Tag: vertical agriculture

Paprika and coffee are also less labour intensive than tobacco and so may be easier to manage for households with a shortage of active workers. On the other hand, these enterprises still require more labour than other traditional smallholder crops including cotton and the need for most households to hire workers if cultivating a large area may discourage some producers. Table 27 compares estimated net profits for non-traditional smallholder crops. As with the results for LSC farmers, these data clearly show that other crops offer a potential for comparable and sometimes higher profits than tobacco. Since these crops also cost less to produce than tobacco, paprika and coffee appear to be excellent alternatives for smallholder growers. As noted, successful development of these crops depends on many things including major investment in support services and infrastructure.Coffee was first introduced in the 1960s on LSC farms in Natural Region I near Chipinge in the Eastern Highlands. Until the mid-1990s, very little coffee was grown outside this area and the Eastern Highlands still account for more 70% of all exports. This situation is now set for great change in that LSC farmers in tobacco growing areas of Natural Region II have been planting large areas to coffee in an effort to diversify their income base. Of the total 9 900 hectares now under coffee in Zimbabwe,vertical growing systems some 5 500 hectares are immature 1-2 year old trees grown on tobacco farms.

When these plantations come into full production over the next 3 years, total annual exports are expected to increase from around 7 000 metric tons of green coffee at present to over 20 000 metric tons. Current export values are in the range of USD 8.1 million; once the trees already planted are fully mature, this could easily increase to over USD 43.5 million, equal to about 7.5% of the gross foreign income from tobacco.15 Zimbabwe grows high quality Arabica coffee that normally attracts a 10% to 20% premium in the world market.Coffee is now one of the fastest growing agricultural sectors in Zimbabwe and is being financed mainly by individual LSC farmers with income from tobacco. As a tree-crop, coffee takes around three years to mature until the first major harvest and few banks are willing to lend for this type of long-term project, especially in the current economic climate.In addition to the trees themselves, other establishment costs include pulping machines, fermentation tanks, drying racks and, ideally, drip line irrigation. These costs can easily add to more than ZWD 10.6 million for a 60 hectare project until the trees are fully mature.Government does not control the coffee industry and individual LSC farmers are free to market their crop directly in US dollars. Coffee prices are based on the New York Futures Market and most growers are able to fix prices up to 12 months in advance. An important advantage of this system is that sales against a forward contract can be used to obtain credit and help smooth individual cash flow requirements. Currently, however, New York coffee markets are suffering from a large oversupply and are at their lowest point for 25 years equal to an average fob export price of only USD 1 360 per metric ton from the Mutare Coffee Mill. Analysts predict the situation will improve over the next 12 months and export prices around USD 2 000 per ton fob Mutare are more indicative of the long-run average.

Therefore the quantitative analysis is carried out using three price scenarios: the current low price , a long-run average price and a middle price.Large-scale commercial farmers. Quantitative results from the analysis of LSC coffee grown in Natural Region II are summarised in Table 28 for a mature, four year old crop.Because coffee is grown to a more or less uniform standard, only two management levels are considered for this enterprise. In this case, the medium input level is based on a conservative yield of 2.0 metric tons per hectare used for most farm budgeting exercises in Zimbabwe. Compared with large commercial farmers in southern Zambia, however, where growing conditions are more or less similar, this is a fairly low yield and it is not unrealistic to expect at least 2.5 tons per hectare for a mature crop with good management. The high input level is based on this expectation.Key results from the quantitative analysis have already been compared with the data for flue-cured tobacco in the overview section above. Without repeating this discussion, it is useful to note that farmer profits improve by proportionately more with higher crop prices than the price increase itself. Furthermore, even though crop profits are very low with current prices , coffee still returns a gross profit and therefore contributes to the viability of a mixed LSC farm system. In other words, even under very difficult market conditions, coffee is still an attractive enterprise with great potential for increased profits as prices improve. In terms of the two management levels, the data suggest there is further potential for even higher profits through better management and increased yields. It may not always be possible to achieve 2.5 tons per hectare because of local agro-climatic conditions, but the potential for significantly greater profits no doubt means that some farmers already target this level.Smallholder farmers.

There are around 2 000 registered smallholder coffee growers in Zimbabwe located exclusively in the Eastern Highlands. Most growers are organised into co-operatives that manage the pulping and milling of smallholder coffee and oversee international marketing, which is handled on commission by the Mutare Coffee Mill. Smallholder farmers currently produce about 30 metric tons of green bean coffee annually equal to about 1.5% of the national total from an area of just over 100 hectares. Although smallholder coffee has not been promoted in the northern flue-cured areas, the quantitative analysis helps to assess the overall viability of this enterprise. As described, the major challenge with promoting smallholder coffee in these locations would be that pulping and processing facilities have to be developed. Simple irrigation facilities, including water furrows and treadle pumps, may also be needed for high yields depending on local conditions. Smallholder coffee, therefore,outdoor vertical plant stands is more likely to substitute for burley tobacco grown in eastern Zimbabwe where there is better access to existing pulping and processing equipment.The quantitative results for smallholder coffee are summarised in Table 29 below. These models assume farmers have access to pulping and processing facilities and no account has been taken of the cost for Zimbabwe to develop these services. Although more through analysis is needed to assess the viability of introducing coffee to a new location, the results here suggest this may be a very attractive proposition. Most encouragingly, the results are especially favourable with low input management where coffee costs less than almost every other enterprise analysed and provides relatively high profits similar to cotton.On this basis, the rates of return are outstanding and indicate that coffee can be a very good low risk investment for smallholder farmers. With more intensive management and/or improved world prices, the returns from coffee rival tobacco. Although somewhat labour intensive because of the time spent picking, crop profits improve significantly with more intensive management and the returns to family labour at these higher levels are similar to the very good daily returns from tobacco. Paprika was first introduced to Zimbabwe in the early 1990s by private investors who sought to promote the crop mainly among large-scale commercial farmers. One advantage of paprika is that the soil types and skills required are very similar to those needed for tobacco so that farmers are already well positioned for success. Compared with tobacco, however, world markets for paprika are relatively small with a total demand of only about 120 000 metric tons per year. Production in Zimbabwe has ranged from 8 500 to 15 000 metric tons annually and, in the years when production peaked, this had a noticeable impact on world prices which discouraged many LSC farmers from continuing to grow the crop. Until recently, paprika buyers typically offered LSC farmers production contracts with a guaranteed minimum price fixed in USD at the start of the season. Deteriorating economic conditions, however, mean this is no longer feasible for most export companies.About 70% of world paprika is used as a condiment in powder form with the balance sent for hexane extraction to derive a colour lipid for industrial food processing. Until now all paprika grown in Zimbabwe has been dried, de-seeded and exported in baled form for processing outside the country. This situation is about to change, however, in that one local firm has nearly completed a new solvent extraction plant with the capacity to process 1 000 to 1 500 metric tons of paprika annually. Once operational, this facility will help add value locally and save on high overland transportation costs for bulk paprika.

Although there has been much private investment in Zimbabwe to promote paprika among LSC farmers , relatively little attention has been given to smallholder growers for the past 2-3 years. Neighbouring countries including Zambia and Malawi have enjoyed some measure of success with smallholder paprika, but buyers in Zimbabwe prefer to work with LSC farmers who are a more reliable source of supply and able to produce higher quality crop that is easier to market internationally. Buyers in Zimbabwe also consider that paprika is a risky enterprise for smallholder farmers because of problems with crop disease and potential for yield loss from localised flooding and other conditions these growers cannot control.Large-scale commercial farmers. The results for LSC paprika are summarised in Table 30. For this analysis, two management levels are considered including a late-season, low-cost crop planted with the rains and a long-season crop aiming for very high yields.As discussed in the overview section above, these results compare very favourably with those for flue-cured tobacco. Although limited world demand means that paprika could never substitute entirely for tobacco, the crop clearly has the potential to provide high farmer profits and so can play important role in a mixed farm system. In terms of employment creation, paprika demands an estimated 70 to 80 days of casual labour per hectare and is among the most labour intensive crops analysed. The rates of return and sensitivity indicators for both short- and long-season paprika are excellent and show this is a robust activity that remains profitable even with a significant reduction in price. Smallholder farmers. Despite limited interest by most private buyers in paprika as a smallholder crop, some promotion and training work was carried out a few years ago in tobacco areas and there are now several thousand smallholder farmers who cultivate the crop each year. Around 75% of smallholder paprika is grown in Natural Region II on very small plots of just 0.1 to 0.2 hectares on most farms. In 1999, a total of 12 500 smallholder households planted paprika over an area of 1 600 hectares for all natural regions; there were 9 800 growers in Natural Region II cultivating a total of around 1 200 hectares. Paprika is priced according to colour and smallholders generally produce a lower-value crop than LSC farmers. Contamination with foreign matters including dirt, stones and even rat hairs because of poor on-farm storage also lead to lower crop value and restricted export opportunities.Results from the quantitative analysis of smallholder paprika are summarised in Table 31. Although production and marketing risks cannot be overlooked, the data for smallholder paprika compare very well with those for smallholder tobacco. Not only are the estimated profits comparable to those from burley and even flue-cured tobacco, but paprika also costs less to grow at each corresponding management level. These are attractive characteristics for smallholder farmers for whom the ability to afford purchased inputs can be a major constraint. Compared with tobacco and all other crops analysed, the data show that paprika offers outstanding rates of return to both cash and total production costs. Furthermore, despite the perception among many buyers that paprika is a risky crop ill-suited to smallholder production, the sensitivity data show that the very good financial results for this crop are extremely robust and suggest that smallholder farmers may be well positioned to compete in the world market even with lower prices.

In the pavement industry, the majority of the materials used for construction, which are primarily composed of gravel, sand and crushed stone, are relatively local materials, unless there is access to water transport. An exception is specialized engineered materials/chemicals such as admixtures and additives. Figure 8 shows the order in which user input/selection is required before the tool outputs a comprehensive summary data of different commodities in tons, ton mile and/or dollar costs in tabular form. In this white paper, examples of domestic flow types are presented. Within the domestic flows, seven tables are presented that require user input. Each table has a drop-down list to select from. The CFS data is being collected every five-years since 1997. Data were also collected for the years 2013, 2014 and 2015. Additionally, users can extract future projection data based on macroeconomic forecasting model starting from 2020 to 2045 for every five-year interval . The origin and destination can be at the country, state or city level. The city and state boundaries in FAF4 are defined as FAF zone specific and the 132 domestic zones are a hybrid of core-based statistical areas that have been defined by the office of management and budget and state boundaries . As noted in the introduction, modeling of climate change indicates that the intensity of rainfall during extreme events will increase significantly, with the amounts depending on the extent of continued human contributions to greenhouse gases . Increased urbanization is one of the factors that has affected urban flooding and storm water runoff due to development in the flood plains and construction of impervious hardscapes. Figure 9 through Figure 11 show qualitatively how degrees of average perviousness of urban surfaces affect storm water runoff,vertical grow shelf infiltration and evapotranspiration.

The Environmental Protection Agency Storm Water Management Model is a simulation program which can perform dynamic rainfall-runoff modeling . The simulation can be conducted for both a single event or over a longer period of time and the program can analyze quantity and quality of runoff from different urban regions. The SWMM model works based on an assembly of sub catchment regions in which precipitation is received and runoff is generated. SWMM then uses simulation of a combination of pipes, storage devices, pumps and regulators, to model transport of this runoff. SWMM can record the quality and quantity of generated runoff in each of these sub catchments. It also can calculate the flow rate, flow depth and quality of water in pipes and channel sections for the duration of simulation. The hydrological processes that can be simulated in SWMM include evaporation, snow accumulation, infiltration, percolation of infiltrated water, water inter flow between groundwater and drainage system, etc. To overcome spatial variability in all these processes, the actual area of interest is divided into smaller sub catchment regions in which the hydrological properties are homogenous. Then, the overland flow can be dispelled between sub catchments, drainage systems or sub-areas. SWMM is also capable of analyzing runoff pollutant loads under different conditions such as dry-weather pollutant buildup, pollutant wash-off during storm incident, contribution of rainfall deposition, and reduction of concentration due to water treatment. The UM-LCA framework developed for this study aims to build a foundation for future assessments of alternative types of urban hardscape. Using materials such as pervious concrete, permeable pavers and porous asphalt and designs for construction of permeable pavements that allow the storm water to pass through the structure into the ground water table is one way to reduce storm water runoff through infiltration and detention, which also increases groundwater recharge and can help improve storm water quality. The water cycle can be quantified using the SWMM model.

SWMM provides additional capabilities relevant to urban hardscapes. There is an additional section called Low Impact Development which defines the properties of low impact technologies such as the permeable pavements. LID is an approach that manages storm water on-site by sustainable land planning and engineering design practices i.e., use of recyclable materials, permeable hardscape designs, and in-place recycling are some of the example practices. The LID module in SWMM includes the following technologies: bio-retention cell, porous pavement, infiltration trench, rain barrel, and vegetative swale. The Atmosphere Compartment contains climate information which determines how much precipitation LID will receive. The Land surface Compartment contains information about LID’s area.The Ground Water Compartment depends whether the LID allows storm water to recharge ground water. For example, the green roof conveys storm water into the drainage pipes and the water does not infiltrate into the ground directly. Therefore, in this case the Ground Water Compartment can be omitted. However, use of Ground Water Compartment is necessary if a structure, such as permeable pavements, allows water to infiltrate into the groundwater table. The climate information and rainfall are required for almost all the SWMM simulations. The data can be collected from National Climatic Data Center . The information about the modelled area is also one of the required inputs. As mentioned earlier, SWMM divides the defined area into several sub catchments according to the land usage . The properties of the subcatchment are: ’X-Coordinate’ and ’Y-Coordinate’ which defines the location of the sub catchment area on the map, ‘Area’ which shows the size of the sub catchment, and ’Rain Gauge’ which determines the duration and intensity of the rain.

The following are input parameters that are used in the LID module: surface layer, pavement layer, storage layer, under drain layer, and subgrade layer. After completion of the analysis, LID provides the following outputs: total inflow volume, total evaporation loss, total infiltration loss, total surface outflow, total under drain outflow, initial storage volume, and final storage volume. These outputs are available for any analyzed time step and can be viewed on the map in the program, tabulated or plotted for the further statistical analysis. Several data sources have been determined that can be used in the SWMM model as inputs. Data from cities in California was taken as an example to illustrate what type of information can be extracted from the sources reported in order to run the SWMM model. The following sources can be used to collect the information of the rainfall and climate for cities and regions in the U.S.Zimbabwe’s economy depends heavily on tobacco. There is concern that global efforts to reduce tobacco use may reduce demand for tobacco and significantly affect Zimbabwe’s economy and tobacco exports. Global tobacco demand has recently been stable or falling very gradually. However,vertical hydroponic public health efforts to reduce demand are offset by aggressive cigarette marketing, rising population and incomes, and the strongly addictive effects of nicotine. But tobacco prices have softened, and the long-term prospects are uncertain. Governments concerned about the future of tobacco are considering diversification options and strategies to develop other high-value crop substitutes . The world’s fourth largest flue-cured tobacco producer and the largest producer of tobacco leaf in Africa, Zimbabwe exports 90 percent of its raw tobacco and has typically earned around USD 600 million in foreign revenue annually, equal to almost 10 percent of GDP, 30 percent of total exports and 50 percent of agricultural exports. Moreover, 250,000 people or 5% of Zimbabwe’s total labor force are engaged in tobacco-related work including tobacco farming, manufacturing and retailing. Most tobacco in Zimbabwe is grown by large-scale commercial farmers, who account for about 87% of the land under tobacco and 95% of the total crop. This study compares the financial costs and returns to tobacco with twelve alternative crops, looking at profitability, costs, labor intensity, financial support, technical infrastructure, land-suitability, marketing difficulties, world demand, and production risks. The information may be useful to agriculturalists and government officials who are interested in exploring the potential for diversification by different sized farmers, as Zimbabwe considers options for the future. The study aims to provide an improved understanding of the trade-offs individual growers face in deciding what crops to grow. The analysis is based on an original set of 91 production budgets estimated in January 2001 specifically for this study, that estimated financial costs and returns in Natural Region II at the time. It should be noted that in 2000/2001, veterans of the independence war occupied several commercial farms and Government subsequently gazetted over 5,300 farms for compulsory acquisition and resettlement. This study took place before the impact of these events on agricultural output was evident, and does not address whether and how they might affect tobacco farming and its contribution to the economy of Zimbabwe. The study shows that tobacco is a highly profitable cash crop for both large and small farmers, generating direct income for large-scale farmers, and indirect income for smallholder farmers.

Dramatic changes in prices and yield are unlikely in the near future. And tobacco would remain relatively profitable, even if prices fell considerably. Farmers in Zimbabwe are aware of the risk of depending on one cash crop and have begun diversifying to lessen their tobacco dependence. Large scale commercial farmers have diversified into coffee and paprika, and horticultural crops – notably roses and supermarket vegetables- using their tobacco profits. These products can be grown in the same soil as tobacco, and generate high profits as well as employment opportunities. The study discusses the limitations of these alternatives, and notes that despite some rapid increases in some crops , they are still grown at a very limited scale compared with tobacco. Tobacco is one of the most profitable cash crops for both large-scale commercial and smallholder farmers. The study shows that tobacco would provide good financial returns even after a large drop in yield or price. Tobacco is likely to remain a very attractive crop for all categories of farmer even under progressively difficult market conditions. There are around 2,000 large-scale commercial tobacco growers, and 16,000 tobacco growing smallholder farmers – less than 1.5% of all smallholder households. Most smallholders grow only very small amounts of tobacco, in part because few smallholders have land that is suitable for intensive farming of tobacco. Even for farmers in suitable agro-ecological areas, however, tobacco is expensive to grow, with high up-front costs that smallholder farmers may not be able to cover. For example, high costs of production of flue-cured tobacco are a barrier to most cash-poor smallholders in tobacco areas. The financial incentives to smallholder farmers with the appropriate land to grow tobacco are, however, strong with the relative returns to cash and labor considerably higher than for most other crops. Most commercial farmers already have diversified sources of income, and have introduced various high-value crops including export roses, supermarket vegetables, paprika and coffee as part of their farm system, specifically to lessen their dependence on tobacco. As with all business decisions, the challenge for farmers is to find the right blend of crop enterprises that works best for them. Beyond the enterprises covered here, for example, the study indicates that, other than the crops discussed in the study, many other niche products also offer diversification potential for smallholder and LSC farmers including mushrooms, flower seeds, medical plants and spices. Citrus crops, including oranges, grapefruit and lemons are an important diversification option with more than 88,000 hectares of orchards planted on LSC farms as of 1999. Game ranching on LSC farms has been another popular diversification activity and can provide an important source of supplemental income. One of the main arguments against measures to reduce the demand for tobacco is the worry that falling demand will cause job losses in the farm sector. Tobacco is a good source of indirect income for smallholder farm households with family members who work as wage-laborers on large-scale commercial tobacco farms. Simple calculations suggest that the total wage bill for tobacco grown on LSC farms is around USD 47.3 million. To the extent that a share of this income is sent as remittance payments to family members in communal areas, tobacco can play a major role in helping to finance the inputs needed for improved management of major smallholder crops including maize and cotton. In countries where most of the tobacco crop is consumed domestically, expenditure switching will create demand for other goods and services, and employment gains in these other sectors are likely to offset the losses in the tobacco sector.

For A. formosa, the most substantial expression difference occurred between DS3 and DS4, although the number of genes DE between DS4 and DS5 is also relatively high . This pattern more or less mirrors our sampling strategy in which finer sampling intervals were used between DS1 and DS4 with a larger developmental gap between DS4 and DS5. Given that there are relatively few changes across the earlier stages, for our first global analysis we focused on the end points of our sampling, bracketing Phase I of development, and identified the genes that increase or decrease in expression between DS1 and DS5. Within each species, between 5568 and 6933 genes were differentially regulated between DS1 and DS5. Counts of genes commonly up- and down-regulated between species are presented in Fig. 3a. A set of 1262 genes was commonly up-regulated across all species through development while a set of 1094 genes was commonly down-regulated across all species through development . Between 498 and 644 genes were uniquely up-regulated in just a single species across development while between 459 and 683 genes were uniquely down-regulated in a single species across development . Gene Ontology enrichment analyses were conducted for the genes commonly up- and down-regulated between DS1 and DS5 in all taxa. Genes related to mitotic activity, including DNA replication, mitotic chromosome condensation, and microtubule ontologies are enriched early in development, while genes belonging to oxidation reduction and fatty acid biosynthetic ontologies are enriched late in development . The sets of genes up- or down-regulated in all of the spurred taxa but not A. ecalcarata were also identified,indoor vertical farming with 521 genes commonly up-regulated in only the spurred taxa between DS1 and DS5 and 318 genes commonly down-regulated between these stages .

Although the petal is generally not considered a photosynthetic organ, GO enrichment analyses on the gene sets DE in only the spurred taxa showed an enrichment of genes with GO terms related to photosynthesis were up-regulated between DS1 and DS5 . Looking across all four species, 11,258 genes showed differential expression between DS1 and DS5 in at least one species, representing a full third of predicted genes in the Aquilegia genome . Conducting a principal component analysis of the expression levels of these genes across all samples showed that most of the variance can be explained by developmental time point . According to PC1 , the comparative developmental staging done across each species is fairly consistent with the expression data across taxa with a few exceptions. The A. ecalcarata samples assigned to DS1 have slightly higher PC1 values than the DS1 samples from the other taxa, suggesting that based on gene transcription, they may be more developmentally similar to the DS2 samples from the other taxa. Judging by PC1, the A. chrysantha samples assigned to DS4 and DS5 may be more transcriptionally similar to DS3 and DS4, respectively, of the other three taxa.In order to identify genes potentially involved in nectar spur development, differentially expressed genes between spurless A. ecalcarata and each spurred taxon were identified at each developmental stage. 15,588 genes are differentially expressed between A. ecalcarata and at least one of the spurred species during at least one developmental time point . Across the first three developmental time points, a greater number of the genes identified as DE between A. ecalcarata and each spurred taxon are expressed at a higher level in A. ecalcarata than the spurred species . By the fourth and fifth developmental stages, an approximately equal number of DE genes are expressed more highly in A. ecalcarata and each spurred species. DE gene sets for each of the spurred species versus A. ecalcarata were then compared to each other to identify genes that are commonly up- or down regulated in the spurred taxa versus A. ecalcarata at each developmental stage .

At each developmental stage, more genes are commonly up-regulated in A. ecalcarata relative to all of the spurred species than up-regulated in all of the spurred species relative to A. ecalcarata. The number of genes commonly up-regulated in spurred taxa gradually increases throughout development . The number of DE genes up-regulated in A. ecalcarata also increases across development, but the greatest increases occur between DS1 and DS2 , and between DS4 and DS5 . In total, 237 genes are commonly up-regulated in the spurred taxa relative to A. ecalcarata at all five developmental stages and 453 genes are commonly up-regulated in A. ecalcarata versus the spurred taxa at all five developmental stages . As a comparison, we also looked at the number of loci that were commonly up- or down-regulated in any one of the spurred species relative to the other three taxa . There was a common pattern in that there were more genes up-regulated in the focal species of the comparison than down-regulated , however, more genes were commonly DE when A. ecalcarata was the focal species. Conducting PCA on the set of genes that were commonly DE between all spurred taxa and A. ecalcarata at any developmental time point shows, as expected, that PC1 captures the difference between the spurred species and A. ecalcarata while PC2 captures common developmental differences across species . PC3 largely captures differences between A. sibirica and the other taxa while PC4 largely captures differences between the two North American species, A. formosa and A. chrysantha. While data points for PCs 3 and 4 generally cluster by species, interestingly, the A. ecalcarata DS5 samples cluster with A. formosa in PC3 and with A. chrysantha in PC4, although we have no hypothesis for why this may be. At each developmental stage, we tested for GO enrichment in the genes identified as either having higher expression in A. ecalcarata or commonly having higher expression in the spurred taxa versus a set of genes expressed in any species at that developmental stage . During the earlier developmental stages , genes related to heme/iron binding and oxidoreductase activity are over-represented in the A. ecalcarata datasets. By DS5, many GO categories related to mitosis are enriched in the set of genes identified as significantly up-regulated in the spurred taxa.

A prior study sought to identify genes important for nectar spur development in the horticultural variety A. coerulea ‘Origami’ by comparing gene expression between two regions of developing petals: the distal tip of the developing nectar spur and the petal blade. A. coerulea ‘Origami’ has relatively long nectar spurs, similar to those of hawkmoth-pollinated species, and these comparisons between developing spur and blade were made at two developmental times points,hydroponic vertical farming when petals were 1 mm and 3 mm long. As this prior study was conducted using an earlier Aquilegia genome annotation, we re-analyzed the data using the A. coerulea “Golsdmith” v3.1 genome annotation . Counts of genes DE either developmentally or tissue specifically are summarized in Supplemental Tables S11 and S12 . Focusing on DE genes between the blade and spur cup tissue, at the 1mm stage, 490 genes were more highly expressed in the blade and 280 genes were more highly expressed in the spur cup . At the 3 mm stage, 1178 genes were more highly expressed in the blade and 767 genes were more highly expressed in the spur cup . The results of this reanalysis are quite similar to the original analysis and exhibit a pattern common to that seen in the comparison of A. ecalcarata to the spurred taxa at early stages : more genes were commonly up-regulated at both the 1 mm and 3 mm stages in the spurless tissue sample than in the sample with spur tissue . These DE gene sets were compared to the lists of genes commonly DE between the spurred taxa and A. ecalcarata at roughly comparable developmental stages . Since A. ecalcarata petals consist primarily of blade tissue, we hypothesized that there would be more genes commonly up-regulated between whole A. ecalcarata petals and the A. coerulea ‘Origami’ blade tissue , and between entire petals of the spurred taxa and the A. coerulea ‘Origami’ spur tissue , compared to the opposite combinations . This is indeed the case, however, there are some genes that are more highly expressed in the blade tissue and the spurred taxa or in the spur tissue and in A. ecalcarata . For the purposes of identifying genes that make up a potential core module for spur development, we were particularly interested in genes that fall into the ‘blade’ and ‘spur’ classes defined above. Comparing genes that are more highly expressed in A. ecalcarata than the spurred taxa at all developmental stages to those that are expressed more highly in the blade than the spur at both the 1mm and 3mm time point reveals only 27 common genes . Looking at the intersect of genes that are more highly expressed in all of the spurred taxa at the stages we assessed with those that are expressed higher in the developing spur at both the 1 mm and 3 mm stages reveals an even smaller set of only 8 common genes . Of the 8 genes more highly expressed in ‘spur’ class, four show a general trend of decreasing through development in the spurred taxa,especially between DS4 and DS5 . These genes encode a dynein light chain protein, a myb/SANT-like transcription factor, a cytochrome P450 protein, and an oxysterol-binding protein. Two genes, encoding a leucine rich repeat protein and a heat shock factor transcription factor, show a pattern of increasing expression in the early stages assessed followed by a decrease, while another gene encoding a C2H2-type zinc finger transcription factor has more steady expression throughout development. One gene, encoding a xyloglucan endotransglycosylase protein, has relatively low expression in the early developmental stages with an uptick in expression moving into DS5 in the spurred taxa, especially in A. sibirica and A. formosa.

Interestingly, the STY homologs, which were among the most strongly differentially expressed loci in the blade to cup comparison and which were later found to be critical to nectary development, were not among the genes overlapping with the spurred to unspurred comparison. During later stages of petal development, however, there is some divergence between the STY homolog expression among the species sampled here, with A. ecalcarata showing lower expression than the other three . Among the 27 genes that are more highly expressed in the ‘blade’ class, several transcription factors stand out. These include a gene in the NAC transcription factor family with similarity to ANAC034 , an OVATE Family Protein with similarity to AtOFP4, and a gene with homology to the cryptochrome 2-interacting basic helixloop-helix transcription factor AtCIB1 .In Arabidopsis, many genetic factors have been identified that influence petal shape. These often function by regulating the timing of the transition from cell proliferation to cell differentiation . Genes identified as playing a role in controlling this transition can broadly be broken down into those that promote proliferation versus those that repress proliferation. The cell proliferation activators start off broadly expressed across the primordium but they become down-regulated in a basipetal wave that is complementary to an opposing pattern of up-regulation of the cell proliferation repressors. As the early phase of nectar spur development in Aquilegia involves prolonging localized cell divisions in the spur, we explored the expression of the Aquilegia homologs to these Arabidopsis candidates in our dataset to see if their expression patterns are generally consistent with a conserved role in Aquilegia petal development or differ between A. ecalcarata and the spurred taxa.Negative regulators of cell proliferation in A. thaliana petals include BIG BROTHER , DA1, the TCP genes TCP4 and TCP5, and the cyclin-dependent kinase inhibitor genes KIP RELATED PROTEIN 4and KRP2 . Although it may be expected that these genes would show the opposite pattern of expression from the cell-proliferation promoters, only a few of the Aquilegia homologs showed a pattern of increasing expression approaching DS5 . Two KRP-like genes, AqKRP2-like and AqKRP-like showed a strong increase in expression, especially between DS4 and DS5. Both of theses genes were also expressed more highly in A. ecalcarata than the spurred taxa. A third KRP-like gene with homology to KRP4 actually sharply decreased in expression during later developmental stages in all taxa assessed. Aside from the two KRP-like genes previously mentioned, AqDA1 is the only other candidate cell division repressor that appears to be increasing in expression toward DS5.

Chapter 3 examines one piece of environmental management that needs to be addressed for deep-seabed mining: biodiversity assessment and monitoring. Multiple approaches are available to conduct biodiversity assessment and monitoring, such as morphology-based taxonomy and metabarcoding . The former involves an expert handpicking organisms out of an environmental sample and identifying them by eye or with the aid of a microscope. The latter involves chemical processing, molecular sequencing, and bio-informatics. Each of these methods are associated with scientific advantages and disadvantages, including how well they inform ecosystem services, and specific economic costs. In addition to discussing these scientific trade offs, Chapter 3 compares their cost-effectiveness in identifying deep-sea organisms, which is relevant to decision-makers when considering assessment and monitoring requirements. Another approach often employed in deep-sea research is the use of remotely-operated vehicles to survey and collect samples, including a wealth of imagery data. In the summer of 2015, Ocean Exploration Trust completed an expedition to explore the southern California borderlands, which included ROV dives at several methane seeps on the continental margin . Methane seeps are habitats where hydrocarbons and other fluids escape from the seafloor, fueling a biological community based on chemosynthesis . In a widely food-limited environment,indoor growers elevated primary production could significantly influence adjacent areas . Chapter 4 uses ROV dive videos from three methane seeps to demonstrate how deep-sea imagery can be used for characterization of ecosystem services. More specifically, Chapter 4 focuses on fisheries services and climate regulating services related to carbon, which have previously been documented at a California methane seep .

It provides the first detailed biological description of two methane seeps, and is the first application of an ecosystem services-based trait approach in the deep sea.Ecosystem services can also be generated by built ecosystems, such as natural storm water treatment systems . NTS are human-made installations that are designed to capture and treat storm water runoff using physical and biological processes . Coastal development and urbanization have altered water flows and introduced contaminants into runoff, which can cause flooding of infrastructure and pollution of local water . NTS provide a low-impact strategy to address these issues by slowing water flows, removing contaminants, and storing runoff for possible reuse . In southern California, where there is a discrepancy between water supply and demand, NTS are becoming more widespread . Chapter 5 reviews potential ecosystem services associated with NTS, such as targeted water infiltration and pollutant removal as well as non-targeted pollination, climate regulation, aesthetic value, and pests . Examples from Los Angeles County illustrate these ecosystem services and practical methods are suggested to begin quantifying and economically valuating them. Chapter 6 investigates one ecosystem service associated with NTS: climate regulation related to carbon. Vegetation within NTS, such as bio-filters and bio-swales, uptake atmospheric carbon dioxide through photosynthesis and can store it as biomass or in soil. Plant and soil respiration, the reverse process that releases carbon back into the atmosphere, is dependent on environmental characteristics such as soil moisture and soil temperature . Chapter 6 provides measurements of carbon fluxes over NTS, in comparison to other urban land uses in San Diego : grass lawns, horticultural gardens, and natural coastal sage scrub.

Data envelopment analysis is used to compare carbon efficiency, i.e. how well each system converts its given environmental characteristics into desirable carbon fluxes. This analysis incorporates both environmental inputs and outputs, which may be helpful when considering urban management strategies. The final chapter of this dissertation summarizes the lessons learned from applying an ecosystem services perspective in multiple systems using multiple approaches. It also provides recommendations for incorporating ecosystem services into environmental decision-making and management.Deep-seabed mining is an emerging industry that could begin commercial production in the near future. It has potential to alter habitats targeted for minerals through physical disturbance, removal of substrate, sediment resuspension and deposition, light, and noise . The International Seabed Authority , the body governing the international seabed and its resources , is tasked with developing rules, regulations, and policies, including those that will “[protect] the marine environment from harmful effects” and “[prevent] damage to the flora and fauna of the marine environment” . This obligation highlights the need for two tasks: establishing a baseline level of biodiversity for potentially impacted areas, and monitoring changes in biodiversity due to mining activities against that baseline. Deep-sea biodiversity supports a range of ecological functions and ecosystem services, such as fish catch, genetic resources for industrial and pharmaceutical products, carbon sequestration and storage, and nutrient cycling . The Clarion-Clipperton Fracture Zone is an abyssal plain area, ranging from approximately 3900-5500 m in depth, that hosts high densities of polymetallic nodules targeted for mining of copper, nickel, and cobalt. The CCZ has 30 exploration claims, each up to 75,000 square kilometers.

It also contains a wealth of biodiversity, some associated with the nodules themselves . Pilot studies and mining simulations have found little recovery of Pacific abyssal plain habitats post-disturbance within 26 years . Common megafaunal taxa in the CCZ include ophuiroids, xenophyphores, and corals, which are found in higher densities in the CCZ relative to other abyssal plain sites . Sediment macro- and meiofauna dominate eukaryotic species richness and densities on the abyssal plains , and include foraminifera, nematodes, polychaetes, isopods, and tanaids . While current ISA draft regulation for commercial exploitation of the Area acknowledges biodiversity as something to measure, the measurement approaches are not specified. Biodiversity can be measured in a variety of ways: number of species in an area, species absolute and relative densities, number of functional roles in a system and species interactions, genetic variation between and among populations, representation of phylogenetic lineages, and number of habitats and ecosystems . Current faunal bio-monitoring programs generally employ morphology-based taxonomy , which requires an expert to manually sort and identify hundreds to thousands of individual organisms. As a result, taxonomic studies in the CCZ require a lot of resources . The spatial heterogeneity of the CCZ biological community necessitates robust biodiversity assessment and monitoring . Large taxonomic gaps in deep-sea benthic communities still exist despite scientists working tirelessly to identify known species and describe new ones. As a result, MBT is limited to animal taxa that not only exhibit distinguishing morphological features , but also those for which expert taxonomists exist. Furthermore, this expertise can be especially difficult to find for deep-sea taxa and, as a result, much of the deep-sea environment remains undescribed . MBT is strongly limited by the amount of time and cost required to generate data, and the lack of taxonomic expertise that limits the breadth of biodiversity it can cover. This severely hampers scaling up both temporal and spatial resolution, and prevents timely adaptive management measures. Although MBT is necessary in order to describe new species , emerging molecular tools can serve to rapidly document biodiversity. Molecular techniques, such as metabarcoding and metagenomics,danish trolley provide a rapid alternative for biodiversity measurements in natural systems . Technical advances have increased our capacity to generate biodiversity data, by moving from DNA extracted from a single individual to environmental DNA obtained from an environmental sample in order to rapidly assess whole biological communities. Here, we focus on the application of metabarcoding: the sequencing of specific genes, used for taxonomic identification, in an environmental sample . A mix of both morphology-based and molecular-based methodologies are advocated to build robust and extended biodiversity inventories in the CCZ .

An example of a combined workflow couples morphological identification of individuals with DNA sequencing . While initially labor-intensive, this combined approach to taxonomy can facilitate later environmental assessment and monitoring by reducing the need for morphological identification as more species are described and sequenced. Additionally, a combined approach can promote standardization for data comparison among CCZ claims. For example, contractors measuring biodiversity within their own claims may be identifying the same species differently from other contractors. With a physical specimen associated with a genetic sequence, this issue could be more readily resolved even without a formal species description. Another example of a combined approach is initial genetic screening of large swaths of the CCZ to prioritize areas of interest for more detailed morphology work. It is likely that a combination of techniques is necessary in order to obtain scientifically robust data for environmental baseline and monitoring requirements set by UNCLOS and the ISA, such as those related to abundance and biomass, and genetic connectivity . This paper aims to evaluate scientific and economic trade offs between MBT and metabarcoding of small eukaryotes in the context of deep-sea biodiversity assessment and monitoring. Specifically, we discuss MBT and metabarcoding for evaluation of deep-seabed mining impacts in the CCZ, where interest in polymetallic nodules is high , small and rare taxa are dominant , and patchiness is substantial . We consider scientific trade offs between approaches in relation to environmental assessment and monitoring objectives, as well as how a combined approach can mitigate each method’s weaknesses. Decision networks are constructed for each methodology to highlight how decisions within each approach can affect scientific outcomes and economic costs. Lastly, we assess and compare direct and indirect costs associated with each methodology in a cost-effectiveness framework.Both MBT and metabarcoding techniques are framed here as a series of choices within a workflow that can influence both scientific outcomes and economic costs. We surveyed deep-sea experts and published protocols in order to determine the steps within each methodology, and how the choices within those steps affect scientific outcomes. In most cases, scientific questions and desired outcomes dictate how choices are made, creating a range of appropriate protocols so only general steps are listed in the results.Cost-effectiveness analysis is an economic approach that evaluates outcomes and their associated costs . An action or policy can be considered “cost effective” if it is the least costly to obtain a desired outcome, or it generates the best outcome given fixed resources. CEA differs from cost-benefit analysis because, rather than answering whether an action should be taken or not, it ranks strategies to maximize their efficiency . Posed with the question of how the ISA and contractors can meet environmental requirements, CEA can facilitate choosing which methods, or combination of methods, are most least-cost cost-effective. For each methodology, experts and published protocols provided estimates of the consumables used and number of work hours taken in order to generate taxonomic data. Total cost is the sum of consumable and labor costs. Fixed costs, such as laboratory equipment and bio-informatics pipelines, were not included in our analysis. Quantity of consumables were summed. Prices were taken directly from supplier websites, and are likely an overestimate because many research institutions receive discounted prices. Common suppliers in the U.S. were used: Fisher Scientific, VWR, and Qiagen, and prices were averaged among them. Labor costs came from best estimates of work hours .In our model, output is the number of “operational species” identified. “Operational species,” or proxies for species, are commonly used in biodiversity assessments because sampled deep-sea organisms are often new to science . MBT can employ morphospecies, individuals grouped together solely by morphology, whereas molecular methods use operational taxonomic units to distinguish species . Using operational species is less costly than describing every new species discovered, which involves writing a detailed morphological description and designating a holotype.Combining morphological and genetic operational species may circumvent the need for formal species descriptions and provide a standard unit of outcome that is relevant to both decision-making and our analysis. To approximate sampling regimes in the CCZ, we looked at a subset of published studies that attempt to characterize CCZ biodiversity of sediment eukaryotes using either MBT or molecular methods . Details from their sampling designs were extracted , as well as their relevant results . This information was used to make more appropriate comparisons between MBT and metabarcoding.Scientific trade offs should be considered while comparing MBT and metabarcoding, including what data are generated and information gained from their interpretation. These are summarized in Table 3.1 and discussed below. CCZ biodiversity is dominated by small and rare eukaryotes , which may favor taking a molecular approach to biodiversity assessments. However, although there is limited deep-sea taxonomic expertise, there is also little robust genetic information on CCZ fauna.

In pre-agricultural societies, zoonotic disease disproportionately affected those who came into contact with animals and their products during hunting and gathering. In Neolithic and post-Neolithic European and Middle Eastern societies, conversely, a greater proportion of individuals came into close contact with concentrated animal pens and therefore became more liable to infection from diseases that followed their byproducts.As Polgar has shown, indeed, the domestication of animals has historically increased the spread of zoonotic diseases including anthrax, tuberculosis, and even types of influenza. Students and researchers should consider the paleo-archeological insights above as they try to understand why Native Americans were often so vehement in their immediate opposition to European agricultural models that concentrated cattle in farms and/or stored grain in newly constructed central warehouses. The development of those models, after all, accompanied the growth of massive disease outbreaks among Native Americans from the 1500s. The primary historical sources unearthed in the work of Anderson and Cronon, for example, would provide one point of reference: in New England, enclosed farms set up by seventeenth-century English settlers seemed to encourage the spread of infectious diseases, whether zoonotically or due to the increased concentrations of peoples.Bovine strains of tuberculosis arrived with European cattle, as did tuberculosis bacilli and influenza strains in Swine,raspberry cultivation pot as well as the trichina worm.Spanish settlers in California and the Southwest created ranches with similar effects, albeit in a slightly different colonial context.

Native Americans, according to one 1674 colonial report, perceived English cattle as “Unwholsom for their Bodies, filling them with sundry Diseases.” As Anderson has suggested in a discussion of human diseases that seemed to correlate with animal outbreaks, those Native Americans “who survived an initial bout of disease often emerged in a weakened state, vulnerable to subsequent ailments that would not necessarily have imperiled a healthy individual.”New England Native Americans caught up in King Philip’s War often “began their hostilities with plundering and destroying cattle,” according to one witness. Large-scale killings of domestic animals continued throughout the war, and Native American hostility to the animals – and their owners – extended to mutilation and torture of cows in particular.Assessing the imposition of domesticated agriculture by Europeans during early contact, Cherokee-American anthropologist and historian Russell Thornton has thus suggested that “the reasons for the relatively few infectious diseases in this [western] hemisphere [prior to European contact] surely include… the existence of fewer domesticated animals, from which many human diseases arise” – unlike those that later grew up due to grain storage and transit patterns which differed from hunter-gatherer lifestyles as well as their own pre-contact forms of land management and crop cultivation.As biological anthropologists have shown, some precontact Native American communities were likely familiar with the association between cultivated animal lots and disease, albeit in more isolated settings. Pueblo Native Americans had witnessed the spread of diseases in areas contaminated by turkey dung, most probably salmonella and Shigella.Turkeys had been domesticated in the northern Southwest, likely from populations of Merriam’s wild turkey. In most cases Pueblo communities demonstrated the capacity for mixed land use between animals and cultivated crops, without zoonotic diseases. But their example at least suggests that some communities may have been aware of the potential disease association, if conditions became less optimal.

To be sure, there remain significant risks in using a conceptual distinction between Paleolithic hunter-gatherers and Neolithic agriculturalists to understand changing Native American health outcomes after European contact. The link between negative Native American responses to European domesticated farming and the problematic health patterns associated with Neolithic agriculture must not be emphasized too specifically, of course; not least because the latter occurred between 8,000 and 10,000 years prior to the Native American encounter with colonial-European agriculture. Yet it is at least possible to draw broad conceptual associations between the two studied periods. Just as in paleo-archaeological records detailing the rise of Neolithic grain production and animal husbandry, source material relating to the Native American-European encounter demonstrates the potential for zoonotic diseases to proliferate in regions that were required to adapt to more concentrated forms of land use . Rather than highlighting a simple biological exchange of disease, then, students could point to the specific interventions of European enclosure and domesticated cattle-raising, which made infectious maladies even more potent and widespread.They would consider the methodologies and conclusions of Armelagos, as well as those provided by other scholars of the move from Paleolithic ecology towards Neolithic agriculture. Having done so, they would be in a position to draw general inferences – and even conclusions – as they assess historical source material for the Native-American-European encounter and the growth of zoonotic diseases, over a relatively wide geographical basis and a relatively broad space of time. The purported difference between Native American hunter-gatherers and European pastoralists becomes rather more problematic if it is used to de-emphasize the role of indigenous agriculture and formal crop cultivation prior to European contact. In order to avoid such a crude assertion, students would do well to assess our developing understanding of the “Hopewell tradition”, which describes the various aspects of the Native American cultures that developed along rivers in the northeastern and Midwestern United States from 200 BCE to 500 CE.

In these regions, tropical domesticate species of gourds, such as Cucurbita pepo, were introduced from Mesoamerica, well before the 2000 to 1000B.C period that scholars once linked to the first domestication of North American plants in the region. Scholars have recognized Curcurbita at the subspecies level and have thus made the convincing suggestion that the Hopewell cultures of North America created a second independent center of domestication for the species, just as they did with other plant species: Pumpkins, marrows, and other gourds were first domesticated in Mesoamerica, while acorn squashes, scallop squashes, fordhooks, crooknecks,low round pots and a variety of gourd species were then cultivated in North America. The domestication of indigenous eastern North American seed plants can thus be categorized in four species: Cucurbita pepo, Helianthus annuus, Iva annua, and Chenopodium berlandieri.During the summer and fall periods, for example, pseudo grain seed-like chenopods became an important starch source for some Native Americans from modern-day Arkansas to eastern Kentucky, at latitudes from northern Alabama to central Ohio, from ca. 1800 B.C. until ca. A.D. 900 or later. Evidence suggests that hunter-gathering practices in these regions were supplemented by the domestication of those and similar chenopods .Thus it is increasingly clear that many North American societies modified parts of their ecological environments even prior to the proliferation of maize production. The latter responded to demographic distress on the one hand, but also may have contributed to certain declining health outcomes, on the other hand . The four indigenous species that were cultivated prior to and even after the introduction of maize, conversely, were likely the product of a much larger context of “stable long-term adaptations and broadscale niche construction efforts that were carried out in the absence of any carrying-capacity challenges or seriously compressed and compromised resource catchment areas.” Their cultivation, alongside other “crops” such as berries and tubers, had required, among other things: differential culling of trees, expanding natural strands of floodplain seed plants, artificial fires, and establishing “orchards” of fruit and berry-producing species.Similar patterns of cultivation could also be detected in other regions, such as California and the American Southwest, as well as the wild rice regions of the central Great Lakes . In 1541, in what is modern-day eastern Texas and western Louisiana, Spanish colonizers such as Hernando de Soto noted that communities of Karanka was cultivated corn, beans, squash, sunflowers, and tobacco.From around A.D. 900 in the riverine “horticultural villages” on the Great Plains, along the Missouri and its tributary rivers flowing into the lower Mississippi, beans, squash, melon, corn, and sunflowers were cultivated by Caddoan speaking Native American communities. Each village also maintained hunting territories where animals were stalked from the late-summer onwards. Thus, here and elsewhere, it is inaccurate to highlight a dichotomy between indigenous horticulture and hunter-gatherer patterns prior to the period of first European contact.Precontact horticultural communities often “retained hunting and gathering practices as regular supplements and insurance against occasional shortages.”After European contact, to be sure, many North American horticulturalists tried to revert to “full time hunting and gathering” as a means for sustenance; even while some in New England, among the Cherokee of the South, and the Navajo of the Southwest, remained as pastoralists.Greater reliance on hunting and gathering after contact represented an indigenous response to the curtailment of horticulture, or its problematic limitation and enclosure, by new European forms of crop rotation and animal husbandry. That response, however, set up a chain of circumstances that resulted in the permanent loss of autonomous access to hunted meats and indigenously cultivated plants by the second half of the nineteenth century. From the late-sixteenth century, Spanish, French, and English settlers in coastal North America tended to overlook the possibility for symbiosis between hunter-gatherer and horticultural methods of sustenance, which often oscillated in relative importance according to the season.

Instead, Europeans noted the Native-American reaction to their presence – a reversion to hunting and gathering and revulsion for their own domesticated agriculture – and erroneously assumed that they solely engaged in nomadic hunting patterns. This attitude “contributed to the notion that removal of eastern nations to Midwestern reservations would solve problems of conflict between expanding Euro-American populations and the Indians’ loss of hunting lands.” Among English colonizers in the eastern seaboard, the view that Native Americans refrained from land cultivation was indeed key in defining the territory as res nullious – empty of privately managed land and thus legitimate for colonial settlement. A similar assessment took place among Spanish colonizers in present-day California and the Southwest, albeit on a different scale.As they moved towards an increasingly central migration trajectory in the face of European colonization, various indigenous communities from the East, the Great Lakes, and the West often adopted European firearms and horses to hunt in new nomadic patterns. Shoshone tribes from the central and northern Mountain West adopted Spanish horses and introduced them to other indigenous communities in the Great Plains. Algonquians such as the Blackfeet, Gros Ventres, and Araphos, as well as some Cree and Ojibwa tribes, “abandoned forest hunting and gathering to become mounted nomadic hunters on the Great Plains” – thereby interacting with other tribes who had adopted Spanish horses as a means to hunt for new food sources. A northern Athapaskan group, the Sarsis, as well as some Comanches, similarly adopted horse nomadism. By the later-eighteenth century, previously horticultural Cheyenne communities entered the Plains, coming to rely on hunted buffalo and bison. Siouan and Caddoan horticulturalists along the streams and rivers of the eastern Great Plains sometimes also switched to horse-nomadic hunting-gathering practices after their traditional forms of plant cultivation and hunting were threatened by the interaction between European settlement and Native American warfare. Pawnee Indians, as well as some Hidatsas on the upper Missouri, moved towards horse-mounted nomadism. Through the eighteenth century, Siouan-speaking tribes gravitated from horticulture towards bison hunting, shifting west and leaving the bottomlands of the Ohio and Mississippi rivers. In most of these cases, as Snow has pointed out, “people were not just responding to the attraction of nomadic hunting. There were by this time [around 1700] also strong direct and indirect pressures from the east, brought on by European settlement and expansion.” Initially, health profiles seemed to increase among various new Plains Native American communities. As Heckel has argued, using data from births between the 1830s and 1870s on the Great Plains, their new proximity to bison and other animals likely benefited the association between greater height and nutritional density. Yet as these communities adopted new forms of hunting, competition with European settlers in the region coupled with over efficiency so as to diminish their access to the area’s sources of animal protein and fat. By the later part of the nineteenth century, therefore, many Native Americans found themselves unable to rely on traditional nutritional interactions between horticultural and hunter-gathering products. Worse, they soon began to lose access to micro-nutrients and macro-nutrients from fast diminishing populations of bison. Relative to other European settlers, indeed, their height advantage diminished somewhat.The narrative above raises further implications for students and researchers, beyond the issue of zoonotic disease and concentrated settlements: if hunting and gathering as well as indigenous forms of crop cultivation were disrupted by the imposition of European agriculture in North America from the late-1500s, how else can we measure the historical symptoms and effects of declining health following the change to the Native American ecological landscape?

The intermittent water seals treatment was applied using a temporary sprinkler system installed in the plots following fumigation and the post fumigation tillage operation; water was applied four times in the first 2 days after fumigation: 0.5 inch after 3 hours, 0.2 inch after 12 hours, 0.2 inch after 24 hours and 0.2 inch after 48 hours. All plastic films were removed 10 days after fumigation. Fourteen days after the initial 1,3-D fumigation, the metam sodium treatment was applied through sprinklers at 160 pounds per acre in 2.75 inches of water. For the dual application treatment, 21 days after the initial treatment, soil was inverted with a moldboard plow and an additional 1,3-D treatment was applied with the previously described Telone rig and rolling operation. Fumigant emissions from eight 1,3-D treatments — two application shank types times four surface seal methods — were monitored in three replicate plots for 10 days following the initial application. Emission of 1,3-D from the soil surface was monitored using previously described dynamic flux chamber techniques . Briefly, a flow-through flux chamber with a 10-inch-by-20-inch opening was installed on the surface following fumigant injection and installation of the films or after the initial water seal treatment . These chambers allow semi-automated,fodder systems for cattle continuous sampling of fumigant concentrations in the air above the surfaces. The cis– and trans-isomers of 1,3-D were trapped in charcoal sampling tubes .

The two 1,3-D isomers were summed as total 1,3-D for data analysis and reporting. Individual tubes were removed from the flux chambers every 3 to 6 hours and stored frozen until laboratory processing. Emission flux and cumulative emission during the 10-day monitoring period were calculated based on surface area and air flow rates through the flux chambers, and treatment differences were compared using analysis of variance . The concentration of 1,3-D in the soil-gas phase was determined 6, 12, 24, 48, 120 and 240 hours after treatment. At each time point, samples were collected using a multi-port sampling probe and a system of gas-tight syringes to draw air from eight depths through charcoal sampling tubes. Samples were stored frozen until analysis. In the laboratory, all samples were processed using procedures described by Gao et al. . Briefly, sample tubes were broken and trapped fumigants were extracted from the trapping matrix with ethyl acetate and analyzed using a gas chromatograph equipped with a micro electron capture detector .Pest control efficacy was evaluated using citrus nematode bio-assay counts, fungal dilution plating, and weed emergence counts and biomass collections from each replicated plot. The pest control data from this research station emission flux experiment were reported in Jhala et al. .In addition to the emission flux and efficacy study conducted at KAC, two field trials were conducted in commercial nurseries to evaluate pest control efficacy and nursery stock productivity. Fumigation and surface treatments in the nursery experiments were the same as in the flux study with minor exceptions. The commercial nursery trials were arranged as randomized complete block experiments with a split plot arrangement of 1,3-D treatments.

The whole plot factor was surface treatment, and the split plot factor was the shank type. Individual plots in these experiments were 22 feet by 90 feet, and each treatment was replicated four times.In 2007, the experiment was established in a garden rose nursery near Wasco. The soil at the rose nursery site was a McFarland loam with pH 6.2, 0.9% organic matter and 74% sand, 13% silt and 13% clay. Treatments were applied on Nov. 7, 2007, when the soil temperature was 64ºF and soil moisture averaged 9.2% w/w from 2 to 5 feet. The experiment was repeated in 2008 in a deciduous tree nursery near Hickman, in a Whitney and Rocklin sandy loam soil with pH 6.5, 0.8% organic matter, and 66% sand, 23% silt and 11% clay. Treatments in the tree nursery trial were applied on Aug. 13, 2008, when the soil was 80ºF and soil moisture ranged from 5.0% to 12.6% w/w in the top 5 feet. Immediately following 1,3-D application, a disk and roller were used to compact the soil and disrupt shank traces and HDPE and VIF were installed using the Noble plow rig. For the water seal main plots, a temporary sprinkler system was installed after the post fumigation tillage operation and intermittent water seals were applied: 0.5 inch after 3 hours, and 0.2 inch each after 12, 24 and 48 hours. The dual application 1,3-D treatments were applied in the garden rose experiment on Nov. 28, 2007, but were not included in the 2008 tree nursery experiment. Metam sodium was applied in 2.75 inches of irrigation water through sprinklers 14 to 30 days after the initial 1,3-D treatment in both experiments.Both nursery trials were managed by the cooperating growers using their standard practices for planting, fertilization, in-season tillage and budding and harvest operations. In the 2007 rose experiment, two rows each of the rose rootstock ‘Dr. Huey’ and the own-rooted garden rose variety ‘Home Run’ were planted as hardwood cuttings in December 2007.

Rose nursery stock was planted 7 inches apart in furrows spaced 3 feet apart, and the field was furrow irrigated during the 2008 and 2009 growing seasons. The own-rooted cultivar was harvested after one growing season in January 2009, and the unbudded ‘Dr. Huey’ root stock was harvested in February 2010 after an additional growing season. At both harvest dates, all plants in one 90-foot row were lifted using a single row undercutting digger, plants were bundled and tagged by plot, and graded in a commercial packinghouse. In the 2008 tree nursery trial, two rows each of the peach root stock ‘Nemaguard’ and the plum root stock ‘Myro 29C’ were planted with 8 inches between plants and 5 feet between rows in December 2008. The tree nursery plots were sprinkler irrigated during the 2009 growing season. Due to the market needs of the cooperating nursery,fodder sprouting system the root stocks in the tree trial were not available for harvest and grading as a part of the experiment. Pest control efficacy and crop productivity were evaluated during the 12- or 26-month nursery production cycle. Nematode control was determined using a citrus nematode bioassay in which two sets of muslin bags containing 100 grams of soil infested with citrus nematode were buried at 6, 12, 24 and 36 inches below the soil surface in each plot prior to fumigation. The initial population of citrus nematodes in infested soil was 4,086 and 3,876 nematodes per 100 cubic centimeters of soil in 2007 and 2008, respectively. The bags were recovered 1 month after fumigation, nematodes were extracted from 100 cubic centimeters of soil using the Baermann funnel protocol, and surviving nematodes were identified and counted. To evaluate the effect of fumigation treatments on soil fungal populations, ten 1-inch-by-12-inch soil cores were collected from each subplot 2 weeks after fumigation. Soils were homogenized, and a sub-sample was assayed for Fusarium oxysporum Schlecht. and Pythium species using dilution plating techniques on selective media. Pythium species samples were plated on P5ARP medium for 48 hours, and F. oxysporum samples were plated on Komada’s medium for 6 days. Emerged weeds in a 1-square-meter area were identified and counted twice in the winter following the fall fumigation and several times during the subsequent summer growing season. Nursery stock establishment, vigor and growth were monitored during the season. Visual evaluations of crop vigor were made on a scale of 1 to 7, where 7 was the most vigorous and 1 was dead or dying plants. Near the end of the growing season, trunk diameter of 10 plants in each subplot was measured 3 inches above the soil surface using a dial caliper. As previously described, rose nursery stock was harvested and graded to commercial standards ratings, but tree nursery stock was not harvested as a part of the experiment.

Data were subjected to analysis of variance, and initial analyses indicated that the shank types did not differ in their effect on any of the pest control or crop growth parameters measured. Thus, data from the two shank type treatments were grouped together within surface treatments and reanalyzed with seven treatments and six treatments . The nematode, pathogen and weed density data were transformed [ln ] to stabilize the variance prior to analysis; however, means of untransformed data are presented for clarity. Treatment means were separated using Fisher’s protected least significant difference procedure with α = 0.05.Within a surface treatment, there were no statistical differences in emission flux between the two application shank types, thus data were combined over application rig. However, significant differences in 1,3-D emission flux were observed among surface treatments . Fumigant emission flux from bare plots was two times higher than from water seals and HDPE and nearly 15 times higher than from VIF within 48 hours after treatment. Emission from water-sealed plots was reduced during the sequential water applications, but flux was similar to bare soil plots after 48 hours. HDPE film continued to give lower emission rates than the bare soil and water seals but was significantly higher than VIF. Throughout the monitoring period, VIF-covered plots had the lowest 1,3-D emissions; maximum flux was 11 micrograms per square meter per second , which was at least 90% lower than that from the bare soil plots. Relative to the bare soil treatment, estimated cumulative 1,3-D emission losses for water seals, HDPE and VIF were 73%, 45% and 6%, respectively, which were similar to reports from a previous field study .Concentration of 1,3-D immediately below the plastic film indicated that 1,3-D retention is much greater under VIF film than under HDPE . Several other studies have shown that VIF can retain substantially higher fumigant concentrations without negatively affecting nematode, pathogen and weed control efficacy or crop yield .Initial analysis of fumigant distribution in the surface 90 centimeters indicated that there were no differences between the application shanks within a surface treatment in this zone; thus data were combined over application shank types . The 1,3-D concentration was highest near the injection depth, at 45 centimeters and lowest near the soil surface, at 5 centimeters , and at 90 centimeters , but this difference diminished over time. The effect of depth on 1,3,-D concentration was most evident in water seals and bare soil plots. HDPE and VIF plots had more uniform distribution of the fumigant through the soil profile than the water seals plots, especially 48 hours after treatment. However, 1,3-D concentration under the VIF tarp was markedly higher than in all other treatments, which suggests that there could also be differences in the top 5 centimeters of soil. These results imply that the use of a highly impermeable tarp can lead to a more uniform distribution of fumigants in the soil profile and may allow satisfactory pest control with reduced application rates .Pest control data from the 2007 KAC emissions trial and a related 2008 emissions trial were reported previously and are not shown here. In general, however, there were few differences in pest control attributed to the fumigant application shanks used in the trial. Pythium species populations were lower in all treatments than in the untreated control, but no statistical differences were noted in Fusarium species populations among treatments. The high 1,3-D rates and well-prepared soils resulted in complete control of citrus nematodes in the bio-assay bags in all treatments and depths. Weed populations were variable among treatments but tended to be lowest in methyl bromide plots and 1,3-D plots sealed with VIF and highest in the water seals and dual 1,3-D application treatments. All treatments of 1,3-D or methyl bromide effectively controlled citrus nematodes in bio-assay bags buried at 12-, 24- and 36-inch depths in each plot. However, these results, which were obtained in well-prepared sandy soils with low pest and pathogen populations, may not apply to more challenging field conditions . Applications of 1,3-D sealed with HDPE or VIF and dual application 1,3-D treatments reduced Fusarium and Pythium species propagules in the soil compared with the untreated plots . These treatments were comparable to methyl bromide in controlling Fusarium and Pythium species.

In immature fruit, enriched pathways were more evident at or after 24 hpi. In contrast, multiple pathways were enriched in mature fruit, as shown by early time points, which suggests an overall activation of stress responses associated with the biotic challenge and tissue breakdown. These time-dependent responses to M. laxa were also evident when quantifying the number of DEGs for enriched categories related to plant defense , which confirmed that immature fruit had the highest gene expression induction at 24 hpi, and that mature fruit had a larger number of genes induced than immature fruit as early as 6 hpi. DEGs related to the plant–pathogen interaction pathway were largely absent from the immature fruit response, with the exception of 24 hpi, but were quite abundant in the mature fruit response starting at 14 hpi . Hormone signaling was enriched early in fruit at both developmental stages, though it appeared to become less relevant in immature fruit at 48 hpi. Cysteine and methionine metabolism and α-linolenic acid metabolism pathways, associated with ethylene biosynthesis and jasmonic acid biosynthesis, respectively, were enriched in both immature and mature fruit, though more prominently in the latter. Pathways related to the biosynthesis of terpenoids were also found to be enriched at early time points in immature and mature fruit , but their enrichment was higher in immature than mature tissue. Other pathways that appeared to be relevant for nectarine responses against M. laxa included the phenylpropanoid and glutathione metabolism,dutch buckets which were highly induced in the mature fruit, likely utilized as antioxidants.

Given the enrichment of genes involved in plant hormone signaling transduction during early infection and the activation of methionine and α-linolenic metabolism in both fruit tissues across time, a targeted analysis of ET and JA pathways was conducted. The transcriptional activation of JA biosynthesis was evident in immature and mature fruit, with special emphasis in the induction of multiple genes encoding the initial biosynthetic steps , from lipoxygenase to 12-oxophytodienoic acid reductase . Later steps of the biosynthesis pathway were only moderately activated in both tissues. In mature tissues at 48 hpi, a down regulation of the JA-amino synthetase gene was observed, involved in the production of the active form of JA, and of the homolog of the JA receptor coronatine-insensitive protein 1 . Two out of the five paralogs of the signaling repressor JA ZIM domain appeared to be activated in immature and mature tissues at multiple time points. The three paralogs encoding the transcriptional activator of JA responses, MYC2, were strongly induced in mature fruit after 14 hpi and upregulated in immature fruit only at 14 hpi and 24 hpi. In fact, the MYC2 gene expression level of the third paralog was significantly higher in inoculated immature than mature tissue, but then, its expression was significantly higher in mature than immature tissue at both 24 and 48 hpi . The steps committed to ET biosynthesis catalyzed by the 1-aminocyclopropane-1-carboxylate synthase and the 1-aminocyclopropane- 1-carboxylate oxidase genes were highly induced in response to M. laxa inoculations, particularly in mature fruit .

Ethylene signal transduction elements showed only moderate changes in gene expression in response to the pathogen. Interestingly, although the negative regulator EBF1/2 was down regulated at 14 and 48 hpi in both tissues, it was highly upregulated in immature tissue at 6 and 24 hpi. However, all three paralogs of the ET response factor 1/2 , which control multiple ET responses and are a point of signal integration for JA and ET signal transduction, were highly upregulated in both tissues. The ERF1/2 gene expression level of the second paralog was significantly higher in mature inoculated than immature inoculated fruit at 14 hpi . In addition, the ET produced by M. laxa-inoculated and control fruit was measured to complement the transcriptional data . Control nectarines followed the ET pattern of a climacteric fruit; low and steady levels of ET in immature fruit and high and significantly increased levels in mature fruit until ripening. However, in inoculated immature fruit, ET production significantly peaked at 24 hpi, corresponding to the peak of transcriptional responses in this tissue, before returning to levels equivalent to the control fruit. In inoculated mature fruit, the ET production was significantly lower than control fruit at 6 hpi, but then significantly increased. These results suggest that nectarine was performing a tightly regulated response of ET.To determine which fungal genes and functions are biologically relevant during M. laxa interactions with nectarine, we performed a functional analysis of the pathogen transcriptome. First, a total of 9581 transcripts were denovo annotated for multiple functional categories, including carbohydrate-active enzymes , fungal peroxidases , genes involved in pathogen–host interactions , membrane transport proteins , and proteins with signal peptides , among others . Then, an enrichment analysis of these large functional categories in the upregulated DEGs across infection was performed to obtain a general picture of specific gene categories induced by the pathogen in immature and mature fruit .

In immature fruit, these large categories were enriched in M. laxa upregulated DEGs at least at one time point when compared to 6 hpi. Particularly at 24 hpi, a significant abundance of CAZymes and PHI genes was observed. Fungal peroxidases were only significantly enriched in immature fruit at 48 hpi. In contrast, enrichment of CAZymes and fungal peroxidases was not observed at any time point in mature tissues. Genes in involved in pathogen–host interactions and membrane transport remained enriched at relatively even levels from 14 to 48 hpi in mature fruit. We identified GO terms related to pathogenicity, virulence, and fungal growth among the upregulated DEGs for each host developmental stage . Among this subset of biologically relevant GO terms, threefold more upregulated DEGs were detected when M. laxa was inoculated in mature fruit compared to immature fruit. Particularly, the number of M. laxa upregulated DEGs in immature tissue increased progressively until 24 hpi and then decreased slightly at 48 hpi, whereas in mature tissue, the upregulated DEGs increased along with infection time. Notably, these gene expression patterns resembled the transcriptional response of the host for each developmental stage . In both stages, M. laxa induced a high number of DEGs related to oxidative–reduction processes and transmembrane transport, although genesinvolved in protein translation and proteolysis were only abundantly expressed in mature tissue. However, genes involved in response to oxidative stress were mainly expressed in immature at 48 hpi, together with the enrichment of fungal peroxidases at this time point . Lastly, the enrichments of Pfam domains were also carried out using the M. laxa upregulated DEGs . In agreement with previous results, Pfam categories were mainly enriched at 24 hpi in immature fruit, with the exception of proteins containing the fungal pathogenesis-related CFEM domain, which were uniquely enriched earlier at 14 hpi. In addition, Pfam domains related to fungal membrane transport were largely prominent in immature fruit, especially at 24 hpi, where up to 53 genes were induced. Less significantly enriched, fungal glycosyl hydrolases, dehydrogenases , and catalases were found at 48 hpi in immature tissues. The number of enriched Pfam domains among M. laxa upregulated DEGs in mature fruit, such as those related to transcription and translation , increased throughout disease progression . However, other relevant domains, such as some related to proteolysis activity , uniquely peaked at 14 hpi. Notably, upregulated DEGs annotated as ribosomal proteins and transcriptional factors involved in growth and cell cycle control were prevalent throughout infection of mature fruit. Later,grow bucket infection time points exhibited enrichments of protein domains belonging to membrane transport and redox functions .To identify potential target genes for the control of M. laxa, a closer examination was conducted of the most highly M. laxa upregulated DEGs from all time points and tissue comparisons . The top five M. laxa-induced DEGs in immature and mature fruit were unique between the tissue types, reinforcing the evidence that the pathogen displays a different behavior according to the developmental stage of the host. Strongly induced DEGs at 14 hpi unique to early infections of immature fruit included fungal phosphate transporters, phospholipases, and oxidoreductases. A member of the glycosidase hydrolase family 31 was highly expressed at 24 hpi in immature fruit, alongside a transmembrane fructose transporter and histidine phosphatase . The highest induced DEGs in immature fruit were detected at 48 hpi and corresponded to an oxido reductase gene , a homolog of the alcohol oxidase from Cladosporium fulvum, and the same transmembrane fructose transporter already found at 24 hpi. Interestingly, M. laxa DEGs with fungal peroxidase annotations, a catalase and a haloperoxidase , were only detected at 48 hpi in immature fruit. In mature fruit, a single protease gene was the highest upregulated M. laxa DEG at all time points. Two polygalacturonases were among the largest induced DEGs during infections of mature fruit; Monilinia_000560 was highly upregulated at 14 hpi, whereas Monilinia_041700 was highly expressed at 24 and 48 hpi.

Another CAZyme was also highly enriched at 14 and 24 hpi. In mature tissue, transporters and hormonerelated genes were among the highest expressed DEGs. An amino acid transporter was significantly expressed at 14 hpi, while a tryptophan 2- monooxygenase was induced at 48 hpi, known to be involved in virulence in another pathosystem. Altogether, these results suggest that targeting of specific genes involved in response to oxidative stress, nutrient transport, and carbohydrate catabolism may reduce quiescent infections, while specific proteolytic genes and additional CAZymes may help inhibit or reduce the severity of disease in susceptible fruit.The first line of plant defense that M. laxa has to overcome is the constitutive physical and chemical barriers present in the fruit surface. The developmental process from immature to mature fruit is characterized by physical and chemical changes in fruit firmness, leading to softening at the onset of ripening. In fact, the flesh firmness of immature fruit was higher than the mature fruit . Monilinia laxa appeared to produce more CWDE in immature fruit, which suggests that the pathogen could be trying harder to overcome the host cell walls in these tissues. Nevertheless, the immature tissue had no visible disease symptoms. Other alterations occurring during fruit development include changes in plant cuticle, sugar accumulation, volatile compounds, and secondary metabolites synthesis, which have been reviewed as promoting susceptibility to pathogens in ripening fruit. Hence, higher soluble solids content and lower titratable acidity on mature fruit could favor pathogen colonization. Plant–pathogen interactions take place when pathogen associated molecular patterns are recognized by the plant’s pattern recognition receptors, which ultimately triggers a defense response known as PAMP triggered immunity. The chitin elicitor receptor kinase 1 was upregulated in the mature tissue at 14 hpi. Also, the expression levels of the transcriptional activator PTI5 were up to 2.5-fold and 5-fold higher in mature fruit when compared to immature fruit, at 24 and 48 hpi, respectively. PTI responses can be suppressed by effector proteins secreted by the pathogen, which in turn, will elicit effector-triggered immunity. In our pathosystem, proteins with the CFEM domain and signal peptides were enriched in the early infection stage on immature tissue. Among the annotated genes with the CFEM domain, the Monilinia_077410 is a homolog of BcCFEM1 from B. cinerea, an effector shared by many Botrytis spp.and described to be important for its virulence. These results suggest that M. laxa may secrete some type of effector proteins in immature fruit. Once the host–pathogen interaction began, both pathogen and host triggered their own transcriptional reprograming. In mature tissue, both nectarine and M. laxa abruptly changed their gene expression profile at 14 hpi, coinciding with the ability of the pathogen to grow and macerate the fruit tissues within 14 h. From 14 hpi onwards, the pathogen started to penetrate and switched toward an aggressive necrotrophic phase, which was retained at later infection times. Functions related to transmembrane transport, oxidation-reduction process, and translation were among the most abundant activities in mature fruit, denoting the growth and spread of the pathogen. In contrast, the number of nectarine and M. laxa DEGs in immature fruit remained somewhat steady through infection time, even when fungal biomass peaked at 24 hpi. Overall, these findings suggest that inoculated mature nectarines displayed an earlier and broader response to M. laxa than immature ones, likely due to the faster pathogen growth and virulence mechanisms activation in these tissues. Both PTI and ETI are able to induce the host hormone signaling transduction pathway, which was found to be enriched, starting at 6 hpi in both tissues.

Hormones are of central importance for the regulation of metabolic processes and plant development in a complex system of interacting hormones and cofactors, the functions of which are closely intertwined and mutually dependent . bio-stimulants developed from humic substances, complex organic materials, seaweeds, antitranspirants, free amino acids , and crude extracts of lower and higher plants have been frequently demonstrated to have an effect on plant hormonal status . While hormone-like compounds may be present in bio-stimulants, it is also possible that de novo synthesis of hormones may be induced by such preparations in treated plants and amino acids, glycosides, polysaccharides and organic acids are contained in many bio-stimulants and may act as precursors or activators of endogenous plant hormones . Hormones or hormone-like effects could therefore be responsible for the action of natural bio-stimulants derived from microorganisms, algae, higher plants, animal, and humate based raw material . Information on currently available bio-stimulants gives some insight into the possible biochemical and molecular genetic effects of bio-stimulants derived from different natural raw materials . Many published reports are available suggesting various bio-stimulants improve plant productivity through increased assimilation of N, C, and S , improved photosynthesis,hydroponic nft channel improved stress responses, Thered senescence, and enhanced ion transport . bio-stimulants are also reported to increase free amino acids, protein, carbohydrates, phenolic compounds, pigment levels, and various enzymes .

The protective effect of many bio-stimulants against biotic and abiotic stresses has been associated with a reduction of stress-induced reactive oxygen species, activation of the antioxidant defense system of plants, or increased levels of phenolic compounds . While it is clear that many biologically derived bio-stimulants contain small molecular weight compounds that are involved in signaling events and may directly influence plant metabolic processes, it remains unclear how an exogenous soil or foliar application of an uncharacterized product can have predictable and beneficial responses in plants. It is well-known, for example, that application of exogenous plant hormones or compounds that disrupt hormone function can have markedly negative effects on plants and that optimization of PGR materials and their applicaitons requires precise information on dosage and timing. Application of bio-stimulants for which the dosage and efficacy of the functional compounds is unknown, cannot, therefore, be expected to result in predictable plant responses and identification of molecules with effects on plant metabolic processes is not, in of itself, a sufficient explanation for the function of a bio-stimulant. It is also uncertain why the application of a bio-stimulant with purported function as a PGR, signaling molecule or other discrete compound would be superior to, or more easily controlled, than a direct application of the purifified product itself. Modern crop production requires a balance of high and consistent productivity with maximum safety for consumers, agricultural workers, and the environment . While some bio-stimulants have been analyzed with regard to unwanted side effects including negative impact on the natural environment most bio-stimulants have not been fully characterized but have been regarded as generally recognized as safe on the basis of the biological origin of their constituents . Generally, bio-stimulants are assumed to be biodegradable, non-toxic, nonpolluting and non-hazardous to various organisms.

While this may be a rational conclusion for many formulations derived from biological materials such as seaweed extracts and their components , higher plants , chitin and chitosan it is not clear that this is a valid assumption for microbial products or products that would not normally be present in agricultural fields. bio-stimulants have been utilized as bio-remediants and have been shown to improve ATP levels and phosphatase and urease activity , and hence increase the rate of degradation of xenobiotics in the soil and to enhance beneficial soil microbial communities under semi-arid climates . bio-stimulants may also help reduce the amount of potentially risky agrochemicals including reducing the use of fertilizers and pesticides . Most compounds contained in bio-stimulants are natural constituents of terrestrial and aquatic ecosystems and metabolites of plant and microbial origin and as such most are generally regarded as safe, particularly at the low rates at which they are typically applied. Thus, it has been proposed that bio-stimulants can be positioned as eco-friendly products for sustainable agriculture . In many countries, however, bio-stimulants are not subject to rigorous toxicological screening and there remains the potential for the persistence of human pathogens in materials of animal origin and for the synthesis of novel compounds of unknown function or toxicology during the manufacturing process. Even though there have been relatively few rigorous demonstrations of the benefit of bio-stimulants, and to a large extent the mode of action of these products remains uncertain, the industry for bio-stimulants is substantial and rapidly growing. Though many recent “market” studies show that the market for these products is growing at a remarkable rate, the validity of these analyses must be considered with care as they frequently do not provide an explicit definition of term “bio-stimulants.” The value of the European bio-stimulants market ranged from e200 to e400 million in 2011, e500 million in 2013 and may grow to more than e800 million in 2018 with annual growth potential in 10% and more . France, Italy, Spain are the leading EU countries in the production of bio-stimulants .

In North America, the bio-stimulant market was valued at $0.27 billion in 20131 , and is expected to grow at a growth rate of 12.4% annually, to reach $0.69 billion by 2018, the USA is the largest producer and consumer of bio-stimulants in the region . In 2014, the USA market was assessed at $313.0 million and is projected to reach $605.1 million by 20192 , at a CAGR of 14.1% . The bio-stimulants market in the Asia-Pacific was valued at $0.25 billion in 2013, and is expected to grow at a CAGR of 12.9% annually, to reach $0.47 billion by 2018. China and India are key countries playing a significant role. The Southeast Asian & Australasian bio-stimulants market was valued at $233.8 million in 2015, and is projected to reach $451.8 million by 2021. The market in Latin America was valued at $0.16 billion in 2013, and is expected to grow at a CAGR of 14.4% annually, to reach $0.32 billion by 2018 . This market is mostly concentrated in Brazil and Argentina. The regional market shares of the global bio-stimulants market6 are: EU—41.7%, North America—21.5%, the Asia-Pacific region—20%, Latin America—12.9%. Globally, it bio-stimulants were valued at $1402.15 million in 2014 and are projected to have aCAGR of 12.5% reaching $2524.02 million by 2019, largely as a consequence of growing interest in organic products. Wu summised that “the global bio-stimulants market is projected to reach $2.91 billion by 2021, with a CAGR of 10.4% from 2016 to 2021. In terms of area of application, the bio-stimulants market is projected to reach 24.9 million hectares by 2021 and is projected to grow at a CAGR of 11.7% from 2016 to 2021” .” The bio-stimulant industry faces many problems and challenges. Until recently bio-stimulant products based on natural raw materials and particularly waste stream has mainly been developed based on observational and less commonly,nft growing system empirical data. While many contemporary bio-stimulants have been shown to be effective in practice, very few bio-stimulants can claim to understand the mechanisms or modes of action . Furthermore, while bio-stimulants can be categorized by source of origin, this is frequently inadequate as very substantial differences can exist between products even within a common feed stock origin. The challenge to bio-stimulant science is further exacerbated since composition and content of active substances in the original plant raw material can be affected by many factors including the location and growing conditions, season, species, variety, organ, and the phase of growth . Similarly, the response of the target crop can be expected to vary across crops and environments. One solution to this problem is to derive the raw materials for the bio-stimulant under highly regulated conditions. This approach has been successfully implemented by leading seaweed producers and fermentation based products that have developed harvesting and manufacturing processes that ensure uniformity of product performance through time. The development of a product with uniformity of response is not, however, a guarantee that the product is optimized for biological efficacy. To address these issues, developments in -omics approaches will be critical in accelerating the discovery of mode of action of bioactive compounds and optimizing their use. Metabolomics, phenomics and agronomics represent the integration of gene expression, protein interactions, and other regulatory processes as they impact on plant productivity and thus are more appropriate tools for discovery in this field than mRNA, transcripts, or proteins analyzed in isolation . Integrative, multidisciplinary approaches using tools from transcriptomics in conjunction with metabolomics and biochemical analysis are necessary to establish the mechanism of action and to identify the active components in the extracts . The difficulty in identifying modes of action and subsequent standardization of composition of multi-component bio-stimulants based on natural raw materials will continue to hamper the use, certification and registration of bio-stimulants. The solution to this problem will require the collaborative efforts of specialists from different fields: chemists, biologists, plant physiologists, industrial manufacture, sales and distribution and those with expertise in practical agricultural production .

Products with a single active substance represent a simpler construct in which the physiological effects and mechanism of action can be more readily determined and hence certification and registration is simpler. The multi-component composition of many preparations, however, are much more difficult to characterize , though they may offer novel insight into biological synergy , multi-functionality and emergence which may be crucial to product efficacy . In the absence of a functional rationale for every constituent in a multi-component bio-stimulant, it is likely that there will be molecules present that may positively or negatively influence plant productivity. Currently, it is almost impossible using available chemical-synthetic, and genetic engineering approaches to reproduce the full suite of molecules and complexes of biologically active substances that are present in most bio-stimulants. Many have noted the state confusion in the field of bio-stimulants and this has resulted in the opinion that much of the bio-stimulant market is not based on science or efficacy and that many products are little more than recycled waste products sold on the basis of pseudoscience and marketing. Indeed, research on several bio-stimulant products has shown them to be ineffective or to contain inactive, unstable or inconsistent properties with several showing negative effects compared when contrasted with well-designed controls . For example, foliar and root application of a product containing amino acids from animal origin have been reported to cause severe plant-growth depression and negative effects on Fe nutrition while a second product containing amino acids from plant origin stimulated plant growth . In another report that tested several bio-stimulant products it was concluded that “none of the bio-stimulant products tested achieved a sufficient degree of pathogen control to warrant replacement of or supplementation with conventional synthetic fungicides” , and there have been demonstrated positive and negative impacts and overall questions of the economic feasibility of the use of humic substances for increasing crop yields . Since biological systems are inherently complex, and given that most bio-stimulant products have not been characterized and have received relatively little replicated and rigorous independent validation, it is perhaps not surprising that many products are ineffective or highly variable in response. Nevertheless, there are a significant number of rigorous independent reports of benefits from some bio-stimulant formulations and market growth data demonstrates that there is a good deal of support for these products within agricultural producer communities. That such market growth has occured, even in the absence of a known “mechanism of function” suggests that there are aspects of plantmetabolism and productivity constraints that are not understood but are potentially important if we are to achieve the goal of increased global food production. The market euphoria that is taking place in the bio-stimulant industry recognizes these unknowns and bio-stimulants are viewed by many innovators and investors as a mechanism to conduct broadscale, if unfocussed, discovery of novel biologically derived molecules. Much as the exploration of marine organisms, and plants and microbes from diverse ecosystems has led to the discovery of novel pharmaceuticals, so too the development of bio-stimulants from the broad range of source materials, holds significant promise of discovery.

Decomposing lower quality litter implies that soil microbes may need to immobilize nutrients to maintain their stoichiometric balance . In addition, under elevated CO2 conditions, available nutrients will progressively move from fast cycling tissues to slow cycling tissues , which induces progressive nutrient limitation that further exacerbates nutrient limitations. Although increased external nutrient inputs and accelerated nutrient mineralization rates under warming soil conditions may enhance soil nutrient availability and partly ease plant– microbe nutrient competition, these additional nutrients may be insufficient to satisfy the enhanced plant nutrient demands . To investigate nutrient competition and its effects on the terrestrial carbon cycle, different theories of plant-soil nutrient competition have been developed and implemented in Earth System Models . However, theoretical justification and observational support for these theories are rarely discussed, which may have resulted in large biases in modeled nutrient and carbon cycling . To reconcile this inconsistency between theory, observations, and models, we focus on one overarching question in this study: Is there an observationally consistent, theoretically supported,ebb and flow trays and mathematically robust theory that is simple enough to implement in ESMs while accurately representing plant–microbe competition for nutrients? To answer this question, we first survey four existing nutrient competition theories and their implementation in ESMs .

In Results, we discuss in detail these four competition theories: CT1, no direct competition; CT2, microbial decomposers outcompete plants; CT3, competition depends on pore-scale soil fertility heterogeneity; and CT4, plant–microbe relative demand controls competition. Then we describe a new theory of nutrient competition based on Equilibrium Chemistry Approximation kinetics . We test our new theory together with other existing competition theories against a unique observational data set of N competition in a grassland ecosystem.To inform the development of ESM land models, observations have to satisfy two criteria. First, observations should capture plant and microbe competition at the whole-soil level, because the significance of microsite heterogeneity diminishes at this spatial scale. Second, measurements should target short-term nutrient uptake, thus enabling relatively clear separation of the instantaneous competitive interactions from other ecosystem dynamics that occur over longer time scales . To our knowledge, the only experiment that meets these two criteria was conducted in an alpine meadow ecosystem with homogeneous vegetation cover using a 15N tracer . In this experiment, the investigators randomly selected 90 10 cm diameter microplots within a 25 × 25 m area and added 0.32 g N/ m2 in the form of 15N−NH4 + or 15N−NO3 − with three soil depths treatments. 15N was injected at 2.5 cm, 7.5 cm, and 12.5 cm depth for 0–5, 5–10, and 10–15 cm treatments, respectively. The isotopic tracer was sampled 24 and 48 hours later, which informed partitioning of added nitrogen between microbes and plants. Furthermore, the grassland system has a very high rooting density, which allows us to isolate competitive interactions in the root zone from transport limitations so that observed competition patterns are directly comparable with theoretical models .

For our model evaluation, we extracted the data points from Fig. 4 of Xu et al. using the matlab script GRABIT .2Among the four existing theories surveyed, the traditional Nutrient Competition Theory assumes that plants and microbes do not compete for nutrients. This theory presumes that plants can assimilate carbon directly from the atmosphere but rely on nutrients released from soil microbial activity, so plants are carbon rich but nutrient limited . Conversely, because soil microbes decompose soil organic matter to obtain carbon and nutrients , they are relatively nutrient abundant but carbon limited . A second reason ecologists hypothesize that plants and microbes do not compete is that microbes can directly use organic N during decomposition , while plants primarily use inorganic N . However, depending on their carbon use efficiency and biomass stoichiometric imbalances against substrates , microbes do immobilize inorganic nutrients and thus directly compete with plants, creating the first contradiction against the CT1 theory. Further, plants may also utilize some low molecular weight amino acids through mycorrhizal fungi associations or direct root uptake , which creates a second contradiction to the theory. However, no existing ESMs apply CT1 to represent nutrient competition . The second theory posits that microbial decomposers out-compete plants in nutrient acquisition. This theory assumes that microbial nutrient uptake is extremely efficient , and microbes assimilate as much nutrients as they can during decomposition, provided they are not carbon limited. When carbon is limited, mineral nutrients are released as a “waste product” . This concept leads to the classic idea that plants can only use “leftover” nutrients after microbial demands are satisfied , which is why measured net mineralization rates are commonly used as a proxy for plant-available nutrients . However, no evidence exists to support its validity at the whole-soil or ecosystem level.

In contrast, 15N labeling studies have demonstrated that plants can continuously acquire inorganic nutrients, even when both plants and microbes are nutrient limited .CT2 has been applied in several ESMs. HadGEM2 and GFDL assume that soil microbial decomposers always outcompete plants and have priority for available nutrients . IPSL and BNU-ESM also assume that microbial immobilization has priority, but apply this priority to the estimated gross mineralization flux in the current model time step, as opposed to the nutrient pool. The third competition theory applies the emerging perspective that plant–microbe nutrient competition depends on the spatial heterogeneity of soil nutrient fertility, and therefore plants do not completely lose the competition at the whole-soil or ecosystem level. In a heterogeneous soil medium, inorganic nutrients move from nutrient-rich microsites toward nutrient-limited microsites , with roots potentially intercepting the nutrients . CT3 has been integrated into very fine-spatial scale models that explicitly consider the role of microsite soil nutrient heterogeneity, nutrient diffusion, root–microbe interactions , and microbe–microbe competition . In these models, plants do not completely lose the competition with microbes because they can take advantage of fine-scale spatial gradients between immobilizing and mineralizing microbes. The emergent responses from these models indicate that nutrient diffusion rates, sink strength , and competitor spatial distributions are the most important factors affecting plant competitiveness. However, these models’ fine spatial resolution is not directly applicable to ESMs. In ESMs, each soil column is assumed to be a well-mixed environment of nutrients and competitors. Such an assumption is currently necessitated, at least, by limited computational power and observations. Although ESM spatial resolutions likely will become finer, simulating microsite-level soil heterogeneity will remain impractical in the near future. In addition, a model based on CT3 may have high explanatory value but low predictive value, because it requires fine resolution observations of soil heterogeneity . The fourth nutrient competition theory has been applied in several ESMs. In these ESMs, plant nutrient demand is simulated based on potential Net Primary Production in the absence of nutrient constraints and the plant C to N ratio ; an analogous approach is taken for microbial nutrient demand. When soil nutrient supply is insufficient to saThisfy these demands,4×8 flood tray both plant and microbial demands are reduced in proportion to their respective demands . The actual NPP is then calculated by rescaling NPP demand with the reduction factor. This “relative demand” theory implicitly assumes that the consumer with higher demand will be relatively more competitive. While being simple, the CT4 predicted plant nutrient uptake is mechanistically inconsistent with measurements , although Goll et al. argued that the “demand-driven” approach requires fewer model parameters. The ESMs that apply CT4 include CLM-CN and NorESM , CLM-CNP , and JSBACH-CNP . The ECA-based nutrient competition theory.—The final competition theory is based on the concept of substrate–enzyme interactions applied to multiple nutrients and consumers . Substrate-enzyme kinetics theory has been extensively applied to model enzyme mediated processes ; however, its potential to represent plant and microbe nutrient competition is under-appreciated. Previous work that used the Michaelis-Menten equation to calculate nitrogen uptake only considered one carrier enzyme and one substrate at a time. To our knowledge, this study is the first that successfully extends classic enzyme kinetics theory to reconcile inconsistencies between observed plant and microbe nutrient competition and predictions from existing ESM competition theories. The substrate–enzyme view of plant nutrient uptake posits that first, extra-cellular enzymes, primarily produced by microbes, degrade polymers into monomers and inorganic nutrients, and second, plants produce nutrient transporter enzymes specialized for each mineral nutrient .

These plant-membrane-bound transporter enzymes react with substrates and form complexes , which are then transported into the root cell, after which the transporter enzymes are freed for the next substrate acquisition. Similar uptake mechanisms have been identified for microbes . This “nutrient carrier enzymes mediating uptake” conceptual framework allows us to extend the classic enzyme kinetics theory to represent plant– microbe competition based on the Equilibrium Chemistry Approximation theory and functional traits of the organisms. The same idea has provided the theoretical basis for the classic Michaelis-Menten type representation of nutrient uptake . However, classic enzyme theory and the resulting MM kinetics only consider one carrier enzyme and one substrate at a time, making them theoretically unable to represent competitive interactions among multiple consumers for multiple substrates. The novelty of the ECA approach is that it systematically considers multiple nutrient carrier enzymes and substrates together, and provides a rigorously derived and theoretically supported mathematical solution . It is also more accurate than the MM kinetics for cases involving only one carrier enzyme and one substrate . No current ESMs apply the ECA theory, although we are integrating it into the ACME Land Model . This work represents our first attempt to demonstrate its utility for plant–microbe nitrogen competition.The ECA representation of nutrient competition provides a theoretical and modeling construct that resulted in very good comparison with the nitrogen uptake partitioning. These predictions demonstrate that integrated across the soil profile, plants were less competitive than microbial decomposers; plant competitiveness against microbes is a spatially distinct property and there is no simple coefficient that can scale their “competitiveness”; the ECA framework offers a theoretically consistent approach to continuously update individual competitiveness; plant competitiveness is controlled by functional and structural traits ; and in the topsoil, plants might out-compete microbes and consequently suppress microbial nutrient uptake. Of course, applying the ECA competition to ESMs comes at the cost of introducing new parameters and additional uncertainty associated with those parameters. However, the ECA approach does not necessarily increase overall model uncertainty . In fact, ECA competition largely reduced the uncertainty in global-scale predictions by considering essential processes that govern system dynamics . We argue that an analogous result occurred in this analysis, i.e., that the uncertainty reduction in model structure overwhelmed uncertainty associated with new model parameters. In addition, most of the ECA parameters are kinetic parameters, which can be directly measured or optimized , implying that targeted experiments and model calibration could further reduce parameter uncertainty.Nutrient competition constantly occurs between plants and microbes in natural terrestrial ecosystems and it will likely intensify under climate change . Therefore, two fundamental questions arise: what controls the partitioning of limited nutrient resources between plants and microbes and how should short-term competition be modeled? Regarding the first question, we highlight the very few observations available to quantitatively partition nutrient acquisition by plants and microbes, and contend that such observations are critical to improve carbon-climate feedback predictions. As we showed here, the detailed 15N tracer experiment used in this study allowed us to evaluate the existing and newly developed plant–microbe N competition hypotheses, because the experiment was conducted at the plot scale and 15N was directly injected in the rooting zone . Thus, most of the observed plant N uptake pattern reflected the direct competition between roots and microbes, via nutrient carrier enzymes quantity and quality. Regarding the second question, we show here that plant and microbial nutrient uptake can be mechanistically explained as different nutrient transporter enzymes reacting with soil nutrients in a competitive manner. By linking plant root and microbial biomass density to nutrient transporter enzyme abundances, our new competition theory produces qualitatively correct competition patterns with literature-derived parameters from other ecosystems, and is easy to calibrate for specific ecosystems. Further, the linkage of nutrient competition with plant and microbial traits will allow a model to represent the competitors’ dynamic allocation of resources to acquire necessary nutrients.

A major advantage of plants in this respect is the ability to test multiple product candidates and expression cassettes in parallel by the simple injection or infiltration of leaves or leaf sections with a panel of Agrobacterium tumefaciens clones carrying each variant cassette as part of the transferred DNA in a binary transformation vector . This procedure does not require sterile conditions, transfection reagents, or skilled staff, and can, therefore, be conducted in standard bio-safety level 1 laboratories all over the world. The method can produce samples of even complex proteins such as glycosylated monoclonal antibodies for analysis ~14 days after the protein sequence is available. With product accumulation in the range of 0.1–4.0 g kg−1 biomass , larger-scale quantities can be supplied after 4–8 weeks , making this approach ideal for emergency responses to sudden disease outbreaks. Potential bottlenecks include the preparation of sufficiently large candidate libraries, ideally in an automated manner as described for conventional expression systems, and the infiltration of plants with a large number of candidates. Also, leaf-based expression can result in a coefficient of variation >20% in terms of recombinant protein accumulation, which reduces the reliability of expression data . The variability issue has been addressed to some extent by a parallelized leaf-disc assay at the cost of a further reduction in sample throughput . The reproducibility of screening was improved in 2018 by the development of plant cell pack technology,flood table in which plant cell suspension cultures deprived of medium are used to form a plant tissues surrogate that can be infiltrated with A. tumefaciens in a 96-well microtiter plate format to produce milligram quantities of protein in an automated, high-throughput manner.

The costs can be as low as €0.50 per 60-mg sample with a product accumulation of ~100 mg kg−1 and can typically result in a CV of <5% . These costs include the fermenter-based upstream production of plant cells as well as all materials and labor. The system can be integrated with the cloning of large candidate libraries, allowing a throughput of >1,000 samples per week, and protein is produced 3 days after infiltration. The translatability of cell pack data to intact plants was successfully demonstrated for three mAbs and several other proteins, including a toxin . Therefore, cell packs allow the rapid and automated screening of product candidates such as vaccines and diagnostic reagents. In addition to recombinant proteins, the technology can, in principle, also be used to produce virus-like particles based on plant viruses, which further broadens its applicability for screening and product evaluation but, to our knowledge, according results had not been published as of September 2020. In the future, plant cell packs could be combined with a recently developed method for rapid gene transfer to plant cells using carbon nanotubes . Such a combination would not be dependent on bacteria for cloning or gene transfer to plant cells , thereby reducing the overall duration of the process by an additional 2–3 days . For the rapid screening of even larger numbers of candidates, cost-efficient cell-free lysates based on plant cells have been developed and are commercially available in a ready-to-use kit format. Proteins can be synthesized in ~24 h, potentially in 384-well plates, and the yields expressed as recombinant protein mass per volume of cell lysate can reach 3 mg ml−1 . Given costs of ~€1,160  ml−1 according to the manufacturer LenioBio , this translates to ~€400 mg−1 protein, an order of magnitude less expensive than the SP6 system , which achieves 0.1 mg ml−1 at a cost of ~€360  ml−1 based on the company’s claims. Protocol duration and necessary labor are comparable between the two systems and so are the proteins used to demonstrate high expression, e.g., luciferase.

However, the scalability of the plantcell lysates is currently limited to several hundred milliliters, and transferability to intact plants has yet to be demonstrated, i.e., information about how well product accumulation in lysates correlates with that in plant tissues. Such correlations can then form the basis to scale-up lysate-based production to good manufacturing practice -compliant manufacturing in plants using existing facilities. Therefore, the cell packs are currently the most appealing screening system due to their favorable balance of speed, throughput, and translatability to whole plants for large-scale production. In any pandemic, the pathogen genome has to be sequenced, made publically available, and freely disseminated in the global scientific community to accelerate therapeutic and vaccine development. Once sequence information is available, a high priority is the rapid development, synthesis, and distribution of DNA sequences coding for individual viral open reading frames. These reagents are not only important for screening subunit vaccine targets but also as enabling tools for research into the structure, function, stability, and detection of the virus . Because many viral pathogens mutate over time, the sequencing of clinical virus samples is equally important to enable the development of countermeasures to keep pace with virus evolution . To ensure the broadest impact, the gene constructs must be codon optimized for expression in a variety of hosts ; cloned into plasmids with appropriate promoters, purification tags, and watermark sequences to identify them as synthetic and so that their origin can be verified ; and made widely available at minimal cost to researchers around the world. Not-for-profit plasmid repositories, such as Addgene and DNASU, in cooperation with global academic and industry contributors, play an important role in providing and sharing these reagents.

However, the availability of codon-optimized genes for plants and the corresponding expression systems is often limited . For example, there were 41,247 mammalian, 16,560 bacterial, and 4,721 yeast expression vectors in the Addgene collection as of August 2020, but only 1,821 for plants, none of which contained SARS-CoV-2 proteins. Sharing plant-optimized SARS-CoV-2 synthetic biology resources among the academic and industry research community working on PMPs would further accelerate the response to this pandemic disease. Screening and process development can also be expedited by using modeling tools to identify relevant parameter combinations for experimental testing. For example, initial attempts have been made to establish correlations between genetic elements or protein structures and product accumulation in plants . Similarly, heuristic and model-based predictions can be used to optimize downstream processing unit operations including chromatography . Because protein accumulation often depends on multiple parameters, it is typically more challenging to model than chromatography and probably needs to rely on data-driven rather than mechanistic models. Based on results obtained for antibody production, a combination of descriptive and mechanistic models can reduce the number of experiments and thus the development time by 75% , which is a substantial gain when trying to counteract a global pandemic such as COVID-19. These models are particularly useful if combined with the high-throughput experiments described above. Techno-economic assessment computeraided design tools, based on engineering process models,rolling benches can be used to design and size process equipment, solve material and energy balances, generate process flow sheets, establish scheduling, and identify process bottlenecks. TEA models have been developed and are publicly available for a variety of plant-based bio-manufacturing facilities, including whole plant and plant cell bioreactor processes for production of mAbs , antiviral lectins , therapeutics , and antimicrobial peptides . These tools are particularly useful for the development of new processes because they can indicate which areas would benefit most from focused research and development efforts to increase throughput, reduce process mass intensity, and minimize overall production costs.The rapid production of protein-based countermeasures for SARS-CoV-2 will most likely, at least initially, require bio-manufacturing processes based on transient expression rather than stable transgenic lines. Options include the transient transfection of mammalian cells , baculovirus-infected insect cell expression systems , cell-free expression systems for in vitro transcription and translation , and transient expression in plants . The longer term production of these countermeasures may rely on mammalian or plant cell lines and/or transgenic plants, in which the expression cassette has been stably integrated into the host genome, but these will take months or even years to develop, optimize, and scale-up. Among the available transient expression systems, only plants can be scaled-up to meet the demand for COVID-19 countermeasures without the need for extensive supply chains and/or complex and expensive infrastructure, thus ensuring low production costs . These manufacturing processes typically use Nicotiana benthamiana as the production host and each plant can be regarded as a biodegradable, single-use bioreactor . The plants are grown either in greenhouses or indoors, either hydroponically or in a growth substrate, often in multiple layers to minimize the facility footprint, and under artificial lighting such as LEDs. In North America, large-scale commercial PMP facilities have been built in Bryan, TX , Owensboro, KY , Durham, NC , and Quebec, Canada . The plants are grown from seed until they reach 4–6 weeks of age before transient expression, which is typically achieved by infiltration using recombinant A. tumefaciens carrying the expression cassette or by the introduction of a viral expression vector such as tobacco mosaic virus , for example, the GENEWARE platform . For transient expression by infiltration with A. tumefaciens, the plants are turned upside down and the aerial portions are submerged in the bacterial suspension.

A moderate vacuum is applied for a few minutes, and when it is released, the bacteria are drawn into the interstitial spaces within the leaves. The plants are removed from the suspension and moved to an incubation room/chamber for 5–7 days for recombinant protein production. A recent adaptation of this process replaces vacuum infiltration with the aerial application of the A. tumefaciens suspension mixed with a surfactant. The reduced surface tension of the carrier solution allows the bacteria to enter the stomata, achieving a similar effect to agroinfiltration . This agrospray strategy can be applied anywhere, thus removing the need for vacuum infiltrators and associated equipment . For transient expression using viral vectors, the viral suspension is mixed with an abrasive for application to the leaves using a pressurized spray, and the plants are incubated for 6–12 days as the recombinant protein is produced.Depending on the batch size , the vacuum infiltration throughput, and the target protein production kinetics, the infiltration/ incubation process time is 5–8 days. The inoculation/incubation process is slightly longer at 6–13 days. The overall batch time from seeding to harvest is 33–55 days depending on the optimal plant age, transient expression method, and target protein production kinetics . Importantly, plant growth can be de-coupled from infiltration, so that the plants are kept at the ready for instant use, which reduces the effective first-reaction batch time from gene to product to ~10–15 days if a platform downstream process is available . The time between batches can be reduced even further to match the longest unit operation in the upstream or downstream process. The number of plants available under normal operational scenarios is limited to avoid expenditure, but more plants can be seeded and made available in the event of a pandemic emergency. This would allow various urgent manufacturing scenarios to be realized, for example, the provision of a vaccine candidate or other prophylactic to first-line response staff.The speed of transient expression in plants allows the rapid adaptation of a product even when the process has already reached manufacturing scale. For example, decisions about the nature of the recombinant protein product can be made as little as 2 weeks before harvest because the cultivation of bacteria takes less than 7 days and the post-infiltration incubation of plants takes ~5–7 days. By using large-scale cryo-stocks of ready-to-use A. tumefaciens, the decision can be delayed until the day of infiltration and thus 5–7 days before harvesting the biomass . This flexibility is desirable in an early pandemic scenario because the latest information on improved drug properties can be channeled directly into production, for example, to produce gram quantities of protein that are required for safety assessment, pre-clinical and clinical testing, or even compassionate use if the fatality rate of a disease is high . Although infiltration is typically a discontinuous process requiring stainless-steel equipment due to the vacuum that must be applied to plants submerged in the bacterial suspension, most other steps in the production of PMPs can be designed for continuous operation, incorporating single-use equipment and thus complying with the proposed concept for biofacilities of the future .