Horticulture

Cultivars Sonora, Foisy, and Chiddam Blanc de Mars were selected. The primary criterion was the total root biomass with Sonora ranked among the highest, Foisy was intermediate, and CBdeM had a low total root biomass. Additionally, the three also have other contrasting phenotypes for traits such as drought tolerance, plant height, days to heading, awn type, and seminal root angle. These parents are what could be considered as “traditional cultivars” in that they were all selected from land races. Sonora was selected from a land race in Durango, Mexico and is known for good drought tolerance but its height makes it susceptible to lodging. Cv. Foisy was selected by Mr. Foisy in Oregon in 1865 and typically yields more than CBdeM and Sonora. CBdeM originates from Ville de Paris, France, and was selected from an English land race. None of these cultivars have a place in commercial agriculture today but still are grown by traditional or artisan farmers as so called heirloom varieties of wheat for bread making. More information about the parents can be found on the UC Davis small grains web page in the 2011 California cultivar descriptions publication . Crosses were made in a triangular manner to form a set of “nested” mapping populations with any given two populations having a single parent in common so that we get the populations Sonora x CBdeM , Sonora x Foisy , and CBdeM x Foisy.

This design provides a built in system for verification of QTLs across populations and genetic backgrounds. For each of the three populations ca. 200 lines were planted on July 11th, 2013 in an air-conditioned greenhouse on the UC campus in Riverside, California,growing lettuce hydroponically in one gallon pots with two plants per pot. These were used for seed increase, leaf tissue for DNA extraction, and for phenotyping of simple traits. Doubled haploid plants in each pair were compared and expected to be identical, however, not all were and any lines with clear differences between the two plants were discarded. A second seed increase was planted on April 4th, 2014 in a similar manner and was also used to collect phenotype data. During this increase plants were grown under 18 hour days with supplemental lighting. In 2015 a two-location field trial was established. Experiments were planted in October 2015 and harvested by May 2015. The two locations were the University of California, Riverside Agricultural Experiment Station in Riverside, California, and at the Coachella Valley Agricultural Research Station . Experiments were set up in randomized augmented designs with three check varieties replicated in each block. The check varieties were Blanca Grande 515, Summit 515, and Cal Rojo.

There were 32 blocks per treatment per location with 16 plots per block. Additional “blank” plots of Summit 515 were planted to make blocks square but were not included in the analysis. Each plot consisted of six rows spaced 20cm apart and 122cm long planted at a density of 560 seeds per plot. Each location had two treatments, one well-irrigated and the other which received limited irrigation after 60% of the genotypes were booting. The well-irrigated treatment received water as needed based upon soil moisture and plant indications. For the limited irrigation treatment water was withheld until plots showed moderate to severe wilting at which point they were irrigated to prevent death.The R statistical package “lmerTest” was used to obtain the predicted mean values for all traits evaluated which included; days to heading, plant height, yield/m2 , and 1000 grain weight . SNP calls from Genome Studio were converted into “A” and “B” genotypes by comparison against parental scores for each population. Markers that were polymorphic between parents for each population were imported to JoinMap 4.1 and used to construct linkage maps. Chromosome and marker index number were used to name markers previously mapped by Wang et al. , for example 5A_6716, and markers that were not previously mapped were named using an underscore and the marker index number with no chromosome indication. Identical individuals were excluded from the genotypes used to construct linkage maps and likely arose as artifacts from the DH procedure or were a result of labeling errors.

Also, individuals with greater than 10% missing data for marker calls were excluded. Initially linkage groups were generated based upon markers mapped by Wang et al. using eight doubled-haploid mapping populations. This was done on a chromosome by chromosome basis including only markers mapped to a given chromosome. Identical markers for the given chromosome were removed prior to mapping. Groupings were made using the default calculation settings for independence LOD and linkage groups were mapped using the default settings for the maximum likelihood algorithm. For linkage groups that failed to generate maps or lacked a sufficient number of markers, additional markers were added from the unmapped pool of SNPs. To do this, all unmapped markers were selected along with the mapped markers for the given chromosome and then the steps listed above were repeated to give new linkage groups with a more suitable number of markers. These newly added markers were then BLASTed against the wheat arm survey sequence to verify their correct linkage group assignment. Phenotypic data for awn type, days to heading, and plant height collected during 2013, 2014 greenhouse evaluations and 2015 field evaluations were used to map QTLs by the software package ICImapping . For greenhouse data, the linkage maps and mean value for two plants of each doubled haploid line were used to map QTLs and for field data the predicted mean values for each genotype were used. The composite interval mapping method with a step of 1 cM was used and the threshold for QTLs detection was determined using 1000 permutations at α = 0.05. Originally, populations SC, SF, and CF consisted of 257, 244, and 214 lines, respectively. During the 2013 seed increase populations were assessed for vernalization requirement, hybrid necrosis,ebb and flow table and uniformity. Of the 200 lines planted for SC, SF, and CF population about 1, 2, and 7%, respectively, showed what appeared to be segregation but could have arisen from multiple unknown reasons. Winter growth habit appeared in 18.5, 7.5, and 1.5% of the SF, CF and SC populations, respectively. This was despite the fact that all three parents are spring wheats and require no vernalization. The appearance of winter growth habit may reflect some combination of recessive alleles of vernalization genes . Hybrid necrosis was rated on a scale of 1 to 10 with 1 being minor and 10 being lethal. Hybrid necrosis was fairly prevalent in SF with 31% of the genotypes showing some level of the phenotype; the CF population had 25% of the genotypes showing some level of hybrid necrosis and the SC population only 6.5%. Any genotype with a score greater than 3 was excluded from genotyping. Because of possible contamination , sterility and with some lines showing winter growth habit and/or hybrid necrosis, populations were reduced in size. Additionally, population sizes had to be limited to genotyping of 150 lines for several reasons, from the cost/practicality issue to future experiment manageability. However, all non-genotyped lines are preserved and can be accessed, if needed. During the 2014 greenhouse evaluations the remaining 150 lines for each population were characterized for vernalization requirement, days to heading, plant height, and awn type . For field trials populations were reduced to 133, 121, and 115 lines for SC, SF, and CF respectively due to winter habit or late flowering of some lines and lack of seed for others.

During the 2015 field evaluations populations were characterized for days to heading and plant height, awn type and 1000 grain weight . However, not all field data could be analyzed and/or were unreliable, thus distributions of trait values in the field for days to heading, plant height at Coachella Valley are shown in Figure 1.3 for SC, SF, and CF.Using phenotypic data collected during the greenhouse evaluations and the field evaluations QTLs were mapped to verify the quality of the genetic maps and provide some basic genetic information about the three populations. Table 1.1 summarizes regions that were mapped in the SC, SF, and CF populations for awn type, days to heading , plant height , and hybrid necrosis . Only the regions consistent through multiple years are discussed. Table 1.2 demonstrates how QTLs can be verified within and between populations by using awn type QTLs as an example. For simplicity of the exercise lines were classified into two groups: awned and awnless. Two genome regions were consistently identified that explained 23.76 – 92.67% of the phenotypic variation within the populations. The first locus was consistently identified in the SF and CF populations on chromosome arm 5AL. The QTL in the SF population covered a 2.2 cM region between the markers 5A_9620 and 5A_6716 with the peak around 266 cM explaining 36-39% of the phenotypic variation across all years and environments. In the CF population, the QTL covers a 3.2 cM region between 5A_9620 and 5A_6716 with the peak around 169 cM. The QTL explains 31-93% of the phenotypic variation observed in the population across all years and environments. This QTL shares the same two markers in common with the QTL identified in the SF population. Additionally, Mackay et al. mapped the same QTL using a wheat MAGIC population and verified it using an association mapping population. They identified the marker BobWhite_c8266_227 as being the closest linked to the QTL which in these populations mapped to the same genetic location as 5A_6716 identified here. The second QTL was consistently identified in the SC and SF populations as being on the chromosome arm 6BL. In the SC population the QTL covers a 4.2 cM region between markers 6B_606 and 6B_1614 with the peak around 103 cM. It explains 62-73% of the phenotypic variation for this population across all three years and two environments. In the SF population the QTL covered a 0.71 cM region in 2013 and 2014, and a 2.1cM region in 2015 with the peak being around 79 and 80 cM respectively. This QTL explains 23-29% of the phenotypic variation in the population. In 2013 and 2014 the QTL was between 6B_45514 and 6B_606, however, in 2015 it shifted by a couplemarkers to 6B_68633 and 6B_84 covered a larger region. However, its peak was still near the same point and the two markers associated with the phenotype in 2013 and 2014 were present in the 2015 region. The QTL shares the 6B_606 marker in common with that identified in the SC population . These results indicate that Sonora carries the dominant allele for B2 on 6BL and that Foisy has the dominant allele for B1 on chromosome arm 5AL. Since CBdeM is fully awned it must have the hd b1 b2 genotype. The trait „heading date‟ or „days to heading” in wheat is determined by several factors, including vernalization requirement controlled by the Vrn genes , the photoperiod genes play a role in determining the sensitivity to photoperiodism and the Earliness per segenes are responsible for controlling flowering time regardless of photoperiod. In the three populations studied here, five major QTLs were found responsible for the heading date character, located on chromosomes 2D, 5A, 5B, and 5D. Two consistent QTLs on chromosome 2D were identified in the SC and CF populations in 2015. In the SC population the QTL covers a 0.67 cM region with its peak around 112 cM between markers 2Dx_32130 and 2Dx_79444. This QTL explains 18.43% of the phenotypic variation seen in the population and has an average additive effect of 7.69 days. In the CF population the QTL covered a 5.8 cM region with a peak around 47 cM between markers 2Dx_7001 and 2Dx_13208. This QTL explains 70.06% of the phenotypic variation in this population and has an average additive effect of -14.24 days. These QTLs are most likely the Ppd-D1 gene described by Beales et al. . Sonora contributed the day length sensitivity allele in the SC population and Foisy contributed the allele in the CF population. This explains why no segregation for the locus was seen in the SF population.

Food is a powerful frame through which to make the climate change problem more concrete and “close to home,” as it implies both a social and essential daily activity. The garden, meanwhile, provides a useful metaphor for the complex global climate system. The curriculum directly connects climate science to community and local action in the garden, thus linking food and climate systems. This systems-thinking lens aligns with Next Generation Science Standards , something that motivates teacher participation if their schools have adopted NGSS. Through local examples, garden activities and guest speakers, the curriculum connects students to other change makers and empowers them with agency to help build a more sustainable food system in their community. Students learn to think of climate change as more than “just” a science problem: it is a social problem requiring action and responsibility from all levels of society—individual to international. Each of the six lessons involves students in activities that translate regenerative agriculture theory into practice . The curriculum provides opportunities for students to learn scientific facts ,hydroponic grow systems share personal narratives , and enact hands-on solutions to climate change via school gardens .

They learn about the negative effects of elevated CO2 in the atmosphere globally and then help lower CO2 locally through increasing plant photosynthetic activity. The pedagogical framework for the curriculum is inspired by Paolo Freire’s critical pedagogy and other more current framings of a signature pedagogy for sustainable food systems education . Educators facilitate collective learning experiences that are often subversive in nature and seek to disrupt inequitable outcomes, both environmental and social. Curriculum implementation followed a co-teaching model. The researcher-teacher partnership draws on complementary domains of expertise: content expertise from the researcher, and classroom management/student dynamic expertise from the teacher. Two symbiotic goals are addressed using co-teaching as an implementation method: 1) students learn climate change from a content expert, and 2) teachers increase knowledge and competence in climate change instruction, allowing future students to benefit from a better-trained instructor and serving as a form of professional development. Studies have shown repeatedly that the best way to improve student performance across a range of subjects is to boost teacher knowledge and competency . This type of participatory, co-teaching implementation inherently limits ability to statistically analyze a large, representative, or randomly generated dataset of students. It is grounded in social science theory of the qualitative, in-depth case study.

Each school required slightly different implementation of the curriculum. In one case snow days canceled several coteaching sessions, which then had to take place via Skype. Taken as a whole, these four cases shed light on important adjustments that can be made to tailor climate change education interventions to site-specific school needs. Pragmatically, meeting unique school needs is a prerequisite for implementing any non-mandatory education intervention in partnership with schools. The study simultaneously investigates student responses to an experiential climate curriculum, and teacher responses to co-teaching as a form of professional development. The methods used for evaluating curriculum efficacy include 1) semi-structured teacher interviews, 2) student surveys , and 3) participant-site observation. Triangulation of these methods improves the validation of results. Deeper understanding can be gained from a small set of cases on CCE, and best practices can then be applied to a larger universe of schools. More specific to each method, teacher interviews followed a six-question interview guide and were semi-structured in nature. Preliminary student surveys provide a baseline for student knowledge and engagement. Compared with post-intervention surveys, this allows basic statistical analysis to define the effect size in the sample population and whether it is significant. The survey assessment includes 10 knowledge-based questions on climate science and food systems applications, as well as 19 engagement questions asking opinion statements measured on Likert-type scales.

This multi-faceted assessment of climate literacy recognizes that “knowledge about climate change can be divided into several general and overlapping categories: knowledge about how the climate system works; specific knowledge about the causes, consequences, and potential solutions to global warming; contextual knowledge placing human-caused global warming in historical and geographic perspective; and practical knowledge that enables individual and collective action” . The engagement questions adapt the Six Americas survey questions to capture students’ change in engagement towards climate change following the curriculum intervention. Participant and site observation over a six week period captures important features of the school climate, both environmental and social, that help contextualize interpretation of results. The quality of the school garden, behavioral norms, and student informal interactions are all variables of interest for understanding other forms of data collection. In climate literacy evaluations, it is important to understand student intention to take action and follow up to document concrete examples of students taking action, which goes beyond simple survey and interview protocols. Certainly, questions can be posed to students asking whether they feel more empowered to seek out their own additional knowledge and participate in climate actions, but ideally these questions can be followed up with evaluation tools documenting actual action outcomes. This was not possible in the contexts of study reported on below but should be a focus for future student climate literacy evaluations. Results presented and discussed below are broadly relevant to climate change education interventions, with some insights as well into the value of food as an engaging entry point or frame for the climate education conversation. Attitude and engagement questions revealed higher levels of concern along the Six Americas spectrum than the national average. The first 10 questions were adapted almost directly from the Six Americas survey, with some modifications for student-friendly language. An additional nine questions were added dealing specifically with food systems, behavior and climate change. Based on the first 10 questions, students were categorized into the six segments from alarmed to dismissive, with almost all students falling in the top three categories . Students demonstrated an overall increase in engagement although this was difficult to measure with precision due to inconsistencies within individual student response patterns. A preliminary analysis is valid for determining directional effect arrows and assessing whether pilot programs show promise, and thus were adequate for this evaluation. Precision could be added in future iterations by simplifying answer scales so they are consistent, and then quantifying student attitudes on a numerical basis. The survey was a bit long to hold student attention, and survey fatigue was a confounding variable in some cases. Work is underway by the Yale Project on Climate Change Communications to create a four-question survey version for teens,vertical grow table which will be a valuable improvement for future studies. Informal observations and conversations reveal a notable curiosity and interest among youth in learning more about climate change. A commonly expressed sentiment, especially at the outset of the curriculum intervention, is that climate change is an important issue that students feel they should know more about. This is mirrored in national statistics reporting that American teens recognize their limited understanding of climate change, and 70% say they would like to know more about the subject . Post-intervention teacher interview themes revealed a widespread appreciation of coteaching as a mechanism for delivering climate change instruction. All teachers interviewed expressed enthusiasm for having a content expert present to deliver instruction on climate, complementing the garden teachers’ expertise in food-related topics, classroom management and student behavior. The positive response from teachers is important to contextualizing student results, as the more enthusiastic and knowledgeable teachers became about climate change connections in the school garden, the more engaging lessons became for students.Teachers were able to learn from the experience and expressed desire to replicate elements of the curriculum on their own in the future, thus meeting one of the process-specific goals of the research. Interviewees also revealed a common theme of searching for hope and action amidst the daunting reality of climate change; the garden and classroom were often identified as key arenas where hope and solution steps exist. Key quotes from interviews are highlighted in Table 15 below.These results, in particular the challenges highlighted by teachers, closely match national findings on climate change education.

In a recent national review of science teachers, the first nationally representative study of science educators to focus on climate change, fewer than half of all teachers reported any formal coursework on climate change, yet over two thirds would like targeted professional development opportunities to allow them to dive in deeper to this complex and emotionally sensitive topic . It is well established that teachers are in need of professional development in order to teach an unfamiliar subject with confidence and competence, and several national leaders in climate education are addressing this . Having a climate science “expert” in the classroom to co-teach a climate change curriculum for the first time is another promising form of PD explored here. Partnerships emerged as a key feature enabling success of food and climate education in schools, mirroring the findings in example 1 above. Partner organizations and individuals are able to provide infrastructure support, outdoor learning environments, guest speakers to reinforce climate education units, and program evaluation assistance. Questions of how to scale impact via partnerships at the district or state level and education policy implications are discussed below. Examining results by school context offers strategies for scaling this type of intervention in rural vs. urban school districts. Students at the Lopez school, with abundant local farm and forest resources to devote to furthering climate curricula endeavors, selected a bio-char experiment as a class climate action project, and will be applying locally produced bio-char to test plots in the school garden to compare with non-treated plots , in partnership with the community. This community-school partnership adds to the body of successful climate change engagement strategies meriting replication, particularly other rural communities where local farmers might be interested in participating in farm to school programming at the school or district level. Experiential food and climate change education is an emerging branch of CCE with great potential, where the school garden provides one context for experiential climate learning while many others are possible . By emphasizing and teaching local forms of food production and consumption, this CCE example seeks to localize climate stewardship and in doing so reduce the carbon footprint of food system products and processes. The food-climate nexus diagram presented in Chapter 1 offers both an impetus for scaling this form of integrated food-climate education, and an example of how to do so while visualizing food-climate interactions. This chapter reports on initial positive results from integrating CCE into both the humanities and school garden classrooms. In the case of humanities-focused CCE, students not only demonstrated gains in climate literacy, but also improved their reading comprehension. Sixth grade students performed at a level equivalent to their eighth-grade peers in terms of listing numerous climate mitigation strategies, and reported both looking up new information and speaking with friends/family about climate change more frequently than all other middle school grades. The examples from school garden classrooms more explicitly adopt and test the hypothesis that experiential CCE is more effective than didactic or lecture-based climate instruction. Results show improvements in student learning and strong student interest in the topic. However, further evaluation methodology development is needed to best capture the impacts on student action and behavior. In order to understand the efficacy of experiential CCE relative to CCE that is not experiential, a controlled experiment would be required that uses the same evaluation methodology for students with and without experiential CCE. This methodology would ideally comprise and observational element where teachers report on student “climate actions” over the course of a defined time period. In future studies, a list of core “climate actions” could be developed as a baseline for evaluators to assess whether students are carrying out these activities . Both food-focused and humanities-focused CCE point at an underlying characteristic of CCE. Rather than being treated as its own subject, or topic to be covered in science classrooms, climate change is an overarching frame that infuses all sorts of school activities, processes, and classrooms, from the transportation that bring students to school, to the food that is served in the cafeteria, to the content students are covering with their mathematics, physics, government, or garden teachers.

Civic engagement advances the idea of creating “public commons” through urban agriculture, an idea related to ecological economics and explored in David Bollier’s book Think Like a Commoner: A Short Introduction to the Life of the Commons. A commons “integrates economic production, social cooperation, personal participation, and ethical idealism into a single package;” it is a paradigm of “self-help and collective gain” and an “alternate self-governance structure for resource management and ‘living well’” . The commons paradigm espouses a political philosophy grounded in grassroots civic activism and proposes different “foundational premises for a new political economy” based on social connections and rediscovering “people’s knowledge” of natural systems in their local contexts . However, we must consider who is able to participate in creating such a space . Who participates, in both policy and urban farming as an activity, is a crucial factor in determining whether outcomes will subvert or reinforce existing power, privilege and structural inequities. As Ramírez states, “While recreating neglected urban spaces into ‘productive’ spaces to grow food is inspiring and beneficial on one level, the prevalence of white bodies inhabiting garden spaces reifies uneven geographies and catalyzes gentrifying forces” . It is the role of inclusive policy processes and watchdog citizen activists to counteract this retrogressive tendency of UA projects. One example of grassroots political action,vertical grow towers working around rather than through institutional channels of policy making, is the Catatumbo Collective’s people-to-people reparations project.

Developed by three immigrant women spearheading an urban agriculture organization in Chicago, the people-to-people reparations map locates minority-run farming projects on a map of the United States, providing a brief description of the project and their specific needs, and then a link or contact info so that visitors can donate directly to the project. They are motivated to publicize agricultural history from a minority person’s perspective and provide a means of public accountability through their mapping project, supporting “those who have borne the brunt of labor exploitation, land theft, and discriminatory agricultural policy” . This project has already led to funding for several farmers’ projects, as well as land gifts to create several minority-owned farms. While the founders recognize the need to continue litigation and action through formal policy channels, they honor the urgency of needing to “start right away” by facilitating “transfers of wealth.” They are also contributing to a more updated database of farmers of color, often underrepresented in USDA farm censuses. There is room for more participatory action research linking researchers to citizens and civic engagement projects . This will allow for data to be shared and transferred more easily, and for the network of UA and food justice participants to strengthen through ties to research institutions and each other. Researchers have an important role to play in addressing data gaps and strengthening the network of urban farmers who have clearly identified needs and are ready to work towards appropriate, measurable solutions.According to the literature, access to urban-produced foods is directly tied to the economic realities of urban farming operations.

Daftary-Steel, Herrera, and Porter make a compelling case for building coalitions to provide the necessary political and financial support to fund UA, as well as tackling the “root causes” of food insecurity through social services. From what limited studies exist, it seems clear that economic viability of urban farms is dependent on income far beyond sales capacity of the urban farm.It is important to communicate to policy makers that urban farms are producing a lot more than pounds of food; they are also “distributing” social goods, creating a “commons”, and providing a connection to nature, community, and education , and these in turn are part of improving community food security. The primary benefits of UA organizations are often education , social integration, economic opportunity, and local environmental quality improvements. Producing enough food to transition a community from “food insecure” to “food secure” is not necessarily going to happen through urban farming alone; however, supplementing food intake with locally produced, healthy fruits and vegetables is an important step in building food security and community health. As such, researchers and UA practitioners may consider generating more robust data on the health, environmental and social benefits of UA to promote among policy makers the idea of UA as a public good, worthy of public investment in the same vein as schools, transportation and education. This is especially true in U.S. cities without strong policies supporting the existence of UA, and with high land values and development pressures.

It is important to acknowledge that urban agriculture is not the only solution to food insecurity and food access and should not be the only forum of support/intervention from policymakers. In fact, in some cases “the emphasis on ‘grow your own’ reinforces self-help and government austerity arguments, absolving government of the responsibility to address the structural and institutional causes of food insecurity” . UA is part of the solutions portfolio to improve food justice and food access, but must be complemented and reinforced by other governance efforts to provide affordable, healthy food through neighborhood groceries, food hubs, cooperative markets, culinary and nutrition education programs, farm to school programs or other means of addressing structural causes of food insecurity . Civic engagement, critical scholarship from multidisciplinary perspectives, and alliances between housing, transportation, and food policy are all necessary components of a UA landscape that improves access and meets the needs of both producers and consumers. In conducting this literature review, using a combination of academic and gray literature, we recognize a significant gap between scholarship and practice. Urban agriculture is not a panacea that will automatically produce all the social, environmental, and economic “goods” attributed in the literature at large without proper structuring or policy frameworks in place. A more realistic, and holistic picture of urban agriculture can be advanced by further rigorous evaluation of what particular organizations are choosing to focus on, how much food they are producing currently , how they are distributing their food, and where they need support. It is not just about whether urban farms have the potential to feed food insecure people, but whether they actually do, depending on locally specific modes of distribution, channels of access, and policy climates. Key ideas from the literature about how to enable socially just, economically viable urban agriculture include a focus on food sovereignty, public investment and/or land rights, “agrihood” developments, and attention to disparate neighborhood race and class dynamics when siting UA operations. Researchers can address key data gaps including the actual tracking and consumption of urban-produced food. We can answer lingering questions including: where does the food go, how much is accessed vs. wasted, what are consumer preferences around accessing urban produced foods, and where do institutions need to fill in gaps in access and/or distribution channels? Results of this literature investigation are next applied to our ongoing study of urban agriculture in the East Bay region of the San Francisco Bay Area,container vertical farming characterized by a high amount of urban agriculture activities , yet undergoing rapid gentrification with persistent high levels of food insecurity and income inequality. Despite growing evidence of the diverse health, education, and environmental benefits of urban agriculture, these vibrant spaces of civic engagement remain undervalued by city policy makers and planners in the United States. Because urban farming takes on many different forms and functions, with intended outcomes that may or may not include yield and profits , thriving urban farms and gardens are under constant threat of conversion to housing or other competing, higher-value land uses due to rising land values, and other city priorities.

This land use challenge and threat to urban farm land tenure is especially characteristic of U.S. cities like San Francisco, one of the most expensive land and housing markets in the country. Under the current urban agriculture paradigm in the U.S., food justice scholars and advocates either try to quantify and highlight the multiple benefits of UA  or pursue a critical theoretical approach, arguing that urban agriculture can yield unfavorable results if pursued without an equity lens, especially in cities with intense development pressures and gentrification concerns . A productivist focus is problematic, because, while urban agriculture can be an important component of community food security, its other social and ecological benefits are just as, and sometimes more, significant . In this article, we suggest that the current debates around “urban agriculture” in the U.S. often lead to an unhelpful comparison with rural farms regarding yield, productivity, economic viability, and ability to feed urban populations, most notably in the policy arena. Defined in these ways, the radical, transformative potential of urban food production spaces and their preservation often gets lost or pushed to the side in city planning decisions in metropolitan regions such as the San Francisco Bay Area, where the threat of displacement is ubiquitous given high levels of economic inequality and extreme lack of affordable land. In order to facilitate what scholars such as Anderson et al. 2018a refers to as the “agroecological transition,” already underway in many urban food ecosystems around the globe , we argue that applying an agroecological approach to inquiry and research into the diversity of sites, goals, and ways in which food is produced in cities can help enumerate the synergistic effects of urban food producers. This in turn encourages the realization of the transformative potential of urban farming, and an articulation of its value meriting protected space in urban regions. Urban agroecology is an evolving concept that includes the social-ecological and political dimensions as well as the science of ecologically sustainable food production . UAE provides a more holistic framework than urban agriculture to assess how well urban food initiatives produce food and promote environmental literacy, community engagement, and ecosystem services. This paper presents a case study of 35 urban farms in San Francisco’s East Bay in which we investigated key questions related to mission, production , labor, financing, land tenure, and educational programming. Our results reveal a rich and diverse East Bay agroecosystem engaged in varying capacities to fundamentally transform the use of urban space and the regional food system by engaging the public in efforts to stabilize, improve, and sustainably scale urban food production and distribution. Yet, as in other cities across the country, they face numerous threats to their existence, including land tenure, labor costs, development pressure, and other factors that threaten wider adoption of agroecological principles. We begin by comparing the concepts of UA and UAE in scholarship and practice, bringing in relevant literature and intellectual histories of each term and clarifying how we apply the term “agroecology” to our analysis. We pay particular attention to the important nonecological factors that the literature has identified as vital to agroecology, but seldomly documents . We then present findings from a survey of 35 diverse urban farm operations in the East Bay. We discuss the results, showing how an agroecological method of inquiry amplifies important aspects of urban food production spaces and identifies gaps in national urban agriculture policy circles. We conclude by positing unique characteristics of urban agroecology in need of further studies and action to create equitable, resilient and protected urban food systems.Agricultural policy in the United States is primarily concerned with yield, markets, monetary exchange, and rural development. The United States Department of Agriculture defines agricultural activities as those taking place on farms. Farms are defined as “any place from which $1,000 or more of agricultural products were produced and sold, or normally would have been sold, during the year” . Urban agriculture has been proliferating across the country in the last decade on both public and private lands, as both for-profit and nonprofit entities, with diverse goals, missions and practices largely centered on food justice priorities and re-localizing the food system. Yet U.S. agriculture policy has been struggling to keep up. In 2016, the USDA published an Urban Agriculture Toolkit, which aims to provide aspiring farmers with the resources to start an urban farm including an overview of the startup costs, strategies for accessing land and capital, assessing soil quality and water availability, production and marketing, and safety and security .

The program uses an “integrated systems approach” to delivering hands-on education in nutrition, ecology, sustainability, and land stewardship . In practice, this consists of educating students in a garden classroom elective for elementary and middle school, hosting a high school farm elective course where students visit local farms, and preparing and preserving food from the school farm in the cafeteria, where local scratch-cooked meals are served year round. Part of the growing national movement around improving the quality of school meals through locally sourced produce, the LIFE program takes advantage of the exceptional quality of both locally produced foods and farm-based educational opportunities on the island. The LIFE program has been funded by a combination of a large private foundation and smaller donations, fundraisers, grants, and in-kind contributions. It is currently working towards a more sustainable, diversified finance model that will expand those invested in the program’s success as well as allow the educational activities associated with LIFE to grow. LCLT coordinates interns to support the LIFE program in the summer , and staff at the Family Resource Center run a volunteer-based gleaning operation on island orchards that yields up to 5,000 lbs. of fruit for the school cafeteria.

Production has grown steadily at the ½ acre school farm, from 1,400 to over 6,000 lbs. between 2009 and 2016 . The program is currently fundraising to purchase two beef cows raised by island teens for the cafeteria meat supply.The results of this research investigation led to the conclusion that students were not yet connecting their experiential learning activities in the school garden to bigger picture environmental themes and challenges, such as climate change,vertical farming companies greenhouse gas emissions, and the environmental impact of conventional food systems. The gaps in environmental knowledge, which forms an important but not complete piece of the environmental literacy equation became clear to teachers at the school, who sought to fill those gaps with new curricula. In partnership with Lopez school and LIFE staff, I developed a food systems and climate change 6-lesson curriculum, designed to engage students in questions at the climate and food nexus. The curriculum walks through the causes, effects, impact monitoring, and solutions to climate change, through the perspective of the food and agriculture sector. The culmination is a class or school-wide climate action project in partnership with the community, sparking informed hope through taking action. This curricular outcome of the LIFE program is evaluated and discussed further as a pathway to farm-based climate education and applied to other school contexts in Chapter 4. In addition to assessing environmental literacy outcomes, environmental impacts of the LIFE program were also investigated in 2015-2016. Using the Cool Climate Carbon Footprint Calculator for food-related emissions, eliminating the need to transport all meat, fruits and vegetables to the school could save 1.4 tons CO2 annually per person.

Adjusting this metric to account for the 9-month school year and multiplying by approximately 120 students who eat at the school cafeteria per day, this yields approximately 120 tons of CO2 savings annually. K-12 education is inherently social. The director of the Organic Farm School finds hope in the very nature of children working together in school gardens, that they might come to see farming as more social and collaborative, and thus more readily forge pathways into cooperative farming.Midnight’s Farm, a 100-acre property located near the center of Lopez Island, heralds the diversity of its operations from the initial entry point down a gravel driveway. A signpost indicates the direction of the compost operations, yoga studio, farm stand selling beef, pork and vegetables, and wood-fired bakery . In the words of the farm owners, “we farm to steward this wonderfully beautiful piece of earth and for the tangible, hands-dirty love of connecting people to the soil and storing a little bit of carbon there, too.” The land was purchased with savings from a previous career as an Alaskan salmon fishing captain, and the past 20 years have seen a progressive investment in land restoration and diversified agriculture operations. From the establishment of hundreds of trees at the property border to rotational grazing plans for cows on pasture and marshland, to fruit trees and ¼ acre home garden with greenhouse, to a blueberry patch being prepared for planting in 2019, biodiversity continues to grow. Revenue streams are accordingly diverse, with the compost and wood chips bringing in the most revenue annually, followed by Field House vacation rentals, beef and pork products, and vegetable sales.

The Field House, available for short term farm stays, hosts visitors year round and is booked throughout busy summer tourism season, capitalizing on the growing market for agritourism opportunities. The farm has typically provided housing for another couple in a barn apartment, in exchange for regular workdays or some combination of paid labor and housing work-trade. Sustainable Agriculture Interns coordinated by LCLT help out during summer months, and the farm is a popular destination for “WWOOFers” as well . Other Lopez Island youth work on the farm several days a week during the summer. At maximum capacity, the farm hosted nine farm employees during the summer 2019 season. Farming practices are the product of decades of experience, and soil fertility is the product of countless yards of compost and mulch application. In the vegetable garden, dozens of crops feed the farm families and neighbors each year. From spring seeding to bed preparation and transplanting, to weeding, irrigation, harvesting, cover cropping and winter greens cultivation in the greenhouse, every activity has its seasonal rhythm. Several planting strips are gradually converting to no-till farming, with compost, mulch, and broad-forking substituting for the mechanical mixing of the soil. Tilling is associated with carbon release and disturbance of the soil biota, so reducing or eliminating tillage is an effort several farmers are working towards, in balance with weed management. Irrigation ponds, dug on most farm properties, fill up with rain in the winter, and provide water to crops through the dry summer months.It is amended with lime and seeded with beneficial plants to boost nutrient quality of forage materials. The cows contribute to the regeneration of pasture soils, providing aeration from their hooves, growth stimulation from grass consumption, and fertilizer from their manure. David and Faith, the owners of Midnight’s Farm,equipment for vertical farming are passionate about researching and implementing agricultural solutions to climate change on their farm.

Their bookshelves are filled with books such as Grass, Soil, Hope; Dirt to Soil; and Growing a Revolution: Bringing our Soil Back to Life, and their social calendar is filled with attending climate talks and hosting climate researchers from University of Washington , WSU, and other institutions. Most recently they are engaged in a carbon footprint analysis of their compost operation, land use, and cattle herd, in order to understand highest impact opportunities for emissions reduction and carbon removal. The results show that currently the farm is contributing to the sequestration of approximately 250 mtCO2e, via forest cover, marshland, managed pastures, compost production and application, which together more than offset emissions from farm machinery, diesel use, and cattle as shown in Figure 9. David and Faith advocate for a “big tent” approach to food systems transition where many different people and groups can see themselves in a process of growing food with a lighter climate impact, and better human health impact. Their vision rests on a premise of developing strong interpersonal relationships, infusing the work with joy, humor, social connection, and opportunities for personal growth. An onsite yoga studio offers space for interns and farming friends to stretch and reinvigorate bodies feeling the effects of hard physical work. David and Faith continue to articulate better and brighter ideas for the future, such as finding long-term land partners and helping launch a climate farm school on the island, pushing forward the vision of a truly regenerative agroecosystem on Lopez. Orderly rows of greens and vegetables lend a sense of efficiency and purpose to the fields of Lopez Harvest. Successional plantings of diverse lettuce varieties march westward across the field, with the largest plants cut for weekly harvests while each neighboring row showcases one fewer week in the field. 500 lettuce plants go in the ground on Wednesdays, and plants are harvested on Tuesdays and Fridays for twice a week deliveries. The humming schedule of running a successful greens production farm serving the two island grocery stores as well as 5-6 island restaurants and food businesses creates a strong weekly rhythm for farm owner and farm workers. Dig, transplant, bed down, repeat.

Six inch spacing, four rows per planting bed. Finish the row, water it in, keep moving. Lopez Harvest sells lettuce mix, a specialty blend of “Island Greens,” chard, microgreens, arugula, herbs, and various seasonal vegetables and specialty crops to most of the for-profit food retail and business operations on the island. Christine, the farm owner, sends out a “pick list” to all customers a week in advance, takes orders by a certain day, and harvests and delivers all orders herself. This is her answer to the question “what does it take to be a successful small-scale farmer on a small island?” She sells her surplus produce directly to retail and restaurant, finding this to be more profitable than selling at the seasonal weekly Farmer’s Market or direct to consumers. She raises additional vegetables for personal consumption, reducing her own need to purchase store-bought foods, and facilitates a meat-share program where costs and benefits from raising meat chickens are shared among participating households. These non-monetary and cooperative forms of exchange are important to the economic viability of her operations. Christine now receives additional revenue from her participation in a beginning farmer mentoring program, where she earns up to $1,000 annually for mentoring younger farmers in their first year of operation . Her farm is on shared land purchased by three couples, and was acquired with family support, a common method for overcoming high barriers to entry for farmland access . While some rows of her field are planted to commercial crops, others are in rye-vetch cover crop mix gaining fertility for next year, or mustards to deter wire worms. The cover crop is mowed down and incorporated into the beds, with some beds serving as experiments for no-till practices where she has also tried occultation techniques to germinate and kill weeds prior to transplanting. This is difficult to enact on her land due to heavy clay soils that need some disturbance to be made ready for tender transplants and is a work in progress. Commercial crops are rotated onto previously cover cropped beds, a dance between production of plants and soil. In Christine’s mind, “good farming is good for the climate;” she adopts practices when they prove beneficial for her land, crops, soil, and business model, and it just so happens that many of these practices are anointed in academic research as climate mitigating strategies. Christine exemplifies a successful independent, woman-owned business model. She receives seasonal labor support through the LCLT intern program and through informal worktrade agreements with friends and neighbors. Christine is a vocal contributor at the monthly farmer coffees, sharing what she’s learned about effective weed control strategies , and a gifted farmer educator. She collaborates with WSU San Extension on a research project to reduce wire worm pest pressure in lettuce crops and is also a collaborator on the Western SARE bio-char cocompost grant, participating in the field trial and soil/crop data collection processes. Christine recognizes the attractiveness of entering into farming cooperatively or with farm partners but struggles with the difficult proposition of supporting multiple households with limited farm revenue streams and land use restrictions. When it comes to sharing land in her current situation, she would love to be able to build and provide more farm worker housing, but is restricted from doing so by county zoning policies that prevent more than two houses from being built on a parcel designated as “farmland7.” The county zoning codes are ripe for reform, but notoriously difficult to get right in terms of regulatory verbiage that protects farmland from becoming housing developments yet allows for ample and affordable farm worker housing.

Transcriptomic and proteomic studies in Fe deficient plants have also reported increases in root transcript and protein abundances, respectively, of enzymes related to the glycolytic and TCA cycle pathways, among others. Iron deficiency also induces an accumulation of organic acids, mainly malate and citrate, in roots. The induction of C metabolism in roots of Fe-deficient plants would not only provide a source of reducing power, protons and ATP for the Fe reductase and H+-ATPase enzymes, but also lead to an anaplerotic root C fixation. Carbon accumulated in roots is exported in the form of organic acids via xylem to leaves, which have otherwise drastically reduced photosynthetic rates when Fe-deficient. The higher energy requirements in Fedeficient root cells are tackled by increasing mitochondrial oxidative processes, and roots from Fe-deficient plants show enhanced respiratory activities and higher O2 consumption rates. On the other hand, the mitochondrial respiratory chain is strongly affected under Fe-deficient conditions, since some of its components are Fe-containing enzymes.Furthermore, an increase in anaerobic metabolism has also been described in Fe-deficient roots,hydroponic vertical farm probably as an strategy to oxidize all the reducing power generated by glycolysis and TCA cycle that can not be easily oxidized in the respiratory chain.

When resupplied with Fe, Fe-deficient plants reorganize its metabolism by readjusting metabolic cycles and changing root morphology towards those typical of Fe-sufficient plants.The most common approach used to study Fe deficiency in roots is to analyze only a small number of genes, proteins and/or metabolites. A more comprehensive knowledge of the processes taking place in Fe-deficient roots has been recently provided by the application of modern techniques allowing for the simultaneous and untargeted analysis of multiple genes or proteins. The aim of this work was to characterize the changes induced in the root tip proteome and metabolome of sugar beet plants in response to Fe deficiency and resupply, in order to provide a holistic view of the metabolic processes occurring in plants under different Fe status.The polypeptide pattern of root tip extracts was obtained by 2-D IEF-SDS PAGE electrophoresis. Real scans of typical 2-D gels are shown in Figure 1; an average number of 141 and 148 polypeptides were detected in Fe-sufficient and Fe-deficient plants, respectively . The total number of spots detected was relatively low when compared to other proteomic studies. Several causes may account for this discrepancy, including i) protein extraction method and amount of protein loaded in the gels, ii) gel size, iii) pI range and iv) sensitivity of the staining method. Averaged 2-D polypeptide maps were obtained using gels of three independent preparations, each from a different batch of plants . To better describe polypeptide changes we built a composite averaged virtual map containing all spots present in both Fe-deficient and control root tip extracts . Iron deficiency caused 2-fold increases in 29 spots and 2-fold decreases in signal intensity in 13 spots . Furthermore, 6 spots were only detected in Fe-sufficient plant samples and 13 spots were only detected in Fe-deficient plants .

All polypeptides in the composite averaged map are depicted again in Figure 1D, to permit annotation of those polypeptides where identification was achieved by matrix assisted laser desorption ionization – time of flight MS . These polypeptides were labeled from a to v as described in Figure 1D, and homologies found are described in detail in Table 1. From the 29 spots that showed increases in signal in root tip extracts of Fe-deficient as compared to Fe-sufficient controls, the 20 more abundant were excised and analyzed by MALDI-MS. Since the sugar beet genome has not been sequenced yet and few sequences are avail-able in the databases, identification was performed by homology searches with proteins from other plant species. From the 20 spots analyzed, 14 proteins were identified . These include proteins related to glycolysis such as fructose 1,6-bisphosphate aldolase , triose-phosphate isomerase , 3-phosphoglycerate kinase and enolase . Three spots gave significant matches to malate dehydrogenase , and two more polypeptides presented homology with α and β subunits from F1 ATP synthase . Other proteins increasing in root tip extracts from Fe-deficient sugar beet plants as compared to the controls were fructokinase and formate dehydrogenase . Also, one spot gave significant matches to a cytosolic peptidase At1g79210/YUP8H12R_1 . Spot n gave significant match to a glycine rich protein, which possibly has a role in RNA transcription or processing during stress conditions. From the 13 spots detected de novo in proteome maps from root tip extracts of Fe-deficient plants , the 6 more abundant were excised and analyzed by MALDI-MS, resulting in only 2 positive matches . These significant matches were found for glyceraldehyde 3-phosphate dehydrogenase and DMRL .Changes in the amount of DMRL as well as DMRL gene expression and flavin analysis were further studied using root tip extracts of Fe-sufficient, Fe-deficient and Feresupplied sugar beet plants .

From the 13 spots showing a decrease in signal intensity in root tip extracts from Fe-deficient plants as compared to controls , 3 were identified by MALDI-MS. Spots q and r gave a significant match to nucleoside diphosphate kinase I and to oxalate oxidase-like germin, respectively. Spot s presented homology with the At4g27270 protein whose molecular function is to interact selectively with FMN, and also presents oxidoreductase activity. From the 6 spots not detected in root tip extracts from Fe-deficient plants as compared to the controls , 3 were identified by MALDI-MS . Proteins matched were oxalate oxidase , peroxidase and caffeoyl CoA Ometyltransferase .Iron deficiency and/or resupply caused significant changes in the levels of 62 out of the 77 identified metabolites. Metabolite level data were normalized to the mean response of the +Fe treatment; response ratios, defined as the level in a given treatment divided by the level in the +Fe control, are indicated in Table 2. Iron deficiency caused significant changes in the response ratios of 26 metabolites. Large increases were found for some organic acids , some sugars , nicotianamine and 2-aminoadipic acid. The response ratio of oxalic acid decreased markedly in -Fe conditions, whereas those of other aminoacids, N compounds, lipid metabolites and others did not show large changes when compared to the Fe-sufficient controls. Twenty-four hours after Fe-resupply, there was a dramatic coordinated increase in the root tip response ratios of galactinol, raffinose, lactobionic acid, cellobiose and nicotianamine when compared to those found in Fe-deficient roots,vertical farm whereas the response ratios of sucrose, myoinositol, citrate and malate decreased. Seventy-two hours after Fe resupply, the response ratios of galactinol, raffinose, cellobiose, nicotianamine and many other compounds had decreased in the YZ areas, whereas in the WZ the response ratios were very low. The response ratio of many of the lipids increased moderately in all Fe resupplied samples. Metabolites in the coenzyme, glycolysis, oxidative stress, pentose phosphate pathway and signalling categories did not show large response ratio changes with Fe resupply.The changes induced by Fe deficiency in the root tip proteome and metabolome from sugar beet plants grown in hydroponics have been studied. More than 140 proteins and 300 metabolites were resolved in sugar beet root tip extracts. Iron deficiency resulted in significant and higher than 2-fold changes in the relative amounts of 61 polypeptides, and 22 of them were identified. Out of 77 identified metabolites, 26 changed significantly with Fe deficiency. In general, our results are in agreement with previous transcriptomic, proteomic and enzymatic studies on Fe-deficient roots.

Our data confirm the increases previously found in proteins and metabolites related to carbohydrate metabolism and TCA cycle pathways. Two major changes induced by Fe deficiency in roots are described in this study for the first time: the increase in DMRL synthase protein concentration and gene expression, and the increase in RFO sugars. The largest change found in the proteome map of root tip extracts from sugar beet plants grown in Fe deficiency conditions corresponded to DMRL synthase, which was detected de novo in Fe-deficient root tips, and is the protein with the highest concentration in these gels . This enzyme catalyses the fourth step of Rbfl biosynthesis, and Rbfl is the precursor of Rbfl sulphates, FMN and FAD, the last one being a cofactor for the root plasma membrane Fe reductase. The expression of BvDMRL decreased drastically 24 h after the addition of Fe to Fe-deficient plants, whereas DMRL synthase protein abundance and Rbfl and Rbfl sulphate concentrations did not change significantly with Fe-resupply in the YZ of root tips , suggesting that the turnover of this protein is slow. Accumulation in Fe-deficient roots of flavin compounds, including Rbfl and Rbfl 3′ – and 5′-sulphate is a characteristic response of sugar beet and other plant species. The exact role of flavins in Fe deficiency is unknown, and it has been hypothesized, based on the similar location of flavin accumulation and Fe reduction and on the fact that the Fe reductase is a flavin-containing protein, that free flavin accumulation may be an integral part of the Fe-reducing system in roots from Strategy I plants [9,28]. On the other hand, these compounds are secreted to the growth media at low pH [6] and, assuming high concentrations at the secretion site, they could mediate extracellular electron transfer between soil Fe deposits and root Fe reductase as it has been described for flavin phosphates secreted by some bacteria. Moreover, excreted flavins could also act as a plant-generated signal that could influence rhizosphere microbial populations, indirectly affecting Fe availability. A major change in carbohydrate metabolism was the large increase in RFO compounds that occurs in roots with Fe deficiency. This increase was higher than that found for sucrose . The total concentrations of raffinose and galactinol were also determined by HPLC-MS, and concentrations of both compounds in the 35-80 nmol g FW-1 range were found in Fe-deficient and Fe-resupplied root tips , whereas concentrations in the +Fe treatment were one order of magnitude lower. The sum of the raffinose and galactinol concentrations in the -Fe, 24h, 72hWZ, 72hYZ and +Fe tissues was 13.9, 7.4, 2.2, 5.1 and 0.6% of the total sucrose, respectively, supporting the relevance of the RFOs changes with Fe status. RFOs have diverse roles in plants, including transport and storage of C and acting as compatible solutes for protection in abiotic stresses. Other explanationfor the large increase in the relative amounts of RFOs could be the ability to function as antioxidants; galactinol and raffinose have hydroxyl radical scavenging activities similar to other soluble antioxidants such as glutathione and ascorbic acid. Since ROS damage and ROS detoxification strategies have been observed in Fe-deficient roots, the increase in RFO concentration could help to alleviate ROS damage produced under Fe deficiency. Moderate increases in sugars commonly found in cell walls such as cellobiose, xylonic acid and arabinose, which may indicate cell wall modifications, were measured in sugar beet Fe-deficient root tips. Changes in cell wall metabolism have been also described in Fe-deficient tomato roots. On the other hand, it has been described that cell wall damage generates oligosaccharides that can act as signalling molecules in stresses such as wounding. The increase in RFOs could also act as a long distance Fe-deficiency signal via phloem sap transport. This is the first description of RFOs accumulation in plants under Fe deficiency, and the physiological implications of this increase deserve further consideration. Most of the proteins found to be up-accumulated in sugar beet root tips by Fe deficiency were identified as carbohydrate catabolism enzymes, including 5 of the 10 glycolytic pathway enzymes , one of the citric acid cycle and fructokinase. Increases in the activities and concentrations of several glycolytic enzymes in root extracts with Fe deficiency have been previously found, including fructose 1,6-bisphosphate aldolase, enolase, triosephosphate isomerase and GADPH . Also, increases in the activities and concentrations of several enzymes of the citric acid cycle with Fe deficiency have been previously reported in root extracts, including MDH. Results are also in agreement with microarray gene analysis in Fe-deficient A. thaliana roots. Increases in the amount of PEPC have been found at the protein level, but this enzyme, with a molecular mass of 110 kDa, was not in the range used in our 2-D gels.

In the absence of major decomposition pathways, PAHs are removed from the atmosphere by dry and wet deposition, which is considered the major source of PAHs in soil . Although PAHs are considered amongst the most widespread organic pollutants in numerous environmental matrices such as soils, sediments, water, and wastewater, the ultimate sink of atmospheric PAHs is soil . Wild and Jones conducted an investigation to quantify the production, cycling, and storage of PAHs in the United Kingdom and determined that more than 90% of the total PAH burden resided in the surface soil. In addition to atmospheric deposition, PAHs can also enter soil systems through disposal of waste materials, creosote use, road runoff, and fossil fuel spills . The fate of PAHs in soil systems is primarily influenced by PAH hydrophobicity and the physicochemical properties of the soil. Due to their strong hydrophobicity and environmental recalcitrance, PAHs are typically associated with nonaqueous phases in soil where they associate into four main compartments: 1) organic matter; 2) the mineral compartment, which includes exposed soil surfaces and surfaces within pore spaces; 3) nonaqueous-phase liquids; and 4) combustion residues such as soot . The degree to which PAHs are retained within the soil system is controlled primarily by soil properties such as organic matter and soil texture as well as the PAH physicochemical properties.

Because of the nonpolar, hydrophobic nature of PAHs, soil organic matter is considered the most important sorbent of PAHs . The organic matter or total organic carbon can also act as a carrier for the vertical migration of PAHs from the soil surface .Polycyclic aromatic hydrocarbons have a very strong affinity for soil organic matter via π-π interactions between the aromatic structure of PAHs and aromatic moieties of organic matter . In soils with low amounts of organic matter or total organic carbon content,fodder systems for cattle the soil texture plays a critical role in the environmental fate of PAHs. For example, Karickhoff et al. reported an increase in pyrene adsorption coefficients with an increasing clay content . In addition, decreasing particle size is typically associated with concomitant increases in the proportion of HMW and decrease in the proportion of LMW PAHs . The greatest PAH soil concentrations in numerous studies have been observed in the silt-sized soil fraction, which was potentially due to the silt fraction containing the greatest concentration of soil organic matter and its associated aromatic structures for binding . In addition, clay fractions are characterized by a very high specific surface area, abundant surface charge, and a high organic matter density, all of which provide a large number of sorption sites for PAHs . Sorption of PAHs to soils generally entails an initially rapid and reversible phase followed by a period of slow sorption occurring over a period ranging from weeks to years, and this slow sorption leads to a chemical fraction that resists desorption and biodegradation . Increasing contact times between PAHs and soil organic matter or fine soil fractions can also result in the “aging” effect or sequestration of PAHs . This process involves the continuous diffusion and retention of PAHs within the solid phase of organic matter and also in nanopores or voids in the organic matrix, thus blocking PAHs from abiotic and biotic loss processes.

As a result of the strong association of PAHs with the nonpolar soil organic fractions, PAH bioavailability, or PAH concentrations in the aqueous phase that are directly available to soil microbes for degradation, is generally low . The bioavailability of PAHs is determined by two main factors, which are the rate of transfer of PAHs from the soil to the living cell and the rate of uptake and metabolism . Bio-availability is an important concept with regards to PAH-contaminated soil remediation and risk assessment as microbial degradation constitutes the major dissipation pathway for PAHs compared to other processes such as evaporation, photolysis, and plant uptake . The extent and rate of microbial degradation of PAHs in the terrestrial environment is influenced by a variety of abiotic and biotic factors which include temperature, pH, aeration, accessibility ofactors, which are the rate of transfer of PAHs from the soil to the living cell and the rate of uptake and metabolism . Bio-availability is an important concept with regards to PAH-contaminated soil remediation and risk assessment as microbial degradation constitutes the major dissipation pathway for PAHs compared to other processes such as evaporation, photolysis, and plant uptake .f nutrients, microbial population, contaminant bio-availability, and physicochemical properties of the PAH . Typically, the rate of PAH biodegradation is inversely proportional to the number of aromatic rings or molecular weight of the PAH . For example, half-lives of phenanthrene in soil may range from 16 to 126 days, while half-lives of HMW PAHs such as benzo[a]pyrene may Mansour, 2016. Some of the major PAH-degrading genera in soils include Mycobacterium, Sphingomonas, Bacillus, Pseudomonas, and Rhodococcus . The ability of soil microbes to degrade PAHs is determined by 1) the ability of bacteria to transport the PAH into the cell, 2) the physicochemical properties of the PAH as a substrate for available microbial enzymes, and 3) the suitability of the PAH as an inducer for the appropriate transport or degradative enzymes .

As shown in Figure 1.3, there are two primary mechanisms involved in the aerobic metabolism of PAHs by soil bacteria . The principal mechanism for aerobic PAH degradation by soil bacteria involves the dioxygenase/monooxygenase enzymes, which incorporates a hydroxyl group derived from molecular oxygen into the aromatic nucleus, resulting in the oxidation of the aromatic ring to form cis-dihydrodiols . This initial ring oxidation is considered to be the rate limiting step of the PAH bio-degradation process in soil systems . The cis-dihydrodiols are stereoselectively dehydrogenated by cisdihydrodiol dehydrogenases to form dihydroxylated intermediates, called catechols . Subsequently, the catechol may then be cleaved by intradiol or extradiol ring-cleaving dioxygenases through the ortho or meta-cleavage pathway to tricarboxylic acid intermediates such as succinic, fumaric, pyruvic, and acetic acids and acetaldehyde. These TCA intermediates are utilized for cell-protein synthesis and energy by microorganisms with the final production of carbon dioxide and water . Soil bacteria can also degrade PAHs via the cytochrome P450-mediated pathway to form transdihydrodiols . Polycyclic aromatic hydrocarbons are biodegraded by soil microbes in one of two ways, either as the sole carbon and energy source or by cometabolism . Because PAHs occur in the environment as complex mixtures of LMW and HMW PAHs, cometabolism is an important interaction that transforms non-growth substrate PAHs, particularly HMW PAHs, in the presence of growth substrates to enhance PAH degradation . Numerous soil microbes have been isolated that utilizeLMW PAHs such as naphthalene and phenanthrene as their sole carbon source and throughout the past decade, multiple soil bacteria have been discovered and isolated that are capable of utilizing HMW PAHs as sole carbon sources . For example, Mycobacterium vanbaalenii PYR-1, an isolate from an oil-contaminated estuary near the Gulf of Mexico,fodder sprouting system has been utilized to determine the complete pyrene degradation pathway using various metabolic, genomic, and proteomic approaches.Pyrene is often used as a model compound for HMW PAH biodegradation because it is structurally similar to several carcinogenic HMW PAHs.The primary pathway for pyrene degradation by M. vanbaalenii PYR-1 is deoxygenation by dioxygenase and monooxygenase at the C-4 and C-5 positions to produce both cis– and trans-4,5-pyrenedihydrodiol, respectively. The metabolite undergoes further metabolization involving more than 20 enzymatic steps utilizing rearomatization, decarboxylation, and oxygenation to produce phthalate that is further transformed to the TCA cycle via the β-ketoadipate pathway . Another pyrene degradation pathway exists for M. vanbaalenii PYR-1 that involves the oxidation of pyrene at the C-1 and C-2 positions to form O-methylated derivatives of pyrene-1,2-diol as a detoxification step . Mycobacterium vanbaalenii PYR-1 is also capable of degrading or transforming biphenyl, naphthalene, phenanthrene, anthracene, fluoranthene, benzo[a]anthracene, and benzo[a]pyrene, thus making this bacterium an effective candidate for the  bio-remediation of PAH-contaminated soils. Because PAHs are toxic, ubiquitous pollutants that are highly resistant to degradation in contaminated soils, remediation of PAH-contaminated sites is critical for protecting human health and the environment. Several physical and chemical PAH remediation technologies such as incineration, excavation and land filling, UV oxidation, and solvent extraction have been used to clean up PAH-contaminated soils.

However, these remediation methods have several negative aspects including cost, regulatory burden, and that some of these conventional methods do not result in PAH dissipation, but rather transfer from one environmental compartment or form to another . These limitations of conventional treatment methods have led to the increased use of bio-remediation techniques at PAH-contaminated sites as they are considered to be safe, environmentally-friendly, and cost-effective . Bio-remediation involves the utilization of biological processes or activity of microorganisms to remove pollutants from contaminated matrices to achieve concentrations that are acceptable according to health and regulatory standards. Bio-remediation technologies can be classified into two main categories, in situ or ex situ. In situ bio-remediation technologies target contaminant removal or attenuation under natural environmental conditions without the need for excavation, whereas ex situ bio-remediation processes involve the physical removal of the contaminated material for remediation off-site . Therefore, in situ remediation practices are particularly effective for widely dispersed contaminants and are typically less expensive than ex situ approaches. Additionally, exposure to site workers to hazardous pollutants is minimal and in situ treatments also allow for remediation in inaccessible environments . Because of these advantages, in situ bio-remediation constitutes approximately 25% of all remediation projects for contaminated sites . However, because in situ bio-degradation does not disturb the contaminated soil, remediation has been found to be more variable due to the natural environmental conditions . Although ex situ bio-remediation is less economical compared to in situ treatments, ex situ bio-remediation methods are less limited by environmental factors that could adversely affect the remediation efficacy and the physical and chemical conditions can be manipulated before and during degradation . Ex situ bio-remediation generally requires less time to achieve efficient contaminant remediation since optimal remediation conditions can be monitored and modified as needed . The successful implementation of PAH-contaminated soil  bio-remediation treatments depends on a multitude of factors that can be categorized into three main domains encompassing PAHs, environmental conditions, and soil microbial communities . Factors involving PAHs include the physicochemical properties of PAHs, concentration and toxicity, and the length of time the PAHs have been in contact with the soil and their associated bio-availability. Because in situ bio-degradation treatments are currently a common type of remediation practice, environmental conditions in soil that must be evaluated for effective PAH bio-degradation include soil type, organic matter content, nutrient availability, moisture, temperature, pH, and presence of oxygen or alternative electron acceptors. Soil microbial transformations are the major process governing degradation of PAHs in contaminated soil, and therefore critical factors related to soil microbes include the presence of a soil microbial community capable of degrading PAHs, which encompasses microbial type, abundance, distribution, acclimation or previous exposure, and metabolic rate. Natural or bio-attenuation is an in situ  bio-remediation technique that involves passive remediation of a PAH-contaminated site without any external alterations . Because there are no site modifications, natural attenuation is considerably cheaper than other  bio-remediation methods. However, one of the major limitations of bio-attenuation is that the process can take extended periods of time to achieve appreciable levels of PAH dissipation. Bio-degradation is especially slow in PAH contaminated sites that have been in contact with non-polar soil domains for a prolonged duration, resulting in decreased PAH bio-availability.Due to the environmental stability of PAHs, the majority of PAH-contaminated sites undergo engineered or enhanced bio-remediation, which involves site modifications to enhance the extent and rate of PAH degradation . Typically, enhanced bio-remediation techniques for PAH-contaminated soils involve bio-augmentation, bio-stimulation, surfactant amendment, phytoremediation, or an integrated combination of bio-remediation techniques.Bio-augmentation is the process in which contaminant-degrading microorganisms are introduced into the soil as single strains or bacterial consortia to increase the rate of PAH bio-degradation. The main advantage of bio-augmentation is the relatively low cost of inoculating soil microorganisms into the soil system.Bio-augmentation is an effective method to increase bio-remediation efficacy for PAH-contaminated soils that possess low numbers of native PAH-degrading soil microbes or when the native soil microbial population does not exhibit sufficient metabolic activity to result in PAH dissipation.

The combination of these approaches is useful for the identification of genes and traits under selection in natural settings, but so far few studies have taken this approach. Eckert et al. tested the association of SNPs with five phenotypic traits and 11 environmental variables across 10 P. lambertiana populations around Lake Tahoe. This study identified six genes associated with phenotypic traits , and 31 associated with environmental PCs. Two genes were associated with both a trait and an environmental axis, including a glucose transport protein associated with d13C and environmental variables linked to water availability. A study focusing on multiple drought response traits and a larger number of SNPs might be able to identify more genes that have variants associated with both environmental gradients and drought tolerance traits. Some traits and processes involved in drought response have been better studied at the genetic level than others . Provenance studies have indicated that differences in stomatal control and shoot growth are often involved in local adaptation to drought, and all other study types have identified the genes likely to be involved . However, although root growth has also been identified as important by provenance studies,nft growing system root-growth-related genes have not been identified.

Conversely, although genes related to resistance traits, such as changes in carbohydrate metabolism, and protective and pathogen defense molecule production, have been identified in expression or association studies, these traits have been largely ignored in provenance studies. Finally, xylem traits, including refilling ability, have not been the focus of any genetic study type.Tree improvement programs that aim to increase growth potential and stress resistance face the challenges of long generation times, the need for large-scale field experiments and the late expression of traits such as wood density . Genomic selection, already routinely used in livestock breeding, has been proposed as a method of speeding up this process by using marker-predicted breeding values for phenotypes of interest . This approach is suitable for species with low LD and for traits with complex genetic architectures as it uses thousands of markers with effects that are estimated simultaneously . As with traditional phenotypic selection, accuracy is likely to be greatest when tests are carried out in environments similar to the target environment, because of the high likelihood of genotype 9 environment interactions . Several recent studies have demonstrated the potential of genomic selection approaches for traits of interest to forestry. Resende et al. carried out an early evaluation of genomic selection in P. taeda, making use of clonally replicated individuals grown on four sites and genotyped at 4825 SNPs. They found that the accuracy of prediction models within sites ranged from 0.63 to 0.75 for diameter and height, and estimated that the breeding cycle could be speeded up by 50% with this method.

Gamal El-Dien et al. used GBS to genotype over 1000 interior spruce trees over three sites that had been phenotyped for yield and wood attributes, and found that the incorporation of genomic information produced more accurate heritability estimates. Genomic estimated breeding values were most accurate when data from multiple sites were used to fit the model. Of even more relevance to selection for drought tolerance, Jaramillo-Correa et al. identified 18 SNPs associated with climatic PC axes in P. pinaster, and found that the frequency of locally advantageous alleles at these loci correlated with population level survival rates in a common garden at the hot/dry end of the species range. Together with the growth trait analyses, these results suggest that association techniques could be applied to predict breeding values for overall drought tolerance or particular drought tolerance traits even though only some of the loci involved have been identified. There is evidence of significant potential for selection approaches to improve drought responses in conifers. Provenance studies have shown evidence of genetic differentiation between populations in drought responses, and genome scan and G2E associations are finding evidence of natural selection on within-species genetic variation. Second, heritabilities for drought tolerance traits, when these have been examined, tend to be moderate to high. The calculation of heritability requires pedigree information: parent– offspring or sibling and half-sib comparisons. Narrow-sense heritability is the fraction of the variance in a trait attributable to additive genetic variation, as opposed to environmental and nonadditive genetic variation. Because heritability depends on both genetic variation in the population assessed and the degree of variation caused by the environment, estimates are not transferable between situations.

In P. pinaster, estimates of d13C narrow-sense heritability ranged from 0.17 to 0.41, depending on how many individuals of what populations were assessed in what sites; and ring width and height growth rates were also moderately heritable . In the same species, heritability of P50 was 0.44, but this was driven more strongly by low levels of other sources of variation rather than high additive genetic variation . Across species, measured heritabilities for d13C range from the very high 0.7 for Araucaria cunninghamii to < 0.1 for P. taeda . Managers of wild forests are often focused on ensuring the resilience and function of the ecosystem rather than productivity. G2E and G2P association studies may help to identify seed sources that could be ‘pre-adapted’ to projected conditions for replanting in wildlands. However, wild trees face a range of challenges, including disease and competition, as well as drought . Stand structure and soil properties may also directly affect how trees experience drought stress. Studies that integrate stand level processes with genetic testing can further bridge gaps between genetic experiments and forest-scale management. Restoration projects could be used as experiments to test genomic predictions of survival and growth in a given environment, as well as the effects of genetic composition and diversity of the planted population on restoration success.Common garden, gene expression and genetic association studies all have different strengths and weaknesses, and none alone will answer the question of how genetic differences affect drought tolerance . As described previously, a combination of different types of association study may help to identify loci that are under selection in the wild and the traits they influence. Similarly, gene expression studies could easily be combined with common garden studies of adults or seedlings to address whether differences in drought responses between populations or genotypes are a result of differences in gene sequences, gene expression patterns or both.Many studies to date have focused on WUE, often using d13C as a proxy. As discussed above, however, WUE is a ratio of changes in photosynthesis and transpiration, which can both vary, and higher WUE may or may not be associated with greater survival or growth in dry conditions. Moreover, different measures of WUE are not entirely consistent. We therefore recommend that future studies use survival and/or growth during and following drought as the metric of overall ‘drought tolerance’,vertical hydroponic nft system and measure photosynthesis and water loss separately if these are processes of interest. The time involved in the measurement of traits for hundreds or thousands of individuals has encouraged the focus on easily measured d13C, but much progress has been made in high-throughput phenotyping techniques . For instance, thermal and long wave infrared sensors can measure leaf temperature or stomatal conductance, near and short-wave infrared sensors can measure leaf water content, and fluorescence sensors can measure chlorophyll content and photosystem efficiency .There are several traits and processes that have been suggested to be important for drought response by physiological studies, but about which there is little genetic information .

Genetic studies frequently identify genes related to carbohydrate metabolism and transport as having altered expression or allele frequencies depending on water availability. It is difficult to make sense of these patterns because the link between these metabolic changes and tree function and survival during drought is still unclear. We also know relatively little about which species can refill cavitated xylem, under what circumstances and by what mechanisms. Thus, it is difficult to determine whether any genes identified by expression or G2E studies are involved in this process. Similarly, how roots and root growth respond to changes in water availability, and what genes are involved in these responses, remain poorly understood. Although the measurement of root architecture can be complex, high-throughput methods are being developed for this as well .Most experimental studies, including those looking at gene expression, have focused on seedlings. There may be important differences in how different life stages respond to drought.

For instance, Pinus nigra ssp. laricio adults have been observed to follow an isohydric strategy, whereas seedlings in a glasshouse experiment did not . Although it is more complicated to impose drought treatments on adults, drought experiments have been carried out on adult trees using networks of rain shields/ gutters to intercept precipitation and direct it away from the trees . This water can also be re-directed to other plots to create ‘well watered’ treatments. For the most part, these studies have been carried out on natural populations. However, if they were coupled with provenance study plantings, one could test for population or genotypic differences in adult drought response. Likewise, apart from some long-term provenance studies , most experiments span a few days to a few months. In order to investigate drought resilience and legacy effects, more multi-year studies are needed.The length and intensity of drought can affect which trait combinations result in greater fitness. In Section IV.1, we mentioned the great diversity of methods used to induce or measure drought stress treatments in gene expression studies. The same diversity is found in G2P and provenance studies as well. There is a need to assess: whether environmental treatments roughly match the range of conditions in the environments in which the target species does or might grow; how environmental treatments relate to plant stress measures ; and whether traits, responses or genotypes associated with drought tolerance in the glasshouse or laboratory predict performance in the field. In addition, studies testing longer term drought treatments are lacking, as are those that explicitly test variable combinations of drought length and severity. Future work should address these gaps.In most of the genetic studies cited above, a relatively high proportion of the genes expressed or linked to phenotypes or environmental gradients of interest either have unknown or poorly defined functions. Behringer et al. , for instance, found that, of the 832 transcripts analyzed for gene ontology, 538 either had no database hits or could not be assigned to a biological process. Although this could be partly addressed with further studies intraditional model organisms, such as Arabidopsis, analysis of loblolly pine and Norway and Sitka spruce genome sequences suggests that there could be thousands of conifer-specific gene families . This shortcoming must be addressed by further development of model systems in conifers.Unsurprisingly, the conifer taxa that have received the most attention in terms of drought tolerance studies or genomic studies are those that are of high economic value, especially those that are frequently grown in plantations.This means that most drought tolerance genetics studies have been carried out on pines , with a modest representation of spruce and Douglas-fir. Although this focus is understandable, this means that a number of ecologically or economically significant taxa have been left out, notably the Cupressaceae . Many Juniperus and New World Cupressus and Calocedrus species are impressively drought tolerant. In the well-studied pinyon–juniper woodlands of the American Southwest, anisohydric juniper tends to exhibit lower mortality than Pinus edulis during severe drought . However, not all pine species exhibit isohydric behavior . Thus, the genes involved in drought response could differ substantially between families, genera or species.In this study, known negative regulators of the drought signaling pathway were knocked down using RNAi to generate increasingly drought tolerant plants for both Arabidopsis thaliana and Brassica napus. Previous studies have shown that knocking out Clade A Type 2C protein phosphatases , which are negative regulators in the drought signaling pathway, resulted in enhanced drought tolerance, but has come at the cost of decreased growth in non-drought conditions. As a mechanism for enhancing drought tolerance without compromising yield in well-watered conditions, RNAi was utilized to knockdown PP2Cs instead of knocking them out. Therefore, we generated stress inducible and constitutive knockdowns of PP2Cs in A.thaliana and stress-inducible knockdowns of PP2Cs in B.napus.

The region of Araraquara has the largest insect Las+ population in summer, while Bebedouro has the smallest, during the whole year. The Asian citrus psyllid is the only known vector of the bacterium, ‘Candidatus’ Liberibacter asiaticus , that causes huanglongbing or citrus greening disease. This insect acquires CLas from an infected citrus tree while feeding as a nymph. Transmission to uninfected trees occurs when infected adults emerge and fly to, and feed on, uninfected trees. Our current understanding of the CLas-psyllid interaction suggests that adults become competent for transmission only after the bacterium moves from the insect gut into the hemolymph and eventually to the salivary glands. We hypothesize that specific molecular interactions between the bacterium and gut epithelial cell membranes are necessary to initiate the movement of the bacterium throughout the psyllid’s body. Furthermore, we hypothesize that if we could block these specific molecular interactions, we may block the bacterium from reaching the psyllid’s salivary glands and therefore block the psyllid’s ability to transmit this bacterium to healthy trees. We tested these hypotheses by first developing an assay to screen and identify psyllid gut membrane binding peptides from a combinatorial peptide library and then feeding selected peptides to psyllids and determining their ability to reduce movement of CLas from the gut to the salivary glands. A total of 7 peptides were identified that bind psyllid gut preparations that demonstrated gut-binding after oral uptake.

These peptides were detected bound to gut epithelial cells in adults that had emerged from nymphs that were fed on the peptides 4 days earlier. Using qPCR assays,ebb and flow table we demonstrated that oral delivery of a combination of three of these peptides apparently inhibited movement of the bacterium into the psyllid’s salivary glands. These results support our above stated hypotheses and demonstrate a potentially new strategy for preventing the spread of citrus greening disease. Research on development of solutions to citrus greening has been hampered by the inability to culture ‘Candidatus’ Liberibacter asiaticus , the bacterium associated with this disease. Further complicating the matter, this bacterium resides within the phloem of the plant and targeting it with bactericides requires penetration into the plant so that the bactericide can reach the bacterium. To overcome these obstacles we developed a rapid screen for both bactericidal activity against CLas and phloem mobility within the citrus phloem In this assay, single CLas+ leaves from Valencia are removed from CLas+ infected citrus, indexed for CLas titer by Q-PCR and Q-RT-PCR of petiole clippings. The leaves are then placed in liquid solutions for 6 days, after which, the CLas titer is determined in both the petiole and leaf midrib. Using this assay we have demonstrated that streptomycin and penicillin are active against the CLas bacterium but that streptomycin bactericidal activity moves more readily into the leaf phloem. We have also used this assay to identify two new antimicrobial molecules more active at reducing leaf midrib CLas than the standard antibiotics when compared at the same concentration .

Using both Q-PCR and Q-RT-PCR we demonstrated that reductions in CLas16S rRNA abundance was more pronounced than reductions in the cognate DNA sequence. Therefore analysis of 16S rRNA is a more sensitive analysis of perturbations in CLas status in the plant. This assay allows concomitant analysis of bactericide activity and plant mobility and is currently being used to screen potential antimicrobial molecules for use in fighting citrus greening. RNA interference , mediated by double-stranded RNA is a gene suppression method that triggers sequence specific mRNA degradation. In citrus, transgenic expression of antisense RNA or hairpin RNA can be used to generate plants with specific genes silenced. However, this technique involves lengthy procedures because of slow growth and long juvenility of citrus trees. Here we set out to develop a quick and convenient method to silence genes in established trees. In one test, commercially synthetized dsRNA was applied weekly to potted Hamlin sweet oranges grown in artificial rooting medium. A 170 base pair dsRNA homologous to citrus phytoene desaturase was able to significantly reduce the expression of the target transcript, at a dose of 200 µg per plant. Photo-bleaching and reduced chlorophyll content, from suppression of CiPDS, were observed in 1 tree at a dose of 200 µg and 2 trees at a dose of 2 mg per plant. In another test, citrus Terminal Flower 1 that negatively regulates flower initiation was targeted in an attempt to induce early flowering. Persian lime plants grown in soil had dsRNA applied as drench weekly, in combination with cold and drought treatment to induce flowering. After 11 treatments, flowers were observed from two individual plants treated with 200 µg dsRNA-CiTFL1, but not from the control treatment .

In summary, initial results indicated root application of dsRNA is capable of triggering gene silencing in citrus. We hypothesize that the magnitude and consistency of the silencing effect can be further improved through optimized application protocols. With appropriate targets identified, we hope that RNAi may be used in citrus plantings to alter physiological/developmental plant responses to improve tolerance to Candidatus Liberibacter, or mitigate negative effects caused by infection.Pathogen-associated molecular patterns -triggered immunity is an important component of the inducible defense system of plants. Citrus species possess varying levels of responsiveness to PAMP flg22 derived from the flagellum of Xanthomonas citri ssp. citri , the bacterial causal agent of citrus canker. Higher sensitivity to Xcc-flg22 is mediated by robust expression of the plant cell surface receptor gene FLS2 and confers an increased level of canker resistance. In this study, we explored the induction of PTI by chitin, another PAMP that is a structural component of insect stylets and exoskeleton, and studied its effect on the feeding behavior of Asian citrus psyllid , the vector of Huanglongbing. The results showed that infiltration of chitin solution into ‘Sun Chu Sha’ mandarin leaves induced expression after 1h of defense genes including WRKY22, GST1, SGT1, RAR1, PAL1, NDR1 and EDS1, a set of genes previously shown to be induced by Xcc-flg22 in the canker-resistant ‘Sun Chu Sha’, but not in canker-susceptible varieties. However, the chitin-induced gene expression was not detected at 18h after infiltration. ACP feeding behavior was monitored using electrical penetration graph to study how induction of PTI by chitin modifies the number and duration of xylem and phloem feeding bouts. When adult ACP started feeding at 1h after infiltration, reduced xylem and phloem feeding were observed in chitin-treated leaves compared with untreated controls. However, this difference was smaller when psyllids started feeding at 18h after infiltration. In summary, our study shows that chitin treatment induced a transient PTI that can affect ACP phloem ingestion,flood table and this inducible defense will be further studied for its utilization on selection and generation of resistant citrus materials. Citrus production has plummeted in Florida by more than 50% in the last 10 years, although many Florida growers still maintain yields in spite of huanglongbing by increasing inputs of insecticides and nutrients. Consequently, focus is shifting to health and profitability of the next generation of trees. Area wide Asian citrus psyllid management and new planting systems are needed to maintain tree health and bring new blocks into rapid production. Systemic insecticides applied to the soil supplemented by foliar sprays provide some protection against ACP but have proven insufficient to adequately control HLB infection during critical the first 3 years after planting. Planting into a bed covered with metalized polyethylene film offers an additional layer of protection by warding off incoming ACP adults through flight disruption. Lack of similar effects with white mulch reflecting only visible light indicates that reflected light in the UV range is required. Use of plastic mulch is a novel practice in citrus production requiring delivery of water, nutrients and systemic pesticides by drip irrigation. Therefore, adoption of this practice to commercial production is necessarily a gradual process. Nevertheless, some large scale trials have been conducted on commercial farms and interest in growing as results come in and technology improves. The metalized mulch must be of sufficient thickness and protected with a clear coat to last 3 years.

Ground must be well prepared and film mechanically installed as a tight 125-145 cm wide strip, higher in the center to shed water and sprays. Drip irrigation can be provided by one or two relatively inexpensive drip tubes placed under the mulch or punch-in emitters at the base of each tree. Trees are typically planted through 15 cm holes cut in the plastic using a post-hole digger. Oil containing sprays should be avoided to protect the polyethylene from undo degradation. Metalized mulch consistently reduces ACP populations and HLB incidence compared to whiteface mulch or bare ground. In addition, the mulch plus drip irrigation system increases soil moisture, reduces weed pressure, and accelerates growth. Chemigation has proved more efficient than soil drenches for delivering systemic insecticide once the root system is established. Yield after 3.5 years in a large scale replicated experiment was improved by 44% from trees planted on UV reflective mulch and treated with insecticides compared to insecticides alone and brix improved by 5%. Thus, metalized mulch can improve growing conditions and augment current insecticide based ACP control measures for young trees. The potential to more than compensate for additional costs by savings of water, fertilizer and herbicides, shortening time to crop profitability and increasing yields has inspired interest in using this technology to protect young citrus plantings threatened by HLB. Huanglongbing is severely impacting Florida citrus. The sweet oranges and grapefruit dominating Florida production appear to be among the most HLB-susceptible citrus cultivars. Transition to resistant or tolerant cultivars will be essential to sustain the Florida citrus industry, with solutions needed in the short and long term. Evidence mounts that useful resistance/tolerance to HLB is present in cultivated citrus and greater resistance may be drawn from more distant members of the gene pool, and both approaches are targeted by the USDA citrus breeding program. Potentially useful HLB- tolerance is apparent in several mandarin hybrids.After six years of growth following almost immediate infection, ‘SugarBelle/Sour Orange’ and ‘Tango/Kuharske’ had the largest trunk diameters, good canopy density, and were progressing into fairly normal bearing. In a planting of seedlings from 85 diverse gene bank accessions there is a strong association between tolerance and pedigrees including citron. Hybridization is ongoing to combine diverse sources of tolerance. There is evidence of resistance to HLB in several Citrus crosscompatible genera. In a study of diverse scions on both unifoliate and trifoliate root stocks, root stock had little effect but scions with Poncirus in their pedigrees had significantly lower populations of the HLB bacterium. Strong resistance and even immunity should be possible using genetic engineering and USDA efforts are also underway to produce HLB-resistant scions using biotechnology. Programmed cell death or apoptosis is a plant defense response that can be triggered upon pathogen infection, and some triggers, such as Reactive Oxygen Species , can activate or prime plant defenses over long distances. There are also some anti-apoptosis proteins in plants which dampen plant defense responses. By suppressing expression of these anti-apoptosis proteins, PCD should occur in a more rapid manner and with augmented defense responses upon pathogen infection. Huanglongbing , caused by Ca. Liberibacter asiaticus , threatens citrus production worldwide. Las grows strictly inside living citrus cells. Citrus cells detect Las, but fail to trigger adequate native defense systems or PCD until the bacteria have multiplied to very high levels. To help accomplish this defense suppression, Las secretes both a functional peroxiredoxin and a peroxidase directly into the citrus cell cytoplasm that significantly dampen both the ROS response and signaling pathway. We have developed a citrus gene silencing strategy to suppress a brake on the natural citrus defense reaction to Las. Five silencing constructs were used to transform Carrizo root stocks. Average silencing efficiencies of the transformed root stocks ranged from 77.30% to 82.35%. Using a Las flagellin protein fragment applied at 10 µM as a proxy for Las infection, expression levels of three citrus defense response genes NDR1, PR1 and EDS1 were significantly higher in silenced lines than in NT controls. Challenge inoculations with Las were then performed by approach grafting selected transgenic Carrizo lines representing all 5 silencing constructs to mature Las-infected nontransgenic citrus scions.

The original smooth-cylinder trap model did not catch many psyllids, <10%, even in high density population exposure; Psyllid trap efficacy also requires correct field positioning ; One design accidentally had a rougher texture on the outside, and it caught more psyllids than similar traps that were smooth; Newer traps were then developed with intentional projections on the outside; Our best trap design so far has yielded a catch rate of 30-40% ACP, compared to psyllid-capture on yellow sticky traps, as determined by a limited number of controlled field trials; 3D-print designs can be modified rapidly from results of field testing, which is currently underway in FL, TX, and CA; 3D-printed traps can also incorporate species cues for light, sound, and odor attractants to overcome field positioning effects. The Asian citrus psyllid is known to obtain nutritional and defensive benefits from its complement of endosymbiotic bacteria. The most abundant of its associated endosymbionts, ‘Candidatus Profftella armatura,’hydroponic nft system has been shown to devote approximately 15% of its predicted genome to polyketide biosynthesis. The interaction between the ACP and Profftella is an ideal target to combat the psyllid since this symbiont is found in all populations of the ACP surveyed thus far and in no other insect.

A dual proteomic-metabolic analysis indicated that the complement of polyketides produced by Profftella plays a significant role in the insect host response to infection with ‘Candidatus Liberibacter asiaticus’ , the bacterial pathogen responsible for Huanglongbing . The primary mechanism of this defense is via production of the cytotoxic polyketide diaphorin, estimated to be produced at 1.3 ± 0.3 mg per 1,000 adult insects. ACP populations reared on CLas-infected citrus trees show significant upregulation of Profftella proteins involved in polyketide biosynthesis as well as quantitative differences in abundance of diaphorin and a related polyketide compared to ACP reared on healthy trees. In this work, we aim to verify previous structural characterizations of diaphorin and probe its potential interactions with receptors in the ACP to gain a better understanding of the interplay between Profftella, diaphorin, and CLas within the insect. We extracted approximately 6,000 adult ACP and verified the presence of diaphorin in the extract using infusion mass spectrometry. Diaphorin is further enriched from the extract using preparative High Performance Liquid Chromatography . Nuclear magnetic resonance is used to evaluate the structure of diaphorin isolated within the fractions, and LC-high resolution mass spectrometry is used to confirm stability of the compound. Future plans include immobilization of purified diaphorin on a solid support resin for use in an affinity purification strategy to identify interacting insect proteins.

This will allow a better understanding of the specific role and localization of diaphorin within the insect, as well as provide insight into the systemic response to CLas acquisition with an aim toward potential control strategies. Citrus Huanglongbing or greening is a devastating bacterial disease that has destroyed millions of trees and is associated with phloem-residing ‘Candidatus Liberibacter asiaticus’ in Florida. In this study, we evaluated the control effect of different antimicrobials including oxytetracycline, streptomycin, and plant defense inducers via trunk injection. Spatiotemporal dynamics of oxytetracycline in planta was evaluated. Las-infected ‘Hamlin’ sweet orange trees on ‘Swingle’ citrumelo root stock at the early stage of decline were treated with oxytetracycline hydrochloride using trunk injection with varying number of injection ports. Spatiotemporal distribution of OTC and dynamics of Las populations were monitored by HPLC method and qPCR assay, respectively. Uniform distribution of OTC throughout tree canopies and root systems was achieved 2 days post injection. High levels of OTC were maintained in leaf and root for at least 1 month and moderate OTC persisted for more than 9 months. Reduction of Las populations in root systems and leaves of OTC-treated trees were over 95% and 99% between 2 and 28 DPI. Conditions of trees receiving antimicrobial treatment were improved, fruit yield was increased, and juice acidity was lowered than water-injected control even though their differences were not statistically significant during the test period. Our study demonstrated that trunk injection of OTC could be used as an effective measure for integrated management of citrus HLB. The Citrus Genome Database is being developed as a one-stop resource for citrus genetics, genomics, and breeding research.

In this presentation we highlight CGD features that provide the citrus community with data and tools to combat Huanglongbing . The database has been redesigned and features a streamlined user interface that allows for quick access to data and tools and is also mobile friendly. It contains curated citrus genetic marker, map, and QTL data, genome data for clementine and sweet orange, as well as annotated reference transcriptomes generated by analysis of published RNA-Seq and EST datasets. The database also has the most up-to-date version of CitrusCyc v3.0 which includes metabolic maps for the C. clementina and C. sinensis genomes. Tools such as BLAST for searches against the genome sequences, GBrowse/JBrowse for viewing genomes, and CMap/TripalMap for viewing and comparing genetic map data are also available on CGD. In addition to the tools to view and search published data in CGD, the Breeding Information Management System allows breeders to upload their data via the web, or directly from the Field Book app, to a private account. The private data than can be analyzed in conjunction with the public data. In addition to citrus data, the genomes of the different ‘Candidatus Liberibacter’ species and psyllid vector are available in CGD in JBrowse and the sequence data is searchable with the BLAST tool. As a community resource, CGD is being developed based on user feedback with the goal of providing the data and tools that will enable citrus crop improvement. CGD is supported by USDA-NRSP10, NSF-PGRP, and USDA-SCRI. First report of delivery of two synthesized oligo nucleotide products into plants. Subsequent feeding on treated plants by insects resulted in ingestion and systemic movement of the two gene-based targeting products: FANA_ASO, _; and a PPMO . Topical sprays onto citrus leaves, root absorption, and tree trunk injections, resulted in cell delivery of the molecules which were designed to: Asian citrus psyllid; Plant pathogenic bacteria of citrus, Candidatus Liberibacter asiaticus , Psyllid endosymbionts, and Diaprepes abbreviatus, weevil.Treatments reduced CLas bacteria within infected citrus trees,nft channel reduced Wolbachia in cell cultures, the insects, while resulting in increased insect mortality. This is the first evidence for successful delivery of FANA_ASOs and PPMO into plants, and use of these molecules for plant delivered strategies to reduced plant pathogens in citrus, and to manage insect pests. Molecules were shown to move systemically through plant tissues as visualized with Confocal microscopy and spectrophotometry.Adult insects showed systemic movement through hemolymph and organs: supra and sub esophageal ganglions, fatbodies, nerves, and alimentary tract. Results suggest a role for these products in the reduction of plant pathogens like CLas, associated with Citrus greening disease; and reduction of insect vectors of pathogens on citrus and other agricultural crops.Two circular prophage genomes have previously been described integrated in Ca. Liberibacter asiaticus strain UF506 genome, and all Florida Las strains have both prophages. Either one or rarely, both, prophages were reported absent in certain recently sequenced Chinese and Japanese Las strains. The SC1 lytic cycle, marked by upregulation of several late genes including a functionally lethal holin , is activated when Las is in planta, but not when infecting the Asian citrus psyllid host. The holin promoter was cloned into the wide host range vector pUFR071, structurally replacing the native lacZ promoter to drive the beta-glucuronidase reporter gene. The holin promoter exhibited very strong and constitutive expression of the GUS reporter gene in Liberibacter crescens , a culturable proxy for Las. We previously reported the presence of an unidentified, heat labile, protease-sensitive repressor in aqueous psyllid extracts that quenches the expression of the holin:GUS reporter. Tandem LCMSMS analyses were used to identify ca. 25 peptide fingerprints in the DNA-binding protein eluate captured by the holin promoter DNA immobilized on magnetic DynaBeads.

A putative repressor protein was identified that was unique to the Wolbachia bacterial endosymbiont found in psyllids and absent in Wolbachia found in the fruit fly . This putative Wolbachia repressor gene was cloned in an expression vector and used for in vitro cell-free protein synthesis. The in vitro translated Wolbachia protein partially repressed the holin::GUS reporter in a dose-independent manner, as compared to the aqueous extract from the psyllid, thus indicating that complete suppression of holin promoter requires an additional partner. Heat-inactivated psyllid extract was unable to enhance the Wolbachia repressor- induced suppression of holin promoter activity, confirming the heat labile proteinaceous nature of the additional psyllid-sourced partner. A conspicuous feature of plant defense reactions in response to an attempted pathogen invasion is the engagement of an oxidative burst. We have previously shown that an SC2 prophage-encoded peroxidase in Ca. Liberibacter asiaticus strain UF506 represents an important lysogenic conversion gene whose expression may increase bacterial fitness and delay symptom development in the host plant. However, absence of either one or both prophages in certain recently sequenced Chinese and Japanese Las strains, prompted a review of H2O2-detoxification mechanisms conserved amongst Liberibacters. Two putative peroxiredoxin genes have been annotated on the Las chromosome, which are conserved and identical among all the sequenced Las strains. The BCP-like peroxiredoxin is predicted to be nonclassically secreted. The expression levels of Las PR2 in both citrus and psyllid were equivalent. Notably, however, the expression of the predicted secreted BCP-like peroxiredoxin was similar to that of PR2 in citrus but nearly undetectable in psyllids, reminiscent of the low level of SC2_gp095 peroxidase expression in psyllids. Despite carrying a native ortholog, Lcr transformed with the BCP-like peroxiredoxin showed a significant increase in tolerance to 100 µM H2O2, as compared to a marginal 4-5 fold tolerance provided by either the PR2 peroxiredoxin or SC2 peroxidase. Most importantly, BCP-like peroxiredoxin augmented Lcr tolerance to 50 µM tert-butyl hydroperoxide by nearly 1000 fold. We hypothesize that both the Las peroxiredoxins may be implicated in alleviating the oxidative burden originating from physiological electron transport reactions. However, the extracellular BCP-like peroxiredoxin may be essential for Las survival in an oxidative host cell environment through a) direct detoxification of host-generated H2O2, b) limiting the catastrophic chain reaction spread of a lipid peroxidation event initiated by H2O2 permeating the bacterial cell membrane, and c) most importantly, akin to the secreted SC2_gp095 peroxidase, dampening the systemic propagation of H2O2-mediated defense signaling in the host plant following an initial colonization event. Las BCP-like peroxiredoxin may represent a critical secreted effector conserved across all pathogenic Liberibacter species that functions to suppress host symptoms, a tactic used by most biotrophic plant pathogens. Detection of HLB-associated Candidatus Liberibacter asiaticus by real time PCR has been widely used in citrus industry since its development a decade ago. However, the methodology is not adaptable to quantitation by digital droplet PCR since it uses a multicopy mitochondrial gene as an internal control. Since there are hundreds of copies of COX gene per cell, inefficient amplification of the low copy target gene can occur. In this study, we sequenced a single copy nuclear gene, malate dehydrogenase from over 100 accessions including several citrus and citrus relatives in the subfamily Aurantioideae. The plant samples selected are known to be hosts of both HLB and the psyllid vector. Primers and probe were developed using conserved regions of MDH gene for Taqman® based qPCR and also for ddPCR analyses. We conducted qPCR assays using various dilutions of HLB infected plant samples to detect CLas in duplex reactions using two different internal controls – COX and MDH in separate reactions. ddPCR assays were conducted using a BioRad QX200 droplet digital PCR System. The advantages of using MDH as an internal control gene in both qPCR and ddPCR will be discussed. Since confirmatory tests to detect the presence CLas cannot be done with plant samples having low bacterial titers, additional confirmatory assays by ddPCR of other regions of CLas genome were also developed. Detection of CLas in samples with low titers is critical for both prevention and management of HLB, especially during early stages of the epidemic, and to slow down the spread of disease. Testing of psyllid vectors for the presence of huanglongbing-associated Liberibacters is known to be an effective method for early detection of HLB in citrus groves.