The agriculture application activities allow for working within an agricultural space, whether a community or school garden, or another designated space for growing food. Each module can be completed within two hours and includes detailed background information and facilitation tips. While training in learner-centered pedagogy is recommended, and frequently provided for Cooperative Extension educators, these features allow for implementation with minimal experience. To accompany the curriculum, a guide for developing and maintaining an agricultural space was written and integrated into the introduction.Agriculture lessons were designed to feature the food system, including different agricultural systems, inputs, and innovations that have contributed to establishing current practices. Aspects of urban agriculture were also incorporated, which comprises smaller form farming in addition to community and school gardening. Agriculture applications for these lessons entail touring a local farm, or having a local producer visit the agricultural space to share information about their production and exploring inhabitants found in the agricultural space to investigate their impact on the growing environment. Another application involves working in the agricultural space with and without modern day equipment in order to understand how innovations within agriculture have shaped modern procedures. Home application activities include interviewing individuals with roles in the food supply chain, learning more about insects and animals that are involved in agriculture,maceta 40 litros growing produce at home, and mapping out one’s own neighborhood to assess food availability and access.

Nutrition lessons begin with practice categorizing foods into food groups, as defined by MyPlate, and then move to identifying macro-nutrients and micro-nutrients and assessing overlaps with the food groups. The other nutrition lessons include learning how to meal plan with the nutrients of concern for under consumption in adolescents and analyzing nutrition messages in media. Agriculture application activities for nutrition lessons involve planning a snack using items grown in the agricultural space, testing soil quality, establishing a compost pile, and making sustainable pesticides using household products. Home application activities include meal planning utilizing recommendations for MyPlate food group consumption and the nutrients of concern for under consumption. Additionally, home application activities entail assessing Nutrition Facts Labels on products found at home and analyzing a nutrition-related advertisement. The first cooking lesson focuses on food safety, including proper hand washing and setting up a safe work space. Each subsequent lesson and culinary application activity begin with a reminder to wash hands that is followed with verification of practicing food safety throughout the experience. Other cooking lessons entail advancing knife skills and practice utilizing basic cooking techniques and equipment. Meal planning in accordance to shopping in season and within budgetary constraints is featured in cooking lessons as well. Recipes provided within the curriculum are vegetarian to limit food safety concerns regarding temperature and to introduce adolescents to plant-based protein sources.Furthermore, all recipes were intentionally developed with low-cost ingredients, including canned and frozen products, and regularly available food items as to not limit low-income communities from preparing the dishes.

In addition to encouraging youth to make recipes, adapting as needed to meet family and cultural preferences, cooking home applications activities entail examining and avoiding potential food safety hazards at home. Additionally, home applications included meal planning utilizing seasonal produce and scaling recipes to feed their families. Teens CAN aims to improve food literacy, and consequently diet quality, of high school aged adolescents, with the ultimate intent to reduce and prevent obesity. Being food literate requires the skills and knowledge necessary to grow, buy, and cook food while considering health, so that empowered individuals can make the healthier choice when given the option. However, food literacy is a relatively emergent concept with accompanying limitations. While improvements in dietary outcomes have been observed in adolescent interventions aimed at improving attributes of food literacy, long-term implications for diet quality and obesity prevalence are lacking. Additionally, food literacy is a complex construct with multiple interrelated factors, making assessment of food literacy challenging. As such, a comprehensive evaluation tool is yet to be developed for this age group. Nevertheless, the potential of food literacy is worth exploring given that youth aware of food growing practices and regionality of produce are more likely to consume fruits and vegetables. Additionally, studies have shown that youth are more likely to consume healthier diets when they are involved in the food preparation process. This was observed during Teens CAN pilot testing as participants regularly harvested and sampled produce growing in their agricultural space. Furthermore, youth who were apprehensive to taste new foods at the beginning were open to trying included recipes by the end. The approach of Teens CAN includes three topic areas of agriculture, nutrition, and cooking with experiential and application lessons aimed at improving knowledge and skills related to healthy eating. Teens CAN is inexpensive to implement, requiring mostly printed materials provided within the curriculum and common school supplies. Additionally, recipes provided within the cooking lessons feature low-cost produce available year-round as well as shelf-stable items.

The curriculum employs activities and concepts that require critical thinking from participants,mobile vertical grow tables which adolescents are capable of completing. In addition to application activities that incorporate an agricultural space, each module includes home application activities to nurture further learning. These activities additionally provide opportunity for appropriate adaptions, such as those for cultural considerations, to make adoption of new practices more viable. Teens CAN was designed and tested following a similar approach to Discovering Healthy Choices for the Shaping Healthy Choices Program, which has similar theming and includes some food literacy components. The Shaping Healthy Choices Program has shown improvements in nutrition knowledge and weight status among other healthy behaviors. Similar agriculture concepts to that of Teens CAN, such as components of the food system and food security, were included in another curriculum, Sprouting Healthy Kids, designed for middle school students in Texas, that was found to improve participant fruit and vegetable intake. As Teens CAN is aimed at improving food literacy of high school-aged adolescents, findings from focus groups where adolescents ranked aspects of food literacy according to importance strongly influenced lesson concepts. Adolescents ranked food and nutrition knowledge among the most important aspects of food literacy for them to develop healthy eating patterns. Focus groups identified that adolescents did not pay attention to food labels or dietary guidance due to not understanding their application. With this, use of Nutrition Facts Labels and recognizing nutrients of concern, as identified within the Dietary Guidelines for Americans, were focused on in the Teens CAN nutrition lessons. The Dietary Guidelines for Americans recommends consuming healthy eating patterns with adequate intake of essential nutrients through a varied diet that incorporates each food group. Though the curriculum was written to align with the 2015–2020 Dietary Guidelines for Americans, recommendations for adolescents did not substantially change with the newest edition. Food cards within nutrition lessons included foods typically considered unhealthy and high in empty calories since adolescents frequently consume these foods, as well as healthy alternatives to allow for comparison. Additionally, whole fruits, vegetables, and grains were heavily featured to encourage consumption as adolescents are well below meeting recommendations for these foods. With adolescence being a time of increased autonomy, all primary nutrition lessons and application activities were written to support adolescents planning for meeting their own nutritional needs. Furthermore, to tailor lessons to adolescents, all characters presented in lesson activities were high school-aged adolescents. Culinary skills education has been called to be incorporated into nutrition education for application of concepts through hands-on food preparation. While adolescents from the previously mentioned focus groups ranked food preparation skills as of low importance, other findings suggest that limited opportunities for hands-on food skills practice are a hindrance leading to low food literacy as young adults Due to this, primary learning concepts involved enhancement of food skills and opportunities to prepare food.

Adolescents also acknowledged that while budgeting and shopping for food were not immediately important in their current life stage, these concepts would be later in life. With this, budgeting and shopping for food was added as one of the cooking lesson concepts. Cooking programs provide an enjoyable experience that introduces youth to preparing and tasting dishes containing new, frequently healthier, foods. Culinary application activities in Teens CAN feature cultural cuisines, advise consumption of produce grown in the agricultural space, and allow opportunities for participants to have independence in ingredient selection. Participation in cooking programs also motivates youth to continue practicing learned food skills at home, which has been associated with more nutritious eating patterns. Adolescents who participate in food preparation at home are more likely to continue enjoying cooking and preparing healthier dishes as emerging adults. The application activities integrated in Teens CAN may indirectly improve adolescent health as community gardening experience was found to be positively associated with willingness to try fruits and vegetables in low-income high school students from an urban community. Additionally, participating in farm to school related activities has been associated with willingness to try fruits and vegetables in addition to improving nutrition knowledge and self-efficacy. Providing opportunities for involvement in agriculture, even if just through gardening, is important given that childhood, in combination with recent, gardening for first-year college students was found to be associated with higher fruit and vegetable consumption compared to those who have never gardened. Teens CAN lessons introduce adolescents to agriculture concepts and encourages growing food at home through application activities. This could perhaps establish a mechanism for adolescents to continue gardening into later adolescence and adulthood. Teens CAN has since been translated into Spanish. Having the curriculum available in Spanish helps reach more participants as almost 40% of the population in California is of Hispanic descent. This allows for the curriculum to be utilized for development of language education, such as district-level programs that encourage multilingualism. Additionally, the materials allow for adolescents to engage Spanish-speaking family members with the home application materials. Planned implementation of Teens CAN was designed to align with recommendations for older adolescent food literacy programs. For example, Teens CAN may be incorporated into classroom instruction, but was conceived with the intention of being employed within existing after school and youth development programs over twelve weeks. Each of the twelve modules feature experiential learning activities intended to cultivate teamwork, which is important for after school educational programs, and build knowledge, skills, and self-efficacy associated with food literacy. With Teens CAN primarily intended for low income adolescents, facilitating the curriculum within after school programs is particularly important for introducing youth to science-based programming applicable to daily living that they otherwise would not be permitted to access. Another recommendation for food literacy programming is to include peer-modeling. One reason for developing Teens CAN was to create a curriculum to be applied in training teen teachers. A study implementing the Shaping Healthy Choices Program curricula within 4-H found that teen teachers were inadequate at facilitating the curricula with satisfactory program fidelity. It was postulated that teen teachers required additional training, especially in regard to curricula content, before they could be competent facilitators.Following participation in Teen CAN lessons, it is anticipated that adolescents will have improvements in relevant knowledge and skills that will enable them to effectively facilitate food literacy programming with younger youth. Adolescents acting as teachers for younger youth, known as cross-age teaching, is a common practice within 4-H. Cross-age teaching can be beneficial for adolescents as it rein forces learning concepts for themselves in addition to building confidence in teaching. In contrast to tutoring, cross-age teaching involves specific training for the teen teachers who then facilitate lessons from a given curriculum over time to a group of younger youth. In particular, cooking education has been successful in a cross-age teaching model. Cross-age teaching perpetuates observational learning and can thus improve self-efficacy for various skills, including those valuable for food preparation. Additionally, these programs allow opportunities for team building and improve peer relationships while also encouraging implementation of cooking skills for younger youth at home. A long-term nutrition and gardening program utilizing teen teachers for elementary-aged youth provides an excellent example and highlights the scalability of a program of this nature. Applying this model employs adolescents that are culturally competent being from the same community and living within the same contexts as the younger youth they are teaching. Adolescents have been found to be as effective, if not more effective than adult educators.

An additional advantage of this strategy is that exogenous ACE2 would compensate for lower ACE2 levels in the lungs during infection, thereby contributing to the treatment of acute respiratory distress. Several companies in the United States and the EU have developed recombinant ACE2 and ACE2-Fc fusion proteins for preclinical and clinical testing, although all these products are currently produced in mammalian cell lines . The impact of plant-specific complex glycans on the ability of ACE2-Fc to bind the RBD has been studied using molecular dynamic simulations and illustrates the important role that glycosylation may play in the interaction between the S protein and ACE2 .Griffithsin is a lectin that binds high-mannose glycans, and is currently undergoing clinical development as an antiviral against HIV-1. However, it also binds many other viruses that are pathogenic in humans, including HSV , HCV , Nipah virus , Ebola virus, and coronaviruses including SARS-CoV and MERS , and as recently determined, also SARS CoV-2. A clinical product in development by University of Louisville is currently manufactured in N. benthamiana by Kentucky Bioprocessing using a TMV vector. The API is also undergoing preclinical development as a nasal spray for use as a non-vaccine prophylactic against coronaviruses, with clinical evaluation planned for 2020 . This candidate PMP antiviral could be deployed under the EUA pathway if found effective in controlled clinical studies. Griffithsin is an interesting example of a product that is ideally matched to plant-based manufacturing because it is naturally produced by a marine alga. Griffithsin has been expressed with limited success in E. coli and tobacco chloroplasts, but better results have been achieved by transient expression in N. benthamiana using A. tumefaciens infiltration or TMV vectors,hydroponic grow system with expression levels of up to 1 g kg−1 fresh mass and recoveries of up to 90% .

A TEA model of griffithsin manufactured in plants at initial commercial launch volumes for use in HIV microbicides revealed that process was readily scalable and could provide the needed market volumes of the lectin within an acceptable range of costs, even for cost-constrained markets . The manufacturing process was also assessed for environmental, health, and safety impact and found to have a highly favorable environmental output index with negligible risks to health and safety.In addition to COVID-19 PCR tests, which detect the presence of SARS-CoV-2 RNA, there is a critical need for protein-based diagnostic reagents that test for the presence of viral proteins and thus report a current infection, as well as serological testing for SARS-CoV-2 antibodies that would indicate prior exposure, recovery, and possibly protection from subsequent infection.The design and quality of the binding reagents , along with other test conditions such as sample quality, play a key role in establishing the test specificity and selectivity, which determine the proportion of false positive and false negative results. Although the recombinant protein mass needed for diagnostic testing is relatively small , the number of tests needed for the global population is massive, given that many individuals will need multiple and/or frequent tests. For example, 8 billion tests would require a total of ~2.5 kg purified recombinant protein, which is not an insurmountable target. However, although the production of soluble trimeric full-length S protein by transient transfection in HEK293 cells has been improved by process optimization, current titers are only ~5 mg L−1 after 92 h . Given a theoretical recovery of 50% during purification, a fermentation volume of 1,000 m3 would be required to meet the demand for 2.5 kg of this product. Furthermore, to our knowledge, the transient transfection of mammalian cells has only been scaled up to ~0.1 m3 .

The transient expression of such protein-based diagnostic reagents in plants could increase productivity while offering lower costs and more flexibility to meet fluctuating demands or the need for variant products. Furthermore, diagnostic reagents can include purification tags with no safety restrictions, and quality criteria are less stringent compared to an injectable vaccine or therapeutic. Several companies have risen to the challenge of producing such reagents in plants, including Diamante , Leaf Expression Systems , and a collaborative venture between PlantForm, Cape Bio Pharms, Inno-3B, and Microbix.Resilience is the state of preparedness of a system, defining its ability to withstand unexpected, disastrous events , and to preserve critical functionality while responding quickly so that normal functionality can be restored . The concept was popularized by the 2011 Fukushima nuclear accident but received little attention in the pharmaceutical sector until COVID-19. Of the 277 publications retrieved from the National Library of Medicine22 on July 9th 2020 using the search terms “resilience” and “pandemic,” 82 were evenly distributed between 2002 and 2019 and 195 were published between January and July 2020. Resilience can be analyzed by defining up to five stages of a resilient system under stress, namely prevent, prepare, protect, respond, and recover . Here, prevent includes all measures to avoid the problem all together. In the context of COVID-19, this may have involved the banning of bush meat from markets in densely populated areas . The prepare stage summarizes activities that build capacities to protect a system and pre-empt a disruptive event. In a pandemic scenario, this can include stockpiling personal protective equipment but also ensuring the availability of rapid-response biopharmaceutical manufacturing capacity. The protect and respond stages involve measures that limit the loss of system functionality and minimize the time until it starts to recover, respectively. In terms of a disease outbreak, the former can consist of quarantining infected persons, especially in the healthcare sector, to avoid super-spreaders and maintain healthcare system operability . The response measures may include passive strategies such as the adjustment of legislation, including social distancing and public testing regimes, or active steps such as the development of vaccines and therapeutics .

Finally, the recover phase is characterized by regained functionality, for example by reducing the protect and response measures that limit system functionality, such as production lockdown. Ultimately, this can result in an increased overall system functionality at the end of a resilience cycle and before the start of the next “iteration” . For example, a system such as society can be better prepared for a pandemic situation due to increased pharmaceutical production capacity or platforms like plants. From our perspective,planta frambuesa maceta the production of recombinant proteins in plants could support the engineering of increased resilience primarily during the prepare and respond stages and, to a lesser extent, during the prevent and recover stages . During the prepare stage, it is important to build sufficient global production capacity for recombinant proteins to mount a rapid and scalable response to a pandemic. These capacities can then be used during the response stage to produce appropriate quantities of recombinant protein for diagnostic , prophylactic , or therapeutic purposes as discussed above. The speed of the plant system will reduce the time taken to launch the response and recovery stages, and the higher the production capacity, the more system functionality can be maintained. The same capacities can also be used for the large-scale production of vaccines in transgenic plants if the corresponding pathogen has conserved antigens. This would support the prevent stage by ensuring a large portion of the global population can be supplied with safe and low-cost vaccines, for example, to avoid recurrent outbreaks of the disease. Similarly, existing agricultural capacities may be re-directed to pharmaceutical production as recently discussed . There will be indirect benefits during the recover phase because the speed of plant-based production systems will allow the earlier implementation of measures that bring system functionality back to normal, or at least to a “new or next normal.” Therefore, we conclude that plant-based production systems can contribute substantially to the resilience of public healthcare systems in the context of an emergency pandemic.The cost of pharmaceuticals is increasing in the United States at the global rate of inflation, and a large part of the world’s population cannot afford the cost of medicines produced in developed nations23 . Technical advances that reduce the costs of production and help to ensure that medicines remain accessible, especially to developing nations, are, therefore, welcome. Healthcare in the developing world is tied directly to social and political will, or the extent of government engagement in the execution of healthcare agendas and policies . Specifically, community-based bodies are the primary enforcers of government programs and policies to improve the health of the local population . Planning for the expansion of a biopharmaceutical manufacturing program to ensure that sufficient product will be available to satisfy the projected market demand should ideally begin during the early stages of product development. Efficient planning facilitates reductions in the cost and time of the overall development process to shorten the time to market, enabling faster recouping of the R&D investment and subsequent profitability.

In addition to the cost of the API, the final product form , the length and complexity of the clinical program for any given indication , and the course of therapy have a major impact on cost. The cost of a pharmaceutical product, therefore, depends on multiple economic factors that ultimately shape how a product’s sales price is determined . Product-dependent costs and pricing are common to all products regardless of platform. Plant-based systems offer several options in terms of equipment and the scheduling of upstream production and DSP, including their integration and synchronization . Early process analysis is necessary to translate R&D methods into manufacturing processes . The efficiency of this translation has a substantial impact on costs, particularly if processes are frozen during early clinical development and must be changed at a subsequent stage. Process-dependent costs begin with production of the API. The manufacturing costs for PMPs are determined by upstream production and downstream recovery and purification costs. The cost of biopharmaceutical manufacturing depends mostly on protein accumulation levels, the overall process yield, and the production scale. Techno-economic assessment models for the manufacture of biopharmaceuticals are rarely presented in detail, but analysis of the small number of available PMP studies has shown that the production of biopharmaceuticals in plants can be economically more attractive than in other platforms . A simplified TEA model was recently proposed for the manufacture of mAbs using different systems, and this can be applied to any production platform, at least in principle, by focusing on the universal factors that determine the cost and efficiency of bulk drug manufacturing .Minimal processing may be sufficient for oral vaccines and some environmental detection applications and can thus help to limit process development time and production costs . However, most APIs produced in plants are subject to the same stringent regulation as other biologics, even in an emergency pandemic scenario . It is, therefore, important to balance production costs with potential delays in approval that can result from the use of certain process steps or techniques. For example, flocculants can reduce consumables costs during clarification by 50% , but the flocculants that have been tested are not yet approved for use in pharmaceutical manufacturing. Similarly, elastin-like peptides and other fusion tags can reduce the number of unit operations in a purification process, streamlining development and production, but only a few are approved for clinical applications . At an early pandemic response stage, speed is likely to be more important than cost, and production will, therefore, rely on well characterized unit operations that avoid the need for process additives such as flocculants. Single-use equipment is also likely to be favored under these circumstances, because although more expensive than permanent stainless-steel equipment, it is also more flexible and there is no need for cleaning or cleaning validation between batches or campaigns, allowing rapid switching to new product variants if required. As the situation matures , a shift toward cost-saving operations and multi-use equipment would be more beneficial.An important question is whether current countermeasure production capacity is sufficient to meet the needs for COVID-19 therapeutics, vaccines, and diagnostics. For example, a recent report from the Duke Margolis Center for Health Policy24 estimated that ~22 million doses of therapeutic mAbs would be required to meet demand in the United States alone , assuming one dose per patient and using rates of infection estimated in June 2020.

In addition, these strains are frequently found in mixed populations within a single host. While we have some knowledge of the symptoms produced by a single strain, there is little understanding of how combinations of strains affect symptom expression and disease severity. To test the effect of population structure on symptom expression, we inoculated Citrus paradisi and C. aurantii with a range of single strains and mixed field populations of CTV. Seedling yellows symptom development was assessed six months post-inoculation. Real-time qRT-PCR was used to assess relative population titre, and these data were compared with symptom severity. We found that there was no obvious correlation between the dominance or presence of specific isolates in the population and the incidence or severity of seedling yellows; there was also no correlation between total viral load within infected tissue and symptom severity. These data further suggest that symptom severity is not due to quantitative differences between strains in a population, but indicates that qualitative differences within and between each population influence symptom development.To minimize losses in the local citrus industry, the South African Citrus Improvement Scheme implemented cross-protection using mild CTV sources to reduce the effect of challenges by endemic severe CTV strains. This management strategy also referred to as “pre-immunisation” or “mild strain protection” was implemented by the CIS at its initiation. The use of cross-protection in South Africa has been mostly successful, but cases of cross-protection breakdown have been experienced and a change in the pre-immunising source for grapefruit cross protection was made to address this. A number of countries apply cross-protection and report diminished expression of disease and improved production including Australia, Japan, Brazil, Argentina, Peru and South Africa.

All pre-immunisation sources used for cross-protection, except the South African sources,hydroponic indoor growing system are single variant sources. CTV is a complex of strains. This insight and the subsequent development of diagnostics for genotyping enabled the analysis of mixed populations. We have expanded on a published CTV genotype testing system and have tested various maintenance sources of the GFMS12 and GFMS35 pre-immunisation sources at 3 different institutions including grapefruit mother trees maintained at the Citrus Foundation Block. Also segregation of genotypes is noted in different multiplications of the two sources and this is in all probability a contributing factor to cross-protection breakdown in the field.Bio-indexing and characterization by CE-SSCP, MMM and phylogenetic analysis of representative samples collected in a highly infected Citrus tristeza virus area of Sicily reveals that two main groups are present: one includes isolates inducing severe seedling yellows and a second is asymptomatic on sour orange. Severe isolates cluster in a single subclade within the group of seedling yellows and stem pitting isolates ; mild ones are similar to T30-like isolates. In order to investigate the phylogenetic relationship of the CTV population with the isolates of respective clades we undertook the complete genome analysis of two of them, namely SG29 and Bau282. SG29 is a severe isolate, from Sanguinello sweet orange, inducing seedling yellows on sour orange and, rarely, stem pitting on Duncan grapefruit, but not on sweet orange. Bau282 is from sweet orange TDV and host susceptibility showed it is asymptomatic on sour orange and Duncan grapefruit. The genomes were obtained after sequencing of the small RNAs and assembly of overlapping sequences by reference alignment from libraries sequenced by Next-generation platforms. The viral small interfering RNAs found were in the predominant 22 and 21 nucleotide-size classes. The complete SG29 and Bau282 genome in length are 19,259 bp and 19,250 bp, respectively, with 12 open reading frames , structurally identical to the other known CTV isolates. Phylogenetic analysis based on 31 full CTV genomes showed that SG29 clustered with the “Asian” VT-lineage in which T318A , AT- 1 , Nuaga and CT11A isolates segregate and has the highest homology identity with T318A and AT-1.

Bau282 clustered within the mild isolates T30 and T385 and BLAST analysis showed a very high identity equal to 99%. Since its appearance two centuries ago, Tristeza has been classified as the most devastating viral disease which affects citrus worldwide. Its causative agent, Citrus tristeza virus , is transmitted by infected grafts or by insect vectors such as the aphid Toxoptera citricida. In all affected citrus growing areas, the existence of genetic variants of the virus with different degrees of severity has been reported. Characterization of CTV isolates can provide epidemiological information and can be useful for disease control. The presence of CTV and its efficient vector has been known in Uruguay since the 1940s. However, there is no data based on molecular biology reflecting genetic variants circulating in the country. In the present study, using RT-PCR amplification of three regions of the CTV genome , we established phylogenetic relationships of the strains in the country at the present time and 20 years ago. The samples used were collected in 1990 and maintained in planta in vector free greenhouses, and also were compared with current field samples. This valuable historical collection provides a sample of past CTV occurrence in Uruguay. The results show that circulating strains in the country are severe and unsurprisingly resemble strains reported in Argentina. However, some of the strains under study are similar to reference strains from Israel or Hawaii. This may reflect the introduction of infected buds or trees in the past.Citrus Sudden Death remains a challenge for citrus production in non irrigated areas in the North and Northwest regions of Sao Paulo State, Brazil. Its incidence has increasingly affecting the drought-tolerant but CSD-susceptible ‘Rangpur lime’ root stock. So far, since the first report of the disease in the 2000s its etiology remains uncertain, but vectored-viruses have been considered as the main hypothesis . Here we checked the occurrence of these viruses in a deep sequencing dataset of total RNA by Illumina platform run for transcriptomic studies of sweet orange.

Pools of reads of Valencia graphed on Rangpur lime and Sunki mandarin, both CSD symptomatic and also from its non-symptomatic pairwise trees growing side-by-side were analyzed. Around 74 million reads for each experimental condition were assembled using the CLCbio platform de novo assembly algorithm using a CTV reference genome . For the NS-CSD dataset only 0.005% of reads matched with CTV virus sequences,macetas 1 litro whereas for the S-CSD dataset 0.014% of reads come from the CTV virus. However, almost three times more reads of CSD disease trees matched with CTV. A careful comparison of CTV reads from S-CSD with NS-CSD allowed us to identify some sets of reads more frequently occurring in diseased trees, as also some CTV reads specifically occurring in the CSD diseased plants. Those genetic differences which make the CTV-reads specific for the CSD symptomatic plants were mapped through the whole CTV genome reference. On the other hand, using the same assembly strategy we observed only few reads matching the Tymovirus genome in the analyzed dataset, independent of whether they come from diseased or non-diseased trees. Despite being poorly represented, the Tymovirus’s reads were shown to be genetically diverse, which was confirmed by Sanger sequencing of amplicons from five different Tymovirus’ genomic regions amplified by specific primers. Citrus tristeza virusis the causal agent of the most important citrus disease and exists as numerous strains which may cause different symptoms. Due to the wide biological diversity of the virus, identification of the actual genotypes present in an area is useful in adopting adequate control strategies. In recent years a plethora of methods for molecular characterization of the virus have been developed. Among others RT-PCR, Real-Time PCR and CE-SSCP have been adapted for discrimination of severe and mild CTV strains. In this work, based on the fully sequenced genomes of the virus available on GenBank, we have developed two quadruplex primer sets, one for simultaneously obtaining amplicons from the 5’UTR, ORF1a, RdRp and p27 regions , the other for p20, p23 and p33, and the Citrus elongation factor EF 1 alpha . One VerechipTM, an electrically active system that integrates a PCR module, consisting of two independent PCR reactors, and a hybridization chamber, is used for both quadruplex reactions. RNA and RT-PCR master mix are combined and loaded to the chip, which is placed in a thermal reactor for rapid end point PCR. Hybridisation mix is then added and the RT-PCR products, with sizes between 73 and 239 bp, flow into the microarray portion of the chip, which is returned to the thermal reactor. Hybridisation complete, the array is then read. The array was represented by 44 probes designed for the phylogenetic groups with type strains T36, T3, T30, VT, NZ-RB, B165 and other emerging clusters such as HA16-5. The distribution of the target genes throughout the genome and the specific probes designed on the nucleotide sequences, enables the identification of any known strain even if recombinant or in mixed infections, and is likely to distinguish new groups.

Historically, decline has been the most devastating disease caused by Citrus tristeza virus , although stem pitting greatly limits production in many citrus industries around the world. Decline has been the major problem caused by CTV in Florida because fortunately severe stem-pitting isolates have been kept out so far. Decline is a man-made disease based on propagation of sweet orange, grapefruit, and mandarins on the sour orange root stock. Although this disease can be controlled by using alternative root stocks, there are soils in which all other root stock choices are less desirable in terms of fruit quality and yield. One of our major goals has been to develop a way to allow growers to use the sour orange root stock in the presence of CTV. Florida has two predominant strains of CTV, a decline strain and a non-decline strain . A first goal has been to map the viral determinant that induces decline. This was done by creating hybrids by substituting T30 sequences into T36. A parallel goal was to use a non-decline T36 hybrid to cross protect trees on the sour orange root stock from decline. This project was delayed considerably because we were not able to definitively assay decline in the greenhouse because we were unable to distinguish decline symptoms from seedling yellows symptoms with these hybrids. In order to examine decline in field trees, we had to obtain permission to do a field test of recombinant DNA produced virus hybrids from the USDA Biotechnology Regulatory Service. Valencia sweet orange on sour orange root stocks were inoculated with T30/T36 hybrids in the greenhouse and transferred to the field in 2010. The test will end August 2013. Results of this field test will be presented showing that the p23 and 3’ non-translated sequences of T36 contain the determinant of decline.A high-throughput sequencing pipeline to characterize Citrus tristeza virus isolates was developed. Three alternative viral templates combined with random RT-PCR amplification were first tested on a single, previously characterized GFMS12 sub-isolate for their enrichment qualities and subjected to Illumina paired-end sequencing. Comparisons between the sequencing data obtained and gene-specific phylogenies were also made in order to test their validity. Double-stranded RNA was found to be most enriching and was utilized for further characterization of additional glasshouse-kept isolates. A novel T68-1-like South African genotype, named CT-ZA3 was assembled de novo and shown to be the dominant component in all GFMS12 sub-isolates tested. This study underlined the effectiveness of next-generation sequencing for genotype discovery as well as whole-genome characterization of CTV isolates to a level of detail not previously attainable with classical methods such as single stranded conformational polymorphism and Sanger sequencing of multiple clones, which have been shown previously to yield incongruences in genotype identification.Next-generation sequencing has been established as a reliable approach to study metagenomic samples. In plant pathology, the application of NGS has not been limited to sequencing for pathogen discovery, but has also been used for applications such as the study of plant-pathogen interactions. In this study, we used metagenomic NGS to establish the virome of citrus samples. Double stranded RNA was extracted from the cambium tissue of virus diseased citrus trees with a cellulose extraction protocol and sequenced in an unbiased manner using Illumina sequencing-by-synthesis technology on a HiScanSQ. Two samples were analyzed; one displayed atypical “psorosis” symptoms and the other was a source plant containing Apple stem grooving virusand Citrus tristeza virus .

These three input delivery schemes have two features in common: payment at harvest, and no payment in case of crop failure. Otherwise the details of input repayment vary a lot from one example to the next in the sharecropping example, costs are paid as a share of harvest; in the agricultural marketing board example, costs are deducted from the output price or paid jointly by villagers; in contract farming, costs are deducted from the value of the harvested crop. This much variation suggests that these contractual details are less important than the two principles listed above. Similar principles can be successfully applied to other technology delivery schemes, such as animal traction equipment.In my book on risk and rural development, I offer a simple extension of the Sandmo model which can account for these observations. Farmers are assumed to worry about out-of-pocket risk: they do not like to finish the year in the red. The addition of this simple assumption is sufficient to account for the success of the above-mentioned schemes even if farmers are otherwise risk neutral . This is important because we have argued earlier in this paper that the expected utility framework which assumes aversion to upside as well as downside risk may not be a very convincing. The question then is: why is assuming aversion to out-of-pocket risk any more reasonable than assuming risk aversion in an expected utility framework? Here behavioral economics comes to the rescue. Ever since Binswangers early work on risk aversion among ICRISAT farmers, researchers working on agricultural technology issues have been aware of experimental economics. But they may not have taken advantage of all its lessons. Results from laboratory experiments have long suggested that what humans fear is not risk but the prospect of loss . This is most easily demonstrated by experiments in which participants are asked to choose among lotteries with identical final payoffs, but a different sequence of events.

While participants often are willing to gamble for future gain, they are less willing to put earlier winnings at risk,maceta 30l even if final payoffs are identically distributed. This could explain why farmers are not willing to put assets at risk by buying agricultural inputs they are not guaranteed to recoup. By eliminating downside risk, the input delivery contracts discussed earlier do not remove upside risk but they deal with loss aversion. Laboratory experiments have also shown that humans have a poor intuitive understanding of low probability events. For instance, it is common for participants to experiments to be willing to pay the same for a risk reduction of one in a thousand or one in a million ñ even though the former should be worth one thousand times more than the latter. People are sensitive to whether they have recently been affected by similar events and can recall similar incidents. Indeed recent exposure to low probability events tend to dramatically raise peoples willingness to pay to protect themselves against the future recurrence of similar events. It follows that people respond to how the risk of future events is framed, and whether they can recognize past experiences in experimental situations. Finally people may be quite averse to small probability events that are beyond their control but not overly worried by high probability events they perceive to be under their control . Taken together, this evidence suggests that people are actually not very rational when it comes to small risks, but also that they are weary of downside risk beyond their control.People often overestimate their chances in risky ventures. As a result, they often want to over invest, provided they are sheltered from downside risk. This may explain why many entrepreneurs whose honesty is not in question seem keen to invest uncollateralized borrowed funds in risky projects. Such findings are in line with our earlier observation regarding the relative success of agricultural input delivery schemes that protect farmers from downside risk but expose them to considerable upside risk.

Taken together, these empirical regularities documented in laboratory experiments may help explain observed patterns of agricultural technology adoption. Recent field experiments add new insights to this body of knowledge. Of particular interest to our purpose is a recent paper by Duflo on fertilizer adoption in Kenya. At the end of the paper, the authors document a series of field experiment investigating the effect of fertilizer vouchers on input usage. They find much higher fertilizer usage among farmers who were offered a voucher for future fertilizer delivery at the time of selling their crop. This finding is broadly in line with experimental findings about quasi-hyperbolic preferences, forced savings contracts, and peoples desire to commit future expenditures . Duflo investigate possible explanations for their finding. Of interest is the observation that fertilizer usage drops significantly if the voucher is sold to farmers only a day or two after they sell their crop. Why this is the case is not entirely clear, however. One possibility is that the money has already found other uses, e.g., paying for debts and social obligations. Another possibility is reciprocity: when the voucher is sold by the buyer of the crop, the seller may feel some sense of obligation to reciprocate by purchasing a fertilizer voucher. More work is underway to disentangle these possible explanations. What they do suggest, however, is that input usage by small farmers in developing countries may be quite sensitive to the method of delivery and sale. Rational models of input purchases are not vindicated as there are strange behavioral responses to commitment devices offered to input purchasers. Peer effects may also matter. Ashaf et al. document an out grower scheme run by an NGO in Kenya. The authors evaluate a program in Kenya that encourages the production of export oriented crops by providing smallholder farmers with credit linked to agricultural extension and marketing services. They use an experimental design in which farmer self-help groups are randomly assigned to either a control group, a group receiving all DrumNet services, or a group receiving all services except credit. Among the services offered by DrumNet, credit is the most important, a finding that is consistent with the significant investment in capital and inputs required to produce the export crop. This result is also consistent with our earlier observation regarding downside risk. These results are to be compared to field experiments that offer crop insurance to small farmers. If Sandmos model is a fair representation of small farmers decision process,hydroponic grow system offering insurance corrects a market failure and is the preferred way to achieve first best. Two separate teams of researchers have experimented with crop insurance in two Indian states. Their results are summarized in a jointly authored paper . Both field experiments have in common the offer of a voluntary insurance contract that compensates farmers in case of deficient rainfall. Payment is based on objectively collected rainfall data.

Farmers purchase insurance in discrete units, with each unit equivalent to set payments conditional on rainfall. Farmers can obtain more insurance by buying more units. The modeling framework presented in Section 2 predicts that more risk averse farmers should purchase more insurance than risk neutral farmers. We also argued that the curvature of the value function V depends on the households capacity to self-insure through the accumulation of liquid assets. This implies that households with more assets need ñ and should purchase ñ less insurance. Since small Indian farmers are often poor, we would therefore expect widespread adoption, with many farmers purchasing enough insurance to protect themselves against much of rainfall risk. This is not what the authors find. Take-up is limited in the Gujarat experiment, only 20% of targeted farmers purchased the insurance but sensitive to price and additional marketing. Although results from the two experiments differ somewhat, risk averse households appear less, not more, likely to purchase insurance. Households do not purchase full coverage; on the contrary, they tend to purchase only one unit of insurance, no matter how large their risk exposure. Furthermore, insurance take-up is higher among wealthy households. None of these results are consistent with the standard Sandmo model. The authors also report that take-up is lower among households that are credit constrained. They argue that these results match predictions of an extended Sandmo model with borrowing constraints. Alternative explanations exist as well, such as lack of familiarity with the insurance product. Other patterns are more difficult to reconcile with the benchmark model. Participation in village networks and measures of familiarity with the insurance vendor are strongly correlated with insurance take-up decisions. While education does not seem to matter, endorsement from a trusted third party does. These results may reflect uncertainty about the product itself, given households limited experience with it. They are to be compared with those reported by Ashaf et al. on the role of farmer groups, and to those of Duflo regarding the possible reciprocity between farmers and crop buyers/input providers. Gine, Yang, Insurance and from Malawi report on another similar field experiment in Malawi. They implement a randomized field experiment to ask whether the provision of insurance against a major source of production risk induces farmers to take out loans to invest in a new crop variety. The study sample was composed of roughly 800 maize and groundnut farmers. The dominant source of production risk is the level of rainfall. The authors randomly select half of the farmers to be offered credit to purchase high-yielding hybrid maize and improved groundnut seeds. The other half are offered a similar credit package but required to purchase a weather insurance policy that partially or fully forgives the loan in the event of poor rainfall. If, as we have argued earlier, farmers are primarily concerned about the downside risk associated with credit, offering the insurance should boost take-up. Surprisingly, the authors find that take up is lower by 13 % among farmers offered insurance with the loan. At primafacie, this seems to reject downside risk concerns as the primary motive for low take-up of agricultural innovations. The authors however find suggestive evidence that the reduced take-up of the insured loan is due to the high cognitive cost of evaluating the insurance: the take-up of insured loans is positively correlated with farmer education levels, but not so for uninsured loan. This brings up another consideration, namely, that people have a complicated relationship with new products. Curiosity may tempt them into trying new products, but such impulse purchases may ultimately prove disappointing. People may therefore steel themselves against large impulse purchases, especially if they are poor. This would be consistent with richer Indian farmers purchasing rainfall insurance, but only one unit, while poorer farmers do not purchase any. Peoples ability to resist impulse purchases may be susceptible to manipulation by marketing efforts. This may explain why fertilizer vouchers in Kenya found more buyers when the purchase of the voucher was combined with the sale of the crop. Given this, adoption of new products may require reinforcement from peers: if others around them are adopting a new product, people may find it harder to resist buying it. This naturally generates threshold effects in adoption, an observation made a long time ago by Griliches . In his study of US farmers, Young and Burke similarly noted the importance of peer effects and conformity in the adoption of certain types of behavior. The emerging economic literature on social network effects has revived interest in diffusion and reinforcement effects. There is extensive circumstantial evidence that social networks matter for the adoption of agricultural technological and institutional innovations in developing countries . In a recent unpublished paper, Caria argues that Ghanaian farmers who are more risk averse are less likely to experiment with new technology. This may explain why risk averse farmers in Carias study look up to risk neutral neighbors for advice on new technology. Taken together, these field experiments suggest that input usage and the purchase of crop insurance are not well accounted for by the standard model presented in Section 2. While an ex tended model that includes credit constraints and downside risk considerations can explain some of the empirical regularities, other results indicate that subtle psychological manipulations affect take-up.

Alternative extrapolations of the same data were subsequently published that accounted for differences in foliage turnover rates between biomes, significantly lowering the global strength of a putative aerobic plant source . To further constrain the potential magnitude of global CH4 emissions from upland plants, we used a foliar VOC emissions model—MEGAN or Model of Emissions of Gases and Aero sols from Nature—to incorporate certain canopy and physical processes that were not considered by Kirschbaum et al. and Parsons et al. . In particular, we used the temperature responses reported by Keppler et al. and accounted for the effects of self-shading within the plant can opy. We used MEGAN with the assumption that the mechanism of CH4 production, if it exists at all, shares some features of the biochemical pathways that produce other VOCs such as methanol. MEGAN includes a detailed canopy environment model that calculates solar radiation and leaf temperature of sun and shade leaves for each of five canopy depths. Driving variables include wind speed, humidity, soil water content, above-canopy direct and diffuse solar radiation, and ambient temperature. MEGAN includes emission factors for light-de pendent and light-independent components of emissions, and irradiances that vary because of self-shading in the plant canopy. Light-dependent and light-independent emissions of CH4 were estimated based on the emission factors recommended by Keppler et al. . Although Keppler et al. did not report light response curves,maceta 30 litros we assumed that emissions increase nearly linearly with irradiance to a saturation point. This is the behavior we observe for other biogenic VOC and is thus a reasonable starting point for the CH4 extrapolation.

The emission algorithm for dark emissions was based on the temperature response shown in Figure 1 of Keppler et al. . A range of global annual CH4 emission estimates was generated using different combinations of the alternative land cover and weather databases described by Guenther et al. . Our parameterization of light and temperature in the MEGAN model is similar to the global model of aerobic CH4 emissions developed by Butenh off and Khalil . The global distribution of CH4 emissions from foliage simulated with MEGAN is shown in Figure 1. Tropical forests are a major source region, which agrees with the predictions of Keppler et al. and the observations of Frankenberg et al. . The annual global CH4 emission from living vegetation estimated with MEGAN ranged from 34–56 Tg year –1, depending on the land cover and weather data used to drive the model. This figure is nearly one order of magnitude lower than the highest estimates provided by Keppler et al. and is consistent with the magnitude of alternative extrapolations provided by Kirschbaum et al. and Parsons et al. , and the global model developed by Butenhoff and Khalil . Our estimates would be about an order of magnitude lower if we had used the mean rate reported by Dueck et al. of 21 ng g–1 h–1.The demand for nutritious and safe food will increase as the human population is expected to reach between 9.4 and 10.1 billion in 2050 and between 9.4 and 12.7 billion in 2100 , along with increasing urbanization and standards of living . Healthy consumption of grains, oil seeds, nuts, and fresh fruits and vegetables is part of an integrated strategy to decrease the risk for diet-related chronic diseases, such as cardiovascular disease, type 2 diabetes, some types of cancer, and obesity . However, the World Health Organization report shows that at global level, 31 hazards caused 600 million food borne illnesses and 420,000 deaths in 2010 . Health concerns exist due to the consumption of mycotoxins produced by fungi that frequently infect grain, oil seed, and nut crops . The health burdens placed on consumers and economic burdens placed on farmers and processors by the presence of these toxins can be severe . Furthermore, heavy metals , allergens , and accumulations of natural molecules and compounds may be detrimental to human health.

At the same time, the number of food borne disease outbreaks related to consumption of contaminated fresh or minimally processed produce has been increasing . In the United States, 48 million illnesses and 3000 deaths associated with food-borne diseases occur annually, with approximately one half associated with crops . In the European Union, during the period 2004–2012, there were 198 outbreaks linked to the consumption of fresh produce . Beyond the burden on public health, food borne illness outbreaks negatively affect the economics of the industry. It is estimated that the overall cost of food safety incidences for the economy of the United States is $7 billion per year, which comes from notifying consumers, removing food from shelves, and paying damages from lawsuits . Furthermore, a single produce-borne disease outbreak can trigger a sharp decrease in the market of the affected crop for years . Following a number of large multistate foodborne disease outbreaks linked to contaminated fresh produce1 , the American Phytopathological Society-Public Policy Board convened the first formal activity in 2007 in a symposium titled “Cross Domain Bacteria: Emerging Threats to Plants, Humans, and Our Food Supply”. A working group on “Human Pathogens on Plants” was assembled to create solutions for this problem and has since convened as a satellite meeting during annual APS meetings. Similar activities have been conducted in Europe through the COST Action on “Control of Human Pathogenic Micro-organisms in Plant Production Systems”. Leafy greens are annually involved in food safety incidents in the United States. From 1996 to 2016, 134 confirmed incidents, including 46 outbreaks, were identified to be linked to products from California that provides one-third of the vegetables and two-thirds of the fruit and nuts in the United States according to the California Department of Agriculture , California Agricultural Production Statistics. During this period, lettuce and spinach were reported as the main vehicles of food safety incidents . After three major outbreaks in 2006, the leafy green industries in Arizona and California created the Leafy Green Marketing Agreement with evidence-based food safety metrics that are updated to incorporate the most current state-of-the-science5 . Likewise, the U.S. Food and Drug Administration subsequently implemented the Food Safety Modernization Act to address the significant public health burden of preventable foodborne diseases.

Under FSMA,frambuesas maceta the Produce Safety Rule established, for the first time, science-based minimum standards that include on farm regulation of fresh fruits and vegetables grown for human consumption. Food safety is a complex issue that requires a concerted effort among scientists, regulators, seed/nursery industry, processors, retailers, and other stakeholders from diverse disciplines and research fields who do not often have the opportunity to meet and discuss global, comprehensive, and objective solutions. On 5–6 June 2019, the University of California, Davis hosted the first workshop on Breeding Crops for Enhanced Food Safety7 to identify knowledge gaps and research priorities in this emerging field to inform the USDA-NIFA and other agencies for funding and research priorities. This workshop connected plant scientists, plant breeders, extension specialists, microbiologists, and food safety experts from industry and academia to discuss collaborative efforts and multidisciplinary approaches geared toward preventing the occurrence of hazardous microbes, mycotoxins, elements, and allergens in crop and food production systems. Together, these pivotal steps by academia, industry, and government groups have laid out the opportunities to enhance food safety with plant breeding and created avenues for unique collaborative efforts and new research directions, which formed the basis for this review. The presence of mycotoxins, elements, and allergens in affected food crops has a high potential for mitigation via plant breeding . These substances, produced by the fungus, the plant itself, or taken up by the plant from the environment, are generally not defense compounds, but can be severely detrimental to the health of humans and animals who consume the crop in which the substances have accumulated. Crop varieties that do not support growth of the fungi that produce mycotoxins have been created in some cases and heritability is sufficiently high for genetic gain in others . Additionally, it may be possible to create host plants that do not allow or create the need for the fungi to produce mycotoxins. The level of allergens in crop plants can also be reduced in some cases via plant breeding, or in others, via genetic engineering or gene editing and breeding for wheat varieties that do not accumulate heavy metals . Furthermore, breeding efforts are conducted for the reduction of antinutritional compounds, such as vicine and convicine in faba bean and the acrylamide-forming potential of potatoes . Mounting evidence suggests that zoonotic bacterial pathogens of humans may have adapted to both animal and plant hosts, enabling them to survive in the food production chain . For example, romaine lettuce and other leafy greens continue to be linked to E. coli O157:H7 outbreaks traced to major leafy green production regions in Arizona and California despite widespread implementation of LGMA food safety practices; moreover, trace back and environmental assessments suggest that contamination is occurring at the pre-harvest level, but root causes remain elusive . A few research groups have discovered phenotypic variability in the interaction between these pathogens and fresh produce, suggesting that plant genetic traits may affect plant susceptibility or tolerance to human pathogen colonization.

A complete description of the methods used in each study is listed in Supplementary Table S1. Similarly to the examples of breeding strategies described above, these reports support the basis for breeding for decreased microbial hazards in several systems. Host plant resistance to the fungi that produce mycotoxins can be a synergistic part of a systems approach to reducing mycotoxins in crop plants . The method is economical for the farmer because it requires no additional equipment or supplies and is integral to the seed itself. It works well with other methods for controlling mycotoxins, including the use of biocontrol agents that farmers can buy and apply to the field, and proper handling and environmental conditions during harvest, drying, and storage, which can also help prevent the growth of the fungi. There has been a long history of breeding wheat for resistance to Fusarium graminearum that produces deoxynivalenol , although complete resistance remains elusive. Significant progress has also been made, for example, in pre-breeding germplasm in maize that does not support the production of aflatoxin or significantly reduces it compared to conventional maize varieties. These traits are being introgressed into U.S. maize inbreds by Paul Williams and Marilyn Warburton at USDA–ARS, Mississippi, and Seth Murray and Wenwei Xu at Texas A&M University. Heritable plant traits that reduce the numbers of harmful human pathogen cells on the edible portions of the plant may also be incorporated into a system designed to reduce risk from these microorganisms without negatively influencing the other components of the system. Similarly, Charlie Brummer and Allen Van Deynze, with support from Richard Smith have identified and are breeding lines of spinach that have reduced accumulation of cadmium, a heavy metal found in some soils in California that can have chronic health effects, especially in children. Wheat varieties that accumulate low levels of cadmium are being developed using the latest genomic and phenotyping technologies . The fresh produce industry faces several major challenges related to controlling risks from in-field contamination of crops by zoonotic enteric pathogens . First, zoonotic fecal-borne pathogens may be widespread in the environment, but rarely detected in field crop, thus making it difficult to precisely define the most important direct and indirect routes of contamination . Second, if bacterial contamination occurs in the field, there is no subsequent “kill step” for many popular produce items such as salad greens that are consumed raw or minimally processed. Third, the infectious dose for these pathogens may be low, especially among vulnerable populations such as young children . Although it may seem improbable that a low level of in-field contamination could result in large numbers of human foodborne illnesses, Danyluk and Schaffner developed a quantitative risk assessment model that predicted that exposure to levels of E. coli O157:H7 in the field—as low as -1 log CFU/g and 0.1% prevalence—could result in a nationwide outbreak in combination with post harvest contributing factors such as cross-contamination during the washing process.

It is important to know how ectomycorrrhizal weathering rates will respond to changing nutrient status of forests to determine what measures must be taken to guarantee forest health as humans continue to alter the global carbon and nitrogen cycles. This review examines what is known about how weathering activity of ectomycorrhizal fungi responds to nutrient demand. First, attention is given to our understanding of how host plant nutrient demand is sensed by the plant and communicated to the roots, then research on ectomycorrhizal weathering is discussed with recommendations for further studies. For the purposes of this review we will focus on nutrient demand of P, K, Mg, and Ca, although S and sometimes Fe are also important nutrients to consider in regards to weathering activity. Ectomycorrhizal weathering activity will be defined as any activity on the part of the fungus that increases the rates of element release from mineral surfaces, such as proton exudation, organic acid exudation, or element removal from the mineral surface.The first is sometimes called “systemic” control and the second and third “localized”, implying that  below ground allocation is controlled by overall plant nutrient status while localized root branching/proliferation and transporter expression and regulation are controlled by the roots, but there is ample evidence that the “localized” characters may also be systemically controlled .Nearly all of the research done to date on plant nutrient status sensing and regulation of nutrient acquisition has been done on model organisms or crop plants, all of which are angiosperms , frambuesa y mor and nearly all of which form arbuscular mycorrhizae. However, in the limited number of cases where ECM forming host plants are examined their reactions to nutrient limitation seem qualitatively similar. Phosphorus deficiency inhibits the growth of new plant tissues, particularly above ground shoots and leaves , leading to a buildup of sugars and starches in leaves which in turn causes increased phloem loading of sugars,frambuesa y mor  and C transport to the roots . Increased P demand has been found in a number of studies to cause increased below ground carbon allocation and increased root: shoot ratios . Increased root growth and branching is generally found in patches of high P availability when plants are P limited . Numerous studies have found increased expression and up regulation of high affinity P transporters in the roots of phosphorous deficient plants covering a wide range of species. Potassium deficiency causes a buildup of carbohydrates in the leaf, but also inhibits phloem loading of carbohydrates . Andrews et al.  found either no effect or a marginal increase in root:shoot with increasing K limitation . In general, however, K deficiency has been found to decrease below ground carbon allocation and root:shoot. Numerous studies find plants unable to respond to local concentration gradients of potassium by enhanced growth or branching in areas of elevated K availability . A number of studies have found high affinity root K transporters are up regulated in response to K deprivation . Mg deficiency causes a sharp inhibition of sugar loading into the phloem, and this is generally found to result in a sharp decrease in below ground carbon allocation in a wide range of plants , even at only moderate levels of Mg deficiency . Ericsson and Kahr also observed that ectomycorrhizal development was strongly suppressed in Mg limited spruce seedlings. Indirect evidence suggests that plant root systems are not able to alter root growth patterns to take advantage of localized patches of high Mg availability. There is very little data available on root Mg transporters . Ca deficiency generally inhibits the formation of new tissues. This may cause an increase in leaf soluble carbohydrate concentrations and result in increased root:shoot and  below ground C allocation. A slight increase in root: shoot with decreasing plant Ca concentrations has been observed in a number of studies , but this increase is always marginal and the increased root:shoot likely reflects growth inhibition of above ground apical meristems and not an increase in below ground C allocation. The majority of calcium is thought to enter the plant with bulk flow of evapotransporation via the apoplastic pathway in parts of the root that lack a casparian band, but it is also known that there are symplastic pathways.The author could not find any data on whether roots are able to increase branching in patches of localized Ca abundance. Overall, our understanding of how plants sense and respond to nutrient status is severely limited. The development of and subsequent research upon nitrate reductase mutants of tobacco plants greatly advanced out understanding of nitrogen responsiveness in plants, but no such milestones have been achieved for P, K, Mg, or Ca. As a result we have very little idea about the mechanisms of plant nutrient status sensing for these important nutrients. More research is needed in this fundamental area of plant physiology for mycorrhizal ecologists to gain an understanding of how mycorrhizae may respond to shifting host nutrient demand in ecosystems undergoing continued acid deposition and/or global change. Increased carbon allocation below ground necessarily correlates with increased carbon allocation to ectomycorrhizae and thus, it follows logically,frambueso maceta  to increased weathering activity by ectomycorrhizal fungi. Roots that can preferentially grow and/or branch in localized areas of high nutrient availability will favor the ectomycorrhizal species which are providing the largest amounts of this nutrient, because by ramifying in those areas they allow those fungal species the opportunity to colonize a larger root area. It is unclear if increased expression and up regulation of high affinity transporters is possible under the ectomycorrhizal mantle. If so, one would assume this greater sink strength on the part of plant root would increase nutrient loss from the fungus,frambueso maceta  and thus increase nutrient transport in ECM rhizomorphs. However, it remains poorly understood how fungi exude nonSN nutrients into the fungal plant interface. A better understanding of this mechanism is necessary to understand how increased expression of nutrient transporters in plant roots will affect ectomycorrhizal activity. It is generally assumed that there is a tit for Stat relationship between the ectomycorrhizal fungus and the plant host. The former providing nutrients in exchange for carbon from the latter. Much research has gone into developing a mechanistic understanding of how this arrangement might be structured at the fungus root interface, nearly all of it focusing on nitrogen as the fungal exchange currency. Nehls et al. suggest that carbon provision by the fungus may be dependant on plant phosphorylation of hexose uptake transporters. This mechanism would allow the host plant to very quickly control carbon loss to the fungus as a function of nutrient provision from the ectomycorrhizal fungus. If these phsophorylation sites are controlled by the supply of nutrients other than N, such as P, K, Mg, or Ca, then this could be a key point of communication between host nutrient demand and fungal nutrient acquisition. A variety of microcosm and field experiments have been conducted to examine how host plant nutrient status affects the weathering activity of ectomycorrhizal fungi. There are trade offs associated with each scale of experimentation. Field studies take into account the full range of biotic and abiotic interactions which govern nutrient demand and mycorrhizal activity. In field studies, plants generally grow under realistic nutrient regimes, and in the presence of dynamic, complex microbial communities. However, it may be very difficult to isolate nutrient deficiency as a single treatment in field studies. Fertilization treatments are one method for examining the effects of nutrient demand on weathering activity; using existing productivity gradients is another. Fertilization may, however, cause other, undesirable effects such as increased pH, and it may take many years for the system to fully react to the altered nutrient status. Natural productivity gradients may vary with regards to a number of factors other than the desired nutrient availability. Both of these methods have been employed in a limited number of field studies to look at how nutrient availability and host plant nutrient status affect ectomycorrhizal weathering activity in&situ. While it is not possible to quantify ectomycorrhizal weathering activity in the field on an ecosystem scale, a number of qualitative measures can be employed to compare weathering activity either between different nutrient regimes or between different minerals. One such measure involves the use of in growth bags. Small mesh bags are filled with either quartz sand alone or sand amended with a particular mineral of interest. The mesh is of a size that allows hyphae but not roots to penetrate the bags. Studies by Wallander et al. , Parrent and Vilgalys , and Kjoller have indicated that the great majority of hyphae found in these bags are ectomycorrhizal fungi. By quantifying the fungal biomass and mineral dissolution in bags one can make inferences about the relative ectomycorrhizal weathering activity in a given soil. One important weakness of this method is that it discriminates against the broad range of ectomycorrhizal fungi that do not produce abundant extraradical hyphae.Hagerberg et al. examined ectomycorrhizal hyphal colonization of mesh bags filled with sand, or sand amended with either a mineral P source or a mineral K source in a variety of Picea&abies forests in southern Sweden encompassing a range of P and K availabilities. Apatite amended bags always had greater mycorrhizal colonization than pure sand bags, but in forests considered to be Plimited there was significantly greater difference in colonization between apatite amended and pure sand bags. No such response was found for forest K status and biotite colonization, although colonization of biotite amended bags was overall significantly higher than purely sand filled bags. Wallander et al. revisited this study, also using southern Swedish P.&abies stands of varying P status, but they focused on P and added a fertilization treatment. Again, they found a significantly greater difference in colonization between apatite amended and pure sand bags when P levels were growth limiting, but when P levels were increased by fertilization there was no increase in mycelial colonization of apatite amended bags as compared to pure quartz sand filled bags. Taken together these studies suggest that ectomycorrhizal colonization of readily weatherable minerals, though not necessarily actual weathering, can respond to forest phosphorous status. In another mesh bag study, Wallander et al. looked at mycelial colonization of quartz filled mesh bags, some of which were amended with wood ash or apatite. The study employed particle induced Xray emission spectra to assess the elemental content of specific rhizomorphs which were also molecularly identified to fungal species. Wood ash amended bags had significantly more mycelia than either sand filled or apatite amended bags. Paxillus&involutus, by far the most utilized ECM in weathering experiments and the one generally shown to have the highest oxalic acid production in microcosm studies was not found on any root tips near the mesh bags, but it was by a large margin the most common ITS phylotype found in the mesh bags. Suillus&granulatus hyphae consistently contained more K than other species and generally had Ca oxalate encrustations on its rizomorphs, while P.&involutus consistently had higher Ca content than any other species. Some trends were also found for Fe and P contents and other ECM species. These results suggest there may be some consistent trends that can be used to place certain ectomycorrhizal species in distinct biogeochemical niches. However, elevated levels of mineral elements do not necessarily indicate increased weathering activity of minerals containing those elements. On the contrary, elevated levels of a given element could indicate that this fungus passes less of this elelement on to its host plant. Another potentially useful method for examining weathering activity of ectomycorrhizal fungi is to look at the density of hyphal tunneling in mineral grains. Jongmans et al. first proposed that regular channels often found in minerals might be fungal in origin. While no “smoking gun” has to date been devised to demonstrate that these tunnels are irrefutably caused by ectomycorrhizal fungi, a number of studies offer circumstantial evidence .  Looking at a topographic fertility gradient in a northern Swedish boreal forest, Hoffland et al. found that the density of tunnels of putative ectomycorrhizal origin had a strong positive correlation with ectomycorrhizal tip density and a strong negative one with soil fertility.

While Nguyen Hatsushiba’s film re presents the slow violence of American weapons and industry wrought upon the poor, forgotten bodies of Vietnamese refugees abroad and in Vietnam, Jae Rhim Lee brings attention to the over 200 toxic chemicals in American bodies including tobacco residues, dry cleaning chemicals, pesticides, flame retardants, heavy metals, preservatives, and more. Bisphenol A , a synthetic estrogen and plastic hardener which causes reproductive and neurological damage, alone is found in 93% of people over the age of 6.Lee’s Infinity Burial Project seeks to end the cycle of toxicity by purifying these pollutants from the body in death while directly trying to dismantle the traditional funerary practice of embalming. Each artist’s work offers some form of purification from the necropolitcal ordering of life on earth. Illuminating the politics of imperceptibility, both artists’ works bring a heightened awareness to the political intersections of waste, “nature,” and death. Jae Rhim Lee’s Infinity Burial Project works against the culture of death denial by offering a re imagination of death that focuses on a re connection to the earth’s nutrient systems. The “infinite” aspect of her work lies in the conceptual distribution of animacy from living mushrooms to the dead body in a way that frames decay in a positive light. Environmental reciprocity is not only achieved through bio physical means since, as Lee explains, “In a time of mounting threat and destruction, the self also needs an offensive strategy,indoor vertical farming one that heals the core of the psyche.”By revealing the cyclical nature of ecological processes of decay within decompiculture, she allows the viewers to face the taboo of death and corpses in a psychologically comforting way.

While green burials options are increasingly popular and offer a wide range of options, her project offers a uniquely natural way to cleanse the body of toxins after death. The Infinity Burial Project features the Infinity Burial Suit that can allegedly cleanse one’s dead body and deliver nutrients to plant roots . Costing $1,500 dollars, the suit is marketed as the “green” alternative to both cremation and embalming, which contaminates living human bodies and the environment. Cremation requires energy and releases the toxins within our bodies into the air while embalming requires the harmful carcinogen called formaldehyde. In contrast, the suit is made with organic, biodegradable cloth that is infused with mushroom mycelia and other organisms in a dendritic pattern that activates upon contact with dead human tissue . Jae Rhim Lee’s Infinity Burial Project seeks to revolutionize funerary practices in ways that protect the living and sustainably connects the body to soil. In 2007, Lee’s wish to remedy cultural death denial and unsustainable funerary practices began at an artist residency in Northern California. During her time there, she visited a permaculture school, farm, and green cemetery located nearby. At the cross section of the remedial abilities of mushrooms, green burials, and food production, she saw the potential for cultural and ecological reimaginations of death through fungi.Upon this revelation, she began researching the possibility of breeding an “Infinity Mushroom” that would be optimal for remediating the toxins in bodies and soil. Lee took inspiration from the entomologist Timothy Miles who coined the term “decompiculture,” the cultivation of organisms that facilitate decomposition, in his research that aimed at breaking down petrochemicals with mushrooms.However, she soon realized that mushrooms are nearly impossible to hybridize and that existing strains are already effective at cleansing toxins. Following her viral presentation at the TED Global conference in 2011, she formed the Decompiculture Society in order to foster public knowledge about post mortem options and cultural ideologies surrounding death through online platforms and workshops .In 2014, Lee became a lecturer and fellow at the Hasso Plattner Institute of Design at Stanford to further her research for the Infinity Burial Project. Following this period, she founded the company Coeio in 2015 and started producing her Infinity Burial Suit to sell.

The name of the company is derived from the Latin word, coeo, which means “to come together.” The name reflects the Infinity Burial Project’s goal of connecting humans and the earth through death. Proving their commitment to this idea, Coeio promises that with every purchase of an Infinity Burial Suit, they will plant 2 trees, compost any manufacturing scraps, and primarily use renewable energy sources. Clearly, this project truly lies at a future oriented intersection of business, biotechnology, ecology, design, and art.Built into the suit is a biomix consisting of mushroom mycelium and other microorganisms that aid in the body’s decomposition in order to neutralize toxins in the body and speedily transfer nutrients to the soil. Mycelium consist of threadlike hyphae, or branches, that facilitate the rhizomatic reproduction of fungi. “A rhizome has no beginning or end; it is always in the middle, between things, interbeing, intermezzo,”and the mycorrhizal fungi that Lee utilizes colonize the root systems of a host plant and provide heightened water and nutrient absorption while the plant provides the fungus with carbohydrates formed from photosynthesis.Although oxygen, soil depth, and temperature variables impact mycelium development, Lee claims that the 4 foot depth used by green cemeteries does not hinder growth.Mycoremediation is the process by which mushrooms remove toxins from the soil or body. Lee uses oyster and shiitake mushrooms for their aptness at consuming various sources of nutrients. These mushrooms neutralize organic toxins by breaking down molecular bonds and bind to heavy metal particles through a process called chelation.While the possibility of eating mushrooms laden with heavy metals seems to be a likely risk, no evidence of fungal surface growth has been shown. Quoting Roszak’s idea of a “narcissistic revolution”, Lee agrees that “a process of self examination, body self integration, reconciliation with death, and a uniting of body with nature” is needed in order to replace the attention demanding, energy economy.Pulling from Freud’s essay “On Narcissism”, Lee wants to meld primary narcissism with ecological stewardship in order to counteract the secondary narcissism which disengages from the world and only focuses on a “false self” –– similar to melancholic processes of mourning.

Secondary narcissism is fed by “mass consumption, warfare, economic decline, environmental degradation,best indoor vertical garden system and the dependence on technologies, [that] pose a threat to the self,” ultimately resulting in psychological and cultural annihilation. Drawing from Foucault’s concept of the “technologies of self,” Lee believes the psychic inwards turn to practice self care purifies oneself from the destructive consumptive practices that degrade the environment, waste human energy, and sideline deep personal needs.In Jae Rhim Lee’s words, “If the body is the first boundary of the self… Death is the eventual distribution of the body into the earth via –– the ultimate formlessness, weightlessness of the body.”Lee recognizes that the dualistic relationship between body and self is connected but fundamentally at odds with each other. As the body is always asserting its mortality, the self is always trying to transform itself into a symbol of transcendence. Concepts of self hood and existential threat occupy a large part of human psychology and constantly affects the makeup of society. Expanding on Erst Becker’s research on how societies manage fears of death, Terror Management Theory looks into the ways in which societal norms are upheld, such as hygiene, in order to buffer death anxiety. Although Lee’s project seems other worldly, her design practice is grounded in feeding emotional and ecological needs. Her Infinity Burial Suit challenges the boundaries between self, “nature,” and other in order to have a closer relationship between self and planet. Lee, like Roszak, advocates for a moderate form of narcissism as a way to reconnect with one’s ecological unconscious and support an environmental stewardship that is desirable and not based on fear or guilt. Like ecopsychology, Lee questions the destructive traits of capitalism and seeks to grow the ecological unconscious which lies in the center of the mind. Capitalism’s “economy of spectacles and desires… [make it] harder to imagine where life should lead and what, besides commodities, should be in it.”By fostering an ecological ego through narcissistic self seeking, one can eliminate the “false self” and focus on the needs of the planet as their own.In line with moderate narcissism, her burial suit offers a “unique option [that is] as unique as [people’s] lives.”By appropriating the sense of self boosted by consumption of commodities, her product offers a desirable example of how one can declare self hood even in death.

Also, from a marketing point of view, the transcendence reached for by the self is still comforted by the sense of “infinity” conferred in the product’s name. By imbuing the seemingly static state of death with the rhizomatic nature of ecosystems, she provides a crucial conceptual linkage between death as a part of sym poiesis: the inter species networks of decay and renewal, rather than auto poiesis, solely sustaining oneself . “Chthulucene” is Haraway’s term derived from the Greek word chthon, meaning “earth”, which focuses on sym poiesis “making with.”She proposes that humans are composts comprised of an infinite variety all different “kinds of assemblages” and that “Right now, the earth is full of refugees, human and not, without refuge,” and “To live and die well as mortal critters in the Chthulucene is to join forces to reconstitute refuges, to make possible partial and robust biological cultural political technological recuperation and recomposition, which must include mourning irreversible losses.”By aiding the body in becoming “clean” compost for the Earth, Lee expresses her utopic vision of death with her Infinity Burial Project.Man’s emissions of carbon dioxide and nitrogen are impacting large portions of the Earth’s forests.In this era of greenhouse gas induced climate change understanding the trajectory of global trends in forest productivity and soil carbon storage is more important than ever. Ectomycorrhizal fungi play a major role in many of the processes that govern how forests will respond to global change. My PhD thesis attempted to shed light on one of the most important questions facing forest ecology today: As nutrient demands by forest trees is altered by human induced global change, how will the functioning of ectomycorrhizal communities respond? In the proceeding introductory pages I will give some background information about ectomycorrhizal fungi and the effects of anthropogenic nitrogen deposition and elevated atmospheric CO2 levels on forests and ectomycorrhizal communities. I will then discuss how my PhD research has contributed to a greater understanding of how forests will be affected by the next century of human alteration of the global carbon and nitrogen cycles. Ectomycorrhizal fungi are important to forest health. The majority of land plants associate with some fungi in mycorrhizal symbiosis . A mycorrhiza is a symbiotic union between a fungus and a plant root, and, while the role of this symbiosis is complex and variable, nearly all mycorrhizae share a common trait of being a mutualistic exchange of plant derived carbon for fungus supplied nutrients. Ectomycorrhizae are one of a number of mycorrhizal types. Ectomycorrhizae are structurally distinct in that the fungus forms a sheath around the root tips, and sends hyphae into the root, but does not penetrate the plant cell walls. This type of mycorrhiza, ectomycorrhizae, associates with a relatively small portion of all plant species, perhaps only about 3%, but this 3% represents the majority of trees of the temperate and boreal forests , so, in terms of land area, the majority of the earth’s forests are dependant on ectomycorrhizal fungi . Ectomycorrhizae have been shown to improve their host plant’s resistance to drought , pathogens , and phytotoxic concentrations of heavy metals , but it is their ability to provide nutrients to their hosts that is generally considered their most growth promoting effect. This enhanced nutrient uptake is a result of ectomycorrhizal fungi’s ability to greatly increase the amount of soil the plants can be in contact with through their extensive mycelial networks as well as their ability to solubilize and take up nutrients from pools not available to plant roots. The majority of research on growth promotion by ectomycorrhizal fungi has focused on their ability to provide nitrogen to their host plants. 

Under flooded conditions, the competition between AsV and P in the rhizosphere deters P uptake and higher concentrations of AsV are absorbed by the plant. Immobilization of As in soils under oxic conditions reduces this competition and P bio availability increases, explaining the higher P concentrations in HS treatments. The nutritional value of rice grain is not typically considered when evaluating the impacts of water management treatments on As uptake, water use, and greenhouse gas emissions. However, potential changes in the grain nutritional content should not be ignored, as rice is a staple food for over half of the world’s population and a critical source of specific nutrients. In this study, in addition to decreasing As grain content, we reveal that II can increase Fe, P, and K levels in rice grain, providing another potential benefit to II management. We confirmed that a single dry down can be a suitable II treatment for minimizing As and Cd concentrations in grain. However, in other rice growing regions different field conditions would exist and II strategies may need to be modified to suit the specific location. To adapt water management regimes to meet specific environmental conditions, a better understanding of the soil and rhizosphere chemistry in rice paddies is crucial. Elucidating the biotic and abiotic changes occurring at the root soil interface and the mechanisms involved in As immobilization and Fe plaque formation, as well as how these are involved in the broader elemental cycling in rice systems,vertical hydroponics can bring us a step closer to defining how we can mitigate the ongoing problem of arsenic in rice while addressing human health concerns for one of the most consumed crops worldwide.

Rice is a staple food for more than half of the world’s population and is particularly susceptible to arsenic accumulation . Elevated As uptake occurs because rice is typically grown under flooded conditions where reduced As predominates and has high mobility and toxicity, leading to accumulation in rice grains and increasing the risk for As consumption by humans . Intermittent irrigation is a water management technique that involves intermittent flooding and draining cycles. Several studies reveal that II is an effective solution for reducing As concentrations in grain, methane gas emissions, and increasing water use efficiency . However, II treatments need additional study to determine their suitability for widespread application in rice paddies. Rice and many other aquatic plants transport oxygen to their roots via aerenchyma, creating an oxidized rhizosphere and resulting in the formation of an iron mineral plaque. This process occurs by oxidation of aqueous FeII present in anoxic soil solution after rice fields are flooded, and the precipitation of iron oxides and hydroxides on the root surface . Iron oxides are extensively studied in numerous disciplines because of their abundance in the environment and high reactivity; they play a vital role in many soil exchange processes. Additionally, Fe plaque formation and composition are affected by environmental factors, such as soil solution composition, pH and redox potential . Fe plaque can account for up to 14% of the dry weight of mature rice roots and can serve as a sink for As . Seyfferth et al., 2017, revealed that Fe and As co occur on the rice root surface. Other studies indicate that rice root iron plaque primarily retains arsenate and someAsIII .

In rice plants grown under continuous flooding, 95% of Fe plaque is composed of ferrihydrite and goethite , and, in lower proportions. lepidocrocite and siderite . The mineral composition of iron plaque is a result of specific biogeochemical factors at the root soil interface, which determine their crystallinity and surface affinity for adsorption of anions and cations . Ferrihydrite is an amorphous mineral that forms via hydrolysis of ferric iron with a large surface area, high adsorptive capacity and a positive charge under most soil conditions, serving as a sink for many oxyanion elements and organic compounds. Ferrihydrite gradually transforms into more thermodynamically stable and more crystalline FeIII oxides, such as goethite, via Ostwald ripening. . Moreover, intermittent irrigation treatments create fluctuating oxic and anoxic conditions in soil, which can alter the speciation of redox active elements at the root soil interface. Iron redox cycling affects the mobility and bio availability of As due to its high affinity for arsenite and arsenate. Oxidizing conditions in the soil can precipitate FeII into FeIII oxides and transform arsenic into AsV , while reducing conditions can lead to the dissolution of iron plaque, resulting in the release of FeII, AsIII and AsV present on root plaque, as well as reduction of AsV . Intermittent irrigation reduces arsenic mobilization by introducing oxic conditions in the soil during the growing season, oxidizing and immobilizing As in soil. Although it is well understood that with redox fluctuations the chemistry of both Fe and As will be affected, we find a gap in understanding how intermittent irrigation treatments of different timing and severity can impact the formation and mineralogy of iron oxides and thus their binding affinity to As. In addition, several authors have studied the sorption of AsIII and AsV on iron oxide surfaces with some evidence for specific binding mechanisms ; however, to our knowledge,hydroponic vertical farming systems a distinction of the binding interactions between AsV and root plaque iron oxides via insitu experiments to evaluate As sorption on the mineral surface have not been conducted.

In Chapter 1, our research demonstrated that II is a suitable strategy to reduce rice As uptake and translocation into the grain, but the chemical changes occurring in the rhizosphere, the mechanisms for As sequestration and the role of Fe plaque remain unclear. The objective of this study is to identify the changes in root plaque chemistry under intermittent irrigation treatments and to understand the binding interactions of synthesized iron oxide minerals present in root plaque with arsenate to facilitate an increased understanding of their impact on rice As uptake.The results from this study demonstrate that the mineralogy of rice root plaque is impacted by water management. Ferrihydrite, goethite, lepidocrocite, and side rite were detected in root plaque samples throughout the growing season, which is consistent with prior studies . In our XRD results, goethite was observed in all plaque samples at the late growth stages . Goethite formation typically follows a mineral ripening process where relatively amorphous and soluble minerals are transformed into a more thermodynamically stable and crystalline phase under oxidizing conditions . Additionally, ferrihydrite was first observed in the XRD spectrum corresponding to the CF treatment, as well as in II treatments at the late growth stages. Several factors can influence the mineralogy of rice root plaque, such as pH and soil solution chemistry. As pH decreases from 7 to 5, ferrihydrite is more likely to form . In the present study, during the 2018 growing season, soil pH increased from 6 to 7.5, making conditions favorable for ferrihydrite formation during the early stages of the growing season. Additionally, this pH range can increase the retention of arsenate by ferrihydrite than goethite . Moreover, Limmer et al., 2018 found that silicon concentrations during the formation of Fe plaque may play a role in its mineralogy. When the ratio of Si/Fe increases, ferrihydrite is more likely to form rather than lepidocrocite . Our SEM results demonstrate that Si and Fe do not co localize in root plaque for all samples. Higher amounts of Si in HS treatment in comparison to CF were found, indicating greater formation of ferrihydrite on roots of II treatments. Under flooded conditions, the transport of oxygen via aerenchyma to the rhizosphere drives the oxidation and precipitation of amorphous iron oxides which are subsequently transformed into goethite. Nevertheless, the identification of ferrihydrite and goethite at different times of the growing season indicates that mineral formation processes are impacted by II, which alters the mineralogy of plaque at different stages of rice growth. Iron plaque formation was studied by Long et al., 2019, revealing that Fe plaque formation occurs progressively; however, it is difficult to identify temporal trends in the mineralogy regarding II treatments when Fe oxide mineral formation begins, during the early stages of rice growth. XRD peak quantification and intensity measurements were limited by the presence of quartz in all samples. Attributed to its highly ordered crystalline structure, absolute intensities of quartz peaks in bulk mineralogy XRD analysis may cause extinction effects and reduce intensities of other mineral peaks, making identification of co existing minerals more challenging .

Additionally, XRD peaks do not often reflect the concentrations of all minerals present in the sample. In some cases, experimental errors or the lattice parameter d= nwill cause the peak positions to vary due to an angle shift. In addition, more crystalline minerals are easier to identify: ferrihydrite being highly amorphous is harder to identify in comparison to a more crystalline mineral such as goethite. For this reason, we focus our discussion on peak identification rather than concentration. Moreover, corresponding XRD peaks from samples collected at the later stages of rice growth, when root plaque mineral composition is less dynamic, are easier to identify in XRD spectra. Furthermore, Liu et al., 2006 found that ferrihydrite and goethite are the iron minerals present in the highest proportion in rice root plaques; therefore, it is critical to understand the binding interactions of these minerals with arsenic. Although the strong adsorption of AsV onto ferrihydrite and goethite has been demonstrated in earlier studies , there is uncertainty about the binding kinetics, and more studies are needed about how the sorption of arsenate compares between these minerals. Thus, insitu ATR FTIR experiments were carried out to elucidate binding mechanisms of AsV to these Fe oxides, which are dominant in the root plaque samples from this study. If specific binding mechanisms can be determined it would improve our understanding of the availability of arsenic for plant uptake under different rice paddy water management. Kinetic analysis results of FTIR spectra for AsV reacted with both goethite and ferrihydrite reveal an increase in peak area as a function of time, indicative of As sorption . These kinetic binding experiments also exhibit a cap on sorption sites of the surface of ferrihydrite based on the peak area change, showing a quick increase of sorption from 15 to 120 minutes. In contrast, for goethite, arsenate sorption increases slower and begins to plateau around 240 minutes. Additionally, we observe a difference in sorption when comparing the delta peak areas of ferrihydrite compared to goethite, being 8 fold higher for ferrihydrite and deducing that it has an increased ability to adsorb arsenate. It is logical to observe that ferrihydrite binds more AsV , as it is a poorly crystalline mineral and its amorphous characteristics are indicative of a greater binding surface and thus can lead to strong affinity for both arsenate and arsenite. These results are in agreement with Huang et al., 2011, which demonstrated higher sorption of iAs for ferrihydrite than goethite, explained by their differences in specific surface area and microporosity. Based on the observed aqueous AsV diagnostic peaks at 907 and 876 cm 1 , we identify distinct peak downshifts in all spectra. When AsV is reacted with goethite IRE coatings, one peak shifts to 871 cm 1 . For AsV reaction with ferrihydrite, a broad peak appears from 883 to 789 cm 1 . A downshift in the location of the FTIR peak for dissolved species upon reaction with a mineral surface is commonly attributed to inner sphere coordination . The reaction of arsenate with iron oxides has been previously studied via H+ /OHrelease stoichiometry titration experiments with strong evidence that arsenate is adsorbed on iron oxide surfaces by forming inner sphere bidentate binuclear complexes . Inner sphere binding, particularly bidentate, is substantially stronger than outer sphere binding and represents a species of Fe that is less bio available. Moreover, prior research has demonstrated that iron and arsenic co occur on the root surface of rice , which is consistent with the positive correlation between Fe and As measured in the DCB extracted plaque for all treatments in the current study .

This research also found that when NRT1.1 was phosphorylated at a low nitrate concentration, it was involved in maintaining the low level primary response; when it was dephosphorylated under a high nitrate concentration, it led to a high level primary response. More recent work has shown that NRT1.1 mediated regulation is quite complex in that it activates distinct signaling mechanisms. Furthermore, a rice homolog of AtNRT1.1has been identified, and variations in this gene in the rice sub species indica have been identified as boosting the absorption of nitrate and the transport of nitrate from roots to shoots, and potentially enhance NUE in rice.Another important nitrate regulator is the transcription factor NLP7, which belongs to the NIN like protein family in Arabidopsis. The NIN protein family was originally found to function in the initiation of nodule development in legume species and these family members are conserved in higher plants and algae. The NIT2 protein is a homologue of the NIN family in Chlamydomonas and can bind to the promoter of the nitrate reductase gene. In Arabidopsis, NLP7 has been demonstrated to be an important positive regulator of primary nitrate response as the induction of the nitrate responsive genes NIA1, NIA2, NRT2.1, and NRT2.2 is inhibited and nitrate assimilation is also impaired in nlp7 mutants. The function of NLP7 in nitrate signaling was further confirmed by the identification of the nlp7 mutant in an effort to explore novel nitrate regulators by using a forward genetics approach. ChIP chip analysis revealed that NLP7 could bind 851 genes including genes involved in N metabolism and nitrate signaling, such as NRT1.1, CIPK8, LBD37/38, and NRT2.1.

A recent study found that NLP7 could regulate the expression of NRT1.1 in the presence of ammonium and bind directly to the promoter of NRT1.1. These findings illustrate that NLP7 works upstream of NRT1.1 directly when ammonium is present. NLP7 can also activate or repress nitrate responsive genes. The Arabidopsis thaliana genome encodes nine NLPs,vertical planters for vegetables all of which contain the conserved RWP RK domain and the PB1 domain. Members of this family can be divided into four subgroups: NLP1 and 2, NLP4 and 5, NLP6 and 7, and NLP8 and 9. Yeast one hybrid screening using four copies of the nitrate response cis element illustrated that all NLPs could bind to the NRE element. In response to nitrate, the transcript levels of NLP genes are not regulated, but examination of an NLP7 green fluorescent protein fusion revealed that localization of NLP7 was modulated by nitrate via a nuclear retention mechanism. Recently, this localization of NLP7 was identified to occur when Ser205 in NLP7 was phosphorylated in vivo in the presence of nitrate. Suppression of the NLP6 function resulted in the down regulation of nitrate responsive genes, indicating that NLP6 is also a master nitrate regulatory gene involved in primary response. Further characterization has shown that the N terminal region of NLP6 is necessary for its activation in response to nitrate signaling. Furthermore, using over expression lines, NLP7 was revealed to significantly improve plant growth under nitrogen poor and rich conditions. Moreover, ZmNLP4 and ZmNLP8, maize homologs of AtNLP7, play essential roles in nitrate signaling and assimilation and promote plant growth and yield under low nitrate conditions, implying that they may be potential candidates for improving the NUE of maize. In addition to NLPs, reverse genetics has identified LBD37/38/39 to be important nitrate regulators. LBD37/38/39 belong to a gene family encoding zinc finger DNA binding transcription factors and are strongly induced by nitrate.

Further characterization revealed that over expression of LBD37/38/39 can repress the expression of nitrate responsive genes including NRT2.1, NRT2.2, NIA1, and NIA2, indicating that the three LBD members function as negative regulators in nitrate signaling. Recently, following advances in bio informatics and global sequencing analysis, systems biology approaches have been developed and successfully applied to plant nitrogen research. The transcription factors SPL9, TGA1, and TGA4 have been sequentially identified by systems approaches. SPL9 was predicted to be a potential regulatory hub and may target sentinel primary nitrate responsive genes. Research has demonstrated that miR156 can target SPL9 and a mutation in the miR156 caused over expression of SPL9. Accordingly, miR156 resistant SPL9 transgenic plants were investigated and it was revealed that the transcript levels of NRT1.1, NIA2, and NIR significantly increased in response to nitrate, demonstrating that SPL9 plays a negative role in the primary nitrate response. TGA1 and TGA4 belong to the bZIP transcription factor family and are induced by nitrate in roots. Interestingly, induction of TGA1 and TGA4 is inhibited in chl15 and chl19 mutants after nitrate treatment, implying that the regulation of TGA1 and TGA4 by nitrate is affected by nitrate transport, but not the signaling function of NRT1.1. Transcriptome analysis of the roots of tga1 tga4 double mutant plants revealed that most of the genes differentially expressed in the double mutant were regulated by nitrate. Among these target genes of TGA1 and TGA4, induction of NRT2.1 and NRT2.2 was substantially reduced in the double mutants. Further analysis demonstrated that TGA1 could bind to NRT2.1 and NRT2.2 promoters to positively regulate their expression . These results all serve to suggest that TGA1 and TGA4 play important roles in the primary nitrate response.Recently, Shuichi’s lab found that nitrate inducible GARP type transcriptional repressor1 proteins act as central regulators in nitrate signaling.

Co transfection assays revealed that NIGT1 clade genes including NIGT1.1/HHO3, NIGT1.2/HHO2, NIGT1.3/HHO1, and NIGT1.4/HRS1 were all induced by nitrate and were redundant in repressing the nitrate dependent activation of NRT2.1. EMSA and chromatin immunoprecipitation–quantitative PCR analysis further showed that NIGT1.1 could directly bind to the promoter of NRT2.1. Transcriptome and co transfection analysis also illustrated that the expression of NIGT1s was auto regulated and controlled by NLPs. In addition, NIGT1.1 can suppress the activation of NRT2.1 by NLP7. Further investigation suggested that the regulation of NRT2.1 by NIGT1.1 and NLP7 is independent due to their distinct binding sites. A genome wide survey discovered the potential target genes that might be regulated by both NLP mediated activation and NLP NIGT1 transcriptional cascade mediated repression or the NLP NIGT1 cascade alone. Furthermore, phosphate starvation response 1 , the master regulator of P starvation response, also directly enhanced the expression of NIGT1 clade genes,vertical farming technology serving to reduce nitrate uptake. CIPK8 and CIPK23 are calcineurin B like interacting protein kinases. CIPK8 is induced rapidly by nitrate and down regulated in the chl15 mutant. Analysis of two independent T DNA insertion lines showed that induction of NRT1.1, NRT2.1, NIA1, NIA2, and NiR by nitrate was reduced in cipk8 mutants indicating that CIPK8 works as a positive regulator in the primary nitrate response. Further investigation revealed that CIPK8 regulated the nitrate induced expression of NRT1.1 and NRT2.1 under higher but not lower nitrate conditions , suggesting that CIPK8 functions as a positive regulator when nitrate is replete. CIPK23 can be induced by nitrate and downregulated in the chl15 mutant like CIPK8. Expression of the nitrate responsive gene NRT2.1 was upregulated in the cipk23 mutants after nitrate treatment, indicating that CIPK23 plays a negative role in primary nitrate response . This gene is essential for the affinity switch of NRT1.1: it interacts with NRT1.1 and phosphorylates NRT1.1 at T101 under low nitrate concentrations to enable NRT1.1 to operate as a high affinity nitrate transporter, while it dephosphorylates NRT1.1 when nitrate is replete so that NRT1.1 functions as a low affinity nitrate transporter. CPK10, CPK30, and CPK32 are subgroup III Ca2+ sensor protein kinases .They have all been identified as master regulators that orchestrate primary nitrate responses. Analysis of the single cpk10, cpk30, and cpk32 mutants has shown that they only trivially affect nitrate responsive genes. However, in the double mutants cpk10 cpk30, cpk30 cpk32, and cpk10 cpk32 and the triple mutant cpk10 cpk30 cpk32, nitrate responsive marker genes were reduced. Transcriptomic analysis showed that CPK10, CPK30, and CPK32 modulated various key cellular and metabolic functions immediately activated by nitrate. Furthermore, CPK10, CPK30, and CPK32 can phosphorylate NLP7 at Ser205 in vivo in the presence of nitrate, and trigger the nitrate CPK NLP signaling network. Recently, three other nitrate regulatory genes NRG2, CPSF30L, and FIP1 were identified using a forward genetics method. Two independent NRG2 T DNA insertion lines showed reduced induction for nitrate responsive sentinel genes , indicating that NRG2 plays an essential role in nitrate signaling.

At the physiological level, NRG2 affects accumulation of nitrate in plants. Further investigation revealed that it regulates nitrate uptake by roots and the translocation of nitrate within plants. These effects might be achieved through modulating NRT1.1 and NRT1.8 as the expression of both genes was altered in the mutants. Genetic and molecular data suggest that NRG2 can regulate the expression and work upstream of NRT1.1, but function independently, with NLP7 in regulating nitrate signaling. In addition, transcriptomic analysis showed that four clusters in the differentially expressed genes in nrg2 mutant were involved in the regulation of nitrate transport and response, confirming that NRG2 plays essential roles in nitrate regulation. Interestingly, NRG2 can directly interact with NLP7 in vitro and in vivo, as revealed by yeast two hybrid and BiFC experiments. All these results demonstrate that NRG2 is an important nitrate regulator. In addition, the Arabidopsis genome harbors 15 members that are homologous with the NRG2 protein. All members of the NRG2 family contain two unknown conserved domains: DUF630 and DUF632. Whether and which other members of the NRG2 family are involved in nitrate signaling and what functions the two domains play are interesting and pertinent directions for future research. The CPSF30 gene encodes 28 kD and 65 kD proteins. The 28 kD protein was identified as a cleavage and polyadenylation specificity factor; the protein contains three characteristic CCCH zinc finger motifs and functions as both an endonuclease and an RNA binding protein. An additional YTH domain, along with the three zinc finger motifs, are contained in the 65 kD protein. A mutant allele of CPSF30, cpsf302 with a G to A mutation in the first exon of gene CPSF30, was identified by genetic screening and used to explore the functions of CPSF30. The expression of nitrate responsive genes can be down regulated in response to nitrate in cpsf302 compared to wild type and restored to wild type levels in a complemented CPSF30L/cpsf302 line, indicating that CPSF30L is involved in nitrate signaling. CPSF30L can regulate nitrate accumulation and assimilation at the physiological level. Transcriptomic analysis showed that genes involved in six nitrogen related clusters, including nitrate transport and assimilation, were differentially expressed in the cpsf302 mutant. Further study revealed that CPSF30 could work upstream of NRT1.1 and independently of NLP7. CPSF30 can also affect NRT1.1 mRNA 30 UTR alternative polyadenylation. All these results demonstrate that CPSF30 plays an important role in the primary nitrate response. FIP1, a factor interacting with poly polymerase 1, was identified as a positive nitrate regulatory gene using the fifip1 mutant and a FIP1/fifip1 line. Nitrate induced expression of NIA1, NiR, and NRT2.1 is repressed in the fifip1 mutant and can be restored to the wild type in the FIP1/fifip1 line. Furthermore, FIP1 can affect nitrate accumulation through regulating the expression of NRT1.8 and nitrate assimilation genes. Further research found that FIP1 could interact with CPSF30 and both genes can regulate the expression of CIPK8 and CIPK23. In addition, FIP1 can affect the 3 0 UTR polyadenylation of NRT1.1, a similar function to CPSF30. CPSF30, FIP1, and some other components such as CPSF100 can form a complex involved in poly processing. Together, these findings suggest that the complex composed by CPSF30 and FIP1 may play important roles in nitrate signaling. In the extant literature, key molecular components involved in primary nitrate responses, covering nitrate sensors, transcription factors, protein kinases, and polyadenylation specificity factors, have been identified. Methodologically, this has been achieved by using forward and reverse genetics as well as systems biology approaches . In summary, in the presence of both ammonium and nitrate , NRT1.1 functions as a sensor. NLP7, NRG2, and CPSF30 have been revealed to work upstream of NRT1.

There was a general pattern that suggested increasing salinity led to decreasing tissue water potential. However, only E. californica, F. salina, and J. carnosa showed a significant change in water potential as salinity increased. The observed change was likely due to increased solute concentration in the tissue to compensate for higher solute concentration in the soil . As soil solute concentrations increase, it becomes more difficult for plants to take up water. In response, plants can concentrate solutes in their tissue, creating a hypertonic state that allows continued passive uptake of water . Lack of response to increasing salinity for some species may have resulted from insufficiently stringent treatments. Our highest salinity treatment was 60% seawater, whereas plants in the low ecotone can experience inundation with full strength seawater. The average low marsh soil has a salinity concentration of 43.9 ppt , while seawater averages about 34.9 ppt, indicating that some species can survive 125.79% seawater. Because our plants only received 60% seawater, or roughly half the concentration plants can experience in the field, it would be useful to repeat this greenhouse experiment with higher salinity treatments. Drought effects can be similar to salinity effects, causing plants to become hypertonic to increase water uptake . Drought significantly affected all species, causing a decrease in water potential when compared to the saturation treatment. It should be noted that although our drought treatment had a significant effect, it is unlikely to replicate true field conditions. To avoid mortality in the greenhouse, plants received water every three to four days, which differs greatly from natural rainfall patterns, even in the wet season. During the dry season, soil moisture is often between 30%  80% of sample weight for marsh and low ecotone soil cores in the high ecotone and upland locations, soil moisture accounts for 0%  30% of sample weight.

We have no data showing whether we achieved similar conditions with potting soil in the greenhouse, and regardless,hydroponic vertical farm we would expect more rapid drying in pots than for in situ field soil. Thus, as for all greenhouse studies, results presented here should be used with caution when predicting performance in the field. To expand on these results, the greenhouse experiment should be repeated using native marsh soil as the substrate and including higher salinity treatments . Response to treatment in marsh soil should provide a more accurate prediction of response to field conditions. Surprisingly, measured differences in water potential did not translate to plant performance. Neither growth nor survival were visibly affected by watering treatment, even in potentially stressful low volume / high salinity treatments. Existing literature suggests that halophytes concentrate solutes to generate low tissue water potential, allowing continued passive uptake of water. In this case, low tissue water potential is not detrimental, since it prevents or reduces water deficits that can impair growth. Another possible reason for the lack of effect on growth was timing of the experiment. We began the experiment in June, when most individuals were beginning to reproduce. Beyond this point, energy is less likely to be allocated to vegetative growth and more likely to be allocated towards reproduction or survival strategies, like salt management . In contrast, younger plants allocate the majority of their energy to vegetative growth . Adaptations, such as salt glands or specialized vacuoles, areenergy expensive and require energy normally allocated to growth . Additionally, decreasing water potential has been shown to inhibit cell expansion , which would disproportionately affect young plants, since the rate of cell expansion in mature plants is reduced. Therefore, by better aligning the experimental period with the natural growth period, and focusing on young plants, treatment effects on growth might become more apparent. D. spicata displayed the greatest variability in tissue water potential, and this variability may have been influenced by factors other than watering treatment. D. spicata was grown in shallower, wider pots in a sandier potting medium. In both volume treatments, water would drain quickly through the pots, leading to uneven soil saturation that likely affected treatment efficacy and making it difficult to draw definitive conclusions regarding the large range in water potential.

However, low water potential values are not uncommon for D. spicata. Other authors have observed sustained, highly negative water potential used to compensate for soil salinity . The highest D. spicata mortality in our experiment occurred in the drought treatments, with three out of four deaths in the 60% seawater drought treatment. Nonetheless, increased drainage and evaporation rates likely contributed to mortality for this species. E. californica was affected by both the drought and salinity treatments, causing lower water potential and a slight negative effect on growth. Interestingly, our results contrast with those from another study. Jong measured E. californica net dry weight when irrigated with a saline Hoagland solution in sandy soil, using artificial sea salt instead of seawater. The water potential of their maximum salinity treatment was similar to our 60% seawater treatment, but the authors found that dry weight of E. californica decreased significantly as salinity increased. This experiment used young E. californica seedlings  the first tissue harvest occurred when seedlings were one month old and continued every 8 days until all plants were harvested, with the authors noting a difference in dry weight between treatments . Since we did not observe a difference in above ground biomass, the contrasting results may be due to the misalignment of experiment start time with the natural growth period. F. salina did not show an effect of salinity and drought stress on total plant growth, since biomass was maintained across treatments. In contrast, Barbour’s and Davis’s results showed a decrease in F. salina’s growth as salinity increased, with total mortality at approximately 89% seawater Hoagland solution . Plants in their non saline control showed the most growth, measured by the length of the main and lateral shoots . The majority of our plants remained constant in size.

The high mortality rate across treatments was driven by aphid infestation, despite attempts to control aphids with Botanigard . The highest mortality occurred in the drought, 60% seawater treatment,vertical farm suggesting that stringent growing conditions may have made plants more susceptible to aphid induced mortality. J. carnosa was the only species that added biomass between the first and final surveys. However, growth did not differ across treatments . One study found that J. carnosa grew best in non saline or minimal saline environments , using recently germinated individuals with stalks that extended 1 10cm above the growing substrate . In contrast, two other studies found that J. carnosa can tolerate salinities twice as concentrated as seawater, but moderate salinity conditions were ideal . St. Omer and Schlesinger used Hoagland solution in a greenhouse experiment to determine that maximum J. carnosa growth, measured by total dry weight, occurred at about 30%  60% NaCl, with growth decreasing above 60% salinity. They did not record plant age . The age of the plants likely impacted the differences in growth among studies due to the difference of energy allocation between mature and immature plants, which would have been exacerbated with higher salinity. Barbour and Davis used younger plants, which may have been more sensitive to treatment effects compared to the St. Omer and Schlesinger experiment , and the results reported here. Our experimental results align more closely with those of St. Omer and Schlesinger , even though our experimental design was more similar to Barbour and Davis . The experiment should also be repeated with younger plants to determine if age has any effect on salinity and drought tolerance. Other experiments that used younger plants observed a decrease in growth or total biomass as salinity levels increased, contrasting with our finding that plants are largely unaffected by salinity. Seedlings are more desirable to use in revegetation operations due to the reduced propagation cost and transplant effort, so it is important to determine the range of conditions young plants can tolerate. Our experiment addressed a knowledge gap regarding halophyte salinity and drought tolerance that could inform the design of future restoration projects and experiments in Pacific coast salt marshes. Revegetation efforts often have low success rates due to the stringent abiotic conditions within the ecotone, which disproportionally affect seedlings . Furthermore, the different natural distributions of halophytes within the ecotone suggest that salinity and drought tolerance could vary among species. In our experiment, treatments had negligible effects on growth or survival – only water potential was affected. These results imply that these five species could survive anywhere within the ecotone, by employing different physiological adaptations – such as succulence, salt glands – to withstand stressful conditions.

However, our results are likely not representative of plant performance in the field due to a variety of factors. The timing of our experiment did not align with the natural growth period of the plants, causing us to use mature plants rather than young seedlings. Additionally, our use of 60% seawater is not representative of the tidal inundation that some of the species may experience in the field. Therefore, future experiments will examine how these factors influence outcomes, using lessons learned during this effort. Taken together, findings from this set of experiments will allow us to 1) identify zones within the ecotone maximizing survival and establishment on a by species basis, or 2) demonstrate that species are flexible enough to compensate for conditions across the ecotone, making careful placement of species unnecessary. In either case, these experiments will provide valuable insight to restoration practitioners. Ultimately, we hope that this work will support rapid and robust strategies to recreate thriving salt marsh systems. Plant growth and yield in natural environments depend on a plethora of interactions with bacteria and fungi. The microbial community associated with roots was proposed to be assembled in two steps: first, the rhizosphere is colonized by a subset of the bulk soil community and, second, the rhizoplane and the endosphere are colonized by a subset of the rhizosphere community. Intriguingly, a set of recurring plant associated microbes has emerged. This review focuses on how plants shape their rhizobiome. On the one hand, common factors among plants likely lead to the assembly of the core microbiome. On the other hand, factors specific to certain plants result in an association with microbes that are not members of the core microbiome. Here, we discuss evidence that plant genetic factors, specifically root morphology and root exudation, shape rhizobiomes. Initial evidence for an influence of plant genotype on rhizobiome composition was that similar rhizobiomes assembled in association with arabidopsis and barley grown in the same experimental conditions, although they displayed different relative abundances and some specific taxonomic groups. A correlation between phylogenetic host distance and rhizobiome clustering was described for Poaceae species, distant relatives of arabidopsis, rice varieties, and maize lines, but not for closely related arabidopsis species and ecotypes. Distinct rhizobiomes were also described for domesticated plants, such as barley, maize, agave , beet , and lettuce , compared with their respective wild relatives. Interestingly, not all plants have a rhizobiome distinct from bulk soil: some species, such as maize and lotus, have assembled a distinct rhizobiome, whereas other species, such as arabidopsis and rice, assembled a rhizobiome similar to bulk soil. The former species display a strong, and the latter a weak rhizosphere effect . The cause of this phenomenon is currently unknown. The strength of the rhizosphere effect varies with the developmental stage of the plant. Similarly, root exudation and microbial communities were found to change with the age of the plant. Furthermore, distinct rhizobiomes were associated with different developmental stages of arabidopsis, rice, and Avena fatua grown during two consecutive seasons. Pioneering studies demonstrated the ability of microbes to alter plant development. Overall, it appears evident that host genotype, domestication, and plant development influence the composition of rhizobiomes. As an alternative to plant developmental stage, residence time of plants in soil was discussed as a hypothesis for successive microbiomes. These contrasting results might be partially explained by differing environmental influences, host plants, or soils, and additional work is needed to resolve these questions. In this review, we discuss root morphology and root exudates as two genetic factors shaping plant–microbiome interactions, and we examine the following aspects: how root morphology and border cells affect rhizobiomes; how plant exudates shape the rhizobiome; and possible plant transport proteins involved in exudation. Figure 1 provides a general overview of exometabolite networks in the rhizosphere, and Box 1 illustrates the interplay between root exudates, border cells, and rhizobiomes in phytoremediation.