Tag: vertical agriculture

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Traditionally, measuring soil quality parameters requires destructive sampling and laboratory analyses that are laborious, slow, or expensive. Similarly, root phenotyping requires time and labor intensive processing and scanning of root tissue to collect data such as root length density and root architecture . Advances in imaging have been able to offset some of these hands on analyses: high resolution RGB imaging can differentiate between soil types facilitating soil type detection, which can improve mapping and hence conservation efforts . New approaches that overcome the limitations of laboratory tests include thermal infrared imaging, which can be used to assess soil moisture distribution and hydraulic properties and inform land surface models . Near infrared spectroscopy has been used for rapid and accurate identification of soil total nitrogen , organic matter , and pH levels in soil that can replace laboratory techniques . Similarly, hyperspectral imaging can be used to accurately provide TN, OM, and organic content information in various soils as well as fungal viability based on pixel spectra specific to browned, damaged, and undamaged tissue types . Because image processing of HSI is more challenging than that of RGB imaging, the two technologies can be used in tandem; for example, to optimize comprehensive analyses of soil and root systems in rhizoboxes . The accuracy of both IR and HSI can be improved by applying extreme learning machine models, which were previously used to increase the accuracy of soil moisture and surface temperature measurements . Because UAVs are scalable and programmable,vertical plant rack we expect that drone usage in phytobiome research will move toward autonomous UAV fleets that can monitor extensive fields with an array of cheaper and more accurate sensors.

We also expect aerial monitoring to be more closely coupled to robotics on the ground that could aid in conducting soil and plant analysis and deployment and maintenance of local sensor networks among various other tasks. Thus far, the development of robotics to measure soil characteristics has primarily focused on applications in environments that are difficult or unsafe to access. For instance, a robot was developed for measuring soil strength over depth, which is normally manually measured using a penetrometer, in unsafe zones . The Mars Phoenix Lander returned in situ measurements of Mars soil temperature, generated a topography map using imaging, and excavated soil samples for downstream testing .Plant microbiome signaling and metabolism rely on exchange of a large diversity of metabolites derived from microorganisms, plants, and the soil environment. Metabolomic methods enable direct characterization of these small molecules from soils and the various biological components. Given the large diversity of compounds that reside intra and extracellularly in these systems, mass spectrometry coupled to chromatography such as liquid chromatography MS and gas chromatography MS have become primary methods for chemical analysis. Both techniques are well suited for identification and quantification of a wide range of molecules found in biological and environmental samples by coupling the physical separation of the compounds using LC with the separation and analysis of ions using MS mass.GC MS typically has higher resolving power and produces richer fragmentation spectra, which makes it particularly well suited for identifying molecules such as small glycans that are often difficult to characterize by LC MS. It is also well suited for volatile molecules and poorly ionizing molecules that are often lost or not detected by LC MS.

LC MS, on the other hand, is better suited for thermally labile compounds and is a technique of choice for analysis of novel compounds. Typically, these approaches are suitable for identification of several hundreds of metabolites based on spectral databases and authentic standards . However, they are currently far from comprehensive, and improving metabolite identification is an important goal of metabolomics research. A number of studies have used MS based metabolomics to examine the chemical exchanges within phytobiomes; for example, the signaling molecules that direct the establishment of bacterial and mycorrhizal pathogens or symbionts with host plants. A number of metabolites have been identified, including sugars, amino acids, organic acids, phenolic compounds, and plant hormones, that are associated with beneficial interactions and are also implicated by single strain and whole community approaches . Exometabolite profiling methods have been used to examine root exudates and their function in recruiting soil bacteria . O’Banion et al. have reviewed the function of the main chemical constituents of plant microbe signaling. Similarly, chemical imaging of solutes in soils has been reviewed . Although MS imaging is a powerful and promising technique , it is extremely difficult to identify organic components from complex environmental samples due to chemical complexity of these samples and the lack of physical separation of compounds prior to ionization. New developments in using ion mobility to separate ions within mass spectrometers have tremendous potential to overcome these limitations and enable direct analysis of metabolites from tissues and environmental samples .It is well known that phytobiomes are affected by plant growth form and life history , plant community composition and habitat of origin, and even host plant species . In fact, there is growing evidence of that intraspecific variability of plant hosts produces variability in phytobiomes . Genetic differences within host species can affect microbe recruitment, community assembly, and, ultimately, the composition of phytobiomes.

As such, the phytobiome can be considered an extended phenotype of the plant that is determined by host genetics, the environment, and their complex interaction. Here, the standard tools of quantitative genetics can be used to study the phytobiome. For example, family experimental designs or kinship based mixed models can be used to partition variation in microbial abundance or composition into genetic and environmental components of variance for an entire assemblage of microbes associated with a particular plant compartment. This approach can provide insight into the host genetic architecture of the plant microbiome and, potentially, help to identify classes of microbes with close affinities for specific genotypes within a population. A number of recent publications have documented genetic variation within plant species for aspects of the microbiome, including providing estimates of heritability for overall microbial community diversity and richness and for the abundance of specific microbial taxa based on counts derived from amplicon sequencing,growing strawberries vertical system for example The majority of such studies have focused on crop plants in agronomic settings and little is known about the heritability of microbes from more natural populations; one exception to this is the outdoor study of Bergelson et al. . We imagine that some of these host genetic effects are related to available habitat for microbial establishment , to resources shared with microbes as root exudates, or from more complex immune responses in the plant. Incorporating host genetics in plant microbiome studies is promising because it will point to mechanisms leading to beneficial or deleterious plant–microbe interactions, as well as leverage the growing resources available in plant genomics. In order to more efficiently develop and deploy improved plant varieties, it is valuable to identify the causal genes or genetic markers underlying agronomic traits and disease resistance . In addition, there is a need to understand the plant genes that influence the composition and function of the microbiome to improve our understanding and in order to maximize plant productivity. Two methods are commonly used to identify genes or markers associated with quantitative traits: quantitative trait locus mapping and genome wide association studies . Both approaches rely on genome wide scans for statistical association between polymorphic genetic markers and quantitative variation in a measured phenotype. In the case of phytobiomes, the phenotype of interest could be a feature of the aggregate microbial community or an estimate of the relative abundance of a specific taxon . A key distinction between these methods is that QTL mapping populations are derived from lines crosses and, therefore, represent experimentally structured populations, whereas GWAS focus on naturally occurring individuals. QTL mapping tends to have more power to detect true associations but reduced ability to localize effects in the genome because of limited recombination in a breeding population. In contrast, GWAS are frequently under powered, given limited sample sizes, but can yield remarkably fine scaled localization due to extensive historical recombination.

It can also be much faster to establish a GWAS population than a QTL population because there is no need to create recombinant progeny through complex breeding designs across multiple generations. However, GWAS requires dense markers and reliable controls for population structure and, at best, yields correlative results rather than causal inference as achieved with QTLs. Because, in QTL studies, fewer alleles and markers are analyzed using a randomized genetic background, statistical analysis can yield causal relationships between alleles and traits . Although both GWAS and QTL analyses establishing relationships between plant genetics and phenotypic traits are common, links between plant genetics and microbiome composition and function have been rare. The earliest studies utilizing this approach focused on plant related microbial diseases , including fungal, oomycete, and bacterial pathogens. More recently, studies utilizing the model plant Arabidopsis thaliana have been published that explore complete microbial communities based on 16S rRNA gene amplicon sequencing. For example, Horton et al. identified host loci that influence fungal and bacterial colonization density on leaves across an A. thaliana population in the field and found that loci encoding defense and cell wall integrity affect bacterial and fungal community variation, whereas loci that influence the reproduction of viruses, trichome branching, and morphogenesis affect bacterial species richness. Similarly, Wallace et al. looked at the leaf microbial communities across maize lines and found that functions related to short chain carbon metabolism, secretion, and nitrotoluene degradation primarily encoded by Methylobacteria spp. are heritable metabolic traits, and that few plant loci were found to be significantly associated. These studies provide an exciting glimpse of the potential importance of host genetic variation in the phytobiome and give a clear path to the identification of candidate genes. Future studies will help to define the groups of microbes with strong host impacts, as well as identify new genetic and metabolic pathways important in plant– microbe interactions. Although aggregate community metrics may be relatively straightforward to generate, they may be difficult to interpret and less meaningful than studies focused on individual microbial species. However, it is also unclear how to best define microbial taxa for counting—what inference can be made from amplicon sequence variants, traditionally defined operational taxonomic units, or gene content abundance derived from enrichment or metagenomic analyses? Finally, genome wide studies carry a heavy multiple testing burden due to dense testing both across genomes and also across multiple taxa or phenotypes. Care will need to be taken to limit false positives and misleading inferences—methods developed for other “omics” based quantitative genetic systems such as expression or metabolic QTL analyses may provide helpful directions as the field matures.In an effort to conduct plant microbiome research across biologically meaningful spatiotemporal scales and with increased control, a range of fabricated ecosystems are being developed. Experimental control and complexity are inversely related in plant microbiome research. At the most extreme, controlled laboratory experiments are often binary , whereas field experiments feature real world complexity that is difficult to replicate year by year. A new generation of experimental platforms of increasing complexity now allows for multi factorial insight, reproducibility, and increased statistical power. The concept of controlled environments for exploring plant ecophysiology dates back to the late 1940s, when Firits Went developed a Phytotron at Caltech , a “Climatron” in St. Louis, MO , and an ecophysiology lab at the Desert Research Institute, University of Nevada, Reno, which is now home to the recently developed EcoCELLs . Went’s work inspired the development of the EcoTron program at Centre National de la Recherche Scientifique, Montpellier, France , and the EcoTron at Imperial College London, United Kingdom . EcoTrons are large, fabricated ecosystems that consist of an above ground dome of approximately 40 m3 and a below ground chamber that contains a lysimeter that can hold 2 to 12 tons of soil . The canopy area is up to 2 m tall and allows work under natural light as well as under controlled or artificial light conditions. Both above and below ground compartments are equipped with arrays of sensors and instrumentation for environmental control. Using the EcoTron, simulations of a wide range of environmental scenarios under realistic conditions can be performed, while measurements important for ecosystem processes such as atmospheric and soil gas composition, temperature, and pH, among others, can be conducted.

Sugars constitute a significant fraction of exudates, and are a main carbon source for microbes. Interestingly, many more sugar uptake than release systems have been described. Sugar Transport Proteins utilize high extracellular proton levels to import sugars, and mutation of STPs leads to higher external sugar levels. Sugars Will Eventually Be Exported Transporters are sugar uniporters, and all root expressed members localize to the vacuole. Due to an alteration of root sugar homeostasis, SWEET mutant plants exhibited higher sugar export from roots compared with wild type plants, and were more susceptible to disease. Intriguingly, no transporters directly exporting sugars into the rhizosphere have been characterized so far, and it is debated whether sugar exudation is a transport driven process at all. Potential evidence for passive sugar efflux was supported by the observation of higher sucrose concentrations around young, permeable root tissue than around older, less permeable root tissue. However, because sugars are synthesized in leaves, they still need to be unloaded either from phloem or from root cells to be exuded into the rhizosphere, a process likely depending on transporters due to the hydrophilic nature of sugars. A further indication of the presence of elusive transporters is the differential sugar exudation in various environments, as shown, for example, for maize grown in potassium , phosphate , or iron deficient conditions.Sugar alcohols are imported by secondary active proteins with broad substrate specificity , whereas the modes of export are enigmatic. Sugar phosphates are involved in intracellular carbohydrate metabolism,hydroponic vertical farming and plastid localized sugar–phosphate co transporters have been reported in several species.

Although sugar phosphates are detected in exudates, neither import nor export mechanisms are currently characterized.Amino acids are recognized by microbial chemoreceptors crucial for the early steps of root colonization, making amino acids an important fraction of exudates. Modulation of amino acid transport could be either a means of communication with microbes, or a response to microbial presence. Amino acid uptake is mediated by several transporter families with broad substrate specificity. Amino acid exudation is affected by several transporters expressed in vascular tissue: mutation of phloem localized UmamiTs resulted in lower amino acid exudation, whereas mutation of xylem localized Glutamine Dumpers caused increased exudation. Although no plasma membrane localized amino acid exporters have been characterized so far, several lines of evidence suggest their presence. First, higher tryptophan exudation from older root zones than younger parts suggests the involvement of transport proteins in exudation, due to the fully formed Casparian strips and thick cell walls in mature root parts interfering with diffusion. Second, concentration differences between amino acids in root exudates and root extracts are not the same for all the amino acids, suggesting the selective transport of at least some amino acids. Third, various transporter families exhibit bidirectional amino acid transport characteristics in heterologous systems , and could be involved in amino acid exudation.Organic acids constitute a large fraction of exudates, and are microbial nutrients. No importers have been characterized so far, but the release of malate and citrate by Aluminium Activated Malate Transporters and Multi drug and Toxic Compound Extrusion families are among the few well understood examples of transporters involved in exudation .

Activity of members of both families is often modulated by metal ions and microbes . Uncharacterized ALMT and MATE family members are primary candidates for exporters of other organic acids due to their similarity to already characterized members, their plasma membrane localization, and their function as proton antiporters.Nucleotides are imported by secondary active transporters, but their exudation remains elusive. It is well established that extracellular ATP has a signaling function, and ABC transporters were proposed to mediate cellular export. Peptide uptake is transporter mediated in heterologous systems, and a role of ABC transporters in peptide exudation has been suggested .Fatty acid transport is necessary for mycorrhizal symbiosis: mycorrhizal fungi depended on their hosts for the synthesis of certain fatty acids, and the current model includes transport of lipids by ABCG proteins in the symbiotic membrane. One ABCG member, STR, was previously shown to be required for mycorrhization. Interestingly, arabidopsis ABCG transporters were similarly shown to export fatty acids for cutin synthesis in above ground tissues . Lipid transport was required not only for symbiotic interactions, but also for pathogen colonization. Fatty acids are detected in root exudates , but the mode of lipid exudation into the rhizosphere has yet to be discovered. A role in lipid exudation could be envisioned for root expressed ABCG members .Secondary metabolites are ubiquitous in root exudates, and ABC transporters are likely candidates for specialized metabolite transport into the rhizosphere. A distinct exudation profile was described for seven ABC mutants, and one mutant line displayed an altered microbial community. Although the causal metabolites could not be identified, the authors noted transport of the same compound by various proteins, and possible broad substrate specificity for some transporters. In a later study, exudates of arabidopsis ABCG37/PDR9 mutant lines were found to be deficient in several phenylpropanoids.

Arabidopsis PDR9 was previously characterized as auxin precursor transporter, which suggests a broad substrate specificity for PDR9. Interestingly, a PDR9 homolog was highly expressed in cluster roots of white lupin devoid of phosphate, illustrating PDR9 involvement in response to various abiotic stresses. These studies illustrate the potential for the discovery of novel transporter functions in the ABC family, an excellent target for future studies investigating root exudation. In addition, MATE proteins transport secondary metabolites into the vacuole,vertical hydroponic garden and plasma membrane localized members could also be involved in secondary metabolite exudation. In summary, more transport proteins involved in metabolite import into roots than in export from roots have been reported so far . The characterization of additional transport families involved in exudation will enable the generation of mutant lines that are devoid of the exudation of specific metabolites. Such lines could be used to investigate the correlation of exudation profiles and microbial communities.Plant derived transporters and exometabolites are intrinsic to plant–mycorrhizal and rhizobial symbioses . We speculate that, although there is paucity of evidence, plants analogously select for a beneficial rhizobiome. Given that plants evolved in the presence of microbes, a subset of which benefits plant growth, we hypothesize that, over millennia, plant exudation via active transport processes evolved with the substrate specificity of plant associated bacteria. In Box 2, we discuss exudates and other steps involved in root microbiome assembly, analogously to the establishment of plant–mycorrhizal and rhizobial symbioses. However, intense future research is needed to reveal the precise mechanisms governing plant microbiome assembly, and the possible beneficial functions of the microbial community. The major mechanisms by which plants are thought to modulate microbial interactions currently include: modulation of their exudate profiles ; root morphology ; and regulation of immune system activities . In turn, mechanisms for successful rhizosphere colonization by soil microbes require that they: are metabolically active ; sense the plant ; move towards the root and successfully compete with other microbes for root niches . In addition, for successful colonization of the rhizoplane or root tissue, microbes must be able to attach to the surface or enter root tissue . Despite apparent parallels between plant microbiomes and the aforementioned symbioses, plant microbiomes have some specific characteristics. First, microbiomes are detected in all environmental conditions, whereas mycorrhizal and rhizobial symbioses are induced in specific circumstances. Second, microbiomes occur on various tissues, whereas rhizobia and mycorrhiza interface with roots only. Third, microbiomes comprise many members, whereas the aforementioned symbioses persist between two predominant partners. Fourth, although most members of the microbiome originate from the environment similar to rhizobia and mycorrhiza, there is evidence that some endophytes may be vertically transmitted via seeds.

Future research should focus on the factors involved in microbiome assembly, the relative contribution of epi and endophytes to microbiomes, and the signaling crosstalk between plants and microbial communities.Rhizobiome assembly and the involvement of the plant in this process are currently enigmatic. Here, we have discussed multiple factors shaping the rhizobiome, including host genotype and development, root morphology, border cells and mucilage, and root exudates. Root exudation is a dynamic process, likely dependent on a plethora or transporters that are mostly uncharacterized. Spatially defined exudation likely results in distinct microbial communities that are observed to be associated with specific root parts. The success of microbial colonization of the rhizosphere depends on several aspects, such as chemotaxis, substrate specificity, competitiveness, and cooperativeness. Furthermore, endophytes likely form biofilms on the root surface, and encounter the plant immune system. Although some factors shaping root microbiomes emerge, many open questions remain .Due to the chemical complexity of soil, exudation is traditionally analyzed in hydroponic culture, an environment distant from the more natural settings of plant microbiome studies. Furthermore, novel technologies enabling high throughput screening of putative transporters against possible substrates are needed to reveal the impact of the respective substrates on the rhizobiome and, in turn, on plant health. An increased understanding of root morphology, exudation, and involved transporters will likely enable the engineering or breeding of plants with altered abilities to interact with specific beneficial microbes or pathogens. This needs to be complemented with an improved understanding of the substrate preferences of plant associated microbes, their interactions, and the mechanisms through which they benefit the plant. A holistic understanding of the functions of a healthy plant rhizobiome would enable the directed design of customized microbial communities. With this, specific plants in a given environment could be tailored to a specific purpose, such as phytoremediation, stress resistance, altered plant development, or increased yield.Interactions between plants and microbes are an integral part of our terrestrial ecosystem. There are several types of plant microbe interactions: competition, commensalism, mutualism, and parasitism. The more common interactions are commensalism or mutualism, where either one or both species benefit from the relationship, respectively . There are several excellent reviews reporting current research on lifestyles and molecular interactions of plant associated bacteria , rhizosphere interactions , plant responses to bacterial quorum sensing signals , endophyte applications , and rhizosphere bacteria responses to transgenic plants . Examination of these interactions helps us to understand natural phenomena that affect our daily lives and could lead to applications resulting in sustainable resources, less impact on the environment, cleanup of pollution and influence on atmospheric gases on a global scale. Advantages of using these interactions for biotechnological applications are many fold. The use of naturally existing plant microbe symbiosis for plant growth and bio control reduces synthetic fertilizer and pesticide treatments leading to cost effectiveness and less impact by nutrients and pesticides on surrounding fauna and flora. The production of useful compounds with pharmaceutical and industrial relevance using plant bacteria symbiosis is energy efficient and diminishes the need to add expensive precursors and catalysts. Remediation through conventional method, such as excavate and treat, is expensive and labor intensive. Conversely, plantmicrobial remediation strategies can be less intrusive and much more economical .Carbon sequestration through plant rhizosphere processes is a potentially sustainable method to lowering atmospheric carbon . This review focuses on recent progress in the fields of plant growth promotion, plant disease control, production of bio active compounds and bio materials, remediation of contaminated sites, and carbon sequestration. The potential of applying these new developments are discussed. Figure 1 summarizes applications resulting from microbe shoot and microbe root interactions and techniques used. Table 1 is a glossary of the techniques mentioned in this review. Plant microbe interactions have been utilized to improve plant growth for the production of food, fiber, bio fuels and key metabolites. The mutualistic interaction can be beneficial in directly providing nutrients to the plant or increasing the availability of compounds such as iron or phosphate. Free living plant growth promoting bacteria also produce compounds that directly affect plant metabolism or modulate phytohormone production or degradation. The phytohormones: auxins, cytokinins, gibberellic acid , abscisic acid and ethylene are signaling molecules essential for growth which mediate a range of developmental processes in plants. Recent studies on each of these areas are presented in the following section. As chemical fertilizers are costly both to the agricultural businesses and the environment, development of biofertilizers is an important and exciting area.

This strategy considers several domains represented by specific tools ranging from relatively simple in the lower tiers to more complex and specific in the higher tiers. The framework aim is to structure information collection and generation for cost efficient risk assessment, compliant with 3R animal use testing principles , which should also be pursued by means of grouping ENMs. A strategy for grouping ENMs based on releases, uses, physicochemical properties, bio accumulation, bio availability, and effects for both human and ecological risk assessment is currently in development across a number of EU research projects such as MARINA, NANoREG, SUN, and GUIDEnano. These efforts have been challenged by the complexity of ENM identity and interactions, but this approach is necessary, as the costs for safety assessment on a case by case basis would be exorbitant.Therefore, a vision on ENM grouping is needed, which should apply in a regulatory context.Applying grouping in regulatory risk assessments should enable read across, that is, filling a data gap by using information on one ENM, or a nonENM, for another substance in the same group.This strategy should be flexible enough to address different assessment goals depending on the user’s needs, considering all data already available as a starting point, contingent upon data quality evaluation and selecting the most appropriate tools to fill existing data gaps. Such a strategy should ideally be exposure driven, starting with identifying the most relevant exposure scenarios in the ENM life cycle, and evaluating completeness and quality of the available data from a risk assessment perspective. This facilitates careful prioritization of ENMs to optimize testing efforts and can inform more realistic ecotoxicological investigations.

Doing so can allow one to screen out irrelevant exposure routes, eliminate unnecessary testing,danish trolley and support prioritization of exposure scenarios. Exposure assessment should begin with an analysis of plausible exposure scenarios; where none is expected, further testing may be precluded for the applicable use patterns and volumes.Researchers and regulators need to understand actual exposures at biological receptors. This exposure driven approach can also provide important information on realistic environmental conditions to affect test designs for improved interpretation of laboratory toxicology studies. Such practices can ensue in the interim, while research continues to discover best hazard assessment practices. Experimental ENM toxicity assessments, using ecologically relevant receptors and across linked biological levels of organization, should inform developing and parametrizing dynamic process based models. Such models should respond to future scenarios and predict impacts. ENM characteristics, exposure conditions, and ENM transformation, dose, and body burden should be used in interpreting biological and computational findings for assessing ENM risks. ENM test results should be bench marked to results for appropriate controls to establish relative hazard . This applies to pinnacle concerns in ecological fate assessment of bio accumulation, bio magnification, and bio persistence.How to develop, interpret, and use pertinent information in ENM environmental risk assessment is a larger issue that should become part of an extended dialogue among regulators, industry, civil society organizations, researchers, and other societal members so that the fundamental research will inform decision making. Collaborative decisions are recommended for focusing ENM ecotoxicology toward relevant scenarios, including testing the most relevant materials throughout ENM life cycles and employing appropriate hazard assessment approaches, toward meaningful ecological risk assessment.

The overarching question motivating this critical review was: how can we ensure that hazard assessment in ENM ecotoxicology is as environmentally relevant as possible? The answer requires considering how ecotoxicity tests are performed, what constitutes pertinent concentration and test conditions for ENMs , the main biotic and abiotic attributes of the environment, how ecologically oriented hazard assessment is undertaken , and how the resulting information should be interpreted. Answering this question yielded three primary insights. First, environmental relevance is informed by a logical consideration of what exposures might occur, to which receptors, and to what outcomes. The consideration should begin with a plausible release and exposure scenario , and use best available knowledge and technologies to develop the full assessment approach. Concerns regarding ENM concentrations used in hazard assessments are paramount, but are not the only concerns. ENM concentrations should be selected to assess potential effects, but overly high concentrations that fundamentally change media conditions should be avoided. Still, concentrations ranging above and below predicted ENM average concentrations must be assessed for understanding potential organismal effects, underlying mechanisms and their concentration dependencies, and for informing process based dynamic biological effects models. In addition to the nanomaterial, the conventional material should be tested. ENM distributions and fates in broad environmental compartments do not equate to concentrations and forms near, or effective at, actual biological receptors.Therefore, research results on ENM effects should not be disregarded on the limited basis of environmentally relevant exposure concentrations when the study conditions were predicated on a broader hypothesis.

In addition to tethering ENM ecotoxicology to exposure initiation scenarios , the concept of employing tiered approaches in hazard and risk assessment resonated . Multistage approaches to ENM hazard assessment are advocated.A highly developed tiered approach for health and safety testing of nanotechnologies has been published and strategies for tiered risk assessment and grouping are underway.Staging ENM ecotoxicology efforts, such that potential interactive impacts at all levels of biological organization are evaluated, could simultaneously inform risk assessment and predictive process based effects model development. As some ENMs can cause biological impacts from ENM properties or characteristics,ENM ecotoxicology should be oriented to logical exposure initiation scenarios based on ENM life cycles, via testing tiers . Finally,vertical aeroponic tower garden coordination is recommended among multiple disciplines in ENM environmental analysis, fate and transport modeling, and hazard assessment, toward rapidly advancing research using tiered approaches around realistic exposure scenarios.Scholars have published extensively on the multifunctional benefits of urban agriculture including: promoting urban sustainability, reducing air and water pollution, building social cohesion, promoting community health and nutrition, teaching food literacy, and creating radical economic spaces for resistance to the capitalist political economy and structural inequities embedded in the “neoliberal city” . Despite growing evidence of these diverse health, education, and environmental benefits of urban agriculture, these vibrant spaces of civic engagement remain undervalued by city policy makers and planners in the United States. Thriving urban farms and gardens are under constant threat of conversion to housing or other competing, higher value land uses due to rising land values, and other city priorities. This land use challenge and threat to urban farm land tenure is especially characteristic of U.S. cities like San Francisco, one of the most expensive land and housing markets in the country. Under the current urban agriculture paradigm in the U.S., food justice scholars and advocates either try to quantify and highlight the multiple benefits of UA or pursue a critical theoretical approach, arguing that urban agriculture can yield unfavorable results if pursued without an equity lens, especially in cities with intense development pressures and gentrification concerns . A productivist focus is problematic, because, while urban agriculture can be an important component of community food security, its other social and ecological benefits are just as, and sometimes more, significant . In this article, we suggest that the current debates around “urban agriculture” in the U.S. often lead to an unhelpful comparison with rural farms regarding yield, productivity, economic viability, and ability to feed urban populations, most notably in the policy arena.

Defined in these ways, the radical, transformative potential of urban food production spaces and their preservation often gets lost or pushed to the side in city planning decisions in metropolitan regions such as the San Francisco Bay Area, where the threat of displacement is ubiquitous given high levels of economic inequality and extreme lack of affordable land. In order to facilitate what scholars such as Anderson et al. 2018a refers to as the “agroecological transition,” already underway in many urban food ecosystems around the globe , we argue that applying an agroecological approach to inquiry and research into the diversity of sites, goals, and ways in which food is produced in cities can help enumerate the synergistic effects of urban food producers. This in turn encourages the realization of the transformative potential of urban farming, and an articulation of its value meriting protected space in urban regions. Urban agroecology is an evolving concept that includes the social ecological and political dimensions as well as the science of ecologically sustainable food production . UAE provides a more holistic framework than urban agriculture to assess how well urban food initiatives produce food and promote environmental literacy, community engagement, and ecosystem services. This paper presents a case study of 35 urban farms in San Francisco’s East Bay in which we investigated key questions related to mission, production , labor, financing, land tenure, and educational programming. Our results reveal a rich and diverse East Bay agroecosystem engaged in varying capacities to fundamentally transform the use of urban space and the regional food system by engaging the public in efforts to stabilize, improve, and sustainably scale urban food production and distribution. Yet, as in other cities across the country, urban farms face numerous threats to their existence, including land tenure, labor costs, development pressure, and other factors that threaten wider adoption of agroecological principles. We begin by comparing the concepts of UA and UAE in scholarship and practice, bringing in relevant literature and intellectual histories of each term and clarifying how we apply the term “agroecology” to our analysis. We pay particular attention to the important nonecological factors that the literature has identified as vital to agroecology, but seldomly documents . We then present findings from a survey of 35 diverse urban farm operations in the East Bay. We discuss the results, showing how an agroecological method of inquiry amplifies important aspects of urban food production spaces and identifies gaps in national urban agriculture policy circles. We conclude by positing unique characteristics of urban agroecology in need of further studies and action to create equitable, resilient and protected urban food systems.Agricultural policy in the United States is primarily concerned with yield, markets, monetary exchange, and rural development. The United States Department of Agriculture defines agricultural activities as those taking place on farms. Farms are defined as “any place from which $1,000 or more of agricultural products were produced and sold, or normally would have been sold, during the year” . Urban agriculture has been proliferating across the country in the last decade on both public and private lands, as both for profit and nonprofit entities, with diverse goals, missions and practices largely centered on food justice priorities and re localizing the food system. Yet U.S. agriculture policy has been struggling to keep up. In 2016, the USDA published an Urban Agriculture Toolkit, which aims to provide aspiring farmers with the resources to start an urban farm including an overview of the startup costs, strategies for accessing land and capital, assessing soil quality and water availability, production and marketing, and safety and security . The 2018 U.S. Farm Bill provides a definition of urban agriculture to include the practices of aquaponics, hydroponics, vertical farming, and other indoor or controlled environment agriculture systems primarily geared towards commercial sales. In both the Toolkit and Farm Bill, non profit, subsistence, and educational urban farming enterprises are not well integrated or included in the conceptualization of UA. While there are many definitions of urban agriculture in the literature from the simplest definition of “producing food in cities” to longer descriptions of UA such as that of the American Planning Association that incorporate school, rooftop and community gardens “with a purpose extending beyond home consumption and education,” the focus of many UA definitions used in policy arenas continues to center around the production and sale of urban produced foods. Accordingly, food systems scholars have recognized that “Urban agriculture, [as defined], is like agriculture in general”, devoid of the many political, educational, and food justice dimensions that are prioritized by many U.S. urban farming efforts. Thus the social political nature of farming, food production, and food sovereignty are not invoked by formal UA policy in the U.S. Many goals and activities common in urban food production, including education, nonmonetary forms of exchange, and gardening for subsistence are obscured by the productivist definitions and can be thus neglected in policy discussions.

The people I spoke with were not ignorant or easily molded, and were justifiably proud of all the learning and hard intellectual work they had put in to becoming eligible olim. As I discussed in the first chapter, however, the materials used in Iquitos’ conversion and migration-preparation courses come from a very limited number of sources, including the Jewish Agency for Israel and a number of self-avowedly Zionist rabbis from Argentina and New York. It does not seem outside the realm of possibility that information about daily life for Jews in Israel, about Palestinians generally, and about Jewish-Palestinian relations specifically, is heavily biased against Palestinians. Much as I found that the educational materials available in Iquitos strongly encouraged adopting love for, belief in, and loyalty to the modern state of Israel as an integral part of being a Jew, these same resources, which contain implicit information about racial hierarchies in Israel, emphasize Jewish-Palestinian strife and the importance of believing in Jewish difference as a core part of Jewish identity. Iquiteño Jews are necessarily being purposefully manipulated by the Israeli government to form Jewish-supremacist ideas before entering Israel. However, many parts of the conversion and migration process encourage a kind of racialized nationalism. In particular, the community focus on the modern state of Israel as central to a contemporary Jewish identity and the need to perform dedication to Israel and a particular form of Jewishness in order to be permitted conversion encourage this. These small nationalisms compile, and the result is that people learn to discriminate without it being a necessity planned by any individual or agency.

Given my observations,flower pots for sale the entire process of Jewish education, conversion, and migration, with all its particularities, contingencies, and administrative requirements, seems to encourage anti-Arabracism as a side-effect. And, as that side-effect is beneficial to the state, it seems evident that it is at the least allowed to continue, even if it was not a premeditated plan. It is important to note that a perspective that puts the burden of creating this internalized hierarchy ignores Peruvian influences. I also suggest that, as most Iquiteño Jews belong to a relatively privileged ethnic group within Peru, most are not accustomed to thinking of themselves as racially marginalized. Rather, the Peruvian field of racial positions in which Iquiteño Jews locate themselves position Afro-Peruvians and indigenous Peruvians as the other points of the triangle, against whom the mestizo Iquiteños define themselves as relatively valorized. Although nobody I interviewed specifically compared Palestinians to any other groups, I noted similarities in the ways in which some Iquiteños referred to their indigenous neighbors. They used racist epithets like “indio” in the way that my young interviewee referred to Palestinians as rats. One might assume, therefore, that Iquiteño Jews find it easy to imagine themselves fitting into a similarly elevated place in Israel, and difficult to imagine changes to their current position of privilege. Historically, futhermore, due to Peru’s large indigenous population, racial censuses and other state documents struggled to clearly demarcate “indigenous” and “not-indigenous” categories based on descent alone; rather, the assumption of European cultural markers became key to classification 24.

Thus Iquiteño Jews might also draw on Peruvian historical dynamics around cultural assimilation as a means of racial advancement as a way of reading their options in an Israeli context. In combining both perspectives, then, a proposed method of transmission for knowledge of the Jewish-Israeli/Palestinian divide begins to appear. The other half of the equation is still missing, however. It is intriguing to consider that, at least in a relatively isolated place such as Iquitos, Israeli racial hierarchies reveal their fundamental dichotomy but not the complexity of intra-Jewish triangulation. Latin American Jews who migrate to Israel might expect to enter the country in a position of privilege, only to find that the truth is more complicated. Thus, it seems apparent that processes that train diasporic Jews to become potential Israeli citizens end up educating these future olim on their likely place within an Israeli field of racial positions — to an extent. Regardless of whether most Iquiteño Jews who wish to migrate to Israel expect to fit into a given field of racial positions in which they gain one enormous privilege, of Jewishness, but might yet face a very difficult battle against many forms of racialized prejudice, and whether their education on these matters is intentional or not, they are involved in an explicit attempt by the Israeli state to maintain and deepen that fundamental Jewish/Palestinian divide. To describe this instrumentalization of Latin American, specifically Peruvian, Jewish migrants, I will use Nadera Shalhoub-Kevorkian’s concept of demographic warfare. Shalhoub-Kevorkian names the strategies the Israeli government uses to surveil and control Palestinian lives via restrictions on settlement, movement, and living spaces “demographic warfare.”

She highlights the ways in which population control is central to Zionist Israeli political projects. Preventing family reunification, return to homes and land owned before the Nakba,tower garden and zoning laws are only some of the ways in which Palestinians are demographically controlled. The goal of this control is to eliminate Palestinians, or at the very least make sure they are outnumbered by an order of magnitude by Jews. I argue that the aggressive advertising and subsidizing of aliyah on part of the Israeli state also represents an act of demographic warfare. Shalhoub-Kevorkian only passingly refers to the Citizenship and Entry into Israel Law , which governs immigration, but controls on immigration directly shape the makeup of a country’s population. Although Iquiteño Jews are construed as racially undesirable, unideal Jews, they are nonetheless Jews. As such, they receive support and encouragement from the Israeli state as they journey towards becoming acceptable Israeli citizens. Thanks to the global reach of Jewish educators and educational material that position Israel as central to Jewish identity, far-flung diaspora communities can both be shaped and shape themselves into better-conforming citizens. After all, an ideal Jew is not needed to be superior to a Palestinian. It is no coincidence that so many Iquiteños have ended up in Ramla: in a city with a large Muslim minority, even undesirable Jews can serve the state’s demographic purpose. How does the Israeli citizenship and immigration regime navigate this complex set of desires? The key is in its power to enforce a specific set of standards for deciding who is a Jew, and therefore who is eligible to come to Israel as a potential Jewish citizen. The split between levels of citizenship and the impossibility of creating a neat and widely acceptable definition of Jewishness reveals how weak and divided the Israeli political consensus is on issues of immigration, race, and Jewish identity. The basis for this incorporation regime is the Law of Return of 1950. Together with the Citizenship Law of 1942, these two pieces of legislation form the basis of Israel as a state; they are arguably more important than any other law in Israel, including the constitutional Basic Laws. Put together, they define Israeli citizenship, and thus who is granted free movement into Israel, in a seemingly very simple way: if a person is a Jew, then they are a citizen. If they are a citizen, wherever they may be in the world, wherever they were born, they may enter Israel to live legally and with full government support. It is, of course, not so simple. Citizenship is always subject to citizenship discourses, or the different “schools of thought” that govern what access to rights citizenship grants to its holders, how people think about citizenship, and who gets to be a citizen. In Israel, these citizenship discourses present a tug-of-war between ethno-nationalist and ultra-religious priorities.

While some scholars add the demands of a liberal democratic regime , this seems like an increasingly over-optimistic characterization as Israel entrenches itself increasingly firmly in the category of illiberal democracy in which the appearance of being reasonably democratic excuses highly undemocratic social and civil rights violations. Iquiteño Jews face two particular and linked difficulties in integrating into this discourse: navigating Israel’s tiered citizenship regime and being recognized as appropriately and fully Jewish.Since the time of the second incorporation regime at the state’s founding, Israel has effectively created tiers of dejure citizenship. On the highest tier are those recognized to be Jews by the state, as well as some minority groups such as Armenian Christians, with a fairly full complement of social, civil and political rights. On the next tier down are those reliant on the increasingly frail liberal framework of Israel’s non-ethnic citizenship. The largest proportion of this group is “1948” Palestinians, those who remained within Israel’s armistice boundaries, who are technically citizens but enjoy many fewer rights, which have historically been suspended at the state’s whim. However, Jewish Israelis also fall into this category if their Judaism is somehow in question. For example, Israeli-born Jewish people who are not strictly halakhically Jewish are denied the civil right of marriage despite being citizens , and the very large demographic of ex-Soviet Jews whose halakhic bona fides are insufficient to merit full inclusion is a perennial source of anxiety to the state. Shifting between these tiers is the non-citizen legal resident, who enjoys a scattershot basket of rights and yet has more security and standing than undocumented or non-citizen Israelis. The situation an Iquiteño Jew wishes to avoid is that of becoming an ambiguous Jew, prevented from accessing full rights despite their citizenship. The fact of this strange bifurcation highlights a cleft between secular nationalists and ethnocentric religious communities apparent in the Law of Return itself: that of the perennially impossible problem of deciding who is a Jew. This is a loaded question regardless of who is asking it. Identity and belonging are fraught with emotional, historical, and familial weight that becomes all the heavier and more complex when an identity is, like Jewishness, a shifting blend of culture, religion, and ethnicity. It becomes truly urgent when the allocation of basic rights depends on fitting this deeply personal and subjective identification into a bureaucratic box and proving it to the satisfaction of an immigration judge. When this is the case, deciding who is Jewish “is not an autonomous problem waiting to be politically and legally resolved but rather a social language that serves the political purposes of social engineering”. According to Jewish religious law, halakha, a Jewish person is someone with a Jewish mother or someone who has converted through an extended community process under the guidance of a rabbi. These rulings postdate the Biblical period and so derive entirely from rabbinical debate. As may be predictable by this point, this seemingly simple pair of criteria are in fact very complex. What if the Jewish mother is herself a convert? What if a child is adopted? What about those whose fathers are Jewish, or those born of Jewish parents who do not practice, or those born to a Jewish mother who have converted to a different religion? What if the conversion is not Orthodox? And whatever the case is, how do you prove it? The Law of Return attempts to avoid these complications for immigration purposes by making the requirements for entry relatively loose. Today, those with Jewish parents or at least one Jewish grandparent and their spouses and children may enter Israel and achieve some level of citizenship. This more permissive approach fits badly with other stricter state applications of halakha, however. They are Jewish enough to enter Israel, Jewish enough to be subjected to the draft, and Jewish enough, in many cases, to consider their Jewishness an indelible part of their identity. However, they are not Jewish enough to fully access the benefits of Israeli citizenship. This divide is exemplary of the clumsy compromises Israel has had to make between secular nationalists and the ultra-religious, compromises visible in the history of the Law of Return. Before 1970, the Law of Return granted entry to those with Jewish ancestry up to three generations back; the major amendment of the Law of Return in that year to its present, more restricted form was the culmination of two decades of court and legislative fights that progressively tightened the criteria for entry across many potential defining aspects of Jewishness.