Tag: vertical farming

The current demand for non-COVID-19 mAbs in the United States is >50 million doses per year27, so COVID-19 has triggered a 44% increase in demand in terms of doses. Although the mAb doses required for pre-exposure and post-exposure COVID-19 treatment will not be known until the completion of clinical trials, it is likely to be 1–10 g per patient based on the dose ranges being tested and experience from other disease outbreaks such as Ebola . Accordingly, 22–222 tons of mAb would be needed per year, just in the United States. The population of the United States represents ~4.25% of the world’s population, suggesting that 500–5,200 tons of mAb wouldbe needed to meet global demand. The combined capacity of mammalian cell bioreactors is ~6 million liters27, and even assuming mAb titers of 2.2 g L−1, which is the mean titer for well-optimized large scale commercial bioreactors , a 13-day fed-batch culture cycle , and a 30% loss in downstream recovery, the entirety of global mammalian cell bioreactor capacity could only provide ~259 tons of mAb per year. In other words, if the mammalian cell bioreactors all over the world were repurposed for COVID-19 mAb production, it would be enough to provide treatments for 50% of the global population if low doses were effective but only 5% if high doses were required. This illustrates the importance of identifying mAbs that are effective at the lowest dose possible, production systems that can achieve high titers and efficient downstream recovery, and the need for additional production platforms that can be mobilized quickly and that do not rely on bioreactor capacity. Furthermore, it is not clear how much of the existing bioreactor capacity can be repurposed quickly to satisfy pandemic needs, considering that ~78% of that capacity is dedicated to in-house products, many to treat cancer and other life-threatening diseases . The demand-on-capacity for vaccines will fare better, given the amount of protein per dose is 1 × 104 to 1 × 106 times lower than a therapeutic mAb. Even so, macetas cuadradas grandes most of the global population may need to be vaccinated against SARS-CoV-2 over the next 2–3 years to eradicate the disease, and it is unclear whether sufficient quantities of vaccine can be made available, even if using adjuvants to reduce immunogen dose levels and/or the number of administrations required to induce protection.

Even if an effective vaccine or therapeutic is identified, it may be challenging to manufacture and distribute this product at the scale required to immunize or treat most of the world’s population . In addition, booster immunizations, viral antigen drift necessitating immunogen revision/optimization, adjuvant availability, and standard losses during storage, transport, and deployment may still make it difficult to close the supply gap. Regardless of the product, the supply of recombinant proteins is challenging during emergency situations due to the simultaneous requirements for rapid manufacturing and extremely high numbers of doses. The realities we must address include: the projected demand exceeds the entire manufacturing capacity of today’s pharmaceutical industry ; there is a shortage of delivery devices and the means to fill them; there is insufficient lyophilization capacity to produce dry powder for distribution; and distribution, including transportation and vaccination itself, will be problematic on such a large scale without radical changes in the public health systems of most countries. Vaccines developed by a given country will almost certainly be distributed within that country and to its allies/neighbors first and, thereafter, to countries willing to pay for priority. One solution to the product access challenge is to decentralize the production of countermeasures, and in fact one of the advantages of plant-based manufacturing is that it decouples developing countries from their reliance on the pharmaceutical infrastructure. Hence, local production facilities could be set up based on greenhouses linked to portable clean rooms housing disposable DSP equipment. In this scenario, the availability of multiple technology platforms, including plant-based production, can only be beneficial. Several approaches can be used to manage potential IP conflicts in public health emergencies that require the rapid production of urgently needed products. Licensing of key IP to ensure freedom to operate is preferred because such agreements are cooperative rather than competitive. Likewise, cooperative agreements to jointly develop products with mutually beneficial exit points offer another avenue for productive exploitation. These arrangements allow collaborating institutions to work toward a greater good.

Licensing has been practiced in past emergencies when PMP products were developed and produced using technologies owned by multiple parties. In the authors’ experience, the ZMapp cocktail was subject to IP ownership by multiple parties covering the compositions, the gene expression system, manufacturing process technology/know how, and product end-use. Stakeholders included the Public Health Agency of Canada’s National Microbiology Laboratory, the United States Army Medical Research Institute of Infectious Diseases , Mapp Biopharmaceutical, Icon Genetics, and Kentucky Bioprocessing, among others. Kentucky Bioprocessing is also involved in a more recent collaboration to develop a SARS-CoV-2 vaccine candidate, aiming to produce 1–3 million doses of the antigen, with other stakeholders invited to take on the tasks of large scale antigen conjugation to the viral delivery vector, product fill, and clinical development.Collaboration and pooling of resources and know how among big pharma/biopharma companies raises concerns over antitrust violations, which could lead to price fixing and other unfair business practices. With assistance from the United States Department of Justice , this hurdle has been temporarily overcome by permitting several biopharma companies to share knowhow around manufacturing facilities and other information that could accelerate the manufacturing of COVID-19 mAb products.Genentech , Amgen, AstraZeneca, Eli Lilly, GlaxoSmithKline, and AbCellera Biologics will share information about manufacturing facilities, capacity, raw materials, and supplies in order to accelerate the production of mAbs even before the products gain regulatory approval. This is driven by the realization that none of these companies can satisfy more than a small fraction of projected demands by acting alone. Under the terms imposed by the DOJ, the companies are not allowed to exchange information about manufacturing cost of goods or sales prices of their drugs, and the duration of the collaboration is limited to the current pandemic. Yet another approach is a government-led strategy in which government bodies define a time-critical national security need that can only be addressed by sequestering critical technology controlled by the private sector. In the United States, for example, the Defense Production Act was first implemented in 1950 but has been reauthorized more than 50 times since then.

In the United States, the Defense Production Act gives the executive branch substantial powers, allowing the president, largely through executive order, to direct private companies to prioritize orders from the federal government. The president is also empowered to “allocate materials, services,macetas cuadradas plastico and facilities” for national defense purposes. The Defense Production Act has been implemented during the COVID-19 crisis to accelerate manufacturing and the provision of medical devices and personal protective equipment, as well as drug intermediates. Therefore, a two-tiered mechanism exists to create FTO and secure critical supplies: the first and more preferable involving cooperative licensing/cross-licensing agreements and manufacturing alliances, and alternatively , a second mechanism involving legislative directives.Many companies have modified their production processes to manufacture urgently-required products in response to COVID- 19, including distillers and perfume makers switching to sanitizing gels, textiles companies making medical gowns and face masks, and electronics companies making respirators.Although this involves some challenges, such as production safety and quality requirements, it is far easier than the production of APIs, where the strict regulations discussed earlier in this article must be followed. The development of a mammalian cell line achieving titers in the 5 g L−1 range often takes 10–12 months or at least 5–6 months during a pandemic . These titers can often be achieved for mAbs due to the similar properties of different mAb products and the standardized DSP unit operations , but the titers of other biologics are often lower due to product toxicity or the need for bespoke purification strategies. Even if developmental obstacles are overcome, pharmaceutical companies may not be able to switch rapidly to new products because existing capacity is devoted to the manufacture of other important biopharmaceuticals. The capacity of mammalian cell culture facilities currently exceeds market demand by ~30% . Furthermore, contract manufacturing organizations , which can respond most quickly to a demand for new products due to their flexible business model, control only ~19% of that capacity. From our experience, this CMO capacity is often booked in advance for several months if not years, and little is available for short-term campaigns. Furthermore, even if capacity is available, the staff and consumables must be available too. Finally, there is a substantial imbalance in the global distribution of mammalian cell culture capacity, favoring North America and Europe. This concentration is risky from a global response perspective because these regions were the most severely affected during the early and middle stages of the COVID-19 pandemic, and it is, therefore, possible that this capacity would become unusable following the outbreak of a more destructive virus. Patents covering several technologies related to transient expression in plants will end during or shortly after 2020, facilitating the broader commercial adoption of the technology. This could accelerate the development of new PMP products in a pandemic situation . However, PMP production capacity is currently limited. There are less than five large scale PMP facilities in operation, and we estimate that these facilities could manufacture ~2,200 kg of product per year, assuming a combined annual biomass output of ~1,100 tons as well as similar recombinant protein production and DSP losses as for mammalian cells. Therefore, plant-based production certainly does currently not meet the anticipated demand for pandemic countermeasures. We have estimated a global demand of 500–5,200 tons per year for mAbs, depending on the dose, but only ~259 tons per year can be produced by using the current global capacity provided by mammalian cell bioreactors and plant-based systems currently represent less than 1% of the global production capacity of mammalian cell bioreactors. Furthermore, the number of plant molecular farming companies decreased from 37 to 23 between 2005 and 2020, including many large industry players that would be most able to fund further technology development . Nevertheless, the current plant molecular farming landscape has three advantages in terms of a global first-line response compared to mammalian cells. First, almost two thirds of global production capacity is held by CMOs or hybrid companies , which can make their facilities available for production campaigns on short notice, as shown by their rapid response to COVID-19 allowing most to produce initial product batches by March 2020. In contrast, only ~20% of fermentation facilities are operated by CMOs . Second, despite the small number of plant molecular farming facilities, they are distributed around the globe with sites in the United States, Canada, United Kingdom, Germany, Japan, Korea, and South Africa, with more planned or under construction in Brazil and China . Finally, transient expression in plants is much faster than any other eukaryotic system with a comparable production scale, moving from gene to product within 20 days and allowing the production of up to 7,000 kg biomass per batch with product accumulation of up to 2 g kg−1 . Even if the time required for protein production in mammalian cells can be reduced to 6 months as recently proposed , Medicago has shown that transient expression in plants can achieve the same goals in less than 3 months . Therefore, the production of vaccines, therapeutics, and diagnostics in plants has the potential to function as a first line of defense against pandemics. Given the limited number and size of plant molecular farming facilities, we believe that the substantial investments currently being allocated to the building of biopharmaceutical production capacity should be shared with PMP production sites, allowing this technology to be developed as another strategy to improve our response to future pandemics.Nutrients, especially nitrogen and phosphorus , affect terrestrial ecosystem carbon cycling through their regulation of plant and soil microbial activity . Natural terrestrial ecosystems are often nitrogen and phos phorus limited , with a general consensus that temperate and boreal ecosystems are commonly N limited while tropical forests are phosphorus limited .

Small datasets were generated for each sample and used in bioinformatic analysis through de novo assemblies and read-mapping. Assembled contigs were identified and classified according to the sequence they aligned to with the highest bit score in BLAST searches against the NCBI non-redundant DNA and protein databases. The Capillovirus, ASGV was identified in the known sample together with several CTV genotypes. However, the atypical “psorosis” sample had a more complex virome that include three viroids , as well as several CTV genotypes. The presence of multiple CTV genotypes was confirmed for both samples by read-mapping to full length reference genomes. The results of this proof of principle experiment indicate that the metagenomic sequencing approach of dsRNA can be successfully implemented to establish the virome of citrus trees with an unknown virus etiology.Research subsequently focused on other aspects of the disease epidemiology: impact of ambient temperatures and graft transmission and multiplication of the pathogens in citrus and orange jasmine Jack as incorrectly referred to in other publications. The research on the impact of ambient temperatures focused on exposing Lam+ve and Las+ve trees to distinct daily temperature regimes . The research was motivated by the already known contrasting responses to ambient temperatures of plants affected by Las or Ca. L. africanus and by the complete lack of information on this subject for Lam. After a series of growth chamber experiments it was demonstrated that Lam is more heat sensitive than Las. Fully symptomatic orange trees affected by Lam exposed to daily regimes of 27 to 32°C, 24 to 32°, or 35 to 38ºC for 60 days were totally cleared of symptoms and of the pathogen,cultivo arandanos en maceta while fully symptomatic trees affected by Las were only partially cleared of symptoms and the pathogen only when exposed to 24 to 38ºC for the same duration.

More recently it was shown that this same temperature regime leads to a decline in Las titers in new flushes on symptomatic branches, an impact which would lead to a significant reduction in pathogen acquisition rates by the insect vectors feeding on them . Although field work will add important information on this aspect of the HLB pathosystem, data so far accumulated indicate that high summer temperatures may restrict rates of spread of the disease and help to explain the irregular dissemination patterns of HLB in SPS. Field and greenhouse experiments involving even higher temperatures for different durations were also conducted, with the aim of curing Las+ve trees, but with limited success . The reasons for the limited success were apparently related to the sensitivity of the citrus tree to high temperatures and to the ability of the pathogen to survive in roots. The temperature-time combinations necessary to kill the bacterium were apparently close to those that would kill a citrus tree, and in the roots, the bacterium remains protected from heat. Studies on graft transmission of Las and Lam were conducted with the objective of comparing graft transmission efficiencies and the ability of both bacteria to multiply, individually or simultaneously, in potted Valencia, Hamlin, Pera, and Natal , under conditions favorable for disease development .The percentage of plants that became infected varied from 10.0 to 23.3% for Lam and 66.7 to 73.3% for Las, and the cycle threshold values varied from 24.14 to 24.97 for Lam and 19.42 to 20.92 for Las. These Cts corresponded to average 106 and 107 cells per gram of tissue for Lam and Las, respectively. Similar values were obtained also when field samples, collected from three distinct regions of SPS, were analyzed . No apparent effect of one species over the other was observed in plants inoculated simultaneously with both pathogens. Lower titers of Lam appear to be the main factor explaining its conspicuous decline over the years in SPS. Lower titer would reduce the chances of pathogen acquisition by the insect vector and its consequent transmission to healthy trees, in a pattern similar to the one observed for Las in new flushes exposed to heat .

This work also showed that, contrary to Lam+ve plants, Las+ve plants harbored the bacterium attiters close to maximal values, three months before symptom expression, an indication that asymptomatic trees may be serving as a source of inoculum, contributing to dissemination of HLB in the field. The research on orange jasmine aimed at determination of the distribution, based on sampling at 76 urban locations over two time intervals, of orange jasmine trees infected by Lam or Las, and determination of levels of genetic and pathogenic similarities among the orange jasmine and citrus liberibacters, based on sequences of the rplJ gene and on cross inoculation experiments . The work was motivated by the detection of Lam in a single mature orange jasmine tree growing in front of the manager’s house in the citrus farm most affected by Lam in 2004, by the detection of Las in 2005 in orange jasmine trees growing in urban areas and, more importantly, by suspicion that infected M. exotica trees may play an important role in the HLB epidemics. In the years 2005/2006 Lam was detected in 56 and Las in 2 of the 477 orange jasmine trees from 10 locations and, in 2009, Lam was detected in an additional 5 and Las in 28 of the 309 orange jasmine trees from seven locations. Lam titers were higher in Lam+ve than in Las+ve trees . As happens with infected citrus under favorable conditions for disease development, symptom severity was stronger on the orange jasmine trees infected by Lam than on those infected by Las. The higher symptom severity in M. exotica may not be related only to the higher bacterium titers in this host since in citrus, Lam reaches lower titers than Las. In Las infected orange jasmine the infection seemed to be transient. This was observed in naturally infected field trees and in graft-inoculated plants. This work also showed that the infected orange jasmine trees were in locations relatively close to each other and, coincidently, in the area of highest incidence of HLB in citrus at that time, a clear indication of pathogen transmission from host to host by D. citri. Similarity among citrus and orange jasmine liberibacters,grow hydroponic in terms of pathogenicity, could not be fully determined due to the strong tissue incompatibility observed between citrus and orange jasmine during the cross inoculation experiments. Most budwood used as inoculum died in heterologous combinations. On those plants in which the budwood survived, only Lam was successfully transmitted and the plants remained infected.

Comparative analysis of the rplJ gene from the liberibacters found in orange jasmine with those found in citrus showed that Lam or Las from both hosts were identical. The importance of orange jasmine and citrus as source of Lam to citrus in SPS was investigated in further work involving the insect vector for bacterium inoculation . Higher Lam transmission rates occurred from orange jasmine than from citrus. As orange jasmine trees infected with liberibacter are not systematically eliminated in urban areas, and vector populations not suppressed, orange jasmine may represent a constant risk to neighboring citrus orchards. Also, since nursery production and sale of orange jasmine are not regulated , asymptomatic orange jasmine trees may be important for distributing liberibacters to distant citrus areas still free from the disease. An overview of the HLB epidemics in Brazil, particularly in SPS, and the main research findings on the HLB pathosystem were briefly presented here. Other field work and studies , and the daily experience of the citrus growers with the disease, have confirmed the necessity of eliminating symptomatic trees and controlling the insect vector on an area-wide basis in order to optimize opportunities for successfully minimizing the spread and impact of HLB. Although many research questions still require answers, research has provided a better understanding of the distinct patterns of spatio-temporal progress of the disease, and knowledge required for official responses and establishment of management practices. Among research outcomes, impacts of high temperatures on Las multiplication in new flushes may have some potential for the development of new, less costly and less insecticide dependent strategies to manage HLB. The impact of the COVID-19 pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 was foreshadowed by earlier epidemics of new or re-emerging diseases such as SARS , influenza , Middle East Respiratory Syndrome , Ebola , and Zika affecting localized regions . These events showed that novel and well-known viral diseases alike can pose a threat to global health. In 2014, an article published in Nature Medicine stated that the Ebola outbreak should have been “a wake-up call to the research and pharmaceutical communities, and to federal governments, of the continuing need to invest resources in the study and cure of emerging infectious diseases” . Recommendations and even new regulations have been implemented to reduce the risk of zoonotic viral infections , but the extent to which these recommendations are applied and enforced on a regional and, more importantly, local level remains unclear. Furthermore, most vaccine programs for SARS, MERS, and Zika are still awaiting the fulfillment of clinical trials, sometimes more than 5 years after their initiation, due to the lack of patients .

In light of this situation, and despite the call to action, the SARS-CoV-2 pandemic has resulted in nearly 20 million infections and more than 700,000 deaths at the time of writing based on the Johns Hopkins University Hospital global database.The economic impact of the pandemic is difficult to assess, but support programs are likely to cost more than €4 trillion in the United States and EU alone. Given the immense impact at both the personal and economic levels, this review considers how the plant-based production of recombinant proteins can contribute to a global response in such an emergency scenario. Several recent publications describe in broad terms how plant-made countermeasures against SARS CoV-2 can contribute to the global COVID-19 response . This review will focus primarily on process development, manufacturing considerations, and evolving regulations to identify gaps and research needs, as well as regulatory processes and/or infrastructure investments that can help to build a more resilient pandemic response system. We first highlight the technical capabilities of plants, such as the speed of transient expression, making them attractive as a first-line response to counter pandemics, and then we discuss the regulatory pathway for plant-made pharmaceuticals in more detail. Next, we briefly present the types of plant-derived proteins that are relevant for the prevention, treatment, or diagnosis of disease. This sets the stage for our assessment of the requirements in terms of production costs and capacity to mount a coherent response to a pandemic, given currently available infrastructure and the intellectual property landscape. We conclude by comparing plant-based expression with conventional cell culture and highlight where investments are needed to adequately respond to pandemic diseases in the future. Due to the quickly evolving information about the pandemic, our statements are supported in some instances by data obtained from web sites . Accordingly, the scientific reliability has to be treated with caution in these cases.The development of a protein-based vaccine, therapeutic, or diagnostic reagent for a novel disease requires the screening of numerous expression cassettes, for example, to identify suitable regulatory elements that achieve high levels of product accumulation, a sub-cellular compartment that ensures product integrity, as well as different product candidates to identify the most active and most amenable to manufacturing in plants . A major advantage of plants in this respect is the ability to test multiple product candidates and expression cassettes in parallel by the simple injection or infiltration of leaves or leaf sections with a panel of Agrobacterium tumefaciens clones carrying each variant cassette as part of the transferred DNA in a binary transformation vector . This procedure does not require sterile conditions, transfection reagents, or skilled staff, and can, therefore, be conducted in standard biosafety level 1 laboratories all over the world. The method can produce samples of even complex proteins such as glycosylated monoclonal antibodies for analysis ~14 days after the protein sequence is available.

The nature of this sample suggests a significant, but not surprising, overlap between those growers more generally willing to work with researchers and those experimenting with various production techniques. Importantly, many growers I attempted to contact were not reach able and/or had gone out of business, and even three of those I did interview had retired or all but exited strawberry production. In the interviews, conducted in 2018 and 2019, I was able to reframe questions that had not quite worked in the surveys, as well as probe on the more difficult questions . Before completing them, I reached saturation, such that additional interviews were no longer producing more themes or deepening understanding, which substantiated that the sample size was sufficient . Research assistants transcribed and coded interview data with NVivo qualitative research software , identifying ideas and themes that further elucidated the more bounded questions asked in the survey. Alongside these two primary sources of data , I reviewed limited discussions about cultivars from my previous project and notes taken from short discussions with growers at field days and follow-up phone calls for the survey. These additional data were thoroughly in keeping with survey and interview data, providing further triangulation of the findings. While the strawberry industry has long enjoyed the benefits of strawberries bred with multiple aims, emphasis in one area often comes at the expense of another . Since UC began its breeding program in the 1940s, growers have generally adopted those varieties with high productivity traits . An important question, therefore, was within the current context of fumigant restrictions and the emergence of novel diseases, to what extent disease resistance had become a desirable trait.To prevent them from choosing all, it asked them for their top three priorities. As seen in table 1, growers mostly wanted high yields, especially if a variation on the same theme, long steady yields,producción macetas 25 litros was included. While interest in resistance to soilborne diseases and in marketability were not negligible, they appeared as secondary priorities. These preferences were corroborated by answers to a question about which cultivars had been planted for the 2016 marketing year .

Of the UC varieties, Cabrillo, Monterey and Fronteras were the most planted and they are high yield performers. In a recent trial involving equal plot sizes, Fronteras produced an average cumulative marketable fruit weight of 11,000 grams per plot, with Monterey producing close to 9,500 per plot. Of these two cultivars, Monterey allegedly has better flavor. San Andreas, the next most widely planted cultivar, is most associated with Fusarium resistance, but in that same experiment yielded only a little over 7,000 grams per plot. The notably flavorful Albion, which is popular among growers selling in farmers markets, although was not often planted by survey respondents , yielded only about 6,500 grams per plot . Answers to a third question further clarified the dimensions of the trade-off between yield and disease resistance. Asked about the maximum decline in yield a grower would accept in a cultivar with high levels of resistance to soilborne diseases and no change in production costs, most growers reported that no or only a minimal yield decline was acceptable . Qualitative responses and interviews provided additional evidence that growers tended to choose yield over pathogen resistance and helped clarify their rationale. Of the 20 growers interviewed, 15 said yield was a high priority, albeit not without some hedging. Many recognized the importance of marketability characteristics, acknowledging that a strawberry that lacks flavor, for example, would turn off consumers. For that reason, they were more likely to grow Monterey than even higher yielding varieties, and some shippers insisted that they grow a marketable variety such as Monterey. Growers who use proprietary varieties because they sell to shippers who require them to have somewhat less choice in what they grow. The shipper sets priorities, and Driscoll’s, in particular, has allegedly prioritized flavor and disease resistance over yield in their breeding. Growers who favor work ing with these shippers do so because they obtain higher prices, making up for the loss of yield. Still, my interview data showed that when given a choice these growers, too, favor yield, especially because they are paid the same no matter what they grow. When I pressed on questions of why yield remained a priority for those selling in wholesale markets when they also complained of low prices, I learned of a significant collective action problem. Most growers recognized that it made sense for the industry to reduce supply but felt that it was folly personally to choose a lower-yielding variety.

This is the technological tread mill problem first identified by agricultural economist Willard Cochrane in 1958. Cochrane noted the tendency of farmers to adopt technologies that reduce costs because early adopt ers make additional profits as their expenses decline . As he also noted, such tendencies eventually negatively affect crop prices because other farmers join in, supply increases overall and price competition ensues, driving some out of business. In the case of adoption of a higher-yielding variety, rather than reducing cost, the output increases with little additional effort, making such a strategy nearly irresistible. As one grower put it, “We’re in a competitive environment. We like to say we don’t grow a commodity, but there are commodity-like characteristics. So if you have a variety and neighbor selling into same market, if he’s more productive he will have an edge.” In addition to low prices, fixed costs such as land leases and land preparation are extremely high and in creasing in strawberry production. Labor costs, though variable, have risen considerably with labor shortages and new minimum wage and overtime laws. Therefore, growers feel they need to sell as many berries as they can to be economically viable. As another grower said, “You could have the best fruit around, but if you don’t have yield you can never make any money. . . . I mean our costs are going through the roof. The only way we can bring some of the costs down is through yield.” At the same time,hydroponic growing systems growers also questioned this logic, asserting that the industry was under mining itself by continuing to breed and grow ever more high-yielding varieties: “So we want these varieties to give out more numbers and last longer, but it’s hurting us in the long run. . . . It seems like people think that if I plant 100 acres and make such amount of dollars, if I put 200 acres in, I’m going to make double that, but it doesn’t work that way.” This observation is corroborated by the most recent statistics on historical trends reported by the USDA National Agricultural Statistics Service. Utilized production of strawberries grown in California increased from 539 million pounds in 1974 to 3,015 million pounds in 2012, an increase of 559%; grower prices increased only from 29 cents per pound to 80 cents per pound, an increase of 276%, in that same time period . It is not that growers were oblivious to the need for disease resistance, but some were making a calculated decision that the yield benefits of a cultivar outweighed the risk of plant loss.

As one grower said, “We can have 30% die out of Radiance due to soilborne pathogens and still beat the yield on San Andreas.” More often, growers had not experienced enough plant loss to make disease resistance a priority: “If we begin to see more Vert or other pathogens, we will worry more. Right now, all is cool.” Some growers, though, who had experienced disease loss were more inclined to let go of leases on diseased land than give up on the yield or marketability advantages of a cultivar. There were exceptions, too. After that, he “switched soils,” but that soil was infested too, and he lost 32% of Monterey that year. He then turned to growing almost entirely San Andreas. Not surprisingly, it was growers with organic fields who were most interested in disease resistant varieties. With fumigation still available, growers with conventional fields remained relatively uninterested in these varieties. Understanding that most growers were unwilling to trade off yield for pathogen resistance because soil fumigants were available, I wanted to explore in more depth what role pathogen-resistant cultivars could play in reducing the use of soil fumigation. The survey included two questions about what prevents growers from reducing their use of preplant soil fumigation and what currently encourages them to reduce their use of preplant soil fumigation. It asked them to choose all answers that applied. Answers to these questions aligned with previous studies and reports . Growers most often chose “crop loss/ potential crop loss” as the condition that prevented them from reducing their use of preplant fumigation . Buyer and lease conditions played a role, as well — for instance, some leases require that growers fumigate so that the lessors, often vegetable growers, get the benefits of fumigation. On the flip side, regulatory pressures, including restrictions on fumigation in the form of buffer zones, were most encouraging growers to reduce fumigation, with opportunities such as en try into organics or land with low disease pressure also playing roles . Qualitative responses and interviews corroborated and nuanced the latter answers. Several growers emphasized how fumigant restrictions had pushed them to find alternative means to grow strawberries and discussed organic certification and the accompanying price premium as a way of offsetting the potential costs and crop losses of forgoing fumigation. In these in stances, they saw disease-resistant varieties as enabling such a transition: “Without disease-resistant varieties, conventional strawberries require the use of fumigants. If they become unavailable, organic is the best alternative.” The trade-off is noteworthy given that growers have to give up other pesticides besides fumigants to be certified organic. A few growers mentioned their willingness to give up fumigation without converting to organics, simply because of fumigation costs. And a few growers noted that organic prices might be too weak to make that trade-off. One wrote in the survey, “If I were organic [I’d reduce fumigation use], but they don’t have the price either right now.” Even the many interviewees who have organic programs were not at this time considering transitioning their entire operation; instead, they were choosing fields for their organic programs where soil conditions make them viable, often areas with low disease pressure. That organic markets were nevertheless the main factor incentivizing fumigant reduction was confirmed by answers to a question about whether there were any conditions in which growers would consider eliminating the use of preplant soil fumigation, not including transitioning to organic. Only 10 growers replied to this question, but seven said no, with two maybes and one yes. When asked to comment about what, if any, conditions might lead growers to eliminate preplant soil fumigation altogether within the next 5 years, surveyed growers mentioned cultivars completely resistant to all major soilborne diseases — not just simply tolerant to diseases, which is what the best cultivars are today. Growers basically wanted alternatives that wouldn’t forgo yield, quality or higher profit — in other words, something foolproof. In an interview, one grower was emphatic on this point: “It has to be proven to me, I gotta see it. . . . But I’m not going to do it because [the UC breeder] says ‘Oh, by the way, I have this variety that’s resistant to Macrophomina, you don’t need to fume.’ Well, let me see that, you know what I mean?” The more personalized setting of the interviews also allowed me to explore what growers would do if fumigants were taken away. Here I learned that while such a possibility heightened interest in disease-resistant varieties, several said that they would leave strawberries or retire early, and many said they would move to soilless regimes. As it happens, one of the challenges of soilless systems is finding cultivars that work in those settings. The performance of existing varieties is reportedly subpar. Those interested in remaining “on ground” clarified that disease-resistant varieties would be helpful, but they would need to adopt other tools as well, such as nonchemical modes of soil disinfestation, making breeding for disease resistance only a partial solution. One grower said, “Just having a variety that is tolerant of x, y or z only does so much. . . . . That would be just like added insurance.”

These challenges underscore the critical need to identify novel approaches to prevent or reduce the public health risk from pre-harvest microbial contamination of fresh produce. Although to date, no breeding program has adopted strategies to control human pathogens on fresh produce, a few studies have taken steps in this direction. For instance, Shirley Micallef is exploring cultivar variability in fatty acid content in tomato fruit as a means to reduce the favorability of tomato fruit for Salmonella . Maeli Melotto is screening lettuce germplasm for susceptibility or tolerance to E. coli O157:H7 and S. enterica to define the genetic basis for the persistence of these pathogens in leafy vegetables . Additionally, in collaborative studies with USDA-ARS, Salinas, CA, United States and FDA-CFSAN, Laurel, MD, Maria Brandl has been investigating lettuce cultivars in relation to basal plant defense responses to plant pathogen infection and to processing for their role in enteric pathogen colonization . Given the complexity of produce safety issues and the need to prioritize efforts for the highest impact, a logical step would be to identify the crop–hazard pairs that create the largest burden on public health and the economy. Typically, the severity of an outbreak is estimated by the number of illnesses, hospitalizations, and deaths. With a hazard × occurrence risk model, one can begin classifying crop/hazard pairs. Although these are relevant metrics, it is very difficult to calculate the relative risk of each crop–hazard pair due to the low re-occurrence of particular pairs associated with outbreak events and the need to accumulate a substantial amount of data over extended periods of time . Nonetheless, potential targets for plant breeding that are being identified may be the basis of future research to reduce human pathogens, mycotoxins, heavy metals, toxic elements,maceta 10 litros and allergens in foods. Currently, the National Outbreak Reporting System of the Centers for Disease Control and Prevention reports disease outbreaks in the United States and maintains a comprehensive searchable database with information spanning from 1998 to 2017.

Using this resource, we have generated a heat map illustrating the relative importance of the major fresh produce in combination with reported etiological agents of outbreaks . Hierarchical clustering analysis of the etiological agents revealed Salmonella, Norovirus, and Escherichia as the three most important biological hazards based on the number of outbreaks, illnesses, hospitalizations, and deaths . In addition, the compilation of these data has enabled the identification of high priority pairs for breeding programs geared toward improving microbial safety of produce . These systems have been studied at the genetic level by Jeri Barak , Maria Brandl , Maeli Melotto , and Shirley Micallef . For instance, it has been discovered that certain varieties of tomato , lettuce , cucumbers , and melons are less likely to support pathogen populations than others, suggesting a plant genetic component underlying these traits .Identifying the molecular mechanisms underlying these interactions can point to promising plant traits to further explore and integrate in plant breeding programs. Encouraging commercial production of plant varieties that carry relevant traits without compromising other aspects of plant productivity and product marketing might help reduce illness from produce. In the area of mycotoxin contamination, Fusarium in wheat is an annual occurrence with prevalence determined by local weather at crop maturity . Aflatoxin in maize is regional and limited to more hot and humid regions, but remains relatively low in the main U.S. corn belt. However, on a global scale, up to 80% of maize seed lots can be contaminated in tropical areas such as Sub-Saharan Africa and India . Peanuts have similar occurrence of aflatoxin in areas such as East Africa. Heavy metals are predicted to continue to be a problem as arable land becomes increasingly scarce due to desertification and urbanization, and lands or irrigation water with heavy metals are more extensively used . These hazards can also be prioritized and paired with the crops in which the highest occurrence makes them the greatest human health hazards .

A multidisciplinary approach will be necessary to develop plant breeding research programs since the occurrence of a contamination event depends on the interaction of several factors such as plant genotype, environmental conditions, the microbe and its community, and plant management practices. Together,these variables may create “The Perfect Storm.” Interactions between enteric pathogens and plants affect all mitigation strategies aimed at inhibiting pathogen growth and survival on crops to improve their microbial safety. Below, we discuss various hurdles and important aspects of these interactions that must be considered to ensure the success of a plant breeding program for enhanced crop safety. One of the most significant challenges in breeding crops to decrease the risk of contamination with enteric pathogens is that they have lower fitness on plants than most well-characterized plant commensal and pathogenic bacterial species. Nevertheless, given the recurrence of food-borne illness outbreaks linked to produce , the ability of enteric pathogens to multiply and survive as epiphytes and endophytes implies that particular plant phenotypes and genotypes can affect their fitness in the plant habitat . For example, the composition of substrates available on fruit and leaf surfaces as well as in their internal tissue ; the density of trichomes, stomata, and veins , which harbor larger pools of substrates than other areas of leaves; and the physical and chemical composition of the cuticle layer on various parts of the plant , which affects water dispersal and hence, water and nutrient availability to microbial inhabitants , may all be relevant traits to investigate in plant breeding efforts for their effect on enteric pathogen colonization. Temperature and humidity conditions, and the presence of free water, are important in the multiplication and survival of enteric pathogens and must be investigated simultaneously with the role of other plant traits. This includes consideration of agricultural practices, such as irrigation type and frequency , which may greatly affect the success of any breeding strategy aimed at reducing surface and internal plant colonization by food-borne pathogens. It is also clear that physicochemical stressors in the plant environment may overshadow other factors in their inhibitory effect on enteric pathogens. Therefore, the role of certain heritable plant traits at microsites that shield the bacterial cells from such fatal stressors should be investigated at the microscopic level as well as the plant or tissue level. Fully elucidating the interaction between food safety-relevant microbes and crops necessitates the consideration of the entire plant microbiome below and above ground. Plant microbiota are complex and strongly driven by plant genetics, plant age, plant anatomical structure,maceta 3l and environmental factors . Identifying conditions that select for members of the plant microbiota able to competitively exclude enteric pathogens, which in general exhibit reduced fitness in the plant niche, can form an important component of this phytobiome approach . In addition, rhizosphere and phyllosphere microbial communities can comprise epiphytes known to affect plant colonization by enteric pathogens or toxigenic fungi either antagonistically through biocontrol strategies or favorably by supporting survival and growth. For instance, phytopathogens that actively degrade plant tissue or trigger plant chlorosis and necrosis may cause changes in pH and nutrient levels that favor the establishment and proliferation of enteric pathogens . Adjustment of management practices and environmental conditions to modulate and exploit microbe– microbe interactions should be actively investigated as part of a holistic approach to inhibit or prevent the colonization of enteric pathogens on/in plants. Certain plant phenotypes may have independent as well as co dependent effects with other plant features so that their role may only be fully revealed by actively investigating and/or selecting for both traits simultaneously.

For example, entry of enteric pathogens into the plant tissue, where they are shielded from external environmental stressors, is thought to increase their survival in the plant habitat . Thus, selecting for genotypes with lower stomatal density and stomatal pore size may prove to be effective in reducing the probability of pathogen survival on plants in the field, provided that plant productivity is not impacted by the selection of that trait. Furthermore, basal plant defense responses to the presence of human pathogens , which can only take place upon exposure of plant cells to, and close interaction with, microbial cells in the plant apoplast, require entry of the enteric pathogen cells into the substomatal space of the tissue. Consequently, the full potential of breeding for a cultivar that is less hospitable to the endophytic lifestyle of an enteric pathogen may require consideration of both plant traits, i.e., traits that affect the entry of the pathogen cells into the plant and those that affect the plant response once the cells have gained entry . The role of the physiological state of plants in their interaction with enteric pathogens cannot be understated. Plant defense responses may vary depending on the age of the plant tissue, the overall plant age, challenge history, and association with other microbes such as plant growth promoting rhizobacteria and plant pathogens . The carrying capacity of plant tissue for enteric pathogens depends on plant species and cultivar, leaf age, fruit ripeness, and root age given that structure and opening density via cracks at the secondary root emergence sites change over time . Evidence is increasing that changes in temperature and rainfall caused by climate change may affect plant physiological and anatomical responses. These include stomatal conductance and density, leaf area and cuticle thickness, plant morphology, and plant nutrient cycling . The level of relative humidity can significantly influence stomatal movement that can affect colonization of the leaf interior by human pathogenic bacteria . It is clear that if these are targets of breeding programs for improving food safety, these traits will have to be resilient under long-term shift in weather patterns. Enteric pathogens vary broadly in their fitness as epiphytes and endophytes in a species-specific manner, and even based on variation at the inter- and intra-strain level . In particular, surface appendages, such as different types of fimbriae and adhesins that act as important plant attachment factors or flagella and other surface molecules that may trigger defense signaling cascades, vary among and within enteric species and strains . Preferential bacterial pathogenic species and even serotype-commodity pairs are not uncommon and the basis for this specificity is still poorly understood. Clearly, phenotypic and genotypic variation among food-borne pathogen targets must also be taken into account while selecting for plant targets to enhance microbial crop safety. Domestication of several crops has resulted in desirable agronomic and organoleptic traits such as shape, color, and prolonged shelf-life, with the unintended loss of other traits . The resulting loss in genetic variation may have reduced the ability of some crops to cope with fluctuating environmental conditions and biotic challenges . Despite this, genetic diversity could still reside in germplasm that is not commercially grown , allowing for the possibility of reintroducing genotypic and phenotypic traits that restore lost properties or establish new ones . The underlying genetic basis for traits that enhance food safety are largely unknown, but as more research uncovers the interactions between plant, pathogen, and the environment, opportunities for identifying these traits will increase. Traits that confer enhanced food safety are likely complex and controlled by multiple genes, presenting challenges to breeding efforts, especially for human pathogen–plant interactions. A starting point could be genome-wide association studies followed by metabolic pathway analysis or functional analysis of mapped intervals . For instance, one could predict various biochemical pathways needed for the synthesis of secondary metabolites with antioxidant and antimicrobial properties that could influence plant-microbe interactions and plant responses to associated microbiota. These interactions may be extremely important in food safety and should be a major focus of pre-breeding efforts. Given the overall challenge of considering numerous aspects of plant genotype × environment × microbe × management interactions, a concerted effort to focus on given pathogen– crop models may be necessary to make headway in utilizing plant breeding as a feasible strategy to enhance produce safety. For effective genetic gain, a systems approach that maximizes consistency and differentiation of the desired phenotypes is essential. These traits must be considered with major traits of crop yield, quality, and resistance to abiotic and biotic stresses.

Assuming that tunneling can be taken as an indicator for overall weathering activity, it remains unclear whether the greater weathering activity in the lower fertility sites is due to lower pH, greater nutrient demand on the part of the host, or greater ectomycorrhizal colonization. Hoffland et al. assessed tunneling activity across a northern Sweden podzol sequence and found that the occurrence of tunnels in feldspar grains coincided with the disapearence of easily weatherable cation sources such as biotite. Taken together, these tunnel studies imply a correlative, but not a causative, link between weathering activity by ectomycorrhizal fungi and host nutrient demand. Wilson at al. used magnetic separation to segregate readily weatherable cation sources such as biotite and orthopyroxene from more cation poor K feldpars. They then used a variety of molecular and microscopic methods to asses the density of microbial colonization and weathering state of these minerals. They found significantly more mycelial colonization of readily weatherable cation sources such as biotite and orthopyroxene than on more cation poor K feldpars, but noticed only slightly increased weathering of the biotite compared to the feldspar minerals. In the aforementioned field studies, there is evidence that ectomycorrhizal fungi may increase mineral foraging and colonization in response to increased demand for phosphorus. There is also evidence that weathered tunnels coincide with increased demand for mineral elements other than phosphorus and that ectomycorrhizal hyphae can preferentially colonize mineral fragments which are good sources of mineral nutrients other than phosphorus. However,frambueso en maceta  there is no direct evidence in field studies that foraging for and weathering of K, Mg, or Ca sources by ECM can respond to demand for these nutrients. There are many reports in the literature of forest ecosystems dominated by ectomycorrhizal hosts which, possibly due to anthropogenic acid deposition, are now limited by base cation availability and not nitrogen or phosphorus. The mesh bag approach employed by Wallander and others in Swedish forests may be a good method for examining how the mycorrhizal role in nutrient acquisition has changed with the changing nutrient status of these forests. Especially good sites to use this approach would be the sharp N depositional gradients near industrial or agricultural sites.Microcosm studies allow weathering to be quantified and can focus on the weathering of a single mineral or any desired mineral mix. Microcosms can be used to examine the weathering potential of individual ectomycorrhizal species and can be employed to isolate the weathering activity of the ectomycorrhizal fungus from that of the plant root. Microcosm studies also allow the researcher to isolate the effects of the availability of just one nutrient on weathering activity. In relatively sterile microcosm experiment it is also much easier to assay for readily decomposable weathering agents, particularly low molecular weight organic acids , and examine how LMWOA production affects weathering rates. In soils, measured bulk solution LMWOA concentrations are generally too low to significantly enhance mineral weathering due to their rapid degradation by soil microbiota. However in semi sterile microcosms and at the fungus mineral interface in natural soils, LMWOA concentrations may be high enough to greatly enhance weathering rates via proton promoted and ligand promoted dissolution. Van Scholl et al. looked at how organic acid production was influenced by nutrient deficiency of Mg, N, P, and K. Decreasing P or N increased organic acid production, while reducing Mg or K either had no effect or slightly decreased overall LMWOA, although reducing Mg did increase oxalate production in some treatments. There were also significant differences between individual fungal species organic acid exudation profiles and how they reacted to different nutrient deficiencies. Paris et al. conducted a series of studies examining how weathering activity of ectomycorrhizal fungi in azenic culture is affected by nutrient availability. They found that Ca , K, and Mg had no effect of weathering activity when one element was deficient, however when Mg and K were simultaneously deficient both phlogopite weathering and oxalic acid production increased. In order to test whether weathering activity can respond to nutrient demand there must be a nutrient sufficient treatment and a nutrient deficient treatment, both with added minerals. The great majority of microcosm studies investigating ectomycorrhizal weathering fail to have both a nutrient deficient and a nutrient sufficient treatment. Only the work by van Scholl et al. and Paris et al. , explicitly tested whether weathering activity can respond to nutrient demand. From these studies it does appear that there is potential for the ectomycorrhizal fungus alone, or the ectomycorrhizal seedling to respond to deficiencies in P, Mg, or K by enhancing weathering activity, however the study by van scholl et al. had no added minerals and thus doesn’t actually measure weathering, and the studies by Paris et al. are azenic pure culture studies. More studies are clearly needed to address this specific question. If the ectomycorrhizal fungus is also below its optimal level for a particular nutrient, then increases in weathering or nutrient uptake observed by ectomycorrhizae in a –nutrient treatment are not necessarily a reaction to host plant nutrient demand. Increased weathering may be a reaction to ectomycorrhizal nutrient demand only. Having separate mycorrhizal and rooting compartments would help to resolve this question as would an additional treatment in which the growth medium is kept very nutrient poor but the plant is foliarly fertilized. The two compartment system used in Jentschke et al. would be a very effective way to segregate the ecomycorrhizal nutrient demand from plant nutrient supply. While they do not explicitly test whether weathering activity can respond to changing nutrient status, a number of other studies can offer insight into the study of ectomycorrhizal weathering and some discussion of them is warranted in this review. Ectomycorhizae have been found to increase weathering in a number of microcosm studies ,planta de arandanos en maceta while others have not found any increase in weathering with ectomycorrhizal colonization . Many of these studies find increased weathering with one ectomycorrhizal species but not another or with one nutrient treatment or mineral type but not another. Generally, studies that deny P or K and add a weatherable P or K source such as apatite or biotite do find increased weathering with ectomycorrhizal colonization. The same cannot be said for Mg; no study has yet looked at how weathering by ectomycorrhizal plants is affected by Ca status.Wallander found that all 3 ECM strains tested increased weathering rates above the non mycorrhizal control, but the mechanism of increased weathering was different for each strain: decreasing solution pH , oxalic acid prodution and greater P uptake . The most commonly proposed mechanism for ectomycorrhizal enhancement of mineral weathering is greater nutrient uptake and transport away from the mineral surface . Organic acid production by seedlings is generally found to be altered, though not necessarily increased, by ectomycorrhizal colonization. Organic acid exudation does not respond in a consistent way to nutrient demand or to the presence of certain minerals, nor is it generalizable across different ectomycorrhizal species. When one LMWOA is linked to increased weathering rates it is most commonly oxalic acid. Oxalic acid is produced in particularly large quantities by P. involutus, which also happens to be the most commonly used ectomycorrhizal species in weathering experiments. Ochs et al. found that there were strong weathering agents in the root exudates of H.& crustiliniforme, present in very low concentrations which were likely not LMWOA’s. The work of Calvaruso et al. and Uroz et al. give convincing evidence for a key role that bacteria may play in ectomycorrhizal weathering. They found that bacteria isolated from the symbiotic mantle of ectomycorrhizosphere of oak mycorrhizas have significantly higher weathering capacity than phylogenetically closely related bacteria isolated from the adjacent bulk soil . Calvaruso et al.  demonstrated that one of these bacteria has the potential to enhance non mycorrhizal seedling growth by alleviating Mg and K limitation by stimulating biotite weathering. These results strongly suggest that further research into the field of mycorrhizal helper bacteria and ectomycorrhizal weathering is warranted. It also suggests that some of the highly reductionist experiments with either no bacteria or a much simplified bacterial community may fail to account for a key mechanism of ectomycorrhizal weathering. Often the rooting area in pot or microcosm studies is quite small such that the roots are far more densely packed than they would be in a natural setting. As a result, the ectomycorrhizosphere is no larger than the rhizosphere in nonmycorrhizal treatments. This eliminates one of the major proposed advantages of mycorrhizal colonization, and possibly a key mechanism by which ectomycorrhizae may confer a greater weathering ability on root systems: greater mineral surface contact and uptake of weathering products directly from mineral surfaces. The majority of microcosm experiments employ an artificial rooting medium and/or an inorganic nutrient solution, both of which may be a poor recreation of the nutrient environment of field settings. Nutrient starvation may be achieved when minor nutrient limitation, more representative of field conditions, is desired. Most microcosm studies also have either no or a highly simplified bacterial community, which may significantly alter weathering dynamics from natural settings. Another key drawback of microcosm studies is that the carbon and nutrient exchange dynamics of isolated seedlings in a laboratory may bear little resemblance to that of seedlings or mature trees in the field. In field settings hyphal networks may allow seedlings to avoid some of the initial carbon investment involved in establishing mycorrhizal colonization. Mature ectomycorrhizal trees are generally considered to be dependent on ectomycorrhizal communities for survival, while seedlings in the lab often experience growth reductions in response to mycorrhizal colonization and uncolonized seedlings can be far larger and more vigorous. Calculating the weathering rates in forest soils is critically important to forest managers, air quality policy, and models of forest productivity. Any removal of timber from a forest represents a removal of mineral nutrients; understanding how quickly those nutrients are replenished by atmospheric deposition or mineral weathering is a key component of a sustainable harvesting cycle . Mineral weathering rates determine a soil’s buffering capacity and are the single most important properties determining an ecosystem’s ability to buffer the effects of acidifying pollutants . Mineral weathering rates in soils are also the single most poorly constrained component of models designed to calculate acceptable airborne pollutant loads of nitrogen and sulfur deposition from power generation, transport, and agriculture . Accurate estimates of net primary productivity of forests over the course of the next century are critically important to global carbon models. Forest productivity is predicted to increase due to elevated CO2 . The extent of this negative feedback to elevated CO2 levels is largely dependant on forest trees’ ability to meet their increased carbon availability and water use efficiency with increased nutrient uptake . As the effects of anthropogenic nitrogen deposition continue to accumulate, large areas of forest are limited by base cation availability , which is a function of mineral dissolution. In coniferous trees, elevated CO2 has been shown to increase the ratio of root to shoot biomass  and allocation to mycorrhizal symbionts . To understand how forest productivity and forest carbon stocks will be affected by global change we must first understand whether increased carbon allocation to nutrient uptake organs actually results in increased nutrient uptake and whether this increased carbon allocation is a result of increased nutrient demand. Most forest trees of the temperate and boreal biomes are dependant on ectomycorrhizae for their survival . Ectomycorrhizal fungi  are symbionts that form an intimate association with the fine roots of trees and some woody shrubs. Increased nutrient uptake is generally considered to be the most beneficial effect of EMF on forest trees , though EMF have also been shown to increase water uptake , provide resistance to aluminum and other toxic metals , and increase pathogen resistance . EMF take up nitrogen from the soil and provide their host plant significant amounts of it; up to 80 % of total plant N uptake is from EMF . 

However, ectomycorrhizae have also been shown to provide their host plants with significant amounts of the mineral derived nutrients calcium , potassium , magnesium , and phosphorous . Studies have shown that ectomycorrhizal fungi may also play a role in the weathering of soil minerals, enhancing mineral nutrient uptake from these otherwise highly recalcitrant nutrient pools . Ectomycorrhizal communities are species rich, with well over a hundred ECM species having been documented in monodominant forests , and dozens or more on individual trees . Our knowledge of the respective ecological niches of ECM fungi is poor, but there is ample evidence that suggests discreet, nonSoverlapping niches of habitat preference and nutrient acquisition exist for some species. As atmospheric CO2 concentrations rise, forest growth and tree’s nutrient demands may increase. The Earth’s atmospheric concentrations of CO2 are increasing due to fossil fuel combustion, agriculture, and deforestation, and are predicted to continue to rise, even if we arrest the increasing rate of anthropogenic CO2 emissions. A number of studies have shown that plants grow faster and fix more CO2 when CO2 concentrations are increased above ambient levels . This increased growth however, is dependant on increased nutrient uptake to support increased standing plant biomass There is evidence that forests are responding to this increased nutrient demand caused by CO2SstimulatedSgrowthSenhancement by increasing root growth and developing a deeper distribution of roots.

Anthropogenic nitrogen pollution threatens to alter the productivity and carbon storage of temperate and boreal forests. Anthropogenic nitrogen pollution from energy generation, transport,growing strawberries vertically and agriculture has more than doubled the inputs of nitrogen to terrestrial ecosystems . Emissions of the other major component of acid rain, sulfur, were successfully reduced in the early 1990’s, and public attention to acid rain has since diminished greatly. ANP however, has either remained steady or increased somewhat in the developed world, and has risen sharply, and is predicted to rise even more sharply in the 21st century in the developing world . Nitrogen put into the atmosphere by transport and energy generation returns to earth as HNO3 and can fall as wet or dry deposition many hundreds of miles from pollution sources . 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 . Beyond a certain amount of accumulated ANP forest productivity may drop sharply as a result of soil acidification and excess N inputs leaching out other essential nutrients, which then become limiting to forest productivity. 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. Thus, the continued productivity of forests sustaining heavy nitrogen deposition will become dependant on the uptake of these mineral derived nutrients. Mineral weathering increases the supply of these nutrients and neutralizes the acidifying effects of nitrogen deposition.Decreased below ground carbon allocation equates to decreased inputs of carbon into deep soil; carbon inputs which may lead to longer Sterm soil carbon retention than above ground litter inputs . This decreased below ground carbon allocation also has profound effects on mycorrhizal relations. Understanding how nitrogen deposition and elevated atmospheric CO2 concentrations will affect forest productivity and soil carbon storage is essential to predicting how future anthropogenic emissions of carbon will affect the Earth’s climate. 

At present, an amount of carbon equivalent to 600 % of our annual CO2 emissions is taken up by the planet’s terrestrial biota each year, the majority in forests . Even small increases in forest productivity could be a major negative feedback to greenhouse gasS induced climate change. There is five times as much carbon stored in soils as there is in living plant biomass, a change of just 1 % in soil carbon pools is equal to 3 years of anthropogenic carbon emissions . There is a wide variety of effects of anthropogenic emissions of N and C that may affect these huge stocks of soil carbon, with the net effect, increase or decrease, very much unclear. Ectomycorrhizal communities may play a major role in determining how forest productivity and soil carbon stocks are affected by anthropogenic carbon and nitrogen emissions. Elevated CO2 has been found to alter ectomycorrhizal community composition and increase mycorrhizal colonization . Anthropogenic nitrogen pollution has been found to alter ectomycorrhizal community composition , decrease ECM diversity  and decrease colonization intensity . 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%.Reductions in C allocation to ECM may significantly reduce soil C storage and serve as a positive feedback to global change. The reductions in carbon allocation to ectomycorrhizae observed under nitrogen limitation may continue as more N deposition occurs or may level off as other nutrients become limiting to forest growth. The increase in below ground carbon allocation observed with elevated CO2 may continue as global CO2levels increase, or may level off or reverse if plants become sufficiently nutrient limited that carbon fixation rates are reduced.

If ectomycorrhizal community shifts observed under elevated CO2 and nitrogen inputs represent a shift towards ectomycorrhizal species that are better able to provide the nutrients most limiting to plant growth, then forests may likely adapt to their shifting nutrient demands and continue to increase in productivity in response to increasing CO2 levels and nitrogen deposition. If, on the other hand, these shifts in community composition, and reductions in mycorrhizal colonization reflect temperate and boreal forests’ adaptation to limitation by nitrogen and only nitrogen,best vetical garden system  then increasing amounts of forest may experience reduced productivity in response to continued nitrogen deposition and the fertilization effect from CO2 enrichment will likely decrease as forests become more severely nutrient limited. My dissertation attempts to shed light on which of these two scenarios is likely to unfold over the coming decades of continued anthropogenic global change. In chapter one, I investigated the effects of nitrogen addition on ectomycorrhizal community composition and colonization in a deciduous forest. Very high levels of nitrogen fertilization significantly changed ectomycorrhizal community composition, decreased ectomycorrhizal diversity, and shifted the ectomycorrhizal distribution more towards the mineral soil. These results suggest that the ectomycorrhizal community may be shifting to meet the changing nutrient demands of the forest and outline a potential mechanism for increased soil carbon storage under anthropogenic nitrogen deposition. Based on the high fungal diversity found in the mineral soil, and the fact that the ectomycorrhizal abundance in the mineral soil increased in response to nitrogen deposition I decided to investigate how the heterogeneous distribution of nutrients in mineral soil determines fungal species distribution. In chapter two, I tried to assess which soil properties determine fungal species distribution. Our sampling design prevented us from assessing the role of some of the chemical properties examined, but carbon content and depth emerged as the most influential soil properties determining fungal community composition. 

Calcium content also appeared to be important in determining fungal community composition. While pure culture studies have shown that ectomycorrhizal fungi vary in their ability to stimulate mineral weathering and take up mineral nutrients, the observed species shifts in ectomycorrhizal communities can only reflect shifting nutrient demands from host plants if plants can allocate carbon to the mycorrhizal fungi that are providing the most mineral nutrients. In Chapter 3, I present a literature review on the current state of knowledge on how plant nutrient demand drives fungal weathering. Within the plant physiology literature there is evidence that plants may be able to respond to phosphorous and potassium limitation with increased carbon allocation to mycorrhizal fungi providing those nutrients. Magnesium limitation reduces below ground carbon allocation, and the effects of calcium limitation on carbon allocation are unclear. In studies of ectomycorrhizal weathering there is a distinct lack of explicit testing of the role of host nutrient status in driving fungal weathering. I conclude by making a number of recommendations for how future studies can address this important question. For the fourth chapter of this dissertation I investigated how elevated CO2 affects plant growth, biotic weathering, and organic acid exudation, as well as the roles of plants, their ectomycorrhizal symbionts, and organic acids in stimulating mineral weathering. Elevated CO2 increased plant growth but did not increase mineral weathering. Pine seedlings but not ectomycorrhizae significantly increased mineral weathering, though there was some indication that one of the two ectomycorrhizal species examined, Piloderma&fallax, may have stimulated mineral weathering. These results do not support the hypothesis that increased nutrient demand by plants, caused by increased CO2 availability, will stimulate weathering, though our system’s departures from forest soil conditions hamper our ability to directly relate our results to processes in forested ecosystems. In my doctoral research I set out to determine whether the ecological, chemical, and physiological nature of the ectomycorrhizal symbiosis will allow for ectomycorrhizal communities to shift their functioning in accordance with the changing nutrient demands of forests experiencing global change. My research indicates that ectomycorrhizal communities may shift to accommodate the shifting nutrient demands of forest undergoing sustained heavy nitrogen deposition, but failed to find an effect of elevated CO2 on mycorrhizal fungi or biotic weathering. I also identified a number of ways in which future studies could address these questions in a more targeted, verifiable manner.Anthropogenic nitrogen pollution is a global problem that threatens the ecological integrity of many terrestrial and aquatic ecosystems. Anthropogenic nitrogen pollution  from energy generation, transport, and agriculture has more than doubled the inputs of nitrogen to terrestrial ecosystems . Emissions of the other major component of acid rain, sulfur, were successfully reduced in the early 1990’s and public attention to acid rain has since diminished greatly. Anthropogenic nitrogen pollution however, has either remained steady or increased somewhat in the developed world, and has risen sharply, and is predicted to rise even more sharply in the 21st century in the developing world . Nitrogen pollution from agriculture either leaches into streams, lakes, and groundwater as nitrate or volatilizes off of fields and manure deposits to return to the earth in the form of ammonium. Nitrogen put into the atmosphere by transport and energy generation returns to earth as HNO3 and can fall as wet or dry deposition many hundreds of miles from pollution sources. As a result of both these processes, but primarily due to the more far reaching HNO3 , large forested regions are receiving inputs of HNO3 that threaten to dramatically alter their ecology and species composition , and, when prolonged severe deposition occurs, reduce their productivity.Ectomycorrhizal fungi are essential to forest health and are particularly important to the nitrogen nutrition of temperate and boreal forests. They form intimate associations with tree roots, providing the roots with nutrients and receiving fixed carbon from their plant hosts. Ectomycorrhizae form symbiosis with less than 3% of the world’s plant species but with many of the dominant trees of temperate and boreal forests, particularly the plant families Pinaceae and Fagaceae, . Ectomycorrhizal communities are species rich, with well over a hundred ECM species having been documented in monodominant forests , and dozens or more on individual trees . Ecomycorrhizae have been shown to transfer significant amounts of P , Mg , Ca , and K to their plant hosts, but their provision of nitrogen is generally considered their primary contribution to plant health.Studies have shown that ECM may provide up to 80% of a host plant’s total N uptake . The extraradical mycelia of ECM greatly increase the volume of soil that roots can exploit . Through the use of a diverse suite of enzymes ECM may be able to solubilize and take up nitrogen from organic N pools that roots cannot utilize .

Furthermore, linear regression shows a decreasing change in slope from CF treatment to MS and HS treatments which is explained by the non linear sorption of As and the different forms of Fe in plaque , demonstrating that Fe in the CF treatment has a higher affinity for As in comparison with II treatments. In Chapter 1, our results demonstrated that total grain As concentrations are higher in the CF treatment in comparison to II. In addition, we found that iron plaque in the CF treatment can bind greater amounts of As . Overall, more As is present and mobilized in rice plants under continuous flooding. Therefore, we postulate that under II the main mechanism for reducing As accumulation in grain is not directly its sequestration in the rhizosphere but rather the oxidation and immobilization of As in bulk soil which drives the observed sequestration in the rhizosphere. Nevertheless, changes in the mineralogy of root plaque play a key role in reducing the accumulation of As in grain. Elucidating the abiotic changes in Fe and As chemistry that occur in soil due to II facilitates our understanding of the mobilization and bio availability of As in rice paddies. However, research on the concurrent biotic impacts on As bio availability and soil health with II is required and this information should be included in a conceptual model for As mobility and bio availability in rice.Rice plants readily take up and accumulate arsenic in the grain when grown in flooded paddy fields, posing a threat to human health . Intermittent irrigation has been a subject of increasing interest due to its efficacy in reducing As accumulation in rice, reducing methane gas emissions, and increasing water use efficiency during rice cultivation . II is characterized by a distinct cycling of flooded and non flooded periods that are accompanied by redox fluctuations, which have a significant impact on the fate and form of certain contaminants and nutrients in soil,container vertical farming as well as soil microorganisms . The cycling of redox active elements in soil is often associated with abiotic factors such as mineral precipitation and reductive dissociation; or biotic factors such as the activity of plant roots and related microorganisms .

The biogeochemical cycle of As is linked to microbial mediated transformations and influences the mobility, distribution, and availability of As species in the environment. In rice paddies, microorganisms play vital roles in both aerobic and anaerobic soil conditions. During continuously flooded treatments, some anaerobic bacteria can use AsV as a terminal electron acceptor in respiration and subsequently reduce it to AsIII, contributing to greater As bio availability in the soil solution . In aerated soils, some aerobic bacteria can transform AsIII to less toxic forms, such as AsV and methylated As . Arsenic reducing and oxidizing bacteria often coexist in the rice rhizosphere, and their abundance and activities regulate As speciation, bio availability, and accumulation in rice paddies . The relative abundance and activity of As transforming microorganisms are key factors that influence the fate of As in paddy soils, and consequently the bio availability of As to rice plants . Moreover, it is well documented that numerous bacteria species are involved in iron oxidation within the rhizosphere, and thus the microbial community may also play an important role in Fe plaque formation . Dissimilatory iron reducing bacteria use FeIII from iron oxide minerals as a terminal electron acceptor during anaerobic respiration. This reductive dissolution reactions facilitates the release of As from iron oxides, increasing its bio availability for rice uptake. On the other hand, the oxidized micro environment created by the oxygen secreted from rice aerenchyma allows iron oxidizing bacteria to couple the oxidation of FeII with the reduction of a variety of electron acceptors, promoting the co precipitation of As with iron oxides .Microorganisms are very sensitive to small changes in their environment and can be influenced by a range of biotic and abiotic factors . It is thus expected that redox fluctuations caused by II events will affect microbial activity and succession in the rhizosphere. II is a promising management strategy for reducing As concentrations in grains but needs to be accepted by rice growers for widespread adoption in rice cultivation. Scarcity of information withholds farmers from making well informed decisions. Although growers are typically most concerned about how changing on farm operations will affect their agronomic systems, there remains a need for mechanistic biotic and abiotic information to explain broad implications of establishing II treatments.

At present, the impact of microbial processes on As cycling in the rhizosphere is not well understood. Thus, there is a need for studies that investigate the association between water management treatments, changes in soil microbial communities, and the influence of bacteria in As and Fe cycling in rice systems. Studies that analyze the impact of II on microbial community composition often report treatments with several dry down events and yield is not always evaluated . Understanding the changes of microbial populations due to paddy water management regimes can provide information about the role of rhizosphere bacteria in Fe plaque formation and As speciation, bio availability, and mobility in rice systems. The primary objective of this study was to reproduce rice field conditions in a series of pot based bio assays with single dry down II treatments of varying severity and to observe the effect of water management fluctuations in rhizosphere soil bacterial communities throughout the growing season. The experimental design aimed to replicate the field conditions from the field based irrigation management rice growth trials conducted at the California Cooperative Rice Research Foundation Rice Experiment Station in Biggs, CA during summer 2017 and 2018 . The current study was conducted in Escondido, CA during summer 2020. The site has a Mediterranean climate with an average temperature of 20.6 °C and average precipitation of 0.813 mm for May  October 2020. Soil from the RES was collected,hydroponic vertical garden mixed to homogenize, and placed into 1 gallon pots. The average As and Fe concentrations in the soil were 3.87 mg kg 1 and 33.39 g kg 1 , respectively. The three II treatments were high, medium, and low severity ; and one continuously flooded as a control. Each treatment was assigned to a 62 L plastic bin that contained 6 replicates of 1 gal pots with paddy soil. Rice seeds were planted evenly on the soil surface and the bins were flooded 10 cm above the soil line. Every 5 days water was added to reach the initial water level, pH and redox potential were measured, and bins were re oriented and repositioned based on a randomized complete block design . For the dry down events, the water from the three II bins was drained and soil moisture was measured on an hourly basis utilizing a Watermark handheld meter and soil moisture sensors set at a depth of 10 to 15 cm. Soil was sampled throughout the season on similar dates from the field experiment presented in Chapter 1. Table 3.1 provides the timeline for sampling, water treatment management, and other events throughout the experiment. Soil was collected using a 1 mL sterile syringe with the end tip cut off to create a miniature soil probe. Approximately 1 g of soil was obtained at a depth of 5 10 cm at the base of the rice plants , and two samples were obtained from each pot. A 0.25 g sub sample was separated, and DNA was extracted with the Qiagen DNeasy PowerLyzer PowerSoil Kit. At harvest, the rice grain was collected, and plants were rinsed to remove soil adhered to the roots. Roots were separated from the shoots and washed thoroughly with DI water to remove any remaining soil. All plant biomass was dried at 65 °C for 24 h. Paddy grain was polished into white grain utilizing a rice huller and mill, and each layer was ground, sieved at 0.5 mm, and stored in airtight containers. Roots and shoots were cut into 2 cm sections and a 5 g sub sample was ground and sieved at 0.5mm and stored for further analysis. DNA extractions and library preparation were performed by the UC Davis Host Microbe Systems Biology Core Facility.

Primers 341F and 806R were used to amplify the V3 V4 domain of the 16S rRNA using a two step PCR procedure. Step one was the amplification procedure where the primers contained an Ilumina tag sequence, a variable length spacer, a linker sequence and the 16S target sequence. In step two, each sample was barcoded with a unique forward and reverse barcode combination using forward and reverse primers. The final product was quantified on a Qubit instrument using the Qubit High Sensitivity dsDNA kit and individual amplicons were pooled in equal concentrations. The library was quantified via qPCR followed by 300 bp paired end sequencing using an Illumina MiSeq instrument and taxonomic groups were assigned using the Silva rRNA database. Data of quantified As and Fe in plant biomass, all treatment effects, and differences between sampling dates were assessed using a two way analysis of variance . All effects with p values < 0.05 were considered significant. P values are presented throughout, but where significance is discussed, Tukey’s multiple comparison test was conducted. Relationships between samples from microbial analysis were visualized using principal coordinate analyses obtained based on Bray Curtis dissimilarity metrics.Consistent with results presented in Chapter 1, II treatments decreased As concentrations in rice grains, shoots and roots, and increasing dry down severity had a greater impact in reducing As bio accumulation . Like As, the concentration of Fe in root samples with plaque was highest for CF treatment and decreased for LS, MS, and HS treatments . It is surmised that dry down events promote oxic conditions in the bulk soil and aqueous FeII is oxidized and immobilized via precipitation of FeIII oxides, decreasing the overall Fe content in the rhizosphere and root plaque compared to the CF treatment. With CF, mobile aqueous FeII persists in the bulk soil and is oxidized only after being transported into the oxygenated rhizosphere. Therefore, greater precipitation of FeIII oxides as root plaque occurs, and a higher total Fe concentration is observed in root samples from the CF treatment. Soil pH continually increased from sowing to harvest by 1.5 units . This increase is explained by the constant consumption of protons in paddy soils during reduction processes associated with flooded rice paddies . Soil redox potential dropped upon initial flooding, increased above 100 mV during dry down events, and declined after reflooding below 300 mV for all treatments . This pattern indicates oscillation between anoxic and oxic conditions. The redox state of Fe and As during these fluctuations can be predicted via the Nernst equation or, simply, by utilizing the pH and Eh measurements and referring to Pourbaix diagrams, which indicate that both As and Fe are present in their reduced forms during flooding and become oxidized during the dry down events of II treatments . During the dry down periods in the three II treatments, soil water potential reached 10, 70, and 120 kPa for LS, MS, and HS treatments, respectively. Based on Carrijo et al., 2018, soil water potentials reached during dry down events are equivalent to volumetric water contents of 40, 35, and 25% for LS, MS, and HS, respectively. These parameters were carefully defined for the field trials, as well as the timing of the dry downs, to ensure that rice plants reach maturity and maintain yields . From 52 identified phyla, the 15 most abundant accounted for 98% of the total sequential reads. The three dominant phyla for all treatments throughout the growing season were proteobacteria, actinobacteriota, and acidobacteriota, which are typically found in agricultural soils . Proteobacteria, the most abundant phylum in our soil samples, is expected tothrive in carbon rich environments, such as rice paddies . Although the mentioned phyla predominated in our samples, there were variations in the relative abundance of these groups. Our samples from the CF treatment express a higher abundance of the phyla actinobacteriota and firmicutes, whereas acidobacteriota, chloroflexi, and myxococcota are more abundant in the HS treatment. These results suggest phylogenetic differences in the bacterial communities are driven by fluctuations of anoxic and oxic conditions.

The Arabidopsis CLE gene was found to be induced by N deficiency, and over expression of CLE inhibits lateral root elongation but not initiation. The peptide sequence of CLE is homologous to CLV3, which binds to CLV1 and the clv1 mutant showed increased lateral root length under low N conditions. The transcript levels of CLE were increased in the clv1 mutant, suggesting a feedback regulation of CLE by CLV1. Transgenic lines with increased CLE levels in clv1 did not inhibit lateral root growth, indicating that the inhibition of CLE3 on lateral root development requires CLV1. Altogether, the N responsive CLE CLV1 peptide receptor signaling module restricts expansion of the lateral root system in N deficient environments. Although nitrate is a crucial nutrient and signaling molecule, its distribution in soils is heterogeneous. To adapt the prevailing nitrate conditions, plants have evolved a systemic response mechanism. NRT2.1 was the first molecular target identified in long distance signaling reflecting root responses to environmental nitrate conditions. Plants were grown using a 1 mM NO3 − solution, then the root was split into two parts, one subjected to N free treatment and the other one treated with 1 mM NO3 −. Both 15NO3 − influx and the transcript level of NRT2.1 were increased in the NO3 − fed root. Recent findings revealed that the C terminally encoded peptide originated from N starved roots; located in xylem vessels, it sends root derived ascending signals to the shoot before being recognized by a leucine rich repeat receptor kinase, CEP Receptor 1 , and then inducing the expression of CEPD polypeptides. CEPD sent long distance mobile signals translocated to each root and upregulated the expression of NRT2.1. The activity and expression of NRT2.1 in plants were inhibited when supplied with high N. Lepetit’s lab configured a forward genetic approach using a transgenic line expressing the pNRT2.1::LUC construct as a reporter gene. The mutant hni9, showing increased expression of NRT2.1 under high N supply,vertical growing systems was isolated and the mutation was found in IWS1, a component of the RNAPII complexes.

Further investigation revealed that the levels of the H3K27me3 on NRT2.1 chromatin decreased, resulting in the upregulated expression of NRT2.1 in response to high N supply in the iws1 mutants. Thus IWS1 is likely to be involved in the transduction of N systemic signals through controlling the expression of NRT2.1 in plants. Another important player participating in root foraging, TCP20, was identified by Crawford’s lab using the yeast one hybrid system to screen the transcription factors that can bind to the fragment of nitrate enhance DNA. TCP20 was found to be able to bind to the promoters of NIA1, NRT1.1, and NRT2.1. The tcp20 mutants exhibited deficiencies in preferential lateral root growth on heterogeneous media in split root experiments, indicating that TCP20 can regulate the preferential growth of lateral roots in high nitrate zones, thus playing an important role in the systemic signaling pathway. Recently, using an electrophoretic mobility shift assay , the DNA binding sites of TCP20 in a 109 bp NIA1 enhancer fragment were found to be in close proximity to NLP7 and NLP6 binding sites. Yeast two hybrid and bimolecular fluorescence complementation assays showed that NLP7 and NLP6 can interact with TCP20 and both the PB1 domains of NLP6&7 and the glutamine rich domain of TCP20 are necessary for protein–protein interaction. Further work will be needed to elucidate the underlying molecular mechanism explaining the involvement of TCP20 in systemic signaling.Root microbiota associate with every land plant and show community compositions and dynamics that are distinct from the surrounding soil microbial community . Both rhizosphere and root endosphere microbiomes affect plant health and soil health via processes such as mineral and nutrient turnover and pathogen suppression . Attribution of specific processes to distinct microbial players or populations is challenging because soil ecosystems are among the most complex environments on Earth . Soils are made up of a multitude of heterogeneous abiotic and biotic components that interact in a dynamic fashion over a range of spatial and temporal scales .

Soil type, together with climatic characteristics, allows for the development and activity of biological constituents that are specific to a given soil in a particular location and can vary dramatically among soils and locations . Those biological constituents can include plants, insects, bacteria, archaea, and fungi, which all contribute to and feed off of the bio geochemical cycles in a given soil. The resulting complex network of interactions is extremely challenging to disentangle due to technological limitations and insufficient information in biological and chemical reference databases . Furthermore, soils contain a vast diversity of microorganisms, which are heterogeneously distributed and engage in frequent horizontal gene transfer. Despite this, most root microbiome studies present data from single time points or single locations and primarily conduct amplicon sequencing combined with limited information on plant or environment. Although the average values provided by such studies may suggest some interactions or mechanisms, few studies follow up with the comprehensive sampling necessary to definitively understand these mechanisms and interactions. In addition, single point studies are difficult to compare or extrapolate to other environments or plants because measured values can vary dramatically over time . Soil and other environmental characteristics can be important indicators of biogeochemical processes that have occurred in the past or are ongoing. Generally, few root and soil microbiome studies take advantage of the relatively inexpensive techniques to measure soil characteristics. Data on parameters such as pH, volumetric water content, temperature, and salt concentration could allow researchers to draw correlations between microbial activity, plant productivity, and environmental parameters and facilitate opportunities to cross reference studies conducted under comparable conditions.

In the last decade, the root microbiome research community has made tremendous progress in understanding the complexity of soil ecosystems through improvements in experimental methods at both laboratory and field scales.This review summarizes recent technological advancements and the resulting research opportunities categorized by ecosystem component and scale ,outdoor vertical plant stands and ends with an outlook and potential applications for phytobiome research.Microbial colonization of the root and rhizosphere can significantly affect root phenology and metabolism. Roots demonstrate enormous phenotypic plasticity with respect to anatomy, shape, cell type, cellular structure, metabolism, and biochemical composition, and these characteristics contribute tremendously to root exudation variation and, as a result, to microbial community differentiation . These reciprocal interactions between roots and microbes are not well understood but their direct link showcases the fact that, for understanding root microbiomes, a foundational understanding of root biology is required. Although hyperspectral imaging of leaves has been broadly applied to monitor plant health, even simple imaging of intact roots has lagged behind due to the challenges presented by the opaqueness of soil . Ideally, imaging of root architecture, microbes, and chemical composition as well as visualization of fluxes such as carbon flow through plant compartments and into the soil would be conducted at multiple temporal and spatial scales. Most current methods for analyzing root growth either require artificial growing conditions , are severely restricted in the fraction of roots detectable , or are destructive . For example, many root phenotypic datasets have employed coring or “shovelomics”, subsequent root picking and washing, and imaging using light imagers such as the RhizoVision Crown platform . This method provides valuable information about root architecture; however, it is extremely laborious, it is often not feasible to excavate deep roots, it can remain unknown how much of the root system was recovered and scanned, and root excavation often times terminates the experiment for the selected plants. All of these methods are severely limited because they are destructive, low throughput, or artificial. The later point is particularly important because root architecture can be significantly affected by plant genetics, environmental conditions, soil type, and root colonizing bacteria and fungi . Magnetic resonance imaging presents a noninvasive modality that addresses some of the limitations of other root measurement techniques. When coupled with an analysis pipeline in an automated system, MRI can monitor root mass, length, diameter, tip number, growth angles , and spatial distribution in a high throughput manner . Similarly, X ray computed tomography scanning can provide a comprehensive picture of root systems as long as the roots have a diameter larger than the instruments’ resolution . Hence, small plants or young roots are not likely to be resolved well. Another limitation common to both MRI and CT technology is that plants must be grown in pots that fit into the imaging machines and the applicability of MRI and X ray CT in three dimensional imaging of root systems across various pot sizes was recently evaluated . Although both MRI and CT were able to resolve high quality 3D images of root systems in vivo, the reconstructed length and image details differed significantly between the two methods. In small pots, CT outperformed MRI and provided more details thanks to higher resolution whereas, in large pots, MRI was able to display root systems more comprehensively than CT.

Soil features such as minerals and burrows can be resolved with CT, while MRI can measure water content in roots and soil. Both CT and MRI, struggled with roots thinner than 400 mm .Using Synchrotron X ray microtomography, Milien et al. contrasted the 3D images of vascular systems of successful and unsuccessful graft interfaces in vine rootstocks. Others have applied synchrotron X ray microtomography to visualize drought induced embolism in various plant species , to correlate root hair with rhizosphere soil structure formation , and to quantify root induced changes of rhizosphere physical properties . Although synchrotron X ray micro CT can render unprecedented detail into the microanatomy of plants and microorganisms, the focus window is relatively limited and biological samples tend to lose viability as a result of the intense X ray radiation. There are various other imaging methods that have been recently developed or applied to phytobiome research, including super resolution confocal imaging, which can enhance 3D mapping of root and microbial or fungal cells and showcase green fluorescent proteins , and correlative confocal and focused ion beam tool with integrated scanning electron microscope, which allows for extremely fine scaled 3D mapping . When applied individually or in combination, the above mentioned imaging methods will provide opportunities to visualize plant tissue and attached or internally residing bacteria, fungi, and viruses at unprecedented resolution, as well provide information about their physical and chemical context. Because root development is vital for plant health, expansion of root image databases and novel correlations between above and below ground plant features will enhance our understanding of plant response to environmental and biological stimuli.An important goal of the plant microbiome field is to discover beneficial or deleterious effects of microbes. This means that recording and understanding plant phenotypes and linking them to microbiome variation is key. Similarly, plant microbiomes are intimately tied to the background soil; hence, monitoring soil characteristics is important but can be challenging and labor intensive at appropriate temporal or spatial scales.Unmanned aerial vehicles equipped with RGB cameras, infrared cameras, multi spectral and hyperspectral cameras, GPS, navigation systems, programmable controllers, and automated flight planning have emerged as powerful tools for nondestructive, high throughput field phenotyping that can be performed throughout the growth season . This has removed a bottleneck in phenotyping but automated processing of this data still presents various challenges, which are discussed elsewhere . Monitoring of agricultural fields using drones has become popular among researchers to more accurately plan and manage their experimental operations. Drones can produce precise maps of soil characteristics and plant characteristics , as well as determine irrigation needs, nitrogen levels, and pest occurrence . RGB, IR, and hyper as well as multi spectral cameras attached to drones can collect images of the above ground portion in a range of wavelengths. The resulting data can produce, for example, a vegetation index describing the amount of wavelengths of light emitted from a crop and, hence, can trigger irrigation systems or evaluate the sensitivity of crop breeds to soil moisture in a high throughput manner . Image data can also provide information about plant health status over time and in dependence on the field location and, thereby, allows the employment of an early warning and response system to plant disease or stress .

Rhizobiomes are influenced by their spatial orientation towards roots in two ways. First, the radial proximity of microbial communities to roots defines community complexity and composition, as described in recent publications, and as outlined by the two step model ofmicrobial root colonization mentioned above. Second, the lateral position of microbes along a root shapes the community, as exemplified by early studies. Importantly, recent microbiome studies take into consideration the former, but not the latter aspect. In this section, we discuss specific microbial associations with various root regions, and the role of spatially distinct root exudation. Root tips are the first tissues that make contact with bulk soil: root tips are associated with the highest numbers of active bacteria compared with other root tissues, and likely select microbes in an active manner. The root elongation zone is specifically colonized by Bacillus subtilis, which suggests a particular role of this zone in plant–microbe interactions.Their community includes decomposers, which could be involved in the degradation of dead cells shedding from old root parts. Similarly, lateral roots are associated with distinct microbial communities, differing between tips and bases, as well as between different types of lateral root. One trait influencing the differential microbial colonization of root tissues could be the differential exudation profiles of the distinct root parts. This is illustrated in the following example. Clusterroots are densely packed lateral roots formed by some plants growing on extremely nutrient poor soils; these roots exude high amounts of organic acids and,nft vertical farming in some cases, protons, to solubilize phosphate.

The low pH and carboxylate rich rhizosphere of cluster roots is associated with a specialized rhizobiome, dominated by Burkholderia species that metabolize citrate and oxalate. Besides organic acids, mature cluster roots also exude isoflavonoids and fungal cell wall degrading enzymes, leading to a decrease in bacterial abundance, as well as fungal sporulation. Taken together, cluster root exudates not only solubilize phosphate, but also regulate microbes in such a way that they do not interfere with phosphate uptake. Beyond this example, spatial patterns of metabolite exudation are largely unexplored.We hypothesize that such patterns exist in all root systems for the following reasons: spatially distinct organic acid exudation is atrait of all root systems ; spatially distinct exudation was similarly detected for strigolactones, amino acids, and sugars; and root nutrient uptake, which is sometimes coupled with proton transport, can also exhibit spatial patterns . Overall, spatially defined metabolite exudation by distinct root parts is likely an important factor in structuring the rhizobiome. Future studies should aim at characterizing spatially distinct rhizobiomes and their functional traits, and at investigating spatially distinct root exudation.Roottips are not only associated with high numbers of bacteria , but also produce border cells and mucilage , crucial for plant–microbe interactions. Depending on the root meristemorganization,border cells are released into the rhizosphere either as single cells or as border like cells .Residence time in the soil is different for the two types of border cell. Single maize border cells stayed alive in soil for months, likely due to the presence of starch deposits, whereas arabidopsis border like cells survived for only 2 weeks. Border cells have a transcriptional profile distinct from root tip wells, with overall lower primary and higher secondary metabolism. ABCtransporters constitute a large fraction of differentially expressed genes, which is consistent with transport of secondary metabolites. Secondary metabolites are likely central to the role of border cells in defense against pathogens.

Pathogen attack can result not only in higher border cell production and release, but also in higher mucilage production by border cells and root tip cells. Mucilage contains proteins with antimicrobial functions, as well as extracellular DNA involved in defense against fungi and certain bacteria. Importantly, mucilage is also produced under nonpathogenic conditions, serving as a lubricant for the root environment and stabilizing soil particles. Interestingly, mucilage also provides distinct carbon sources for microbes, thus influencing rhizobiome composition. Border cells similarly interact with nonpathogenic microbes : they release flavonoids that attract rhizobia, uncharacterized compounds that induce branching of mycorrhizal hyphae, and arabinogalactans that trigger biofilm formation of specific beneficial bacteria. The full extent of how border cells and mucilage shape root–microbe interactions remains unclear. It is tempting to speculate that the specialized metabolism of the border cells results in a distinct exudation profile of not only proteins and mucilage, but also low molecular weight compounds that could serve as microbial nutrients or as signaling compounds. Further research should focus on the genetic and physiological differences between border cells and border like cells, as well as on the transport proteins involved in exudation of low molecular weight compounds, DNA, and proteins.Plant–microbe interactions are not only defined by plant root morphology and plant derived exudates, but also by microbe–microbe interactions . Thus, we focus further here on microbial communities. Specififcally, we discuss: how plant exudates influence microbial diversity; how plant responsive microbes are identified; how microbes interact and how mycorrhizal fungi influence root–bacteria interactions. The rhizosphere serves as carbon rich niche for the establishment of microbial communities, in contrast to bulk soil, which is rapidly depleted in carbon and other nutrients by heterotrophic microbes.

Given that the ability of microbes to metabolize plant derived exometabolites might determine their success in the microbial community, several studies have investigated whether the diversity of plant exudates correlates with microbial diversity. Some studies found higher plant diversity was associated with higher microbial diversity, and that the addition of a diverse exudate mix to plant monocultures increased microbial diversity. Interestingly, isolates from soils with a diverse plant community consistently exhibited less narrow niches and displayed less resource competition than did isolates from low plant diversity environments. Although on a global scale, environmental factors had a larger impact on microbial diversity than did plant diversity, we can conclude that, on a local scale, high plant diversity likely promotes a diverse microbial community.The large diversity of microbial communities is a current challenge for plant–microbe research, because it is impractical to study questions such as how members of a community interact, and what specific traits a microbial community has. Therefore,indoor vertical farming many studies currently aim at identifying the subset of microbes responsive to plants. Strikingly, only 7% of bulk soil microbes increased in abundance in the rhizosphere compared with bulk soil, which reduces the number of taxa to investigate from thousands to hundreds. Other approaches to identifying plant responsive microbes have focused on transcriptional profiling. Compared with soil abundant microbes, plant associated microbes exhibited distinct transcriptional responses to plant exudates and, intriguingly, displayed distinct phylogenetic clustering. Network analyses further revealed that rhizosphere microbes displayed higher levels of interactions than did bulk soil microbes. These studies illustrate the potential for the identification of a distinct set of plant responsive microbes. The above points highlight how plants influence microbial communities. However, the members of microbial communities also interact with each other. Compellingly, it is still unclear whether microbe–microbe interactions are predominantly positive or negative. Network analyses reported predominantly positive intrakingdom interactions. By contrast, laboratory growth assays identified competition as the major factor in shaping isolate communities, and cooperation could only be detected for 6–10% of the isolates. One major difference between the two experimental approaches is that the former investigates a natural system, whereas the latter is based on the ability to culture microbes. Isolation of microbes introduces a bias, since it can select against cooperators, precluding obligate syntrophs. Further evidence that at least some microbes avoid competition was provided by co cultivation experiments.

Environmental isolates: displayed high substrate specialization; did not necessarily take up the compound with the highest energy; and diverged in substrate use when cultivated for several generations. In addition, some metabolites exuded by microbes could be metabolized by others, suggesting potential cross feeding between community members. The above findings suggest complex interactions of microbes. It remains to be resolved in which situation competition or cooperation dominates communities. However, it is evident that microbial interactions are based on altered gene expression. Microbes responded to competing bacteria or even close relatives by differentially regulating genes involved in metabolite exudation and transport processes, making the study of microbial transporters a compelling topic for future studies. Thus, metabolite uptake, release, and sensing are important factors in shaping microbial communities. Metabolite turnover in soil is influenced not only by plants, but also by functionally diverse bacteria, fungi, and animals. Plant–fungal and plant–animal interactions in the rhizosphere go beyond the scope of this review, and are discussed elsewhere. Here, we provide a few brief examples focusing on the impacts of mycorrhiza on rhizobiomes and exometabolite turnover. Endomycorrhizal fungi receive a significant fraction of the carbon fixed by plants . Interestingly, these fungi also exude sugars, shaping a distinct bacterial community. Likewise, Ectomycorrhiza receive carbon from plants, and form a dynamic bacterial community; they even participate in plant to plant carbon transport. The field of fungal microbiomes is nascent: if and how fungi control exudation, whether fungal microbiomes have beneficial functions, and how plant and fungal microbiomes influence each other are all unknowns. Although many questions remain, these recent findings already suggest that a holistic view of rhizosphere nutrient cycling and signaling exchange via exometabolites requires a whole community approach including all domains of life.Plant exudates shape microbial communities. Overall, plants exude up to 20% of fixed carbon and 15% of nitrogen, which includes an array of simple molecules, such as sugars, organic acids, and secondary metabolites, as well as complex polymers, such as mucilage . Although every plant produces exudates, the amount and composition of root exudates varies. First, exudation is defined by the genotype of the host, as observed in the distinct exudation patterns of 19 arabidopsis accessions. Strikingly, the amount of variation between the accessions depended on the metabolite class investigated. Glucosinolates displayed most, flavonoids medium, and phenylpropanoids low variability. Second, exudation changes with plant developmental stage: with increasing age, arabidopsis sugar exudation decreased, and amino acid and phenolic exudation increased. Third, exudation is modulated by abiotic stresses: the amounts of exuded amino acids, sugars, and organic acids changed in maize grown in phosphate , iron , nitrogen , or potassium deficient conditions. In addition, phosphate deficient arabidopsis plants increased coumarin and oligolignol exudation, heavy metal treated poplar induced organic acid exudation, and zinc deficient wheat increased phytosiderophore exudation. Differential exudation is a plausible mechanism by which plants could modulate their interaction with microbes, as exemplified by the correlation between exudation patterns and rhizobiome variation reported for eight arabidopsis accessions. Differential exudation modulated by transport proteins is discussed below.Plant derived exometabolites need to cross at least one membrane to transit from the cytoplasm of root cells into the rhizosphere. There is considerable discussion as to what degree plants are able to regulate this transport. In general, different modes of transport could be envisioned. First, small, hydrophilic compounds could diffuse from the root into the rhizosphere, driven by the large concentration gradient. Second, channel proteins could facilitate such diffusion. Third, active or secondary active transporters could shuttle compounds across membranes against a concentration gradient. Diffusion of compounds can only be relevant in young root tissue, which is still devoid of Casparian strips or suberized endodermis that both block apoplasmic flow in adult tissues. Transport proteins involved in exudation are mostly elusive. From a conceptual point of view, plasma membrane localized exporters likely have a direct, and vacuolar transporters an indirect effect on exudation. The vacuole is a major storage organelle for many metabolites detected in exudates, such as sugars, organic acids, and secondary metabolites. Alteration of vacuolar transporter levels impacts vacuolar and cytosolic concentrations and, thus, can influence metabolite exudation into the rhizosphere. The few characterized transporters involved in exudation are essential for the transport of specific compounds, and are presented in Table 1. Since only a few transporters involved in exudation have been characterized, we suggest additional families that might be involved in the process. To complete the picture of metabolite exchange between roots and soil, Table 1 additionally contains a few important plasma membrane localized metabolite uptake systems. Below, we discuss the evidence for transport processes involved in the import and exudation of compounds detected in root exudates, such as sugars, organic acids, and secondary metabolites.

Multimedia models for ENMs can predict environmental concentrations based on sources of continuous, time dependent, or episodic releases and are similar to multimedia models that predict environmental concentrations of organic chemicals and particle associated organic chemicals.For ENMs, predicting particle size distribution as affected by particle dissolution, agglomeration, and settling is desired for various spatial and temporal end points. For one integrated MFA and multimedia model , user defined inputs are flexible around product use and ENM release throughout material life cycles.It is noted that although validation of multimedia models is a formidable task, various components of such models have been validated as well as model predictions with such models for particle bound pollutants. Most far field models of ENMs have major challenges. First, the quantities and types of ENMs being manufactured are unknown to the general public due to issues surrounding confidential business information, leading to a reliance on market research.The resulting public uncertainty will persist while nanotechnology continues a course of rapid innovation, as is typical of new industries.The rates of product use and ENM releases at all life cycle stages are also not defined.There are challenges associated with modeling transport processes through specific media and across media , highly divergent time scales of processes, lack of required input parameters, and the need for validation of results .Several multimedia models developed for conventional chemicals could be adapted around ENMs,vertical grow but few account for fate processes specific to nanoparticles .In addition, various transport models for a single medium and in the multimedia environment could be adapted for far field analysis of ENMs, but few account for fate processes distinctive to ENMs .

Moreover, their validation, which would require ENM monitoring data, is a major challenge. The lack of understanding of many fundamental ENM behaviors under environmental conditions propagates into broad uncertainties, for example in predicting ENM removal to solids or aqueous fractions in WWTPs.ENM surface chemistries fundamentally affect ENM agglomeration or dispersion and likely affect bio availabilty.Some species on ENM surfaces may degrade in the environment,while other adsorbates can be acquired.Carbonaceous ENMs may be transformed or degraded by environmental processes such as photo,enzymatic,chemical,and bio degradation.Redox and other environmental conditions will affect nanomaterial surfaces, which for nano Ag includes formation of sulfide that inhibits dissolution.Surface chemistry also affects transformation rates of primary particles and aggregates .For many ENMs such as nanoceria,reactivity is highly size dependent. To accurately model material fates thus requires understanding how material surface properties affect integrity, how both change under varying environmental conditions such as pH, clay content,and organic matter content, and how surface properties and particle reactivity affect physicochemical processes that are parametrized in far field models. This is especially true for ENMs used as pesticide delivery mechanisms, including carbon nanotube composites with specifically reactive surface monomers. Yet only recently has modeling attempted to address differing properties of a material’s structural variants .Evaluating computational model predictions is a challenge for ENMs, which presently are estimated to occur in the environment at low concentrations.Also, detection methods for ENMs in environmental media and distinguishing ENMs from natural chemical analogs are still under development,with more evaluation strategies needed including a framework for validating new ENM analytical detection methods.

Fullerenes from incidental sources were quantified in river sediments collected from locations across the globe and quantified in the atmosphere over the Mediterranean Sea.Perhaps related to a viable exposure scenario, fullerenes were quantified at relatively high concentrations in treated wastewater effluent and at ng/L to μg/L concentrations in river waters receiving effluent discharge. While not necessarily nanoscale, similarly high concentrations of TiO2 were reported for sediments sampled near a WWTP outfall.The greatest uncertainty in ENM exposures is near field , at the receptor where toxicant dose manifests as internal dose. Heteroaggregation is a dominant fate process for ENMs when they interact with natural colloids.Given sufficient residence time for ENMs in environmental matrices, heteroaggregation and to a lesser degree homoaggregation will affect localized compartmentalization, including stability in the water column and therefore, sedimentation.However, these processes do not preclude biological impacts under simulated environmental conditions, as has been shown for nanoceria in a complex aquatic mesocosm.Exposure can be confirmed by quantifying receptor body burdens, thereby allowing for quantitatively relating near field exposure to biological effects.Thus, in the absence of detailed, biologically complex, near field models for local exposures to environmental receptors, the ability to trace ENMs to biological receptors sampled directly from the environment becomes the best available approach to relate far field exposures to biological impacts.Overall, material flow models and multimedia modeling of ENMs have advanced to inform ENM ecotoxicology. Available far field modeling frameworks are adaptable to changing inputs despite uncertainties in production volumes. Major uncertainties remain at the nexus of ENM surface and core chemistries as related to nanomaterial transport, aggregation, and degradation characteristics.

However, fundamental research is needed to discover and parametrize complex fate processes. New approaches, such as assays that can be used to rapidly probe surface associations,demonstrate how to populate far field models and how to determine near field exposures associated with effects. Although existing models can simulate particle movement, deposition, and some transformations, the knowledge state regarding ENM environmental exposure conditions via measurements or modeling simulations cannot be assumed to accurately represent actual conditions at biological receptors.Many of the outstanding research issues and recommendations for evolving ENM ecotoxicology are echoed in the discourse for other chemicals of emerging concern .These include the need for systematically understanding ENM and decomposition product toxicity across various receptors within linked levels of biological organization,quantifying actual exposures and uptake into environmental receptors,gaining mechanistic insights into and biological markers for acute and chronic low level exposures,and understanding how environmental factors including cocontaminants affect ENM transformation and biological impacts. Still, how can the potential for exposure and impacts of ENMs be anticipated, prevented, managed, or mitigated? Further, what data and tools do decision makers need to inform their work? While no formalized process for incorporating all exposure conditions and concepts of ENM transformation, dose, and body burden into risk assessments currently exists, a proposed framework approach to risk characterization over the life cycle of ENMs has been published and is available.This framework advocates an initial decision cutoff in regards to exposure; in the absence of exposure,indoor growers the need for further assessment is diminished or negated.In this available framework, ENMs that are certain to rapidly dissolve into ionic components in a destined environmental compartment would be assessed for risk based on the released components rather than the original nanoparticles.Persistent ENMs are expected to accumulate in matrices such as sediments.The consequences of ENMs to successive generations, biodiversity, and ecosystem services are not addressed by model organism specific assays of discrete growth and mortality.Nonetheless, in this available framework, toxicity end points associated with standardized testing protocols for sediment, aquatic, and terrestrial standard population level end points over short and long time frames are advocated for assessing hazards of simulated ENM concentrations in the environment.In this framework, sunlight is an environmental variable, bio accumulation is measured, and ENM modifications during product and material life cycles that may change bio availability are considered.While such a framework has broad organizational appeal, priority setting within the framework is required and thus could focus on tests that are relatively well aligned with likely exposure scenarios. Even with a risk assessment framework that considers ENMs across product life cycles and considers sediments, water, and soil in testing end points,major hurdles hinder regulatory agencies, and research scientists, in using concepts such as exposure conditions, ENM transformation, dose, and body burden in interpreting biological and computational findings for assessing risks. Toxicity tests developed for dissolved chemicals typically require significant modification for use with ENMs.Tests may not apply to ENMs if they are not appropriate for solids.Additional scientifically based hazard information from the peer reviewed literature may or may not be available for consideration. ENMs used in ecotoxicity tests, which are sometimes laboratory synthesized to overcome uncertainty regarding proprietary coating or other commercial formulations, may be insufficiently analogous to allow for extrapolating information or risk comparisons.

Issues include the need to know test material characteristics and how they relate to testing results and the ENM life cycle. Even if an initial risk assessment considers ENM solubility,ENM dissolution is not instantaneous; therefore, at what stage of dissolution does the contaminant no longer pose a hazard as an ENM? Also, where biological impacts stem from ENM surface characteristics, how can mass concentration be used to judge hazards? Environmental ENM effects in benchtop experiments can be indirect, stemming from physical nutrient depletion,or amplifying organism uptake of cocontaminants.Other indirect physical effects derive from ENMs adhering to the organism surface,light shading,or internal food displacement.Near field exposures can result in biological hazards from specific ENMs based on their properties .By definition, ecological risk assessment is “the process for evaluating how likely the environment will be impacted as a result of exposure to one or more environmental stressors.”ERA involves predicting effects for individuals, populations, communities and ecosystems, and concerns itself with valuable ecosystem services such as nutrient cycling.Thus, conducting ERAs for ENMs could benefit from an ecological outlook. All levels of biological organization, and interactions between them, would be considered when assessing responses to ENM exposure . Release and exposure scenarios , use of functional assays for assessing environmental compartmentalization ,and combined life cycle and multimedia modeling have important roles in focusing ENM ecotoxicology. Less recognized is that mechanistically based models of dynamic biological effects are informed by hazard assessment research. Different types of process based, dynamic models allow for predicting effects from exposures stepwise, starting at sub cellular levels, into individuals, through populations, and conceivably to communities and ecosystems. Developing process based models requires researching key effects processes and ecological feedbacks.Models are formalized to describe interactive processes culminating in toxicity such as reactive oxygen species generation and cellular damage. Process based mathematical expressions evolve with empirically based discoveries or through model reconciliation with experimental data. Parameters are independent of toxicity testing protocols, although models could be informed by standard test results. Thus, ENM ecotoxicity research could support predictive toxicology by informing and populating process based, dynamic ecological effects models. A comprehensive fate and effects research agenda is needed for addressing ENM quantification in complex media.Such an agenda has allowed for assessing experimental compartmentalization,and sensitively assessing environmental persistence,toxicity, bio accumulation,trophic transfer,and indirect effects from the uptake of ENMs coated in other hazardous materials.Such research could substantially inform ENM risk assessment for a relevant environmental exposure scenario. However, most ENMs have not been studied comprehensively along the entire exposure and effects continuum . Further, the approach is not sustainable. Rather, the need is to develop efficient approaches applicable within an overall approach to rapidly evaluate the large number of ENMs under commercialization . A research agenda that focuses on distilling key determinants of exposure and hazard for ENM environment systems that can be measured experimentally would be most compelling. Thus, while the science of ENM ecotoxicology and exposure characterization has advanced, there are disconnects between how regulators review ENM based products for environmental safety and the research that is conducted to evaluate hazards. Except for results published in open source outlets or directly reported, research may be unknown to government bodies. Ongoing synthesis of published research results is challenging due to high variability across study conditions and ENMs tested, and due to effort needed to regularly update such comparisons. Moreover, there is a systematic resistance to publishing “no effect” studies in the peer reviewed literature.As a result, relying only on published research to inform regulatory decisions can present challenges. A life cycle based framework facilitates exposure modeling and hazard testing to support risk assessment. However, extrapolation of effects to untested concentrations, study, or environmental conditions, and across biological levels of organization, requires understanding dynamic biological process based effects, which current standard tests neither deliver nor sufficiently inform. Ultimately, exposure scenarios are useful for framing and focusing ENM ecotoxicology, and some version of a tiered intelligent testing and risk assessment strategy is needed. Such a conceptual tiered strategy considering the impact of the ENMs’ varying properties on ecological risks at different life cycle stages was proposed in the EU FP7MARINA project and is being further developed in the EU NANoREG program.