Month: March 2023

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

With product accumulation in the range of 0.1–4.0 g kg−1 biomass , larger-scale quantities can be supplied after 4–8 weeks , making this approach ideal for emergency responses to sudden disease outbreaks. Potential bottlenecks include the preparation of sufficiently large candidate libraries, ideally in an automated manner as described for conventional expression systems, and the infiltration of plants with a large number of candidates. Also, leaf-based expression can result in a coefficient of variation >20% in terms of recombinant protein accumulation, which reduces the reliability of expression data . The variability issue has been addressed to some extent by a parallelized leaf-disc assay at the cost of a further reduction in sample throughput . The reproducibility of screening was improved in 2018 by the development of plant cell pack technology, in which plant cell suspension cultures deprived of medium are used to form a plant tissue surrogate that can be infiltrated with A. tumefaciens in a 96-well microtiter plate format to produce milligram quantities of protein in an automated, high-throughput manner. The costs can be as low as €0.50 per 60-mg sample with a product accumulation of ~100 mg kg−1 and can typically result in a CV of <5% . These costs include the fermenter-based upstream production of plant cells as well as all materials and labor. The system can be integrated with the cloning of large candidate libraries,growing lettuce hydroponically allowing a throughput of >1,000 samples per week, and protein is produced 3 days after infiltration.

The translatability of cell pack data to intact plants was successfully demonstrated for three mAbs and several other proteins, including a toxin. Therefore, cell packs allow the rapid and automated screening of product candidates such as vaccines and diagnostic reagents. In addition to recombinant proteins, the technology can, in principle, also be used to produce virus-like particles based on plant viruses, which further broadens its applicability for screening and product evaluation but, to our knowledge, according results had not been published as of September 2020. In the future, plant cell packs could be combined with a recently developed method for rapid gene transfer to plant cells using carbon nanotubes . Such a combination would not be dependent on bacteria for cloning or gene transfer to plant cells , thereby reducing the overall duration of the process by an additional 2–3 days . For the rapid screening of even larger numbers of candidates, cost-efficient cell-free lysates based on plant cells have been developed and are commercially available in a ready-to-use kit format. Proteins can be synthesized in ~24 h, potentially in 384-well plates, and the yields expressed as recombinant protein mass per volume of cell lysate can reach 3 mg ml−1 . Given costs of ~€1,160 ml−1 according to the manufacturer LenioBio , this translates to ~€400 mg−1 protein, an order of magnitude less expensive than the SP6 system , which achieves 0.1 mg ml−1 at a cost of ~€360 ml−1 based on the company’s claims. Protocol duration and necessary labor are comparable between the two systems and so are the proteins used to demonstrate high expression, e.g., luciferase. However, the scalability of the plant cell lysates is currently limited to several hundred milliliters, and transferability to intact plants has yet to be demonstrated, i.e., information about how well product accumulation in lysates correlates with that in plant tissues. Such correlations can then form the basis to scale-up lysate-based production to good manufacturing practice -compliant manufacturing in plants using existing facilities.

Therefore, the cell packs are currently the most appealing screening system due to their favorable balance of speed, throughput, and translatability to whole plants for large-scale production. In any pandemic, the pathogen genome has to be sequenced, made publically available, and freely disseminated in the global scientific community to accelerate therapeutic and vaccine development. Once sequence information is available, a high priority is the rapid development, synthesis, and distribution of DNA sequences coding for individual viral open reading frames. These reagents are not only important for screening subunit vaccine targets but also as enabling tools for research into the structure, function, stability, and detection of the virus . Because many viral pathogens mutate over time, the sequencing of clinical virus samples is equally important to enable the development of countermeasures to keep pace with virus evolution . To ensure the broadest impact, the gene constructs must be codon optimized for expression in a variety of hosts ; cloned into plasmids with appropriate promoters, purification tags, and watermark sequences to identify them as synthetic and so that their origin can be verified ; and made widely available at minimal cost to researchers around the world. Not-for-profit plasmid repositories, such as Addgene and DNASU, in cooperation with global academic and industry contributors, play an important role in providing and sharing these reagents. However, the availability of codon-optimized genes for plants and the corresponding expression systems is often limited . For example, there were 41,247 mammalian, 16,560 bacterial, and 4,721 yeast expression vectors in the Addgene collection as of August 2020, but only 1,821 for plants, none of which contained SARS-CoV-2 proteins. Sharing plant-optimized SARS-CoV-2 synthetic biology resources among the academic and industry research community working on PMPs would further accelerate the response to this pandemic disease. Screening and process development can also be expedited by using modeling tools to identify relevant parameter combinations for experimental testing.

For example, initial attempts have been made to establish correlations between genetic elements or protein structures and product accumulation in plants . Similarly, heuristic and model-based predictions can be used to optimize downstream processing unit operations including chromatography . Because protein accumulation often depends on multiple parameters, it is typically more challenging to model than chromatography and probably needs to rely on data-driven rather than mechanistic models. Based on results obtained for antibody production,maceta de 30 litros a combination of descriptive and mechanistic models can reduce the number of experiments and thus the development time by 75% , which is a substantial gain when trying to counteract a global pandemic such as COVID-19. These models are particularly useful if combined with the high-throughput experiments described above. Techno-economic assessment computer aided design tools, based on engineering process models, can be used to design and size process equipment, solve material and energy balances, generate process flow sheets, establish scheduling, and identify process bottlenecks. TEA models have been developed and are publicly available for a variety of plant-based bio-manufacturing facilities, including whole plant and plant cell bioreactor processes for production of mAbs , antiviral lectins , therapeutics , and antimicrobial peptides . These tools are particularly useful for the development of new processes because they can indicate which areas would benefit most from focused research and development efforts to increase throughput, reduce process mass intensity, and minimize overall production costs.The rapid production of protein-based countermeasures for SARS-CoV-2 will most likely, at least initially, require bio-manufacturing processes based on transient expression rather than stable transgenic lines. Options include the transient transfection of mammalian cells , baculovirus-infected insect cell expression systems , cell-free expression systems for in vitro transcription and translation , and transient expression in plants . The longer term production of these countermeasures may rely on mammalian or plant cell lines and/or transgenic plants, in which the expression cassette has been stably integrated into the host genome, but these will take months or even years to develop, optimize, and scale-up. Among the available transient expression systems, only plants can be scaled-up to meet the demand for COVID-19 countermeasures without the need for extensive supply chains and/or complex and expensive infrastructure, thus ensuring low production costs . These manufacturing processes typically use Nicotiana benthamiana as the production host and each plant can be regarded as a biodegradable, single-use bioreactor . The plants are grown either in greenhouses or indoors, either hydroponically or in a growth substrate, often in multiple layers to minimize the facility footprint, and under artificial lighting such as LEDs. In North America, large-scale commercial PMP facilities have been built in Bryan, TX , Owensboro, KY , Durham, NC , and Quebec, Canada . The plants are grown from seed until they reach 4–6 weeks of age before transient expression, which is typically achieved by infiltration using recombinant A. tumefaciens carrying the expression cassette or by the introduction of a viral expression vector such as tobacco mosaic virus , for example, the GENEWARE platform . For transient expression by infiltration with A. tumefaciens, the plants are turned upside down and the aerial portions are submerged in the bacterial suspension.

A moderate vacuum is applied for a few minutes, and when it is released, the bacteria are drawn into the interstitial spaces within the leaves. The plants are removed from the suspension and moved to an incubation room/chamber for 5–7 days for recombinant protein production. A recent adaptation of this process replaces vacuum infiltration with the aerial application of the A. tumefaciens suspension mixed with a surfactant. The reduced surface tension of the carrier solution allows the bacteria to enter the stomata, achieving a similar effect to agroinfiltration . This agrospray strategy can be applied anywhere, thus removing the need for vacuum infiltrators and associated equipment . For transient expression using viral vectors, the viral suspension is mixed with an abrasive for application to the leaves using a pressurized spray, and the plants are incubated for 6–12 days as the recombinant protein is produced.Depending on the batch size , the vacuum infiltration throughput, and the target protein production kinetics, the infiltration/ incubation process time is 5–8 days. The inoculation/incubation process is slightly longer at 6–13 days. The overall batch time from seeding to harvest is 33–55 days depending on the optimal plant age, transient expression method, and target protein production kinetics . Importantly, plant growth can be de-coupled from infiltration, so that the plants are kept at the ready for instant use, which reduces the effective first-reaction batch time from gene to product to ~10–15 days if a platform downstream process is available . The time between batches can be reduced even further to match the longest unit operation in the upstream or downstream process. The number of plants available under normal operational scenarios is limited to avoid expenditure, but more plants can be seeded and made available in the event of a pandemic emergency. This would allow various urgent manufacturing scenarios to be realized, for example, the provision of a vaccine candidate or other prophylactic to first-line response staff.The speed of transient expression in plants allows the rapid adaptation of a product even when the process has already reached manufacturing scale. For example, decisions about the nature of the recombinant protein product can be made as little as 2 weeks before harvest because the cultivation of bacteria takes less than 7 days and the post-infiltration incubation of plants takes ~5–7 days. By using large-scale cryo-stocks of ready-to-use A. tumefaciens, the decision can be delayed until the day of infiltration and thus 5–7 days before harvesting the biomass . This flexibility is desirable in an early pandemic scenario because the latest information on improved drug properties can be channeled directly into production, for example, to produce gram quantities of protein that are required for safety assessment, pre-clinical and clinical testing, or even compassionate use if the fatality rate of a disease is high . Although infiltration is typically a discontinuous process requiring stainless-steel equipment due to the vacuum that must be applied to plants submerged in the bacterial suspension, most other steps in the production of PMPs can be designed for continuous operation, incorporating single-use equipment and thus complying with the proposed concept for biofacilities of the future . Accordingly, continuous harvesting and extraction can be carried out using appropriate equipment such as screw presses , whereas continuous filtration and chromatography can take advantage of the same equipment successfully used with microbial and mammalian cell cultures . Therefore, plant-based production platforms can benefit from the same >4-fold increase in space-time yield that can be achieved by continuous processing with conventional cell-based systems .

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

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

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

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

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

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

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

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

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

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

They also take up P, Ca, Mg, and K from the soil and provide their host plant significant amounts of these nutrients . In return for this key role in plant nutrition the host plant transfers significant amounts of fixed carbon to their EMF. A recent review by Hobbie suggests an average of approximately 15% of total fixed carbon is allocated to ectomycorrhizae, but studies have found more than 60% of recent carbon assimilation and net primary production may be allocated to EMF.In addition to their instrumental role in tree nutrition EMF are believed to be instrumental in forest biogeochemistry. They have a strong effect on processes that govern soil carbon residence time and have been the subject of much interest over the last 15 years of biogeochemistry research due to their ability to stimulate mineral weathering. A Web of Science citation search finds that since 1995, 74 articles have been generated on the topic of ectomycorrhizal weathering. Many have found that EMF do stimulate weathering, and the proposed mechanisms include acidification , nutrient uptake , production of siderophores , and production of low molecular weigh organic acids .  Numerous studies point to a significant biotic contribution to mineral weathering in forest soils from prokaryotic , fungal saprotrophic , mycorrhizal , and plant components of the biota. The relative importance of these different groups in mineral weathering as well as the effects of elevated CO2 on biotic weathering remain poorly understood. Low molecular weight organic acids are actively exuded by biota in response to nutrient demand and have been proposed to be key drivers of biotic weathering . Work by Fransson & Johansson ,arandano cultivo  and Johansson et al. suggests that LMWOA production by both plants and EMF may increase in response to elevated CO2, either as a result of source sink relationships within the plant caused by increased carbon fixation or as an active response to increased nutrient demand. Assessing the relative contributions of these microbial and plant components of forest biota to LMWOA production, the effects of CO2 on LMWOA production, and the effects of these LMWOA production levels on mineral weathering rates are necessary elements for a better understanding of the importance of biotic weathering. Previous work by van Hees et al. demonstrated a slight increase in weathering rates and organic acid production in mesocosms with pine seedlings compared to those without seedlings but limited effects of ectomycorrhizae on weathering rates or organic acid production. In that study pine growth responded poorly to EMF due to low nutrient levels, EMF did not persist in one of the treatments, and overall weathering was very low because a highly weathered substrate was used which was likely well coated with resistant secondary mineral coatings. Here we focus on the role of LMWOA’s in biological weathering by using a column microcosm system to elucidate the role of Scots pine seedlings, their associated EMF and the effects of elevated CO2 on weathering rates. In contrast to earlier work that used a similar system we have increased nutrient levels, used a rooting substrate derived from primary minerals, used different fungal symbionts, and incorporated an elevated CO2 treatment.Seeds of Pinus&sylvestris were surface sterilized for 30 min in 33 % H2O2, rinsed in sterilized water and sown in sterile, autoclaved water agar for 6S8 weeks for germination . Ectomycorrhizal and non ectomycorrhizal seedlings were prepared in petri dishes of peat:vermiculite:Modified Melin Norkrans media as detailed in Fransson and Johansson . The ectomycorrhizal fungal species Suillus&variegatus O. Kuntze and Piloderma&fallaxStalpers , growing on half strength Modified Melin Norkrans media were used for EMF inoculum. After 12 weeks seedlings were removed from petridishes and planted in 10 cm X 10 cm X 10 cm pots filled with a 1:10 sterilized, autoclaved peat:quartz sand mixture. The collected soil was subjected to sequential centrifugation in winogradsky salt solution , followed by nicodenz extraction at ultra high speed . The resulting bacterial suspension was tested for the presence of culturable fungi by plating on potato dextrose agar, which yielded no observable fungal colonies. The bacterial suspension was used for inoculation within 2 days of extraction and was stored at 4S8°C in the meanwhile.A sand culture system nearly identical to that of van Hees et al. was employed with opaque plexiglas tubes serving as vertical growth columns. Each column was filled with 405 grams of a mineral mix comprised of 50% quartz sand, 28% oligioclase, 18% microcline, 1.8% hornblende, 0.9% vermiculite, and 0.9% biotite. The quartz sand was acid washed overnight and washed with DI water until the solution pH was >6 before being mixed with the other ground minerals. The complete mix was approximately 40% silt size class and 60% sand size class . The tubes were drained by applying suction at the base of the columns with a ceramic lysimeter cup and column leachate was collected in opaque 250Sml glass bottles. When tested before planting, this drainage system maintained the mineral mix at a moisture content of 5±2%. Rhizon SMSSMOM suction lysimeter samplers were inserted horizontally 10 cm below the soil surface for the purpose of extracting rhizosphere soil solution to measure LMWOA production. The columns were maintained at 20 ± 0.5°C during the “daytime” and 14S16°C during the “nighttime” by the use of plexiglass chambers placed over the column system with peltier coolers used to regulate temperature. CO2 levels were maintained at 330S380 ppm and 700S750 ppm under each chamber. Light was supplied by a high pressure sodium lamp with an intensity of 300 PPFD at the seedling tops . The columns were watered 3 times a week with 24S36 ml nutrient solution . The watering solution contained 33 µmol 2HPO4, 407 µmol NH4NO3, 27.5 µmol K2HP04, 55 µmol Ca 2, 27.5 µmol K2SO4, 5.5 µmol H3BO3, 1 µmol FeCl3, 0.1 µmol Na2MoO4, 0.1 µmol ZnSO4, 0.1 µmol CuSO4, 55 µmol Mg2. The pH was adjusted to 5.0. The molar ratio of N/K/P was 100:10:6, which is comparable to the optimal nutrient use efficiency values for conifers of 100:15:6,frambuesa maceta  as determined by Ingestad , except it is slightly depauperate in K. After addition of the mineral mixture to each column the columns were allowed to equilibrate with the nutrient solution for three weeks. In April 2008, one seedling was planted in each column . At this time 2 seedlings of each colonization treatment were dried for future analysis. Black plastic beads were placed on top of the soil after planting to prevent the growth of algae. One week after planting, each column was inoculated with 5 ml of the fungus free bacterial inoculum described above.Treatments were factorial with +/seedlings, +/S EMF , and +/SCO2 . N=4 for the non mycorrhizal and non-planted treatments, and 5 for the EMF treatments; in total there were 36 columns, 28 of which were planted. The experiment was run for 9 months. Organic acid and phosphate concentrations were measured in rhizosphere soil solution, and elemental concentrations and pH were measured in column leachate collected during the experiment. Upon harvest,the cation exchange capacity of the mineral mix pre and post 9 months of growth, the weight and elemental contents of seedlings,and the chitin contents of the roots, and mineral mix were assessed. This information was used to construct whole column nutrient budgets. Rhizosphere soil solution was extracted for low molecular weight organic acid analysis by applying suction for up to 3h with a 50Sml plastic syringe to lysimeter samplers. LMWOA samples were collected five times from each column at 5, 6, 7, 8, and 9 months post planting, with sample volumes ranging from 2 to 12 ml. Sampling was performed 24–36 h after watering, and immediately frozen at S20oC for later analysis. Column leachate was sampled every 3S4 weeks. Total volume in each bottle was measured and 2 duplicate 15 ml aliquots from each bottle were sampled and frozen for future elemental analysis. Leachate was sampled a total of 11 times for each column. Upon harvest seedlings were removed from the mineral mix, and all possible adhering mineral particles were shaken/brushed off the roots under dry conditions.Each seedling was separated into roots, stem, and needles and dried at 60oC for 24 S72 hours until no more mass loss was noted, and we then measured dry weights . The plant DW is the sum of the three compartments and the root:shoot was calculated as root DW / . Seedlings sampled before planting into columns were given the same treatment. The mineral mix from the columns was separated into three fractions: bottom, top, and rhizosphere. The bottom fraction consisted of the portion of the column below the extent of lowest roots in that column , the rhizosphere fraction consisted of all of the soil which remained adhering to the roots when the roots were gently removed from the columns, and the top fraction was the remainder. Each fraction was weighed moist and a sub-sample of approximately 50 grams  was collected and dried at 60°C for 24S72 h until no further mass loss was noted. Additional smaller sub-samples were freeze dried for chitin analysis. Low molecular weight organic acids were determined by capillary electrophoresis by the method of Dahlen et al. . Briefly, LMWOA’s were analyzed on an Agilent 3DCE capillary electrophoresis system . The concentrations of 12 different LMWOA’s were analyzed: acetate, butyrate, citrate, formate, fumarate, lactate, malate, malonate, oxalate, proprionate, succinate, and shikimate, as well as phosphate. To determine oxalate and citrate, EDTA was added in a separate run to eliminate interference from Al and Fe ions. LMWOA data is presented in µmol/L solution collected from rhizosphere lysimeters and as µmol/L/gram plant DW. Acid digestion of plant material was undertaken following the procedure of Zarcinas et&al. as follows: 0.1 gram of each seedling component was separately digested at room temperature overnight in 2 ml concentrated HNO3 , heated up to and refluxed at 130oC with a funnel lid for 5S7 hours and subsequently diluted with 12S15 ml deionized water. Exchangeable ions were measured for each of the three postSharvest mineral fractions for each sample as well as for 9 replicates of the pre-experimental mineral mix. Extractions were performed in a 1:10 mineral mix:1M NH4AOc suspension by shaking for 5 hours at 100 rpm at room temperature. The supernatant was separated by centrifugation and filtered through a pre-washed 0.45 µm NaAcetate filter syringe. Before cation exchange, the pre-experimental mineral mix was equilibrated with the experimental nutrient solution 3 separate times for 12 hours each to mimic the period that the columns were allowed to equilibrate for three weeks before planting. Plant digests, CEC extracts, and column leachate were all analyzed for elemental contents of Al, Ca, Fe, K, Mg, Mn, Na, P, S, and Si on a Perkin Elmer atomic optical emission inductively coupled plasma emission spectrometer . A set of four standards was established based on preliminary analysis for each sample type. In addition to hourly rerunning of standards, duplicates and an internal scandium standard were run to ensure an accuracy of elemental contents to +/S 1%. Elemental loss though column leachate was calculated from the leachate concentration and the total volume of leachate. Plant roots and growth substrate were assayed for chitin content post harvest to assess fungal biomass. Chitin was extracted and analyzed by HPLC at the Department of Forest Ecology & Management, SLU , according to the method in Ekblad and Näsholm . Chitin concentration of each column fraction was multiplied by the mass of that fraction and these sums were added to obtain total chitin content per column. Significant quantities of chitin were not found in any of the bottom fraction samples. To relate fungal biomass to plant biomass the total chitin content was divided by the total plant biomass in each column.Except where explicitly stated all data are presented as the mean per column. LMWOA and chitin were also presented as mean per column per unit seedling mass. In this experiment we investigate 2 different independent variables: CO2 and seedling treatment . If significant interaction effects were found they were indicated. Otherwise, when looking at the effects of seedling treatment the two CO2 treatments were combined . 

Anthropogenic nitrogen pollution threatens to alter the productivity and carbon storage of temperate and boreal forests. Soil nitrogen status is, for temperate and boreal forests, the dominant edaphic factor controlling forest productivity and shaping species composition . Anthropogenic nitrogen pollution has facilitated invasive species establishment in many forests of the temperate and boreal zone, and contributed to widespread species loss . There is ample evidence that moderate levels of ANP may significantly increase the net primary productivity of temperate forests .This is the core of the “nitrogen saturation hypothesis” developed by Aber et al. , and it is used by many as a theoretical framework to understand the ecological processes at work over the course of prolonged periods of ANP . According to the Nitrogen Saturation Hypothesis, the tipping point and proceeding drop in forest productivity is a result of soil acidification, and excess N inputs leaching out other essential nutrients, which then become limiting. This shift from nitrogen limitation to limitation or coSlimitation by phosphorous , potassium , or calcium due to prolonged ANP has already been observed in a number of forests in Eastern North America. Increased nitrogen availability decreases below ground carbon allocation . Decreased below ground carbon allocation involves decreased inputs of carbon into deep soil; carbon inputs which may lead to longer Sterm soil carbon retention than above ground litter . This decreased  below ground carbon allocation also has profound effects on mycorrhizal relations. Anthropogenic nitrogen pollution has been shown in fertilization and deposition gradient studies to have large impacts on ectomycorrhizal communities. 

N fertilization and deposition is commonly associated with a shift in ECM species composition with some species increasing in abundance while others disappear under high N fertilization or deposition . Many studies have also observed a decrease in both ECM diversity and colonization intensity  with nitrogen additions.The effects of ANP on ectomycorrhizal communities may have serious negative implications for ecosystem integrity. Our knowledge of the respective ecological niches of ECM fungi is poor,vertical farming equipment  but there is ample evidence that suggests discreet, nonSoverlapping niches of habitat preference and nutrient acquisition exist for some species. The potential loss of ECM species from nitrogen deposition reduces forest biodiversity and may represent a reduction in forests’ resiliency to future environmental change. ECM represent a very large sink for fixed carbon; studies have found more than 60% of recent carbon assimilation and net primary production may be allocated to ectomycorrhizal symbionts, though most estimates are closer to 15% . Ectomycorrhizal biomass may be much more recalcitrant than fine root biomass . Reductions in C allocation to ECM may significantly reduce soil C storage and serve as a positive feedback to global change. The great majority of studies on nitrogen fertilization and ECM have been conducted in conifer stands and have focused on the organic horizon, yet there is evidence that forests dominated by broad leafed angiosperms may react differently than coniferous gymnosperms to ANP . Very few studies examining the effects of ANP on ECM communities or even on ECM communities for any purpose have looked at the ECM community in the mineral soil. Over half of all ECM biomass may be in the mineral soil and ECM community composition varies significantly with soil horizon .

To examine the effect of N enrichment in broad leaved forests and in mineral and organic horizons we investigated the ECM community in a mixed broad leaved forest at the Harvard Forest Chronic N Enrichment research site. This National Science Foundation sponsored Long Term Ecological Research facility is the longest running nitrogen fertilization study in the US.Ectomycorrhizal sampling was performed in July, 2005. We randomly selected 4 5m X 5m subplots from the interior 16 subplots. We used 30cm X 2.5cm PVC pipe to collect soil cores. We sampled 4 subplots from each of the control, low N addition, and high N addition hardwood plots . Six cores were taken from each subplot.Because sampling was conducted over the course of a month during which time rooting dynamics may change, sample collection was divided evenly between different subplots and nitrogen treatments over time. Each core was divided into organic and mineral soil. There was typically a distinct border between the organic horizon and mineral horizon, and to prevent any crosscontamination 0.5S2 cm of soil at the interface between the cores was not kept. While the depth of the organic horizon varied somewhat, the organic horizon was generally around 5 cm thick, and the mineral horizon we sampled 25cm. After each core was divided, each portion of the core was washed over a 2mm sieve with distilled water. Fine roots were then collected and put into a petridish filled with water for ectomycorrhizal root tip sampling and quantification. Roots were examined under a dissecting microscope and roots that were not turgid or that appeared senescent were removed, as were any roots that were higher than second branching order. Any roots that appeared to be red maple were also removed.  Red maple associates with arbuscular mycorrhizal fungi, not the ectomycorrhizal fungi that this study focused on. Fortunately, red maple roots are quite morphologically distinct as they have a much lighter color and a unique beaded morphology that makes them easy to distinguish form the ectomycorrhizal roots. 

The live, ectomycorrhizal fine roots from each core were placed in a water filled petridish for community characterization and quantification. A subset of all samples was examined for percentage root length colonized before they were assessed for community composition. Petridishes were placed over a piece of transparency paper with a black grid. The dish was illuminated from underneath and the roots were examined under a dissection microscope at low magnification. Each line of the grid was followed visually and every intersection with a root was recorded as either mycorrhizal if the grid intersected the root at an ectomycorrhizal root tip or nonmycorrhizal if the grid intersected the root at a point on the root that was not an ectomycorrhiza. If the grid crossed a coralloid cluster of mycorrhizae each discrete intersection of the line with a seprate branchlet of the cluster was counted. All gridlines,what is vertical growing  vertical and horizontal were counted for each dish and then the grid was rotated, the roots were mixed and the count was done again. For each sample 5 counts were done, and the percentage root length colonized  for each sample was calculated as the average amount of total mycorrhizal intersections/divided by the average amount of total root intersections.The ITS region was selected for sequencing, and PCR amplification was done with the primer pair ITS 1F and ITS 4 . The ITS region is the most commonly sequenced region for fungal identification and has a large database of vouchered sequences. Its high variability makes it a suitable region for species identification . PCR products were treated with ExoSAP IT to remove primers and inhibitory salts. PCR amlicons were sequence directly without cloning using ABI Big Dye version 3.1 and pre sequencing cleanup was performed with the ABI recommended ethanol/EDTA precipitation. Single pass sequencing was conducted on an ABI 3100 16 capilary Sanger sequencing machine. Sequences were analyzed using Sequencher 4.2 . Sequences were edited to remove priming sites and poor quality portions of the sequences at the 3’ and 5’ ends. Only sequences with at least 200 clear distinct base pairs were used. Many were disqualified due to apparent contamination or cooccurrence of other fungal PCR product. Acceptable sequences were identified by comparison with the sequence database at the National Center for Bioscience Informatics using the basic local alignment search tool to identify rough phylogenetic identity. Sequences were then grouped according to these approximate phylogenetic groupings and clustered using Sequencher 4.2 with a minimum overlap of 50% and minimum sequence identity of 97%. High quality sequences were then selected from clusters for BLASTing against the NCBI database again. When matches at 97% or higher were found, the best match to a vouchered sporocarp sequence was used as the taxa name. In many cases no match at 97% or higher for a vouchered sporocarp was found and we felt that we could only reliably identify the genus of these taxa. The similarities between communities were assessed with ordination methods using the statistical software PCSORD . For ordination, each community consisted of all successfully identified ectomycorrhizal sequences from the 6 cores taken for a specific horizon in a specific subplot . The communities were compared across nitrogen treatments or between soil horizons . Mantel tests were independently conducted to assess whether the communities in different horizons or nitrogen treatments were significantly different. Differences were visualized using non parametric multi dimensional scaling . NMS is a suitable method for comparing complex microbial communities because it does not assume a normalized distribution of species or equivalent variance between communities . For NMS ordination an initial run was performed using the “medium thoroughness” default settings to identify the optimum dimensionality of the ordination. After that, the ordination was performed again . To account for unequal amounts of root tips between communities, the abundance of each taxa in each sample was divided by the total number of root tips in that sample such that the total abundance of all taxa within each sample equaled 1. Only the 45 most abundant taxa were used for community analysis. The effect of horizon and nitrogen treatment on the abundance of the 25 most common taxa was assessed using a two way ANOVA followed by post hoc comparisons using the student’s ttest or Tukey’s HSD test . The effect of horizon and nitrogen treatment on groups of taxa was assessed for monophyletic groups that had at least 3 species, each of which occurring on at least 3 subplots. For such groups of species the standardized species’ abundances were used with the additional standardization step of equalizing the total abundance of each species, so that the total abundance of each species was set to equal 1; this was done so that one very abundant species did not bias the whole genus or family. All ANOVA were done with JMP v5.0.1 .The 495 ectomycorrhizal sequences were grouped into 65 OTU’s, which we will henceforth refer to as species. They varied in abundance from 62 tips to a number of species for which only one tip was found. The rank abundance curve for the ECM community has the shape of a typical soil microbial community with a few abundant species and many rare species. The 5 most abundant species account for 42% of all tips. The most abundant genera were Lactarius, Russula, Cenococcum, Thelephora/tomentella, and Amanita, which comprised 23.2%, 22.6%, 12.5%, 8.2%, and 6.9% of the total number of tips sampled, respectively. A complete list of species, and their affinity for nitrogen treatment and horizon is detailed in tables 1 and 2. Four nitrophilic and 5 nitrophobic species could be identified amongst the most abundant species, as well as three species that had significantly higher abundance in the low N treatment . Four of the most abundant species exhibit a clear preference for the organic horizon, and 3 for the mineral horizon. There does not appear to be any interaction between horizon preference and reaction to nitrogen fertilization but the number of suitable candidate species was too low to allow significance testing. When we scale up and look at species groups at either the genus, family, or order level we see that the Agaricales , Russulaeae , and Calvulinaceae exhibit strong preference for horizon, while only the Clavulinaceae has a consistent reaction to nitrogen fertilization .Many studies have demonstrated that high levels of nitrogen addition significantly impact ectomycorrhizal communities, though very few of them have looked at deciduous stands, and none of those have looked at the mineral and organic horizons separately. In this study, we found that high levels of nitrogen fertilization have significantly altered the ectomycorrhizal community in the Harvard Forest NSF LTER Chronic N Enrichment study. There is no evidence that the community composition, diversity, or colonization intensity have been appreciably affected by “low” levels of nitrogen fertilization . 

Accordingly, the order anaerolineae of the phylum chloroflexi has been identified as an obligate anaerobe, and the order flavobacteriales of the phylum bacteroidota are aerobic chemoorganotrophs . The sequence reads of anaerolineae after dry down events are highest for the CF treatment, followed by LS. In contrast, flavobacteriales follow the opposite trend, increasing in sequence reads after the dry down event for HS and MS treatments. Given that the identified aerobic and anaerobic orders in our rhizosphere soil samples correlate with the oxic and anoxic conditions introduced by dry down treatments and follow an expected trend with regards to the severity of the treatments, we can infer that II treatments of a single dry down event have the potential to shift the microbial communities of the rhizosphere in rice paddies. As we aim to understand how changes in the abundance of microbial communities under II treatments throughout a rice growing season affect the chemistry and cycling of elements in the rhizosphere, we recall that in Chapter 2 and in Seyfferth et al., 2017, a positive correlation between As and Fe precipitated in rice root plaque was found. Now we must determine if bacterial communities are contributing to the interactions between these elements in the rhizosphere of rice. Bacteria of the family geobacteraceae of the phylum desulfobacterota, anaeromyxobacteraceae of the phylum myxococcota , as well as the genus ferribacterium of the class gammaproteobacteria ,vertical vegetable tower have been found as iron reducing bacteria in paddy soils. In our study, geobacteraceae was found in a lowest amount in the HStreatment, and anaeromyxobacteraceae in the MS and HS treatments.

Both taxa increased in sequence reads between 60-90 days after sowing. Reads of the genus ferribacterium were present in all treatments before dry down events and decreased progressively to zero following dry down treatments for HS, MS, and LS. Additionally, some commonly reported Fe oxidizing bacteria, which may also oxidize As, include genera acidovorax and thiobacillus of the class gammaproteobacteria , and the genus nitrospira of the phylum nitrospirota . The sequence reads of thiobacillus were higher for the HS treatment, followed by MS throughout the growing season. Similarly, acidovorax expressed higher reads for the HS treatment after a dry down event and decreased after reflooding; it was not identified in the CF treatment. Unexpectedly, nitrospira, despite being aerobic, did not follow this trend; higher sequence reads were identified for the LS and CF treatments after dry down treatments. This genus has been related to different processes involved in Fe, N, and S cycling in the rhizosphere of rice, which could explain a contrasting trend given that it may be impacted by proton exchange processes in these elemental cycles . Our results show changes in aerobic and anerobic, as well as iron oxidizing and reducing, taxa related to II treatments, confirming that oxic and anoxic fluctuations in soil due to a single dry down event impact the community structure of rice rhizosphere soil bacteria. Alpha diversity results revealed 20 to 25% more diversity in features within a CF soil sample compared to dry soil before sowing. In contrast, we learned from our PCoA results that the changes in microbial community composition expressed in variability between samples is higher for II treatments than CF throughout the growing season.

These changes in the diversity of the II samples are explained by the variability factors such as taxonomic differences and abundance. Our pot experiment was conducted as a replication of the 2017 2018 rice growing field trials at Biggs, CA explained in Chapter 1. Although water management treatments, as well as plant care were carefully planned, it is necessary to consider that this experiment cannot fully imitate field conditions in terms of scale, hydrology, and other environmental factors. In fact, it may be considered as a closed system given that each replicate consists of 1 gallon of soil, thus the movement of dissolved constituents of the soil solution is limited, as well as the space for root growth and the balance of air and water in soil . In addition, dry down treatments were not performed in the precise way as field trials, given that our system cannot imitate the evapotranspiration and percolation rates of a paddy field. Instead, bins were drained for 1, 3.5, and 5.5 days for LS, MS, and HS, respectively. Despite having reached the water potential of the II treatments at the field trial, the period of drainage was shorter. Given these differences, we cannot expect that the observed changes in microbial communities from pot trials will necessarily be the same at the field scale. Additionally, As concentrations in the soil used for this experiment represented northern California conditions and are low in comparison to paddy soils in Southeast Asia and other rice growing regions. Consequently, As levels were too low to be the main factor shaping the bacterial communities in this study . It is important to consider that higher concentrations of As may significantly affect the microbial community diversity in paddy soils . Seasonally dry tropical forests are dominated by deciduous species coexisting with a small number of evergreen species . Trees withstand the dry season through two mechanisms of drought resistance: desiccation delay and desiccation tolerance . Two important traits related to desiccation delay are leaf shedding which reduces water loss, and depth of rooting , which determines the sources of water and nutrients used by vegetation . Although previous reports suggested that evergreen species access relatively deeper water sources than deciduous species ,more recent reports suggest that access to water is more related to tree size than phenology .

However, there is relatively little information regarding differences among deciduous species having different timing or leaf shedding behavior, even though it is well known that leaf senescence behavior varies greatly among tropical dry forest tree species. Flushing and leaf abscission result from complex interactions between plants and their environment; in many species, the main abiotic factors driving these processes are solar radiation, air relative humidity, vapor pressure deficit,vertical farming equipments precipitation and soil water content . Four main categories of leaf shedding phenology have been proposed by Williams et al. : evergreen species, which retain a full canopy throughout the year; partially deciduous species, which lose up to 50 % of their canopy during the dry season; semi deciduous species, which lose more than 50 % of their canopy during the dry season; and deciduous species, in which all leaves are lost during the dry season as they remain leafless for at least 1 month. Most tropical dry forest species are thought to deploy the majority of their root systems relatively deep in the soil profile where moisture tends to be greater and of longer duration . However, in northern Yucatan the hard upper limestone layer, beginning immediately below the shallow soil, impedes root growth, limiting downward growth to crevices and rhizoliths, and the occasional cavities filled with soil material .Thus, in the seasonally dry tropical forests of northern Yucatan, the ability of tree species to grow deep roots and access additional sources of water beyond topsoil could be a crucial characteristic related to variation in phenology and the relative abundance of contrasting tree species. Sources of water used by trees can often be identified by comparing the isotopic composition of water from stems with potential water sources, because there is usually no isotopic fractionation of either hydrogen or oxygen isotopes during water uptake . When trees take water from more than one source, the proportion of water absorbed from each source can be calculated using isotope mixing models . Such models were developed to cope with multiple sources and allow the input of ancillary data that are known about the system to constrain model outputs, thereby providing results that are restricted to real possibilities. Sources of water used by native trees in northern Yucatan have been studied using these approaches, and large variation in the depth of water uptake among deciduous and evergreen species has been observed . Furthermore, using these same isotopic approaches along a forest age chronosequence in northeastern Yucatan, evergreen trees were found to access deeper water sources than deciduous species in early succession . Thus, integrating rooting depth as a component of tropical dry forest tree strategies appears especially promising in complex karstic Yucatecan soils. Water use efficiency , the ratio of carbon gained in photosynthesis relative to water loss during transpiration , is another key factor when considering the costs and benefits of a deep rooting system.

Leaf carbon isotopic composition can be used to assess WUE in certain circumstances, and is often positively related to WUE because a high photosynthetic rate per unit stomatal conductance is usually associated with relatively low internal CO2 concentration and reduces discrimination against 13CO2 by rubisco . Although d13C has been used alone to infer WUE, its combination with analysis of isotopic composition leaf organic oxygen improves interpretation of leaf d13C values by allowing analysis of whether variation in d13C is due to changes on the photosynthetic activity or stomatal activity . When humidity increases, the isotopic enrichment of leaf water decreases, causing a reduction in d18O . Theory and empirical data also demonstrate that d18O correlated negatively with stomatal conductance . In shallow soils of northern Yucatan, Querejeta et al. showed that individuals of the same tree species differing in age had different WUE, with younger trees having greater WUE than older ones, indicating that these techniques hold promise for integrating potential differences in water sources with leaf physiological activity. This study focuses on phenological variation between two dominant tropical dry forest species in relation to the depth of water uptake. We hypothesize that the late deciduous habit in P. piscipula and the early deciduous habit in G. floribundum may be determined by their ability to take water from different sources. P. piscipula may have access to deeper sources than G. floribundum. However, due to the restrictions for root growth imposed by the hard bedrock, both species will likely extract most of their water from shallow sources. We also hypothesize that differential use of water sources is linked to key ecophysiological measures of plant performance, including the timing of leaf fall, leaf size, leaf water potential and the balance of carbon gain and water loss as interpreted by leaf stable isotopic composition.Our results show that the greatest variation in stem water d18O and plant water sources occurred during the frontal season and initiation of the dry season in February, whenG. floribundum was shedding old leaves and growing new leaves, but P. piscipula maintained its leaves from the previous wet season . Contrary to what was expected, P. piscipula took water primarily from shallow sources regardless of the month, although some contribution from deeper sources has the potential to occur. Rain also appeared to be an important source for this species. This implies that P. piscipula could have a very well developed shallow root system that allows rapid water uptake after a precipitation event. On the other hand, G. floribundum took water from topsoil and bedrock, the latter being a more important source in the dry season. This suggests a deeper root system than G. floribundum. Overall, our results indicate that the contrasting early and late dry season leaf loss phenology of these two species is not simply determined by rooting depth, but rather a more complicated suite of species based characteristics based on opportunistic use of dynamic water sources, the balance between carbon gain and water loss, and maintenance of water potential at the end of the dry season. These results are consistent with other studies demonstrating a broad array of coordinated strategies for dealing with seasonal drought in tropical forests . A primary factor determining differences in leaf loss phenology between the two studied species appears to be the maintenance of water potential. G. floribundum consistently exhibited more positive water potential values than P. piscipula, suggesting that G. floribundum has a limited capacity to tolerate negative water potential and moderates water use in a manner that maintains bulk leaf water potential at relatively more positive values compared to P. piscipula . This could provide an advantage of maximizing carbon gain during the dry season when light availability is high .