Chile, the disease has spread throughout the entire national citrus zone. Tthe major damage has been caused in the northern region where isolates capable of causing severe stem pitting have been detected. The CTV genome is close to 20.000 bp and encodes three suppressors that block the natural defence strategy of the plant, the “silencing mechanism”. This research work proposes a strategy to generate transgenic rootstocks resistant or tolerant to the disease through posttranscriptional gene silencing in which the silencing signals will move from rootstock to scion preventing future infections. We have developed two hybrid genetic-constructs CTV-p25-p20 and CTV-p20-p23 based in the T36-strain. These transgenes were synthesized and integrated in hairpin mode in the pHellgate12 plasmid. These constructs were used in transformation of Citrus macrophylla in order to obtain rootstocks resistant or tolerant to CTV. The methodology employed was the transformation of epicotyl tissues; the transformation was mediated by Agrobacterium tumefaciens, strain EHA105. The transformed explants were incubated at 25ºC in co-cultivation medium in the dark for 72 hrs and then the explants were regenerated in a medium with kanamycin selection. We have obtained transgenic shoots that show transgene integration into the plant-DNA. In the near future these shoots will be grafted with a CTV infected Lime scion to challenge the transgenic rootstocks and evaluate the silencing process in the scion.

Twenty four samples from non-symptomatic Pummelo trees as well as trees expressing virus-like symptoms such as inverted leaf cupping and/or stem pitting were collected randomly across citrus-growing regions in the Philippines between 2012 and 2013. Samples were propagated on calamandarin rootstock and maintained under standard greenhouse conditions at the Bureau of Plant Industry in Davao City, Philippines. Pummelo budwood obtained from the greenhouse propagations was shipped to the University of California, Riverside under permit. Total RNA was extracted and analyzed by reverse transcription polymerase chain reaction using primers targeting the major coat protein gene of the Citrus tristeza virus . All samples from Luzon Island tested negative for CTV while eleven samples from Mindanao and Visayas Island were CTV positive. The CTV genotype of the positive samples was determined using multiple molecular markers targeting different CTV genome regions. The predominant CTV genotype identified was VT in single or mixed infections with T30. A single sample contained the T36 CTV genotype. Preliminary phylogenetic analysis based on the full length sequence of the CP gene indicated that ten Philippines CTV isolates with VT and VT+T30 genotypes clusThered in one clade closely related to VT isolates from China and Japan . The single Philippines isolate with a T36 genotype clusThered with CTV isolates from India , Hawaii , and Taiwan . To minimize losses in the local citrus industry, the South African Citrus Improvement Scheme implemented cross-protection using mild CTV sources to reduce the effect of challenges by endemic severe CTV strains. This management strategy also referred to as “pre-immunisation” or “mild strain protection” was implemented by the CIS at its initiation. The use of cross-protection in South Africa has been mostly successful,mobile vertical rack but cases of cross-protection breakdown have been experienced and a change in the pre-immunising source for grapefruit crossprotection was made to address this. A number of countries apply cross-protection and report diminished expression of disease and improved production including Australia, Japan, Brazil, Argentina, Peru and South Africa. All pre-immunisation sources used for cross-protection, except the South African sources, are single variant sources. CTV is a complex of strains. This insight and the subsequent development of diagnostics for genotyping enabled the analysis of mixed populations. We have expanded on a published CTV genotype testing system and have tested various maintenance sources of the GFMS12 and GFMS35 pre-immunisation sources at 3 different institutions including grapefruit mother trees maintained at the Citrus Foundation Block. Also segregation of genotypes is noted in different multiplications of the two sources and this is in all probability a contributing factor to cross-protection breakdown in the field.Bio-indexing and characterization by CE-SSCP, MMM and phylogenetic analysis of representative samples collected in a highly infected Citrus tristeza virus area of Sicily reveals that two main groups are present: one includes isolates inducing severe seedling yellows and a second is asymptomatic on sour orange. Severe isolates cluster in a single subclade within the group of seedling yellows and stem pitting isolates ; mild ones are similar to T30-like isolates. In order to investigate the phylogenetic relationship of the CTV population with the isolates of respective clades we undertook the complete genome analysis of two of them, namely SG29 and Bau282. SG29 is a severe isolate, from Sanguinello sweet orange, inducing seedling yellows on sour orange and, rarely, stem pitting on Duncan grapefruit, but not on sweet orange. Bau282 is from sweet orange TDV and host susceptibility showed it is asymptomatic on sour orange and Duncan grapefruit. The genomes were obtained after sequencing of the small RNAs and assembly of overlapping sequences by reference alignment from libraries sequenced by Next-generation platforms. The viral small interfering RNAs found were in the predominant 22 and 21 nucleotide-size classes.

The complete SG29 and Bau282 genome in length are 19,259 bp and 19,250 bp, respectively, with 12 open reading frames , structurally identical to the other known CTV isolates. Phylogenetic analysis based on 31 full CTV genomes showed that SG29 clustered with the “Asian” VT-lineage in which T318A , AT- 1 , Nuaga and CT11A isolates segregate and has the highest homology identity with T318A and AT-1 . Bau282 clustered within the mild isolates T30 and T385 and BLAST analysis showed a very high identity equal to 99%. Since its appearance two centuries ago, Tristeza has been classified as the most devastating viral disease which affects citrus worldwide. Its causative agent, Citrus tristeza virus , is transmitted by infected grafts or by insect vectors such as the aphid Toxoptera citricida. In all affected citrus growing areas, the existence of genetic variants of the virus with different degrees of severity has been reported. Characterization of CTV isolates can provide epidemiological information and can be useful for disease control. The presence of CTV and its efficient vector has been known in Uruguay since the 1940s. However, there is no data based on molecular biology reflecting genetic variants circulating in the country. In the present study, using RT-PCR amplification of three regions of the CTV genome , we established phylogenetic relationships of the strains in the country at the present time and 20 years ago. The samples used were collected in 1990 and maintained in planta in vector free greenhouses, and also were compared with current field samples. This valuable historical collection provides a sample of past CTV occurrence in Uruguay. The results show that circulating strains in the country are severe and unsurprisingly resemble strains reported in Argentina. However, some of the strains under study are similar to reference strains from Israel or Hawaii. This may reflect the introduction of infected buds or trees in the past.Citrus Sudden Death remains a challenge for citrus production in nonirrigated areas in the North and Northwest regions of Sao Paulo State, Brazil. Its incidence has increasingly affecting the drought-tolerant but CSD-susceptible ‘Rangpur lime’ rootstock. So far, since the first report of the disease in the 2000s its etiology remains uncertain, but vectored-viruses have been considered as the main hypothesis . Here we checked the occurrence of these viruses in a deep sequencing dataset of total RNA by Illumina platform run for transcriptomic studies of sweet orange. Pools of reads of Valencia graphed on Rangpur lime and Sunki mandarin, both CSDsymptomatic and also from its non-symptomatic pairwise trees growing side-by-side were analyzed. Around 74 million reads for each experimental condition were assembled using the CLCbio platform de novo assembly algorithm using a CTV reference genome . For the NS-CSD dataset only 0.005% of reads matched with CTV virus sequences, whereas for the S-CSD dataset 0.014% of reads come from the CTV virus. However, almost three times more reads of CSDdisease trees matched with CTV. A careful comparison of CTV reads from S-CSD with NS-CSD allowed us to identify some sets of reads more frequently occurring in diseased trees, as also some CTV reads specifically occurring in the CSD diseased plants. Those genetic differences which make the CTV-reads specific for the CSD symptomatic plants were mapped through the whole CTV genome reference.On the other hand, using the same assembly strategy we observed only few reads matching the Tymovirus genome in the analyzed dataset, independent of whether they come from diseased or non-diseased trees. Despite being poorly represented, the Tymovirus’s reads were shown to be genetically diverse, which was confirmed by Sanger sequencing of amplicons from five different Tymovirus’ genomic regions amplified by specific primers. Citrus tristeza virusis the causal agent of the most important citrus disease and exists as numerous strains which may cause different symptoms. Due to the wide biological diversity of the virus, identification of the actual genotypes present in an area is useful in adopting adequate control strategies. In recent years a plethora of methods for molecular characterization of the virus have been developed. Among others RT-PCR, Real-Time PCR and CE-SSCP have been adapted for discrimination of severe and mild CTV strains. In this work, based on the fully sequenced genomes of the virus available on GenBank, we have developed two quadruplex primer sets, one for simultaneously obtaining amplicons from the 5’UTR, ORF1a, RdRp and p27 regions , the other for p20, p23 and p33, and the Citrus elongation factor EF 1 alpha .

Surface area-proportional growth could arise if nutrient/waste exchange across the plasma membrane is a limiting factor for growth. We observed that red light-illuminated optoBem1 cells also exhibited a change in DNA content over time. While most cells maintained a ploidy of 2N or less during the first 3 h of Bem1 disruption, a population of 4N cells appeared following 6 h of arrest , consistent with prior reports suggesting that after Bem1 disruption, some arrested cells eventually leak through the cell cycle block and undergo DNA endoreduplication. To ensure that the surface area-proportional growth was not an artifact of increased ploidy, we set out to generate ‘giant yeast’ via a second, non-optogenetic method: disruption of Cdk1/Cdc28 using the temperature-sensitive allele cdc28-13 . Unlike optoBem1 cells, nearly all cdk1-ts cells at the restrictive temperature arrest in G1 without undergoing further DNA replication. We found that cdk1-ts cells grown at the restrictive temperature to induce arrest in G1 also exhibited a linear increase in cell diameter, consistent with growth proportional to surface area . However, cdk1-ts were unable to maintain this rate of growth over the entire 12-h time-course: After reaching a volume of 500–700 μm3 , cell growth stalled . Taken together, our results from both optoBem1 and cdk1-ts cells indicate that the isotropic growth rate during G1 is proportional to surface area over a wide range of cell sizes.

DNA endoreduplication does not appear to affect this overall growth rate but may be required to sustain it beyond a critical cell size, giving rise to the robust continued growth of optoBem1 cells. It has been shown in other organisms, for example, that DNA endoreduplication enables large increases in cell size. One possibility by which our findings can be reconciled with prior observations of exponential growth in wild type budding yeast is that cells become surface area-limited at sizes just above that of wild type cells, thereby inducing a shift from volume proportional growth to surface area-proportional growth.Cell size control pathways exist to correct for deviations from a set-point size, yet most previously-identified size control pathways specifically operate on cells that are born too small, delaying cell cycle progression to enable further growth to occur. Because the light and temperature-shift stimuli with which we prepared ‘giant’ yeast are fully reversible, we reasoned that we could monitor the return to a steady-state size distribution after releasing giant cells from their block. We prepared giant optoBem1 cells by incubating them in red light for 8 h and monitored them by live-cell microscopy after releasing them into infrared light. Strikingly, we found that cell populations rapidly returned to their unperturbed state , with individual daughter cells reaching the set-point volume in as few as three rounds of division .Return to the set-point size is not driven by cell shrinking,hydroponic nft system as giant mothers maintained their maximum volume over multiple rounds of budding .

Instead, the giant mothers are eventually diluted out as successive generations are born, an effect that is especially prominent in cell populations at least 10 h post-Bem1 release . In these populations, size distributions have a single mode near the set-point volume but exhibit long tails towards larger volumes . Our observation that cell size recovers after only a few generations strongly supports the existence of size control acting on large cells and demonstrates that size homeostasis across a cell population is robust even to extreme increases in cell volume.Quantitatively monitoring cell growth in yeast—as well bacterial, archaeal, and mammalian cells—has shown that the behavior of many organisms is consistent with an adder that monitors size across an entire cell cycle to correct for deviations in cell size and maintain size homeostasis in the population. However, a recent study argued that in budding yeast, the adder behavior could arise from independent regulation of pre- and post-Start events, without a cell needing to keep track of its added volume across all cell cycle phases, and may fail under various perturbations. To test whether adder-based mechanisms could account for size control in giant yeast, we quantified inter division volume change in successive cell division cycles after releasing optoBem1 cells into infrared light. For this experiment we prepared optoBem1 cells that also expressed fluorescently-labeled septin rings, which enabled us to time both bud emergence and cytokinesis and thus separate pre-Start and postStart size regulation . The ‘adder’ model predicts that the cell volume at division should be proportional to cell volume at birth with a slope of. Indeed, for unperturbed cells, we found that cell volume at division was linearly related to volume at birth with a slope of 1.19 . However, we found that the adder model poorly explained the cell size relationships in our giant cells, where the volume at division was related to volume at birth with a slope of 1.73 .

This relationship was also evident when individual cells were tracked over time: the interdivision volume change, Δ, was positively correlated with the volume at birth . This size-dependent volume change occurred entirely during S/G2/M phase, as cells added a minimal volume during G1 that did not vary with cell size . We also performed analogous experiments in cdk1-ts giant cells that were shifted back to the permissive temperature. These experiments revealed a similar relationship: large cells grew more than small cells, exhibiting a linear relationship between volume at division and volume at birth with a slope of 1.70 . These results are broadly consistent with recent work showing that although size control in unperturbed cells resembles an adder-based mechanism, no mechanistic adder regulates volume addition across the entire cell cycle. Our data also suggest that any size regulation limiting the growth of large cells is likely a consequence of regulation in S/G2/M, as growth during G1 is negligible.If an adder is unable to explain size homeostasis in giant yeast, what regulatory mechanisms or growth laws might operate on the daughters of giant cells during S/G2/M? Two possibilities include a bud ‘sizer’, where bud growth would be restricted after reaching a critical size; and a bud ‘timer’ in which cytokinesis would occur at a fixed duration following the beginning of S/G2/M . Such ‘sizers’ and ‘timers’ have been proposed to operate in a variety of biological systems. To distinguish between these possibilities, we tracked the timing of bud emergence and cytokinesis by septin ring appearance and disappearance, respectively,nft channel following reactivation of Bem1 in giant optoBem1 cells . Daughter volume strongly correlated with mother volume , inconsistent with a bud sizer mechanism. Our prior observation that the inter division volume change scales positively with cell birth size further argues against a bud sizer for cell volume control. In contrast, our data were consistent with a timer specifying the duration of S/G2/M: the time from bud emergence to cytokinesis did not vary as a function of mother cell volume and took average 95 min across cells of all volumes .Similar experiments performed using cdk1-ts cells were consistent with our observations in optoBem1 cells, revealing a size-independent duration of budding. However, we observed one notable difference: the duration of the size-invariant bud timer in giant cdk1-ts cells was substantially longer than that of giant optoBem1 cells . As Cdk1 is a key driver of mitosis in eukaryotes, the increased duration of the bud timer in cdk1-ts cells may arise from the need to refold or synthesize new Cdk1 molecules to complete S/G2/M following a shift from the restrictive to permissive temperature. Furthermore, even when grown at the permissive temperature, the doubling time of cdk1-ts cells is longer than an isogenic wild type strain , suggesting that cdk1-ts may not be able to fully complement CDK1. In summary, we find that a timer specifying a constant budding duration describes how a cell population founded by ‘giant’ cells returns to their set-point volume. Although mother and daughter sizes are correlated across a broad size range, daughters are always born smaller than mother cells. After cytokinesis, daughter cells remaining larger than the set-point volume exhibit a G1 phase with virtually no growth and bud rapidly, leading to a geometric shrinking in successive generations . Indeed, a back-of-the-envelope calculation demonstrates that if newly-budded daughters are each 50% smaller than their mothers, a 32-fold decrease in cell volume can be achieved in 5 generations .

Assuming a 100 min doubling time , a return to the set-point size would take ~8 h. A fixed budding time, even in the absence of active molecular size sensors in S/G2/M, is sufficient to buffer against persistence of abnormally large cell sizes in the population. We also note that the bud duration timer we describe is quite complementary to G1-phase size sensors such as Whi5, which compensate for a small size at birth by elongating G1 phase.Our conclusions are derived from cells prepared using two independent perturbations: optogenetic inactivation of the Bem1 polarity factor and a temperature-sensitive cdk1 allele. Importantly, each of these perturbations targets distinct cellular processes and thus produces distinct physiological defects. Cells lacking Bem1 activity exhibit weakened cells walls and undergo successive rounds of DNA endoreduplication following their initial arrest in G1 . In contrast, loss of Cdk1 does not produce such defects but its disruption requires incubating cells at 37˚C, which may broadly activate environmental stress response pathways. Furthermore, cdk1-ts may not fully complement CDK1, even at the permissive temperature . That each of these perturbations reveals similar mother-daughter size correlations as well as a size-invariant bud timer strongly supports the generality of our conclusions. The bud timer we describe here is consistent with prior work suggesting that the duration of budding tends to be invariant to changes in growth rates. However, such a timer need not be a dedicated biochemical circuit to sense budding duration, compare it to a set-point, and dictate the transition to cytokinesis. Its existence could simply arise due to the time required by independent cellular processes that coincide with bud growth, such as the combined duration of S-phase or mitosis. Nevertheless, one observation suggests more complex regulation: the duration of the size-invariant bud timer is markedly longer in enlarged cdk1-ts vs. optoBem1 cells , yet mother-daughter sizes are nearly identical in these two backgrounds . These data suggest that the duration of the bud timer may be inter-related to Cdk1 activity and cells’ growth rate during S/G2/M. Recent work has found that mitosis and bud growth rate are closely coordinated and that cells may extend the duration of mitosis to compensate for slow growth that occurs under poor nutrient conditions. Dissecting the dependencies between growth rate, Cdk1 activity and the duration of post-Start events presents a promising direction for future study.All yeast strains used are isogenic to an ‘optoBem1’ strain which was created in the w303 genetic background and contained exogenous PhyB-mCherry-Tom7 with endogenous Bem1 C-terminally tagged with mCherry-PIF, as previously described. The cdc28-13 strain was a kind gift from David Morgan. A pACT1-CDC10-eGFP expression vector was created by Gibson assembly, with the CDC10 expression cassette inserted between the NotI and XmaI sites of the pRS316 vector. For the experiments described in Figs 3 and 4; Fig D, E, F, and G in S1 Fig; and S2 Fig; the indicated vector was transformed into our optoBem1 or cdk1-ts strain and selection was maintained by growing yeast in synthetic complete media lacking uracil . For all other experiments, yeast were cultured in synthetic complete media .Preparation of yeast prior to optogenetic experiments was performed, in general, as previously described. Yeast undergoing exponential growth in synthetic media were treated with 31.25 μM phycocyanobilin and incubated in foil-wrapped tubes at 30˚C for a minimum of 2 h. For all microscopy experiments, yeast were spun onto glass-bottom 96-well plates coated with Concanavalin A and washed once with fresh PCB-containing media to remove floating cells. Cells remained approximately spherical following this procedure, as assessed by Concanavalin A staining .Imaging was performed at room temperature. For experiments where isotropic growth was measured , yeast were plated and imaged immediately following PCB treatment. For experiments where growth following Bem1 reactivation was examined , PCB-treated yeast were first placed in clear culture tubes and incubated at room temperature for >6 h while undergoing constant illumination with a red LED panel .