Category: hydroponic agriculture

Populations Sonora x CBdeM , Sonora x Foisy , and CBdeM x Foisy have 146, 141, and 128 lines respectively. The DH lines were phenotyped using a modified cigar roll method of Zhu et al. . The system is similar to the Cyg germination growth pouches and the gel based system of Bengough et al. . It consists of two plexi-glass plates 20 cm x 30 cm fitted with spacers, germination paper, racks holding the plates upright and tubs used to hold water. One hundred seeds were counted and weighed to estimate average seed weight. Seeds of similar size and weight for each genotype were imbibed in water for 24 hr prior to planting. Germination paper wetted with deionized water was placed on one of the two plates, and two seeds of the same genotype were placed with embryos down 5 cm below the top edge of the paper and 8 cm apart. This set up was covered by a second sheet of wet germination paper and a second sheet of plexiglass. The entire set-up was clipped together and placed upright into tubs of water about 8cm deep; this water level was maintained constant throughout the experiment. Seedlings were grown for 7 days at room temperature without supplemental lighting in a head house at the University of California, Riverside between February 2014 to May 2014 and November 2014 to February 2015. Experiments were setup in a randomized complete block design with four replications where replications were treated as blocks. Each replication had two plants from every genotype. After 7 days plates were removed from the tubs, disassembled, 4x8ft rolling benches and seminal roots were imaged using a hand held digital scanner set to 300 DPI.

To acquire images, the top sheet of germination paper was carefully removed and the top plexi-glass plate was laid over the seedlings so the scanner could be passed over from above. Seminal root angles were measured using the angle tool in ImageJ . For each plant, the angle between the first pair of seminal roots was measured at approximately 3cm below the embryo of the seed , as shown in Figure 1. The analysis of variance for seminal root angle and number was based on mean values of the experimental units. Broad sense heritability was calculated on a mean basis across four replications. Genotype means were used to calculate Pearson‟s correlation coefficients for seminal root number, seed weight, and seminal root angle. Genomic regions associated with traits of interest were detected by the software package IciMapping using linkage maps for these populations as described previously and the mean value of 8 seedlings of each genotype from four replicates. The composite interval mapping method with a step of 1cM was used and the threshold for QTL detection was determined using 1000 permutations where α = 0.05. Markers in the linkage maps were renamed using the index number provided by Wang et al. preceded by the chromosome designation. QTL consistent between years within populations and/or consistent between populations were considered as verified QTL and named according to McIntosh Catalogue of gene Symbols for Wheat . Following the format of previous publications an uppercase “Q” in the name signifies strong verification of the QTL and lowercase “q” was used to name QTL that were consistent but warrant further investigation. In most cases associations of root system characteristics with specific genome region varied between populations and within populations from one year to the next. Over two years of the experiment and with all three populations taken together, a total of 31 genomic regions showed statistically significant associations with the seminal root angle and number . Seminal root angle was associated with 12 chromosome regions in the SC population, located on chromosomes 2D, 3B, 4A, 5A, 6A, 6B, 6D, and 7B. In the SF population, five regions on 2D, 5B, 6B, and 7B were identified and another five regions were identified in the CF population, on chromosomes 5B, 6A, and 7A.

The chromosome region with the single largest effect for the seminal root angle was located on chromosome 2D in the SC population. Its estimated effect was equivalent to 7.33° of the total root angle, and it was responsible for 21.42% of the population variation. The region with the lowest, but statistically significant effect for root angle was identified in the CF population, accounting for an estimated 2.90° of the root angle and explaining 9.40% of the variation observed in this population. For the seminal root number, nine genomic regions were identified in the three populations. Of these, four were identified in the SC population, on chromosomes 4A, 5B and 7A. The SF population had only one region, on chromosome 4B. The remaining three regions were identified in the CF population on chromosomes 1B, 6B, and 7D. The region with the largest effect was on chromosome 4A in the SC population, with an estimated effect of -0.25 roots per seedling explaining 17.32% of the total variation. The region with the lowest but statistically significant effect was identified on chromosome 6B in CF, with an estimated effect of 0.15 roots per seedling, explaining 8.41% of the population‟s variation. For the purpose of this study, only those genome regions that showed consistent associations with specific traits within a given population over both years were considered as verified QTLs . In the SC population three such regions were identified, located on chromosomes 2D, 6A, and 7B . The region on chromosome 2D was 4.17 cM region with a peak at 113 cM between markers 2Dx_79444 and 2Dx_77420 in 2014. It accounted for 25.99% of the phenotypic variation seen in the population that year. In 2015, the region was located between markers 2Dx_32130 and 2Dx_79444 covering a 0.67 cM with a peak at 112 cM. That year it explained 21.42% of the phenotypic variation seen. The allele for wider seminal root angle was contributed by Sonora. The second QTL was located on chromosome 6A. In 2014 it was between markers 6A_72189 and 6A_55084 covering a 4.90 cM region with a peak at 151 cM. It explained 7.04% of the phenotypic variation that year. In 2015, this QTL formed a peak at 155 cM between markers 6A_55084 and 6A_21174, it coved 1.35 cM and explained 7.21% of the variation for the trait. The allele for wider seminal root angle was contributed by Sonora.Each of the three tested population showed large phenotypic variation for both seminal root traits measured in this study.

The largest range in seminal root angle was between Sonora and CBdeM with average seminal root angles of 108.73° and 63.31° respectively . The least difference, but still statistically significant,flood and drain table was between CBdeM and Foisy which have more similar seminal root angles of 63.31° and76.95° respectively. The distribution patterns among progenies imply considerable trait complexity.All three parents typically had five seminal roots with few variations between replication giving averages of 4.36, 4.38, and 4.49 seminal roots for SC, SF, and CF respectively. The occurrence of less than five seminal roots is likely explained by environmental interaction and associations with seed weight. Since all parents typically develop five seminal roots it is not surprising that the three populations have similar means and ranges. As will be discussed later, the lack of consistent QTLs for seminal root number may suggest that this trait is heavily influenced by the environment and seed weight. However, one consistent QTL was identified which also suggests that there is a genetic component as well. Additionally, heritability values were relatively high for both traits in all populations but it does not seem to promise any ease of selection for breeding efforts. As will be discussed it certainly doesn’t hint at simplicity for the genetics of these traits.The 90K SNP array was used on eight mapping populations of doubled haploids to order SNPs along individual chromosomes and 44,345 of those were mapped to one or more of 46,977 loci . Due to differences in polymorphism among different sets of parents, only a fraction of all mapped markers can be expected to be useful in any given pairwise combination. Moreover, as distribution of crossover can vary substantially between different pairs of parents the actual genetic map position of any given marker may also differ . To facilitate utilization of the maps generated using the 90K SNP chip, Wang et al. 2014 created a consensus SNP map of wheat, based on the tested eight populations. In essence, this map provides average marker positions for all polymorphic markers of their study and may be used to coordinate maps generated for different populations. As it was explained in an earlier chapter, total lengths of maps for each of the three populations here varied but more importantly, at times very few common markers were present in specific chromosome regions. For verified QTLs, that is for consistent associations between specific DNA markers and genome regions consistently showing up in replications, the consensus map was used to allocate those to specific regions and used DNA sequence data of the closest associated marker to blast against the wheat sequence survey on the URGI database and determine its actual location. In this fashion, relative locations of QTLs identified in this study can be compared to all previous results and can be verified in the future. This approach makes it possible to use even those DNA markers that were not polymorphic between two parents of a given population increasing the resolution of a mapping exercise. This study identified 31 genomic regions associated with seminal root angle and seminal root number in three populations. Most of these regions were unique to specific populations and varied from year to year. This implies that these traits are far from simple, as proposed by Oyanagi and do not appear to be controlled by single loci. It must be pointed out that compared to other studies on seminal root traits, the results presented here appear to be better supported by experimental data. Using a single population of 103 doubled haploids Hamada et al. were unable to identify a QTL for seminal root angle; two QTLs for deep root ratio appeared on chromosomes 1B and 5D. Another QTL, for seminal root, was found on chromosome 5A. None of the regions consistently identified in this study appear to be located on chromosomes of Hamada et al . In another study, Christopher et al. identified 12 QTLs for seminal root angle and number in a single mapping population of bread wheat consisting of 184 individuals. The QTLs for seminal root angle were located on chromosomes 2A, 3D, 5D, 6A, and 6B; those for seminal root number on chromosomes 1B, 3A, 3B, 4A, and 6A. While some chromosomes are the same as those identified in this study, none are on chromosomes verified as valid QTL in this study: 2DS, 6AL and 7BS for seminal root angle and 4BL for seminal root number. In another study Liu et al. again identified a total of 12 QTLs for seminal root angle and number. Seven of those, for seminal root angle were on chromosomes 1A, 2B, 3A, 3B, and 7D and five for seminal root number on 2B, 3B, 3D, 5A, and 7A. Again, there are some genome regions in common with this study but none appear to be are similar to our verified QTL. Most studies employ a single mapping population. Beavis demonstrated that in populations numbering 100 progeny, the QTL effects were greatly overestimated, in populations with 500 progeny the QTL effects were slightly overestimated while populations with 1000 individuals produced estimates close to the actual magnitude of QTL effects. That study highlighted the necessity for larger populations and the need for verification of QTL across populations. Beavis did not address the issue of mapping in parallel populations sharing common parents. To the best of our knowledge only a couple of studies made use of two or more populations in studying root system traits: Zhang et al. used three related recombinant inbred line populations with a single common parent and Kabir et al. used two unrelated populations. Zhang et al. identified QTLs for seminal root number on chromosomes 1D, 2A, 2B, 2D, 3A, 3B, 4A, 4D, 5A, 5D, 6A, 6B, and 7B.

This discrepancy could be due to misannotations in the genome or to the existence of alternative biosynthetic pathways. In any case, the addition of phenylalanine and histidine greatly improved the growth and phosphite oxidation rate of FiPS-3 cultures. The growth benefit conferred by these two amino acids was similar to that of yeast extract or casamino acids and was probably due to their ability to serve as organic carbon and nitrogen sources. FiPS-3 has previously been characterized as a strict anaerobe, and the presence of cytochrome c oxidase genes in its genome has been attributed to an uncharacterized mechanism of nitrite resistance, although this hypothesis was never tested . Here I show that FiPS-3 is not only capable of growing under aerobic conditions, but indeed grows better in the presence of oxygen than in controls containing no electron acceptor. This observation, coupled with the presence of cox genes in its genome, suggests that FiPS-3 is capable of oxygen respiration. This is an unexpected result given that oxygen tolerance is rare among sulfate reducing bacteria and that the capacity for aerobic respiration is rarer still, with only a handful of oxygen-respiring sulfate reducers having been documented so far . However,hydroponic indoor growing system the fact that FiPS-3 grew at a similar rate in the presence of oxygen as it did under sulfate-reducing conditions is puzzling, since oxygen is a substantially more favorable electron acceptor from a thermodynamic point of view and should, therefore, allow for faster growth. This may be indicative of a failure on the part of FiPS-3 to adequately cope with oxidative stress, which imposes a growth cost under aerobic conditions.

Its inability to grow in a well-aerated culture lends support to this hypothesis and highlights the fact that the actual dissolved oxygen concentrations it was exposed to in unmixed cultures were likely well below the concentrations present in the head space. As such, it may be more accurate to regard FiPS-3 as a microaerophile rather than a true facultative aerobe. Furthermore, it is also possible that FiPS-3 was not actually growing by aerobic respiration in oxygen-containing cultures but rather growing by sulfate reduction with the oxygen serving to re-oxidize any sulfide produced back to sulfate. Even though sulfate was not added to the aerobic cultures, even a small amount carried over from the inoculum could have supported the observed growth if the sulfide produced was continuously reacting with oxygen to replenish the sulfate pool available to the cells. Further experiments, such as quantification of cox gene expression levels during aerobic growth or assessment of aerobic growth in the presence of complex IV inhibitors, are needed in order to distinguish between these two scenarios. In my FiPS-3 cultures, growth by DPO led to the precipitation of struvite or hydroxyapatite crystals when magnesium or calcium were added to the media. Schink et al. have previously observed the formation of struvite crystals during phosphite oxidation by FiPS-3 cultures. They attributed this phenomenon to the accumulation of inorganic phosphate to high concentrations in the extracellular milieu and the subsequent reaction of this phosphate with magnesium ions present in the medium to form crystalline mineral precipitates. This process of DPO-dependent biomineralization exploits the large difference in solubility between phosphite and phosphate and could potentially be applied to the development of bio-concrete. Bio-concrete refers to ‘self-healing’ concrete that incorporates bio-mineralizing microorganisms in order to help seal cracks that develop over time and thus improve durability and strength .

Most of the research conducted in this area has so far focused on the precipitation of calcium carbonates by heterotrophic bacteria . However, the precipitation of calcium and magnesium phosphate minerals by means of a chemolithoautotrophic DPO-capable bacterium may present an alternate approach for the advancement of this technology. Although upregulation of the PtdF protein in the presence of phosphite has been previously reported , this is the first evidence of increased expression of the entire ptx-ptd gene cluster under phosphite-oxidizing conditions. This finding affirms the connection between the ptd genes and DPO and lends further support to the hypothesis that these genes are necessary for growth by means of phosphite oxidation. It is important to note that 1 mM phosphate was present in both growth conditions, so that the increased expression of these genes was not due to phosphate starvation, which is known to induce expression of ptx genes in APOcapable organisms . However, the substantial difference in expression levels seen between the ptx and ptd genes was unexpected and implies that ptxDE and ptdFCGHI may represent two distinct functional modules that are differentially regulated. The fact that ptxDE are also present in APO-capable organisms in combination with an alternate phosphite transporter also supports the notion that the ptx and ptd genes represent separate modules. As discussed in Chapter 1, PtxD is known to be the enzyme responsible for phosphite oxidation both in APO organisms and in FiPS-3, while the function of the predicted transcriptional regulator ptxE remains a mystery. It is possible that the role of ptxE is actually to promote transcription of ptdFCGHI in the presence of phosphite, which could explain the higher levels of expression seen for these genes. Given that PtxD is highly efficient at turning over phosphite , the transport and energy conservation steps may in fact represent the bottlenecks for growth by DPO. Increased expression of the ptd genes could, therefore, be a way to compensate for the relative inefficiency of these processes compared to the phosphite oxidation step itself. Furthermore, the exceptionally high level of ptdF expression compared to the other ptd genes suggests that this enzyme may catalyze the ratelimiting step in the pathway.

Overall, my RNAseq results highlight the importance of the ptd genes during DPO-dependent growth of FiPS-3, but exactly what their functional roles are and how they are regulated remains to be elucidated. It is essential, going forward, to determine the minimal set of genes that are necessary and sufficient for growth by DPO,vertical rack system either through targeted knockouts in FiPS-3 or through expression of candidate genes in heterologous hosts. DPO enzymes could then be purified and characterized in vitro in order to determine their mechanisms of action. Unfortunately, my attempts to carry out targeted gene deletions in FiPS-3 as well as to heterologously express the full ptx-ptd gene cluster in D. balticum SaxT and in E. coli have sofar proven unsuccessful. Nonetheless, I hope that my efforts to improve the growth of FiPS-3 under both anaerobic and aerobic conditions will aid in the development of genetic tools in this organism that will allow future researchers to address some of these unanswered questions. Furthermore, the ability of FiPS-3 to precipitate phosphate minerals as a byproduct of DPO holds great promise as a potential mechanism for bioconcrete production. However, before this process can be applied on an industrial scale, further research must be undertaken in order to establish the optimal conditions for biomineralization by FiPS-3 as well as to assess the material properties of the different mineral products.Killed controls showed no phosphite oxidation even after 29 weeks and the rate of phosphite oxidation increased over time. The persistence of phosphite under abiotic conditions is congruent with its kinetic stability resulting from the high activation energy needed to break the P-H bond in the phosphite molecule . Moreover, the extent of phosphite oxidation observed in our enrichments is evidence of a dissimilatory process involved in energy metabolism as opposed to a purely assimilatory process. Phosphate accumulated in the media over time at a rate that corresponded to that of phosphite depletion and reached a final concentration that matched the amount of phosphite consumed . Assuming a phosphorus content of ~30 fg/cell , a bacterial culture would have to assimilate only about 300 µM phosphate in order to reach a density of ~1×109 cells/mL. This means that the phosphite consumed in our enrichments far exceeded what would have been required for use as a phosphorus source. In fact, there was more than enough phosphate initially present in the cultures to fulfill the phosphorus needs of growing cells in the absence of phosphite supplementation. The fact that microbial growth rates were significantly higher in phosphite-oxidizing cultures compared to no-phosphite controls provides further evidence that phosphite was acting as an electron donor and energy source for cellular metabolism. Phosphite oxidation and growth only occurred in media amended with CO2 and/or HCO3 – indicating that DPO was coupled to CO2 reduction, as has been shown for FiPS-3 . The rates of phosphite oxidation seen in our rumen fluid amended enrichments are comparable to those seen in cultures of FiPS-3 . However, our enrichments were incubated at 37o C and FiPS-3 can only grow at temperatures between 15 and 30o C . Indeed, our microbial community analysis confirmed the absence of any 16S rDNA reads belonging to the Desulfotignum genus in our enrichments.

There was, however, a clear shift in community composition in the presence of phosphite, with OTU 33 , OTU 21 , and OTU 28 being the most enriched taxa. Addition of rumen fluid to the enrichments greatly enhanced the rate of phosphite oxidation and also substantially altered the microbial community composition. Interestingly, OTUs 33 and 28 were no longer observed in the community. Instead, OTU 21 was further enriched to about 40% of the total community. The tight correlation seen between phosphite oxidation and Phox-21 abundance strongly supports its functional role in DPO. It remains unclear why OTUs 33 and 28 were enriched in our original cultures but not in the presence of rumen fluid. Given that rumen fluid was added to the cultures in an attempt to enhance the growth of OTU 33, its decline was particularly surprising. It is possible that these organisms benefited from the presence of phosphite under low-nutrient conditions, but were then outcompeted when nutrient-rich rumen fluid was added. Also, the fact that these taxa had very low initial abundances means that even moderate amounts of growth would have led to large increases in fold change, which would have magnified the effect of any ancillary growth benefits that these organisms may have received. In any case, the addition of rumen fluid did enhance the growth of Phox-21, which was an unexpected outcome, but perhaps not too surprising given the rich variety of nutrients and cofactors present in rumen fluid .The strong inhibitory effect of molybdate on phosphite oxidation and Phox-21 abundance in a culture not dependent on sulfate reduction was likewise unexpected, since molybdate is known to be a potent and selective inhibitor of sulfate reduction. It acts as a futile substrate for the enzyme ATP sulfurylase, resulting in depletion of the cell’s ATP pool . However, the addition of sulfate did not enhance phosphite oxidation, stimulate sulfidogenesis, or enhance the growth of Phox-21. It is possible that molybdate is targeting the phosphite oxidation pathway directly or affecting some other unrelated pathway, but further work is needed in order to confirm the mechanism of inhibition. Sulfite also appears to inhibit phosphite oxidation, which is further evidence of a potential incompatibility of the sulfate reduction pathway and phosphite oxidation in our enrichment. It is not clear whether the inhibition by sulfite was due to a direct interaction with the phosphite oxidation pathway or to a broader toxicity. PtxD, the phosphite oxidase found in FIPS-3 as well as in most APO-capable bacteria, is known to be inhibited by sulfite in vitro . This enzyme, if present in Phox-21, would be a likely target of sulfite inhibition. However, sulfite is also known to have a broad antimicrobial activity at concentrations as low as 600 µM , which may have also played a role in its inhibition. Phox-21 belongs to the GW-28 candidate order, a poorly studied clade within the Deltaproteobacteria with no known cultured representatives. Its closest relatives are all uncultured clones from different anaerobic waste treatment sites. Clone GW-28 was found in a household biogas digester in China , clone POMEbac42 was found in a food waste digester in Singapore , clone De3155 was found in alkaline landfill leachate sediment in China , and clone QEEB2BG06 was found in a mesophilic wastewater digester in Germany .

The survey was distributed to the 5th, 6th, 7th, and 8th grades, representing 116 students. The 6th grade results represent the “experimental” group in this semi-experimental design, while the 5th and 7th grade students represent “control” groups who did not receive the climate change curriculum focus in their humanities classes. The 8th grade students are used for additional comparative analysis as they study climate change for 12 weeks in science class, and are thus a form of comparison between science and social studies-based climate curriculum.Interview subjects represented school staff , teachers and the partner non-profit Climate Generation , for a total of 5 in-depth interviews of approximately 1 hour in length. By the nature of this study as a participatory research partnership, one of the interview subjects is also a co-author of this paper. Rather than muddying the waters of analysis or representing a conflict of interest, it is our belief and intention that co-authorship provides necessary clarity and depth of perspective on the curriculum pilot by partnering a climate education researcher with an educator interested in critically analyzing, improving, and disseminating a climate curriculum innovation. Interviews were semi-structured, and focused on the following topics: a) impacts of teaching climate change through humanities, b) student and parent responses to curriculum, c) process of developing curriculum and/or delivering instruction , and d) advice and recommendations for other schools. The classrooms observations took place towards the end of the school year, in April, and notes were recorded for analysis by one co-author . The other co-author contributed insights from her observations and experience working in the school over the course of the year in her interview . Observational research methods bring critical insights and context to inform interpretation of results,vertical grow tables revealing personal motivations and helping to uncover process dynamics leading to end results. Survey results were compared across grades and analyzed using Google Forms data analysis as well as basic statistical analyses.

Google Forms is a commonly used educational technology, and thus using this method has the advantage of making the process easily replicable to teachers, schools, and education organizations seeking to gather their own data on similar interventions. Disentangling confounding variables and adjusting for student baseline academic performance and demographics are additional avenues for future research but were not the focus of this study. Interviews were manually coded for themes and analyzed by type to understand differences in reaction between teachers vs. other adults involved in curriculum development and piloting. Interview themes inform next steps for Lowell as well as other schools seeking to implement climate change curriculum through a humanities focus, and guide researchers studying effective strategies and evolving trends in climate education. They reveal strategic improvements possible for the curriculum, as well as hypotheses for testing in larger CCE research explorations .The climate literacy survey was administered to 30 5th graders, 36 6th graders, 27 7th graders, and 23 8th graders, for a total of 116 students. The 6th graders averaged 74.4% correct response on the nine multiple choice questions, while the 5th graders averaged 60.4% correct, 7th graders 69.5% correct, and 8th graders 74.8% correct . The 6th graders had higher correct response rates for five out of the nine MC questions; in the remaining four, the 7th grade had the highest response rate on two questions, and the 8th grade had the highest response rate in the other two. Asked about the global temperature rise limit specified at the United Nations Conference of the Parties talks in Paris, the 6th graders outperformed their peers by the greatest margin, perhaps due to the focus on UN climate conferences leading up to a mock UN climate negotiations activity included in the curriculum . In the open response questions regarding solutions for lowering CO2 levels and mitigating climate change, the 6th grade students matched the 8th graders in terms of total number of responses across various categories , and were the only grade in which a student mentioned the single most impactful individual action to mitigate climate change: have fewer children .

While the sample sizes are too small to merit statistical significance, there are other conclusions of significance to be drawn from these results .The curriculum coordinator, who worked most closely on the development process along with Climate Generation staff, recognized the need from the beginning to build a wide platform of support for the idea from stakeholders both within and beyond the school. Several parents as well as staff, teachers, and external partners were present at the very first curriculum planning meetings. According to school staff, the reaction from parents has been overwhelmingly positive, and there is reported evidence of students discussing climate change with their parents. In the Director’s words, “any push back we were expecting has not happened,” .The conversation has spread among parents at the whole school, who are aware of what is going on, and are motivated to keep their students at Lowell because of the new curriculum focus. It is additionally used as a recruitment tool to attract new families– 2018/2019 6th grade recruitment gains are related to the new curriculum, according to staff.The curriculum coordinator summarized the first year implementation as follows: “Students learned how climate change affects a variety of people around the world, and how different people are responding to it. They read fiction and non-fiction texts featuring climate change and studied the interaction of geography, civics, and economics with climate change. Next year we are hoping to add a substantial service-learning aspect to the course, as well” . When asked if middle schoolers are “ready” to learn about climate change, addressing a common concern among educators about an age threshold for talking about a topic as overwhelming as climate change, all respondents answered in the affirmative. In the words of one teacher, students learn about the Holocaust and slavery in middle school, so they’re already dealing with emotionally charged content.

One 6th grade teacher initially felt inclined to avoid the topic due to “gloom and doom” connotations and possible negative emotional responses, preferring to focus instead on developing a love of nature and the outdoors among students. However, he reports having his mind changed by the curriculum pilot experience, and notes that his skepticism about the climate fiction novel in particular proved unwarranted. The students had incredibly positive responses to the cli-fi novel chronicling a girl and her family’s flight from their homeland as climate refugees and struggle to start a new life. Students felt catalyzed to take action: “The Cli-fi novel was groundbreaking for me. The kids really enjoyed it. It’s a weird way for doom and gloom to be exposed to them, but yet their reaction is incredibly surprising, I thought they’d turn off, but it almost seems motivating and inspiring them to learn more” . The Director of the Middle School brought up several noteworthy outcomes from the first year not captured in the student climate surveys. From the school’s internal standardized testing, he noticed a dramatic improvement in the 6th graders’ reading comprehension scores. Students increased by almost two Standard Deviations from their scores last year, an unusually high rate of increase,flower pot wholesale with top students maintaining their performance and the bottom third reaching grade level standards . One key growth point identified in several interviews was the need to expand and improve the solutions focus within the curriculum and provide opportunities for students to take meaningful action locally on climate mitigation options. For example, when students read “The Boy Who Harnessed the Wind,” students build windmills in science class. During the focus on renewable energy, they could also do a community action project around solar incentive programs: Maryland has a program for homeowners to put solar on their roofs, and students could advocate for D.C. to adopt a similar program as the same publicly owned utility covers both jurisdictions. Another idea brought up by staff is to change the end-of-year field trip from New York City to an island in the Chesapeake Bay threatened with disappearance due to sea level rise. The solutions piece is the most complex part to develop, as the content must be laid down first and authentic solutions built on top of that. This is a clear area of focus for future years . During this visit, 6th grade students articulately expressed the difference between climate change and global warming, which they acknowledged they had not known previously.

One student clearly explained the process of hydraulic fracturing in incredible detail, down to the underground wells with cement lining, injecting water at high velocity to release the oil stored in underground pores, producing a lot of wastewater and creating seismic activity . Students were generally excited to share what they had learned, and recounted memorable topics and projects of particular interest to them. This included the “CliMojis” art project, where they created personal Climate Emojis after reading a Washington Post article about a company designing climate change-based emojis, so that texters could communicate their frustrations and anxieties around climate change pictorially . The students read and discussed the article, and then designed their own climojis, synthesizing learning in a visual form. Other significant learning experiences reported were the Fossil Fuel Museum and learning about how bees will be impacted by climate change while reading a novel called The Hour of the Bees. When asked what students were hoping to do about climate change based on their newfound knowledge, students responded with ideas such as spreading awareness, creating more things from plant materials , farming in a way that supports the environment, and putting more plants in the ground. Many students seemed to have clear ideas around how to take action and expressed feelings of hope and empowerment when describing their collective “climate solutions.” As a whole, the Lowell middle school demonstrated much higher levels of knowledge and engagement around climate change than the average American teenager or adult. Based on a 2010 nationally representative survey of American teenagers, knowledge of climate science basic facts was found to be very low . 59% of American adults fall into the “Alarmed” or “Concerned” categories of the YPCCC Six Americas spectrum as of December 2018 compared to 82% of Lowell middle school students. What remains a challenge both nationally and at Lowell is building optimism around our ability to solve climate change: only 8% of youth agreed that we can and will do something to mitigate climate change in a recent study , and a mere 5% of Lowell students indicated they believe their generation will solve climate change. While acknowledging the receptive audience for implementing the curriculum, the results relating to increased student engagement, increased reading scores and favorable response to a humanities-focused climate curriculum are nevertheless significant and worth building on as an approach to middle school climate education. Further hypotheses are generated such as the claim that climate change as an engaging topic can help boost student performance in core academic disciplines , requiring further testing via controlled experiments. The time period between 6th and 8th grade is a significant youth development stage during which students develop capacity in knowledge retention and empathy and gain exposure to many new topics, and yet the 6th graders performed equal to or above the 8th graders on most climate knowledge and engagement questions. They shared information learned with families and friends more often than their 8th grade peers learning about climate change through science only, generating important hypotheses for CCE/IGL scholars . Results and best practices from this case study should be applied intentionally to other classrooms and school contexts. The web of support is a crucial enabling factor as well as the participation of key influencers, which must be identified in other contexts. The Lowell School curriculum coordinator suggests several vehicles for integrating similar curricula into more structured, state-mandated public school subject matter: through choice of reading materials in civics classes, suggested options for student independent research projects, and current events classes at the high school level.

Mack et al. find that 68 urban gardens in Phoenix, AZ are currently serving just 8.4% of “food desert” residents, and through spatial analysis, 53 gardens sited strategically could serve 96.4% of such residents. From these studies, it is clear that UA projects are not necessarily occurring where they are most needed to increase food security. When it comes to spatial analyses, “while a macro-level quantitative study of the potential in terms of land availability shows that it would be feasible to grow the basic daily vegetable needs for the urban poor in the United States, current evidence from urban farms located within lower-income communities shows that such farms are not necessarily feeding the communities in which they are located,” due to a variety of factors including cost of produce and cultural desirability.Barriers to access are not just due to geographic distance, but rather an array of intersecting factors including the high costs of some urban produced foods, especially from commercial or for-profit operations. Fresh, local produce from vertical or rooftop farms such as Gotham Greens , Plenty Higher Ground Farm , Freight Farms or AeroFarms are often sold at a premium to restaurants and grocery stores, and thus unaffordable to low income households . Despite claims that vertical farms can “feed the world in the 21st century” , it remains to be seen if vertical farms can address food access and food justice. Such farms are often following a corporate food system model of profit maximization and resource use efficiency, subscribing to capitalist logics rather than alternative, social-justice-oriented practices. Among for-profit farms, “the few profitable operations tend to be those selling to high-end restaurants and consumers, not to lower-income residents” . The cost of food, especially healthy fresh produce, is often in tension with other high costs of living in urban areas , causing low-income residents to become dependent on emergency food services and food pantries. This intersects with poor nutrition and diet-related diseases- according to the Alameda County Community Food Bank Hunger Study report,vertical towers for strawberries “food is often the most critical factor in our clients’ health”, and 40% of clients are in fair or poor health .

Food banks and food pantries fill important “access gaps” that urban farms could better supplement or address if cost of urban produced food was made more affordable, or through donations to food banks . Low-income households can circumvent the high costs of urban produced food from commercial farms by establishing their own backyard gardens , or adopting plots in community gardens. Through direct participation in UA, in particular food insecure individuals can offset significant percentages of fresh vegetable expenditures , and enhance food security through improved healthy food access . Access via UA participation is certainly enabled when urban farms and gardens are physically proximate to low income neighborhoods, demonstrating the intersection of cost and geography in expanding access. There are abundant examples of non-profit farms that give food away for free or at reduced rates , yet there is little scholarship on the consumption or impact of donations/discounted offerings specifically.High costs of land and development pressures also play a significant role in limiting access to both farming and locally-produced foods, as seen in studies of Chicago, New York City, and the San Francisco Bay Area . High cost of land prevents community gardens from being established in the urban core in Chicago, leads to hundreds of community gardens in NYC slated for redevelopment annually, and drives gentrification and displacement in neighborhoods around urban farms. Land tenure insecurity directly contributes to lack of access as many urban farms formerly serving minority and immigrant populations have been forcibly closed due to development priorities for privately owned lots . A recent article on land security indicators among California urban farmers showed that farms with higher land security also had “more financial and institutional support, and are located in census tracts with higher economic opportunity” . This highlights the necessity of devoting publicly owned lands to urban agriculture in low income and minority neighborhoods, as private lands are highly vulnerable to development pressures, thus jeopardizing any gains realized by social justice oriented urban farms. In contexts where urban farms strive to provide living wage jobs and career or educational opportunities for low-income communities, youth, or formerly incarcerated individuals, it is often challenging to also provide food access to these same communities.

Unless significant grant funding or donations exist, the goals of boosting food security are in tension with capitalist economic realities to pay living wages and sell the product at below-market costs . This speaks to the “unattainable trifecta of urban agriculture,” that is the idea that UA can simultaneously achieve community food security, provide on-the job training and fair living wages, and generate revenue through sales to cover these costs without substantial outside investment , as well as the tension between farm security and food security . In examples such as City Growers and Higher Ground Farms in Boston, organizational efforts to provide jobs and job training lead to marketing of produce to high-end restaurants, retail establishments, farmers markets, and CSAs at prices unaffordable to food insecure households .A fourth important food access barrier cited in the literature relates to cultural acceptability and nutrition education, widely accepted as part of food security definitions . Access to culturally appropriate foods is known to be an important factor , yet little is understood about the effects of urban farms growing culturally relevant foods and its relation to food access. More qualitative research is needed on the cultural acceptability of urban produced foods and how that might correlate with improvements in access. There is increasing evidence of the importance of culturally relevant educational materials around nutrition, food literacy, and culinary skills for improving access and actual consumption of healthy, fresh, urban-produced foods among low income, minority, or immigrant households . Culinary skills and food literacy are becoming focal points of school garden programs , and innovative organizations such as the Green Bronx Machine show how urban agriculture embedded into high-needs schools can directly improve food education,vertical growing which translates into increased access and consumption . Additional research is needed to quantify the impact of educational school gardens on community food security. Recent urban foraging literature is exploring stewardship practices and culturally relevant products gathered by foragers in cities around the world, as well as the sociocultural benefits that result .

From Mien immigrants gathering dandelion bud-shoots in urban parks , to informal urban foragers helping maintain trees and parks in Seattle, WA ranging in age from 23 to 83 , to the value of edible weeds urban foraging is an activity that recognizes certain agroecosystems as “commons” for public access and management. Urban forest justice scholars “recognize the rights of local people to have control over their own culturally appropriate wild food and health systems, including access to natural resources and to the decision-making processes affecting them” . The potential to address food insecurity with foraging and gleaning activities is being explored by organizations such as Ample Harvest and The Urban Farmers in Northern California; Ample Harvest’s online platform supports over 42 million backyard and community gardeners in ending food waste by channeling excess produce to 1 out of every 4 food banks across the country . While some food justice scholars conclude that current shifts toward local, organic, sustainably produced foods are only accessible and affordable to those with higher economic means “or at least the cultural cachet necessary to obtain such foods through barter, trade, or other means of exchange” , the examples above illustrate successful alliances of food justice advocates and local government working to enable sustainable, healthy food access for all urban residents. Through strategic planning and policy design, it may be possible to move beyond ad-hoc successes in linking urban agriculture with food access. The articles reviewed in this section provide a mix of academic studies, theoretical arguments, and policy literature. Additional empirical evidence and longitudinal studies are needed to demonstrate the ability of UA to significantly improve nutrition and food insecurity among urban low-income households over time. Furthermore, consumer preference surveys of urban produced foods are a conspicuous absence in the reviewed access literature. We turn next to food distribution, and the question of how urban produced foods get from the farm to the consumer through various distribution mechanisms.While many articles reviewed mechanisms for channeling rural or peri-urban produced foods into urban areas to increase fresh produce access , very little scholarly data exists on the distribution and accessibility of urban produced foods, and what does exist is largely under-theorized. In fact, very few sources reviewed explicitly name “food distribution” as a key term. Urban agriculture remains a relatively small, yet important percentage of the larger food distribution system in cities: “few, if any, urban agriculture projects, are intended to replace traditional food retail or would claim to lead to food self sufficiency for individuals or for cities” . As such, very little is understood about where and how urban farmers distribute their food including modes of transportation delivery, either individually or in aggregate, and to whom .

It is important to focus on the means through which food produced by different types of farm operations travels from farm to consumer, and the processes through which that food is exchanged , as this directly impacts access and consumption. The scholarly literature as well as media stories describe various modes by which fresh produce is distributed in the city to address fresh food access including both formal and informal distribution channels . Applying a distribution lens to the existing literature yields similar results to the food access analysis in that several articles theorize idealized distribution systems, showing the capacity of hypothetical urban and peri-urban farms to supply distribution networks that meet most urban food demands . Others highlight barriers and challenges farmers face in practice around distributing their produce to those in need while maintaining their operations . None, in our search, focus analysis on distribution flows of urban produced foods across a city. Rather, a more common focus is on which distribution channels are best for getting produce, not necessarily urban produced, into the hands of food insecure households or residents of “food deserts” . Is it a corner store, a large supermarket, or small local farm stand within a mile radius that households need to access fresh produce?In the case of corner stores, several studies have built on analyses of the prevalence of corner stores and liquor stores in low-income census tracts and endeavored to study the effects of providing fresh local produce in these stores otherwise carrying largely processed foods and sugary beverages. Results have been mixed, with some cases of pairing urban farms with corner store retailers yielding increases in sales of fresh produce , but others showing no increase and even resistance from corner store operators who feel that this produce will not sell and therefore become a waste disposal issue . Small Farmers markets as distribution sites receive critical assessments in the literature for their ability to serve as distribution channels to low-income consumers. Alison Hope Alkon writes about the closing of a farmers’ market in West Oakland, a historically African American neighborhood, juxtaposed with the white spaces of farmers markets that are thriving in neighboring Berkeley in her book Black, White and Green: Farmers Markets, Race and the Green Economy . She theorizes the promise and limitations of the “green economy” and chronicles the food movement’s anti-capitalist roots yet ultimate manifestation as reproducing capitalist inequalities. Lucan et al.’s study of farmers markets in the Bronx took issue with limited hours of operation, seasonality, affordable common produce, and availability of predominantly healthy foods among farmers markets compared to nearby stores . Accepting Electronic Benefit Transfer payments is a basic prerequisite for farmers markets to be considered accessible to low-income consumers, a concept pioneered by the GrowNYC’s Greenmarket program . While farmers markets in all 50 states now accept food stamps , the price of offerings such as a bunch of kale still exceeds the price of nearby fast food options that may offer a more filling but less nutritious meal option. Some states are moving in the direction of matching EBT funds through various “market match” policies, a step towards improving food distribution and access at farmers markets .

Settlers from the Midwest arrived with cattle, sheep, pigs and chickens, and by 1908 Lopez had a commercial creamery shipping 1,500 lbs of butter each month to the mainland. By the 1920s the islands reach a high in number of farms and farmland, with 566 farmers and over 68,000 acres in production, largely for homesteading and subsistence purposes . Important crops included fruit orchards, strawberries, peas, and beef cattle eventually taking over from dairy herds after the 1948 milk regulations. Homesteading and horticultural production continued through the mid to late 1900s; total farmland acreage fell to its lowest point in the 1970s and increased again up until 2007. Of concern for soil fertility, hay production has been a significant component of farmland increase, contributing to soil depletion over time. Despite its small population size and total agricultural acreage, San Juan County ranks in the top half of Washington counties for value of sales from hogs/pigs and sheep/goats .Development pressures building up in the 1990s and 2000s began to adversely impact farmland and housing access. In 1989, the Lopez Community Land Trust formed in response to a rapidly emerging affordable housing crisis on the island, as home prices rose 190% in one year. The Land Trust immediately focused energies on fundraising and building affordable housing communities, breaking ground on the first set of homes in 1990, and eventually completing the award-winning net zero Common Ground community in 2006, recognized for its innovative integration of straw bale construction, local materials, rainwater catchment system, solar hot water heating, and community solar array. The Land Trust adopted sustainable agriculture as core to its mission from the outset,vertical farming supplies recognizing the need to “provide permanently affordable access to land for such purposes as quality housing, sustainable agriculture and forestry, cottage industries and co-operatives by forever removing the land from the speculative market” . In 1996 LCLT collaborated to bring the nation’s first mobile meat processing unit to the islands to humanely slaughter animals, managed by the newly formed Island Grown Farmers Cooperative .

The mobile processing unit is USDA-inspected and greatly reduces costs from transporting animals off island for slaughter. LCLT helped establish the Lopez Island Farm Education program in 2006, as well as a sustainable agriculture internship program that has funded and placed over 65 interns on island farms to learn regenerative practices for farmer-educators. LCLT initiated the Lopez Island Farm Trust in 2018 to preserve parcels of farmland in perpetuity, starting with the purchase of the historic Stonecrest Farm property for $1,000,000. LCLT’s accomplishments are summarized in Figure 6. Today, farmers specialize in crops such as grass-fed meats, berries, tree fruits, diverse vegetables, grains, fiber, lavender and herbs, as well as value-added products including preserves, cheeses, ciders, and wines . Construction of greenhouses and hoop houses and commercial kitchens has enabled year-round production and preservation of the agricultural bounty. The average size of farms has decreased to 58 acres as the focus is more on small vegetable production than meat operations. Average market value of products sold per farm has decreased as well to just over $13,000, although once farm expenses are factored in, net farm income is -$6,293 . Small scale heritage grain production has re-emerged on several islands, which represents an exciting step towards relocalizing important food supply chains and reclaiming sovereignty that has been taken away from communities through consolidation of food “commodities” . Grains comprise the largest acreage of certified organic crop production in San Juan County at approximately 200 acres in production. Grains also represent new revenue stream for farmers taking advantage of growing interest in sourcing local grains among local bakeries and restaurants. A talk at the San Juan Agricultural summit in 2019 on farming history in the San Juan concluded with the statement that “it is a myth you can’t make a living farming in the islands, but the successful people have been those who have innovated and shown their savvy at investing in new varieties or types of crop and in contacting distant specialty markets” . Looking to the future, continued access to farmland remains a strong concern for the agricultural community, as the island faces heightening pressure for development serving the summer tourism and second home markets, and the ability of the Land Trust to purchase other pieces of island farmland is limited by fundraising capacity.

A 2011 report titled “Growing Our Future: An Agricultural Strategic Action Plan for San Juan County, WA” goes on to state that “appropriate state or local policies and regulations, as well as training, support, and resources are necessary to promote effective transfer, succession, and leasing of farmland for a new generation of farmers. Housing for these new farmers is a major issue as there are no code allowances for adding housing for succession farmers on existing farms, nor is there affordable housing available for new farming efforts on leased land.” Changes to code language are needed to allow for farmland transitions to occur that provide affordable housing options for new farmers. Additional incentives must be put in place to facilitate the active and regenerative farming, rather than degradation and neglect, of farmland parcels. When San Juan County farmland value is estimated at over $13,000 per acre , aging farmers are facing retirement decisions, and farms are operating a net loss financially, the continued economic viability of sustainable agriculture is called into question. Challenges of aging farmers, attracting new farmers with interest in regenerative practices, and affordability of land and farm worker housing are familiar to farming communities nationwide. Lopez has an advantage in facing such challenges through its support network at the island and county levels: LCLT, the County Agricultural Resource Committee, Ag Guild, and WSU San Juan County Extension are active proponents and providers of technical assistance, policy support, financial resources, and outreach geared towards supporting current farmers and attracting new skilled agriculturalists to the island community. Such supports are essential to confronting these and additional challenges related to building a resilient local food system on a chain of islands. When it comes to protecting farmland in the San Juans,vertical lettuce tower the strategic action planning process in 2011 came to the conclusion that “success in protecting farmland will ultimately be defined not only by the amount of farmland conserved, but also by the productive, profitable, and sustainable use of that farmland by local farmers, thereby contributing to a strong, diversified economy that benefits farmers and their community, while also building a viable and resilient local food system” . Supporting “productive, profitable, and sustainable use” of farmland will require action steps outlined as report recommendations, including: 1) Adopt and promote scale-appropriate state and local regulations in order to foster farm businesses and support a thriving local farm economy; 2) promote opportunities for new farmers to establish successful farms; 3) develop adequate access to infrastructure necessary to process and maintain diverse agricultural operations; and 4) expand local and regional marketing opportunities .

Members of the Ag Guild, ARC, and local agricultural stakeholders are working towards many of these goals, discussed further in the section below. Further partnerships are sought with conservation organizations to buy, conserve, and sell farmland parcels to those intending to farm the land regeneratively, meeting both conservation and food production objectives.The literature on agroecology and sustainable food systems highlights critical factors for success in the domain of farming or food production, starting with affordable access to good quality farmland. Other essential “success factors” for sustainable and regenerative agriculture include cultivating soil health, minimizing external inputs, educating farmers to perform knowledge intensive practices, and cultivating human resources to support agroecological transitions . Jules Pretty describes five forms of “capital” that are important to building sustainable agricultural systems: physical, natural, social, human and financial . Following his presentation of agricultural sustainability, each of these forms of capital is considered and analyzed in the case of the Lopez Island food system in the sub-sections below which evaluate land access, soil health, use of inputs, farmer education, and human resources present in the form of farmers practicing regenerative agriculture. The analysis of physical, natural, social and financial capital is based off of informal interviews and conversations with local agriculture organization leaders as well as participation in island agricultural education events. The aging farmer population and farmland transition dilemmas on Lopez are challenges mirrored in agricultural communities nationwide, encompassing both large industrial and smaller scale operations. Several of the island’s most successful farms are led by farmers in their 50s, 60s, and 70s, without a clear plan of who will take over as the current owner-operators seek to retire. The primary mechanisms for farm transfer and new farm establishment are through LCLT, the San Juan Islands Ag Guild, and the real estate market for island farmland. LCLT works towards three goals related to land access: affordable housing, sustainable communities, and farmland conservation. Their most recent initiative, the Lopez Island Farm Trust , was formed in 2018 to spearhead farmland conservation work. LIFT aims to strengthen the local food system and provide affordable access to land through a “comprehensive legal, ethical, and economically viable land lease system.” LIFT seeks to acquire, lease and manage new and historical farms; provide education for beginning farmers; foster business opportunities for regenerative agriculture operations; and encourage multi generational living on the land.

LCLT plans to use the affordable lease template as a model for securing and transitioning other farmland parcels, whether gifts or purchases, to the next generation of regenerative farmers. Ensuring the success of the newly leased Stonecrest Farm operation is essential to the continuation of this work, as facilitating a smooth transition to a new family operation is inherently challenging. It remains to be seen how replicable the Stonecrest Farm purchase is, or the degree to which it can serve as an affordable land access model, due to the difficulty for the land trust to raise large sums of money on a regular basis; “it was a big lift for us,” says LCLT Community Liaison Rhea Miller, of the fundraising effort to purchase Stonecrest. The Ag Guild recently received a three-year Beginning Farmer and Rancher Development grant from the USDA to research and improve access to farmland for new and beginning farmers. The grant collaboration included WSU SJC Extension, the Northwest Ag Business Center, Whidbey Island Conservation District, and the Organic Farm School farmer training program on Whidbey Island. As part of the grant activities, staff at the Ag Guild conduct outreach with beginning farmers interested in accessing farmland and establishing operations in the San Juan Islands, and posts opportunities for farmland access on its website’s “Farmers-to-Farmland” page . The outreach process includes connecting farmers to suitable farmland parcels and providing relevant information about available water sources, markets, local contacts, and housing options. Rather than seeing this as a failure, ensuring opportunity to opt out is an important part of the farmland succession process and ultimately setting up new farmers for success . Recognizing and overcoming challenges of a specific context is an essential part of farmland transitions, with some challenges more easily overcome than others . In most cases, the land tenure for new farmers would be through lease agreements, rather than ownership models, as much of the farmland available in the county is owned by the Land Bank, Preservation Trust, or private individuals open to leasing arrangements with aspiring farmers. There is a divide between the landowning and land leasing populations, with many young people not able to afford to buy into an ownership arrangement. Pathways to cooperative and collective ownership5 of farmland as a land access opportunity are largely absent in the Lopez case study and throughout the Pacific Northwest. Ag Guild staff are very open to the idea of supporting more farmers, both current and new, in establishing cooperative enterprises. Organic Farm School directors are similarly encouraging of this idea, arguing that many new farmers might not be ready to take over an 80-acre parcel of land and put it to productive use immediately, but it might be more appropriate for a group of five to divide up vegetable production, flower production, poultry production, grazing and value added products.

For instance, the pre-side dress soil nitrate test is used widely in conventional systems to indicate plant available N just before the exponential growth phase of the crop. However, low soil NO3 – pools can occur even when N availability is high if soil NO3 – turns over rapidly, such as when high input and high output fluxes occur simultaneously. The higher soil carbon availability often resulting from organic management can increase both microbial N demand and gross soil N transformation rates, thereby increasing plant-soil-microbe soil N cycling and turnover of inorganic N. Thus, new indicators of N availability are needed that take into account active C and N processes in organic systems. Good candidates are labile soil organic matter fractions, which will benefit from more on-farm validation and standardization. Expression levels of genes involved in root N uptake and assimilation may also indirectly indicate plant available N in soil and provide a complement to bio-geochemical indicators of N availability, especially when soil NO3 – turnover is high. Plant N uptake and assimilation systems respond to wide variation in external N availability and internal N metabolites that reflect plant N status through regulatory mechanisms that optimize capture of limiting nutrients. Recent work has expanded knowledge of plant root transcriptional responses to N availability from laboratory-based systems into natural soil conditions, thus providing a basis for selecting candidate genes as indicators of soil N processes. These genes include high-affinity transporters of NH4 + and NO3 – ; nitrite reductase, responsible for reduction of NO3 – to nitrite; and glutamine synthetase and glutamate synthase,vertical agriculture which are involved in NH4 + assimilation into amino acids. Analyzing expression of these genes in roots may provide a “plant’s eye view” of soil N availability, and show how root Nassimilation is high even when soil inorganic N pools are low, i.e. in situations of tightly coupled and rapid N cycling.

If working organic farms can achieve both tightly-coupled N cycling and high crop yields, then how do farmers do it? Are there indeed bio-geochemical or plant-based indicator measures that will help organic farmers learn about their systems and provide the basis for adaptive management? Tomato , a model species for plant N metabolism and plant genetics, is widely grown on organic farms in California, where organic farmers use a variety of management practices. This provides a unique opportunity for a landscape study on how variability in SOM and management relate to yield and N cycling on working organic farms and how root expression of N metabolism genes could indicate rapid plant-soil-microbe N cycling . The overall hypothesis of this study is that tightly-coupled N cycling will be associated with higher levels of total and labile soil C and N and more diverse nutrient inputs . In turn, expression of root N metabolism genes will be elevated and more closely related to soil bio-assays for N availability than inorganic N pools in such fields. A landscape approach was used to assess crop yields, plant-soil N cycling, root gene expression, and the potential for soil N retention across a representative set of organic fields growing Roma-type tomatoes in one county in California, USA.The study took place during the tomato growing season to focus on the synchrony between soil N availability and crop N demand. The participatory framework in concert with GIS-based evaluation of land in organic tomato production was designed to provide real world context for evaluating novel combinations of indicators for N cycling in organic systems. The specific objectives were to: 1) identify different N cycling patterns in organic fields representative of the local landscape based on a suite of plant, soil, and soil microbial variables; 2) examine how root expression of key N metabolism genes relates to biogeochemical indicators of plant-microbe-soil N cycling; and 3) evaluate trade offs among ecosystem functions in N cycling scenarios.The organically-managed fields in this study were on similar parent material in Yolo County, California, which is situated along the western side of the Sacramento Valley.

Annual precipitation in 2011 was 403 mm, and the mean maximum and minimum temperatures were 21.7 and 7.3°C, respectively, compared to 462 mm, 23.1°C, and 8.4°C for the previous 20 years . From 1989–2011, certified organic acreage in Yolo County, California increased 15-fold while production value increased nearly 30-fold to >$30M.Farms growing organic Roma-type tomatoes in 2011 in Yolo County were identified using the California Certified Organic Farmers directory and farmers were contacted during the winter of 2010–11. CCOF is the primary organic certifier in this region of California. Widespread interest among organic farmers in this region to improve N cycling and increasing concerns about N loss due to state-level policy initiatives related to greenhouse gas emissions and water quality provided an entry point to engage a variety of farmers in this study. Eight growers expressed interest in the project and identified the fields in which they expected to transplant tomatoes in early April 2011 . Through multiple one-on-one meetings with these farmers we learned management practices and following the study, we discussed biophysical and management data from their field relative to data from other fields in the study and potential reasons for differences.GIS analysis of the land in organic tomato production was performed in order to ascertain how well the 13 fields that were sampled compared to the range of variability in organic tomato fields in Yolo County. Soil, landscape, and management attributes of all fields in organic tomato production in Yolo County were characterized with a landscape regionalization approach. A set of 103 points were randomly assigned to all such fields based on a 2008 field-scale county survey, representing one point every 4 hectares. For each of these points, the values of 12 variables were compiled from several sources. Categorical variables included soil great group and soil drainage class from the SSURGO database, the number of crop rotation types in a one mile surrounding square, and an agricultural sub-region classification. Continuous variables from the SSURGO database included percent sand, silt, and clay, organic matter, elevation, and the Storie index .GIS data were subjected to a clustering algorithm, partitioning around medoids , based on a distance matrix derived from Gower’s dissimilarity algorithm. PAM analysis with five clusters returned the best defined clusters yielding an average silhouette width of 0.499. The proportion of the landscape in organic tomato production represented by each cluster was calculated by performing a Voronoi tessellation of the 103 points, assigning eachpolygon of the tessellation to a cluster type,vertical farming aeroponics and then intersecting the tessellation with the field boundaries to allow determination of cluster areas.

Based on a lack of grower interest, cluster 2 was not represented.Soil and plant sampling was designed to capture indicators of ecosystem functions related to plant-soil N cycling at times corresponding to key agronomic and phenological events, including immediately prior to tomato transplanting , peak tomato growth period , and tomato harvest . In each field, plots were established at six random locations within a 0.25 ha area. Pre-transplant measurements took place several days prior to tomato transplanting but after other field operations, such as tillage, incorporation of organic amendments and/or vetch cover crops, and bed formation. Tomatoes were transplanted in all fields between 6 April and 20 April, 2011. In each of the six plots, three soil cores for each depth were removed from tomato beds and composited in the field, separately for each plot.For mid-season measurements, fields were all sampled within two weeks of one another, an average of 68 days after transplanting.A soil core was removed in each plot, situated between two tomato plants 15 cm from the planting row. Three 50–150 mg sub-samples of roots were promptly removed from the soil core in the field under minimized/indirect light, rinsed, patted dry, and flash frozen in liquid nitrogen for subsequent RNA extraction .The two plants adjacent to this core were cut at the base and petiole samples from recently matured leaves were removed. Plants were rinsed and dried at 60°C for two weeks before grinding and analyzing for C and N . Tomato yields were sampled just before the farmer’s harvest. In each plot , two 1m × 2m sub-plots were established. At each of these subplots, individual tomato plants were cut at the base and ripe fruit was separated by hand from green and decayed fruit . This process uses criteria similar to that of machine harvested tomatoes as well as those harvested by hand for fresh market sales. Biomass of fruits and shoots were weighed in the field then sub-sampled and dried at 60°C for 2 weeks, before grinding and analyzing for C and N . Soil cores were also taken from each subplot and composited in the field for measurements described below.Soil samples were kept on ice and processed within several hours of field extraction by thoroughly homogenizing by hand. Soils from the 0–15 cm depth were analyzed for a variety of soil C and N fractions, bio-assays for N availability, and soil properties, while deeper depths were analyzed for inorganic N and gravimetric water content only. Inorganic N was extracted from moist soils with 2M KCl and analyzed colorimetrically for NH4 + and NO3 -. Potentially-mineralizable N was measured as NH4 + liberated during a seven-day anaerobic incubation at 37°C. Chloroform fumigation-extraction followed by UV-persulfate oxidation and alkaline persulfate oxidation was used to measure microbial biomass C and N , respectively. K2SO4 extractable organic C and N were quantified in non-fumigated samples. Permanganate oxidizable C , which reflects a processed soil fraction that is sensitive to management was measured according to standard procedures. Gravimetric water content was determined by drying at 105°C for 48 h. Air dried soil samples were sieved to 2 mm, ground, and analyzed for total C and N at the UC Davis Stable Isotope Facility. Shoots and fruit were analyzed for total C and N, δ13C, and δ15N at the UC Davis Stable Isotope Facility. Petiole NO3 – , an indicator of recent N status in conventionally-produced vegetables, was measured in the most recently-matured leaves. Petiole NO3 – changes rapidly with growth stage, so the data are graphed by post-transplanting growing degree day to account for phenological differences among fields as a result of slightly different sampling times relative to transplanting.Root RNA was extracted using Trizol reagent according to the manufacturer’s guidelines followed by DNase digestion using RQ1 RNase-free DNase . Total RNA was purified using the RNeasy Plant Mini Kit . RNA concentrations and quality were assessed using the Agilent Nano drop and the RNA 6000 Nano Assay . Only RNA samples with RNA integrity numbers of at least 7.0 were used for subsequent analyses. These RNA were used for cDNA synthesis for qRT-PCR analysis. cDNA was synthesized from 0.5 μg DNase-treated total RNA using the Superscript III kit .Expression of cytosolic glutamine synthetase GS1 in roots was more strongly related to indicators of plant-soil N cycling than were the other six key genes involved in root N metabolism . Of the soil variables, GS1 was more strongly related to soil bio-assays for N availability than to inorganic N pools . Microbial biomass N and PMN were most strongly associated with expression of GS1 in roots, followed by soil NO3 – . Permanganate oxidizable C and MBC, both indicators of labile soil C pools, also had significant associations with GS1 expression in roots, but soil NH4 + did not. Expression of GS1 also was positively associated with shoot N and petiole NO3 – , as was glutamate synthase NADH-GOGAT. Inclusion of GWC as a covariate in multiple linear regression models improved the proportion of explained variation in GS1 expression .PCA of 28 indicators of yield and plant nutrient status, root N metabolism, and soil C and N cycling showed strong relationships among suites of variables, which clearly differentiated fields along the first two principal components . The first principal component explained 28.3% of the variation; on the left side of the biplot are higher values of most variables, including yield, soil bio-assays, expression of root GS1 and NADH-GOGAT, and labile and total soil C and N pools . Soil NH4 + and NO3 – concentrations from all three sampling times as well as AMT1.2 were associated with one another and with positive values along principal component 2, which explained 19.4% of the variation.

Using weighted Principal Coordinates Analysis , the differences between the soil microbial communities of the two soils can be seen by the two distinct clusters, regardless of surfactant amendment . Due to their broad occurrence in numerous contaminated sites and diverse metabolic pathways for xenobiotic degradation, genera of the Sphingomonadaceae family such as Sphingomonas are considered effective PAH-degrading soil microorganisms . Bastida et al. evaluated PAH biodegradation in a semiarid petroleum-contaminated soil amended with compost and concluded that Sphingomonadales played a dominant role in the initial steps of PAH biodegradation, suggesting that Sphingomonadales were primarily responsible for the conversion of the aromatic hydrocarbons into cis-dihdyrodiol via dioxygenases as well as in the metacleavage pathway to catechol. Kaistobacter has only recently been linked with PAH degradation and their role in PAH biodegradation is still unclear; however, Li et al. utilized 13C-phenanthrene and stable isotope probing in activated sludge and suggested that Kaistobacter was among the primary native microorganisms responsible for phenanthrene degradation. Wang et al. utilized KEGG functional prediction and PICRUSt analysis of PAH-contaminated sediment and concluded that Kaistobacter contributed to the “Polycyclic aromatic hydrocarbon degradation” KEGG pathway, specifically, the process of metabolizing pyrene to 3,4-dihydroxyphenanthrene. Although Firmicutes and Proteobacteria phyla comprised a substantial proportion of the soil microbial community, the effects of Brij-35 and rhamnolipid surfactant application, particularly at the high rates, on soil microbial dynamics was apparent. In the pyrene-contaminated clay and sandy loam soils, the OTU numbers and Shannon diversity index were not different from the surfactant-amended treatments in both native and bio-augmented soil treatments,hydroponic bucket except for the addition of rhamnolipid at the high rate, which resulted in a dramatic decrease in OTU number and Shannon diversity index .

The Shannon diversity index of the Native+Pyrene clay and sandy loam soils decreased from 6.53 and 7.74 to 2.54 and 4.18, respectively, in the soils amended with rhamnolipid at the high rate. Notably, the abundance of Bacillus present after the 50-d incubation of the pyrene-contaminated clay soil, with or without bio-augmentation, was less than 2% when rhamnolipid bio-surfactant was amended at the high rate . In the native clay soil amended with rhamnolipid at the high rate, the most dominant genus was Mycoplana . The ability of Mycoplana to effectively use the rhamnolipid bio-surfactant as a carbon source likely resulted in a substantial decrease in the abundance of known PAH degraders, such as Bacillus, Sphingomonas, Kaistobacter, Mycobacterium, and Rhodococcus that were present in other soil treatments . Although it has been shown that some species of Mycoplana such as Mycoplana sp. MWVMB2 were capable of effective PAH biodegradation in soils contaminated with phenanthrene up to 200 mg kg-1 with or without the use of surfactants such as Span 80, Tween 20, cetyl trimethyl ammonium bromide, sodium dodecyl sulfate, and Triton X-100, the Mycoplana sp. that was the dominant genus identified in this study was not able to mineralize pyrene after 50 d . In contrast, the native sandy loam soil amended with rhamnolipid at the high rate did not follow this trend and Bacillus comprised approximately 58% of the genera relative abundance . It should be noted that at the end of the 50-d mineralization study, the native sandy loam amended with rhamnolipid at the medium or high rate was just commencing pyrene mineralization, suggesting that the rhamnolipid biosurfactant was potentially exhausted as a preferential carbon source by the soil microbes . A study by Wang et al. considered the influence of rhamnolipid biosurfactant, Tween 80, and sodium dodecyl benzenesulfonate at 5, 10, 50, 100, and 1,000 mg kg-1 on soil microbial dynamics and PAH biodegradation in aged PAH-contaminated soil. The researchers reapplied the surfactants after 42 d due to surfactant adsorption onto solid matrices as well as partial surfactant biodegradation based upon surfactant degradation results by Cserháti et al. . Wang et al. observed similar results to the sandy loam soil amended with rhamnolipid at the high rate in this study, with Bacillus abundance being three to five times as high as that of the other surfactant-amended PAH-contaminated soils.Additionally, the native sandy loam soil amended with rhamnolipid biosurfactant at the medium rate as well as the bioaugmented sandy loam soil amended with rhamnolipid at the high rate contained a substantially greater relative abundance of Pseudomonas compared to the unamended and bioaugmented sandy loam soil .

Pseudomonas are known PAH-degrading soil microorganisms and have been shown to effectively degrade PAHs such as naphthalene, phenanthrene, pyrene, and anthracene in crude-oil contaminated soils. The PAH biodegradation by Pseudomonas was also shown to be enhanced in the presence of surfactants such as Tween 80, Triton 100, and rhamnolipid biosurfactant . Cébron et al. used DNA stable isotope probing in 13C-phenanthrene-contaminated soil to assess the effects of ryegrass root exudates on PAH biodegradation and concluded that Pseudomonas sp. was one of the few soil microorganisms activated by the root exudates because the easily degradable carbon source addition provided by the root exudates favored the development of fast-growing rstrategists and copiotrophic soil microorganisms belonging to Gammaproteobacteria . Rhamnolipid biosurfactant, which is composed of a β-hydroxy fatty acid connected by the carboxyl moiety to a rhamnose sugar molecule, has the potential to also be utilized by Pseudomonas as an easily degradable carbon source similar to root exudates . Colores et al. investigated the effect of Witconol SN70 nonionic surfactant on the soil microbial community as well as the biodegradation of hexadecane and concluded that Pseudomonas populations in the soil could utilize both the surfactant and hexadecane for growth, which could have important implications on remediation efforts. The effect of rhamnolipid at the high rate can also be seen using weighted PCoA, where the treatments in both soils clustered separate of the other unamended and surfactant-amended treatments . Additionally, Brij-35 surfactant at the high rate resulted in a cluster separate from the unamended and surfactants amended at the low and medium rates, which were clustered together, indicating no substantial difference in the soil microbial communities . The amendment of either surfactant at various rates, except rhamnolipid at the high rate, to the sandy loam soil resulted in an increase in Brevibacillus abundance compared to the unamended native or bioaugmented sandy loam soil . Wei et al. evaluated Brevibacillus in liquid culture spiked with pyrene and showed that Brevibacillus was able to degrade 57% of pyrene as the sole energy and carbon source; however, these findings have yet to be repeated in a soil system and warrant future research,stackable planters as the increased abundance of Brevibacillus may be attributable to growth due to surfactant degradation in the sandy loam soil and may have important implications for surfactant-enhanced  bio-remediation.

The addition of Brij-35 at the low rate to the native or bio-augmented sandy loam soil resulted in a dramatic increase in Bacillus compared to the unamended native or bio-augmented sandy loam . The greater Bacillus abundance in the native sandy loam soil amended with Brij-35 at the low rate may have contributed to the increased pyrene mineralization compared to the native unamended sandy loam soil . The inoculation of PAH-degrading bacteria in a wide range of contaminated soils has been successfully implemented for the removal of priority PAH pollutants and continues to be a promising remediation method due to its low cost, lack of secondary pollution, and environmental safety . The bio-augmentation of M. vanbaalenii PYR-1, an isolate from an oil-contaminated estuary of the Gulf of Mexico, Redfish Bay, near Aransas Pass, has previously been shown to significantly enhance the initiation and rate of PAH mineralization in both PAH-contaminated soils compared to the native soils.The effectiveness of the bioaugmentation of M. vanbaalenii PYR-1 on pyrene mineralization was evident in the unamended, Brij-35 amended at all rates, and rhamnolipid biosurfactant amendment at the low rate, in both soil systems . Mycobacterium vanbaalenii PYR-1 has been studied in detail with respect to the molecular genetics of PAH degradation and has been shown to encode PAH ringhydroxylating oxygenases nidAB/nidA3B3, which are utilized in the oxidation of HMW PAHs such as pyrene . Additionally, M. vanbaalenii PYR-1 has a complex and very hydrophobic rigid cell envelope that is enriched in mycolic acids and the mycolic acid wall monolayer acts as a biosurfactant to enhance PAH solubility and bio-degradation . Because of these characteristics, M. vanbaalenii PYR-1 is considered an excellent candidate for bio-augmentation in PAH-contaminated soils. The 16S rRNA gene analysis was used in this study to determine if M. vanbaalenii PYR-1 was capable of successfully acclimating after the introduction in both soil systems with or without the addition of the surfactants at different rates. As shown in Figs. 3.2 and 3.3, the bio-augmentation of M. vanbaalenii PYR-1 in all clay and sandy loam soil treatments, except for rhamnolipid at the high ratein both soils and rhamnolipid at the medium rate in the sandy loam soil, resulted in an increase in Mycobacterium compared to the native treatments. This increase in Mycobacterium was especially evident when comparing the Native+Pyrene and PYR- 1+Pyrene treatments . Additionally, LEfSE software was used to determine which soil microorganisms were differentially abundant between the bioaugmented and native soil systems. The abundance of Mycobacterium was found to be significantly greater in the bio-augmented soil treatments compared to the native soil treatments . Functions of different OTUs and prediction of the functional composition of the metagenome in both soils was accomplished using the 16S rRNA gene data, Greengenes reference database, KEGG pathways, and PICRUSt to evaluate the effectiveness of surfactant addition as well as the bio-augmentation of M. vanbaalenii PYR-1 on pyrene mineralization.

By analyzing soil functional genes, contributions of different bacteria involved in the biodegradation of PAHs were assessed. For instance, according to the “Xenobiotics biodegradation and metabolism” list on the KEGG website , pyrene and phenanthrene can be degraded to 3,4-dihydroxyphenanthrene via “Polycyclic aromatic hydrocarbon degradation”, which can then be further metabolized into the TCA cycle via “Naphthalene degradation” and “Benzoate degradation” . These KEGG pathways include numerous predicted PAH-degradation-related KOs, such as PAH oxygenase large subunit , PAH oxygenase small subunit , and extradiol dioxygenase and determine whether bioaugmentation or surfactant addition had any significant effect on these genes and thus, PAH biodegradation in the two soils. Upon analysis of the “Polycyclic aromatic hydrocarbon degradation” KEGG pathway, M. vanbaalenii PYR-1 bioaugmentation in both soils significantly increased the KOs associated with the PAH biodegradation pathway compared to the native soils . The same trend of bioaugmentation of M. vanbaalenii PYR-1 resulted in increased PAH-biodegradation-related KOs in the “Naphthalene degradation” and “Benzoate degradation” compared to the native soil systems . Additionally, PICRUSt was utilized to assess which taxa contributed to the PAH-related KOs. For example, M. vanbaalenii PYR-1 substantially contributed to PAH oxygenase large and small unit; however, other soil microbes in addition to M. vanbaalenii PYR-1 contributed to the increased extradiol dioxygenase in the bioaugmented soils compared to the native soils . These results were in agreement with Niepceron et al. who evaluated phenanthrene biodegradation potential by assessing the PAH-ring hydroxylating dioxygenase sequences in PAH-contaminated soil and showed that PAH-RHDα was closely related to either Burkholderia or Mycobacterium. Wang et al. also used PICRUSt to investigate the successions of bacterial communities in PAH-contaminated soils undergoing  bio-remediation and concluded that bacteria in the Mycobacterium genus contributed substantially to functional genes in all PAH-degradation pathways for metabolizing pyrene to the TCA cycle.Target shooting is an increasingly popular recreational sport with over approximately 100,000 shooting ranges worldwide . In the United States , there are an estimated 12,000 shooting ranges, consisting of 9,000 civilian and 3,000 military shooting ranges . Over 18 million adults participated in any type of clay target shooting on these civilian shooting ranges in 2014, which was a 3.6% increase compared to participants in 2012 . Accordingly, there have been vast numbers of clay targets used for these outdoor clay target shooting activities. Baer et al. determined that clay target use in the U.S. since 1970 has averaged approximately 560 million targets/year. Until recently, clay targets were composed of approximately 67- 70% clay or dolomitic limestone, 30-32% coal tar or petroleum pitch used as a binding agent, and fluorescent paint . The coal tar or petroleum pitch binding agent is a large source of PAHs with concentrations up to 3,000-40,000 mg/kg clay target . Each clay target used in these outdoor shooting activities weighs approximately 100 g each and spread into fragments of various sizes when shot.

In addition, most of the Cu was sequestered in root tissues. Therefore, the likelihood of Cu over-accumulation in fruit is low. According to US Department of Agriculture, annual per capita consumption of fresh cucumbers in the United States is 3.0 kg in 2013, which means average daily cucumber consumption is ∼8.2 g per person-day . The average cucumber water content is 95%, so the daily consumption is 0.41 g dry weight per person-day. Thus, daily personal Cu intake from cucumber used in this study would be 10.0, 11.2, 11.8, and 12.9 μg from control, low, medium and high treatments. According to the Food and Nutrition Board at the U.S. Institute of Medicine of the National Academies, the recommended average requirement for Cu is 700 μg per person-day, with a tolerable upper intake level of 10 mg per person-day.Therefore, Cu intake from consumption of cucumber exposed to nCu enriched soil would be within the recommended Cu levels, even at the higher application level. Hence, consumption of nCu treated cucumbers, even at the high level, represents no significant added risk to consumers. Fruit quality is affected by, among others, sugars and fatty, amino, and carboxylic acids. The profile alteration of these nutrients may result in flavor and nutritional supply changes induced by exposure to nCu.Leaves growing in sunny locations have comparatively high photosynthetic capacities, Rubisco activity, rates of electron transport, and rates of dark respiration . Some species are restricted to sunny or shady locations, and the leaves of these plants are often genetically adapted to their characteristic light environment. The leaves of other species, including those that are naturally exposed to particularly variable light environments,fodder system acclimate to local conditions . Acclimation to extended changes in light enhances net assimilation and nitrogen use efficiency while decreasing vulnerability to high light stress . Either anatomical or biochemical mechanisms may be involved in acclimation .

The local light environment influences the morphological development of leaves in many species, resulting in comparatively thick leaves in bright locations . Fully expanded leaves have a limited capacity for morphological change , and acclimation by these leaves requires biochemical changes in carboxylation, electron transport, and light harvesting, as well as modifications to chloroplast structure and orientation . Monocotyledons with basal meristems, long leaves, and dense canopies may represent a case where photosynthetic acclimation by biochemical change is particularly advantageous. The grass Lolium multiflorum exhibits a strong capacity for local photosynthetic acclimation along the length of a leaf . The leaves of plants like Lolium are produced in dark or dim conditions at the base of plants, and, over time, are pushed to the upper part of the canopy. Typha latifolia , atall monocot that forms dense and highly productive monospecific stands in wetlands , may provide an even more extreme example. T. latifolia ramets originate from rhizomes that are buried in sediment, submerged under water, and often shaded by a dense layer of litter and existing plants. Initial leaf growth is supported by carbohydrates that are either mobilized from rhizomes or translocated from older leaves. Depending on sediment thickness and water depth, and the density of the litter layer and existing canopy, the lower 50–100 cm of a Typha leaf may experience almost total darkness . These characteristics make Typha a useful experimental system for investigating the acclimation capacity of morphologically mature leaves. Basal growth in Typha allows the separation of leaf age from light environment; the oldest segments of Typha leaves are exposed to the brightest light, as opposed to plants with apical meristems, where the youngest leaves are in bright conditions. We investigated the photosynthetic capacity of T. latifolia leaves over time following step changes in shading at different locations along leaves. We hypothesized that morphologically mature Typha leaves have a strong ability for local acclimation, and that individual leaf segments acclimate to the local light level autonomously from the rest of the leaf.T. latifolia rhizomes and crowns were collected in April 2004 at the San Joaquin Freshwater Marsh , located in Orange County, California . Rhizomes with an average weight of 49.7 ± 2.5 g were planted in 20-l pots filled with sand. Plants were grown in a greenhouse under either low light with a mean PPFD of 2.7 mol m−2 d−1 and a maximum of 159 mol m−2 s−1, which was created with 80% neutral shade cloth, or high light with a mean PPFD of 19.4 mol m−2 d−1 and a maximum of 990 mol m−2 s−1.

Each pot had one plant, and the pots were widely spaced. The lower segments of leaves were unshaded by either neighboring plants or upper leaf segments, and the light levels were approximately constant along the length of leaves. The water level in the pots was maintained 5 cm above the sand surface with daily additions of deionized water. The pots were fertilized every 2 weeks with Flora Grow and Flora Micro following the manufacturer’s instructions . The pots were drained before fertilization to avoid salt buildup.Two-month-old sun and shade grown plants with several fully expanded leaves were placed on a bench under high light, and a pair of fully expanded leaves from each plant were selected for experimentation. Individual leaf segments between 20 and 45 cm from the tip were exposed to either sun or shade during the 15-day transfer experiment using cylinders of 80% neutral shade cloth, creating the full combination of segments exposed to constant low light , constant high light , low to high light , or high to low light . Additionally, a set of segments on the same leaves were exposed to either constant high light or low to high light . All treatment combinations and locations were replicated six times. The photosynthesis rate under bright light , stomatal conductance and dark respiration rate were measured every two or three days for two weeks in the middle of the sun and shade segments , on six replicate plants using a portable gas exchange system . Afull sun was measured at a PPFD of 2000 mol m−2 s−1 and Rd was measured in darkness after allowing 3–5 min for equilibration. Leaf temperature was controlled at 25 ◦C and reference CO2 concentration at 370 mol mol−1. The leaf to air vapor pressure deficit ranged from 0.6 to 1.5 kPa. Photosynthetic light response curves were measured after leaves had fully acclimated to a change in light . The light response curves were fit using a non-rectangular hyperbola . Afull sun was calculated as the photosynthetic rate at 2000 mol m−2 s−1; Amax was calculated by extrapolating the regression to infinite light; Rd was calculated as the y-intercept; the apparent quantum yield was calculated as the slope extrapolated to darkness. The light response curves were started at high light , and assimilation was measured in response to stepwise PPFD decreases until full darkness.

Stomatal conductance decreased gradually in response to light decreases, and increased gradually in response to light increases. This sluggish stomatal response either led to lower rates of photosynthesis for light curves run from dim to bright conditions relative to curves run from bright to dim conditions, or forced unreasonably long equilibration times. Moreover,fodder system for sale midday field and greenhouse observations showed that leaves exposed to a continuous PPFD of 2000 mol m−2 s−1 for ∼15 min exhibited a steady CO2 assimilation. We therefore opted to carry out light curves from bright to dark conditions, but acknowledge that lags in stomatal adjustment may have resulted in somewhat higher Ci for the light curves than would have been observed for fully equilibrated leaves. Nonetheless, we emphasize that our study is comparative, and the key is consistency across treatments; we executed the light curves the same way for all treatments and leaf segments. Nitrogen concentration , and leaf mass per area , were measured on the leaf segments used for gas exchange. Nitrogen was determined using the micro Kjeldahl technique; samples were oven dried, ground in a Wiley mill, weighed, digested, and nitrogen concentration was determined with an auto analyzer .We characterized the vertical gradients of light and photosynthetic characteristics during midday sunny conditions in August 2004. The PPFD profile was measured through the canopy at 48 different locations in the SJFM using a horizontal quantum sensor mounted on a 2 m handheld pole. Each profile consisted of ten individual measurements recorded with a data logger at 0.0, 0.6, 1.2, and 3.0 m above the sediment surface. The 3.0 m measurement was above the canopy. LAI was measured at the base of the canopy with a LI-COR LAI-2000, assuming non-clumped leaves and without distinguishing between live leaves and litter. Photosynthetic light response curves were measured on three segments of fully expanded leaves from 5 different plants. The cross section of leaves changed from flat at the tip to triangular at the base, and it was not possible to seal the chamber on leaf segments further than 100 cm from the tip.The parameters derived from the light response curves, the nitrogen content, and the leaf mass per area, were compared between treatments using Univariate ANOVA or t tests. The effect of light treatment was analyzed by Student’s t-test. Univariate ANOVAs and Tukey tests were used to compare Afull sun, Amax, gs and Rd between the light treatments within each sampling period. The effects and interactions of treatment and time following transfer were analyzed with multivariate analysis of variance ; this analysis corrected F values due to temporal auto correlation. MANOVA does not require the response variables to be equally correlated, assuming an unstructured variance–covariance matrix . The effect of leaf position on the photosynthetic parameters of leaves growing in natural conditions was analyzed with three paired t-tests, because of the high variation among leaves.

Statistical analyses were performed with JMP software version 7.0 and Minitab statistical software version 15.Fully expanded T. latifolia leaf segments exhibited strong acclimation to a change in light. The light response curves of individual segments of shade grown leaves that were transferred to high light were similar to those of segments that remained in high light throughout the experiment . This acclimation was highly localized; the response curves of SH-SH segments did not change, even as adjacent SH-SU segments acclimated to high light. Likewise, the response curves of SU-SU segments did not change, even as adjacent SU-SH segments acclimated to shade . Photosynthesis in bright light , maximum photosynthetic capacity , dark respiration , and stomatal conductance differed significantly between sun and shade segments following acclimation, regardless of initial growth conditions . In contrast, intercellular CO2 concentration and apparent quantum yield showed no significant differences. When compared within plants that started in sun, Afull sun and Amax of SU-SU segments differed significantly from SU-SH segments. The same pattern was found for Rd and gs, but not ˚y and Ci, where no significant differences among treatments were found. When light treatments were compared within plants that started in the shade, SH-SU segments and SH-SH segments were significantly different for most parameters except ˚y and Ci .Acclimation to a change in light occurred over a 10 to 15 day period. The MANOVA showed significant effects of light treatment and time, as well as an interaction, on leaf gas exchange . The rate of Afull sun by SH-SU segments increased over time, and was significantly greater than that of adjacent SH-SH segments beginning on day. The Afull sun observed for the SH-SU segments after ∼10 days was comparable to that of SU-SU segments . Broadly similar, or somewhat faster, responses were observed for the SU-SH treatments; Afull sun decreased, reaching a rate that was comparable to that of SH-SH segments . Sun grown segments exposed continuously to high light alsoshowed a decrease in Afull sun over time , though this trend was smaller than that observed for the SU-SH segments, and Afull sun by the SU-SH segments was significantly less than that by the SU-SU segments beginning on day 8. Stomatal conductance paralleled the changes in Afull sun; the gs of SH-SU segments increased over time, becoming significantly different from that of SH-SH segments on day 4, and reaching a maximum after ∼10 days that was comparable to that of SU-SU segments . Likewise, the gs of SU-SH segments decreased significantly, reaching a rate that was comparable to that of SH-SH segments .

However, it is unclear to what degree trees truly avoid drought stress relative to plants that go dormant. ‘Drought resistance’ is the ability to withstand drought exposure, whereas ‘drought resilience’ is a measure of how quickly a tree can resume normal growth when conditions improve .Conifers manage tissue water potential in two main ways: isohydric trees close stomata to maintain water potential, whereas anisohydric species allow water potential to drop . Isohydric trees use increasing abscisic acid concentrations as a signal to keep stomata closed, whereas anisohydric trees use low leaf water potential itself as a signal to close stomata . Anisohydric conifers include many Cupressaceae and some Taxaceae . Xylem architecture affects how changes in stomatal conductivity influence cavitation risk, and anisohydric trees tend to have xylem that is more cavitation resistant . Wider tracheids increase conductivity and the risk of hydraulic failure , whereas those with smaller inter-tracheid pits or more lignified walls are less vulnerable . The reduction of leaf area with branch die-back, reduced needle number or smaller needles can also reduce water loss. Anisohydric species often exhibit branch die-back during drought, whereas isohydric trees typically retain a full canopy until death . Some conifer species can refill xylem following cavitation. This is thought to be an energy-intensive process that depends on carbon reserves . This may explain why droughtstressed trees can exhibit lower refilling capability . Picea abies refills freezing-cavitated xylem before soils have thawed by taking up water through its needles . This could explain why other conifers can refill xylem in the absence of positive root pressures, unlike co-occurring angiosperms . However, refilled xylem may be less resistant to future drought stress, a characteristic known as ‘cavitation fatigue’ .Loss of water potential in cells is associated with cell turgor loss, denaturation of proteins and changes in membrane fluidity. To avoid cellular damage, plants synthesize molecules that act as osmotic balancing agents. These reduce cellular solute potential,gutter berries and may increase turgor at lower water potentials. In addition, hydrophilic compounds can prevent the membranes from leaking .

Other compounds stabilize proteins or detoxify reactive oxygen species. These protective molecules include proteins such as chaperonins and dehydrins , the amino acid proline and various carbohydrates .We hypothesize that protective molecules may be produced earlier during a drought in anisohydric species because leaf water potential drops more quickly .As a result of reduced stomatal conductance , oxidative damage and other factors, photosynthetic rates and chlorophyll concentrations often decline during drought . Therefore, in addition to protecting cells from damage, increased allocation to nonstructural carbohydrates may help to avoid carbon starvation by keeping energy resources in easily mobilizable forms. The patterns of change in overall NSCs and starches seem to differ between species and drought length and severity . Clear evidence of death as a result of carbon depletion is still lacking . However, carbon storage and allocation patterns do vary under drought stress , demonstrating implications of water limitation on carbon availability.The number of branches and leaves affects total transpiration. Rooting depth affects access to deep soil water and is probably crucial for seedlings as well as adult trees in areas with seasonal drought . Deep roots may also redistribute water from deep to shallow soils . More small diameter roots, with high surface area : volume and a lower vulnerability to cavitation, may aid drought resistance . Structural changes can have long-lasting effects. Decreasing soil moisture can induce greater root production, but extended drought reduces root mass , which limits responsiveness to precipitation pulses . Lumen width and cell wall thickness of tracheids are plastic, with those produced in moist seasons and years generally being wider, more numerous and thinner walled than those produced in dry periods . Xylem is often functional for multiple years , and so current drought responses can affect water transport during future drought. The production of protective molecules typically drops soon after normal water potential is restored . However, transcriptional and physiological ‘memory’ in stomatal guard cells has been observed, with stressed plants maintaining smaller stomatal apertures when re-watered . There may also be ‘legacy effects’ on NSC production and traits such as growth and xylem anatomy . Plants that quickly return to normal could gain a growth advantage. In areas in which recurring drought is common, however, we hypothesize that this memory effect reduces mortality risk. There are multiple traits involved at different stages of the drought response .

Stomatal control and patterns of root and shoot growth affect the degree to which a plant avoids drought stress. These traits plus xylem morphology, protective molecule production, changes in carbohydrate metabolism and pathogen defenses influence drought resistance. Finally, the recovery rate of photosynthesis and other processes, the degree of persistent changes in structure and the ability to refill xylem affect drought resilience. In the next two sections, we first review the methods used to date to examine genetic controls on ecologically important traits, and then explore how these methods have been and can be leveraged to test for genetic variation in, and identify the genetic basis of, the traits and processes addressed above.Gene expression or transcriptome studies examine changes in the amount of RNA transcripts to identify genes that are upregulated or down regulated under different conditions. Changes in the amount of a gene product can result in different phenotypic responses, even if all individuals have the same gene sequence. Such changes are responsible for plasticity, and may involve temporary or heritable epigenetic modifications . Gene expression studies may involve a variety of techniques, but most recent studies have used microarray chips – DNA probes to which cDNA or RNA hybridize, resulting in fluorescence – or cDNA sequencing . The latter avoids the need for probe and microarray design and can survey whole novel transcriptomes . Real-time quantitative polymerase chain reaction is highly sensitive, but is most often used to target specific candidate genes or to confirm a subset of expression changes . All techniques are sensitive to which tissues are sampled at what time . Moreover, unless expression responses in different genotypes or populations are explicitly compared, this approach does not address local adaptation.Provenance or common garden studies, where seedlings from many different sources are planted in a common environment, began to reveal heritable differences between tree populations long before the availability of genetic marker data . Provenance studies established in the mid-20th century to identify seed zones for replanting or highly productive genotypes have been re-purposed to investigate potential responses to climate change .

Many recent studies have also used seedling common gardens . Studies conducted across multiple sites, or incorporating multiple treatments, can estimate the plasticity of traits, allowing the fitting of transfer functions that predict performance based on source and planting environments . However, such studies do not reveal which genes are responsible for observed differences unless paired with other techniques. It should be noted that there is usually substantial variation within tree populations . The third set of approaches can be used to investigate the causes of heritable variation between populations and individuals.These approaches aim to identify genes or genomic regions related to a trait or to adaptation along environmental gradients. QTLstudies are a classic way to identify the loci involved in continuous trait variation. However, although QTLs for a number of traits have been identified in trees, this approach has had limited success for a variety of reasons, many of which are reviewed in GonzalezMartınezet al.. For instance, a great deal of time and space is needed to cross parental tree lines and raise a sufficient sample size of progeny. Conifers also have very large genomes with low linkage disequilibrium and, without enough genetic markers available,strawberry gutter system most QTLs are undetectable . In addition, high-resolution genetic/physical maps or positional cloning is needed to identify causal genes/mutations . By contrast, genome scan and association studies make use of large numbers of newly available markers , and are carried out in highly diverse out crossing natural populations . Genome scans identify loci that differ more or less between populations than expected by chance . For instance, outlier Fst values can be used to infer the type of selection: balancing selection results in low Fst and shared alleles, and divergent selection in high Fst with segregated alleles. Genome scans can also identify patterns suggestive of a selective sweep. These studies do not automatically provide information about which, if any, environmental variables are responsible for the pattern. One can test whether patterns of differentiation match an environmental gradient, but this is necessarily a post-hoc interpretation . Association studies use a regression approach to identify loci in which genetic variation is associated with variation in trait values or home environment. Such analyses can be carried out at the individual or population level. Genotype-to-environment association studies identify loci that vary along environmental gradients . An association between an SNP and aridity, for example, suggests that the gene or its regulatory region affects performance in wet vs dry environments. This does not reveal how the locus affects phenotype, and careful interpretation is needed as a result of correlation between climatic variables. Genotype-to-phenotype association studies identify loci correlated with a particular phenotype , but the phenotype may or may not be relevant for fitness in the field. Most association studies in conifers to date have used SNPs in a limited number of candidate genes . This ensures that genes suspected of involvement are surveyed, but limits the ability to identify additional loci. However, with the decreasing cost of sequencing, approaches that generate large numbers of SNPs are increasingly being used for genome-wide association studies . One set of approaches, including RAD-seq and genotyping-by-sequencing , involves the use of restriction enzymes to cut and sequence a small subset of the genome .

This can produce tens of thousands of SNPs with high coverage . Many of these SNPs will be in noncoding regions, which is good for the potential discovery of regulatory regions, but can limit the number of gene associations detected. Another approach involves the creation of a transcriptome or full genome sequence for a species, and the development of probes for all or most of the putative genes to identify SNPs . This approach can also yield useful gene expression data if multiple tissue types or treatments are included in the development of the transcriptome .Most drought gene expression studies in conifers have focused on pine seedlings, with a few investigating other Pinaceae genera . The direction of expression responses to the environment, including dry conditions, is highly conserved between Pinus contorta and Picea glauca 9 engelmannii, even though average expression levels often differ . It is unclear whether this is true across conifer families. No expression studies have focused on adult drought responses. The methods used to induce drought stress vary. Studies have withheld water for a specified period , until soil moisture reached a threshold or needles wilted , or needle water content declined to a certain level . Some have used chemically induced water stress . Caution must therefore be used in interpreting differences across studies, as these could be methodological artifacts . Genes related to signaling and gene transcription are frequently upregulated in drought-stressed seedlings. Changes in signal cascades must precede changes in their targets, and such expression shifts often occur within the first week of drought stress. Those in the ABA pathway are well represented . In addition to being involved in stomatal closure, ABA signaling can affect shoot growth and water uptake . However, there are also ABA-independent pathways in most taxa, which may use leaf water potential as a signal . Upregulation of genes in the ethylene pathway could be related to reduced shoot growth or leaf area . Genes related to protective molecules are also frequently upregulated . Late-embryogenesis-abundant proteins, named for their role in seeds, appear to stabilize proteins and membranes and prevent protein aggregation . Dehydrins, a subgroup of LEAs, often protect against drought stress, although some are induced by other abiotic stresses . Heat shock proteins, detoxifification enzymes and genes in the synthesis and transport pathways of osmoprotective carbohydrates and proline may also be upregulated. Genes involved in pathogen or biotic stress defenses are often upregulated during drought stress, but those involved in growth, including cell division and wall construction, are often downregulated .

Adult psyllids were monitored for 6 h on nursery citrus trees treated with two kaolin formulations at 3 and 5% w/v. The two kaolin formulations have a repellent effect on D. citri, causing an overall reduction of 40% of psyllids settled on treated seedlings compared with untreated control. Moreover, both formulations disrupt D. citri probing behavior, with a significant reduction in the proportion of psyllids that reach the phloem compared with untreated nursery citrus trees. In general, there were no differences between the kaolin formulations and among the concentrations tested in both experiments . Then, both formulations could be used in an integrated D. citri management program. These findings reinforce the recommendation of kaolin application on young citrus planting as a useful strategy for HLB management, mainly on the edge of the farms. One of the most crucial steps to find a long-term cure for Huanglongbing is to culture the causative agent which is known as Candidatus Liberibacter asiaticus. In order to culture this bacterium in vitro, it is critical to know the physiochemical conditions in citrus phloem and psyllid organs where CLas present.Micro-electrodes are needle-shaped sensors with tip size of less than 20 µm. The use of micro-electrodes allows minimally-invasive measurement of the micro-scale gradients within native biological system. In this study, oxygen and pH micro-electrodes were used to determine the local oxygen concentration and pH inside citrus phloem and psyllid including intestine and hemolymph. Measurements are done both in healthy and unhealthy psyllids and citrus plants. The results are compared together and with the previous available measurements done with chemical extract from those targets. Results of this study will benefit further design the suitable strategies to culture Candidatus Liberibacter asiaticus in vitro. Many phytophagous hemipteran insects feed by penetration of a stylet bundle into plant tissues to feed on phloem or xylem tissues. Among these are agriculturally important insect vectors of plant diseases which include : white flies, aphids, mealybugs, scales,mobile vertical farm leaf hoppers and psyllids, including the Asian citrus psyllid -the vector of the devastating citrus greening disease.

A common trait of plant vascular feeding hemipterans is ability to form a ‘stylet sheath’ structure that encapsulates the stylet bundle while they penetrate into the plant tissues to feed. The exact function of stylet sheaths in hemipteran feeding are not known; however, trait conservation across diverse phytophagous hemipterans implies a biological importance. It is speculated that sheaths may function to ‘cloak’ the insect feeding process to evade host plant defenses; it also may provide structural stability to the stylets during penetration. We have previously developed a method to isolate pure sheath structures and have used these sheaths to determine gross composition. The primary component of the sheaths is a polymeric glucosyl polysaccharide consisting primarily of “starch-like” alpha-1,4-glucosyl bonds but also a small portion of cellulose-like beta-1,4-glucosyl bonds. This was determined both by mass-spectrometry analysis and demonstration of degradation with amyloglucosidase and cellulase enzymes that are specific for starch and cellulose, respectively. Another major structural component was determined to be proteinaceous as demonstrated by the detection of proteins within purified sheaths and the ability to degrade polymerized sheaths using various proteases. We have further demonstrated that inhibitors of sheath formation can be topically applied to citrus leaves and that this results in inhibition of psyllid feeding on the citrus. This work is presented as a broadly applicable new concept in pest-insect control based on the use of molecules that block the insect’s ability to establish a successful feeding site on the host plant. Huanglongbing , caused by the phloemlimited bacterium Candidatus Liberibacter asiaticus , poses one of the largest threats to citrus production. In ten years, it has spread to most citrus trees in Florida. Citrus production during the 2015/16 crop year is down 54% from the pre-HLB era. After initial leaf infection by Diaphorina citri , Las moves to, colonizes, and damages the entire root system, however Las and HLB symptoms remain sectored in the canopy. Canopy sectoring is thought to occur because of limited lateral connections of phloem sieve tubes. To investigate if lateral movement of Las between sieve tubes is distance or tissue specific, root systems of grafted and seedling trees were split to three different heights: below crown, above crown, and above graft union .

The trees were then graft inoculated at a consistent height above one side of the split root system. Infection of both halves of the root system was monitored weekly. Our results indicate the graft union and crown did not play a unique role in lateral movement. No difference was observed in infection between grafted and seedling trees. Greater variability in the time between Las detection between the inoculated and opposite side in trees with the highest trunk split suggests that vertical distance is an important factor in lateral movement of the bacterium. CLIBASIA_03135 is a highly induced gene in CLas mRNA samples obtained from infected citrus plants. The expression of this gene is undetectable in samples obtained from CLas infected psyllids. The encoded protein could play an important role in helping the bacteria to thrive in the harsh conditions of the plant phloem. To evaluate the biochemical characteristics and its biological significance, we have cloned the gene and purified the encoded protein . Coimmunoprecipitation assays allowed us to identify the GroEL chaperone as the main interacting protein. The specific interaction between LotP and GroEL was confirmed by using a two-hybrid system in Escherichia coli. LotP is a dimer in solution with a native molecular weight of 44 KDa and has ATPase activity in vitro. It displays remarkable structural homology, but no sequence conservation with the amino-terminal region of the Bacillus subtilis LON protease. LotP was systematically annotated as an ATP dependent LON protease, however, the B. subtilis LON protease is an ATP dependent aminopeptidase composed of 6 units each of which are 90 KDa. The results obtained in our analysis allowed us to define that LotP belongs to a different family of proteins involved in protein refolding. LotP is the first member of this large family yet to be biochemically characterized. Our results suggest these proteins modulate the activity of stress-response proteins by direct physical contact depending on the stress conditions present in the environment, hereby opening a possible way to combat HLB disease. The biological agent associated with Huanglongbing in citrus is a phloem-restricted Gram-negative alpha-proteobacteria that belongs to the genus ‘Candidatus Liberibacter’. Among three species of Candidatus Liberibacter, Ca. L. asiaticus has recently invaded several citrus growing regions of the Americas, causing major economic losses. Early HLB detection is necessary in implementing HLB disease management strategies to mitigate the disease.

The major source material for HLB qPCR diagnostic test is symptomatic leaves, but a combination of uneven distribution of the bacteria and the slow development of leaf symptoms which can resemble those induced by various biotic/abiotic stresses, reduces the probability of HLB early detection. We have previously shown that CLas is more evenly distributed in the root system of an HLB-positive tree than in the leaves, suggesting that root tissue may be an alternative source for qPCR diagnosis. We selected about 100 young citrus trees located in an orchard with a low level of infection that is adjacent to an orchard with heavily infected trees from TX and FL, respectively. Leaf and fibrous root samples for HLB qPCR test were collected monthly from these trees since January, 2016. For the current study,vertical farming racks we developed efficient DNA extraction and a new qPCR primers and probe system that target CLas 16s rDNA for root samples. The HLB qPCR data showed that the number of HLB-positive trees confirmed with leaf tissue maintained the steady state during the course of the experiment while the number of HLB-positive trees detected by root tissue has started increasing drastically since May, 2016. The qPCR data showed that about 65% of TX and 80% of FL HLB positive trees were tested positive only with root samples while about 8% of TX and 6% of FL HLB-positive trees were tested positive only with leaf tissue. About 27% and 14% of HLB-positive trees from TX and FL, respectively, were tested positive both with root and leaf tissue. The survey results indicated that the HLB qPCR detected CLas among root tissue substantially more often than among leaf tissue. In response to a pathogen attack, multiple defense mechanisms are triggered in the host plants, including basal defense and gene-for-gene resistance. In particular, Las infection, causes extensive changes in gene expression for several major biological processes including stress responses, signal transduction, transport, cell organization and carbohydrate metabolism. Las deploys effectors that target mitochondria and chloroplasts, which are responsible for the synthesis of adenosine triphosphate and have a critical role in in the creation of reactive oxygen species for both pattern-triggered and effector-triggered immunity signaling in the plant. In this work we investigated HLB disease in relation to ROS and ATP regulation through the analysis of genes correlated to the production and detoxification of H2O2, since H2O2 is one of the major and most stable ROS regulating basic acclamatory, defense and developmental processes in plants. We found that Las infection increased the level of ATP and H2O2 in citrus leaves suppressed the H2O2 detoxification system, resulting in an overall increase in the level of H2O2, which becomes toxic for the plant and initiates damage to the tissues. Trees severely infected by huanglongbing present increased fruit drop, and the harvested fruit are usually smaller and compromise juice quality by being sour, bitter and having off flavor.

It was shown that fruit drop is a result of secondary infection at the calyx zone by the fungus Lasiodiplodia theobromae. The objective of this study was to identify whether those fruit that are ready to abscise have a different quality than fruit that do not have a developed abscission zone. ‘Hamlin’ and ‘Valencia’ orange trees, both healthy and HLB-affected, confirmed by qPCR analysis for Candidatus Liberibacter asiaticus , from two harvests and one harvest were shaken, the dropped fruit collected, and the retained fruit harvested. The harvested fruit were washed, juiced using FMC commercial extractor, pasteurized and frozen for later chemical and sensory analyses. The juice was tasted by 55 untrained panelists in multiple paired-comparison tests where samples were presented as pairs of either “drop/retain”, “drop/drop” or “retain/retain”. Juice from healthy and HLB-affected trees were presented in separate pairs. Panelists could not differentiate juice from healthy fruit retained on the tree or dropped for all three harvests. However, for juice made with HLB-affected fruit, panelists could differentiate juice from fruit that had a developed abscission zone and dropped on the ground after shaking in comparison with fruit that was retained on the trees. The test was statistically significant for all three replications of Hamlin harvested in December, and in two of three replications of Hamlin harvested in January and Valencia harvested in April. A trained descriptive panel also found differences between juice made with fruit having different levels of abscission, with juice from HLB-infected dropped fruit having the most negative off flavor attributes, including sourness, bitterness, astringency, off flavor. Sensory data confirm chemical data and both sets of data were correlated, bringing further insight into off flavor induced by HLB in orange juice.The implications of different control strategies is critical when developing viable disease management plans. Cost, efficacy, disease/pest prevalence, and perceived consequences/benefits, among many other factors, play a role in deciding what control methods to utilize. Additionally, these control methods tend to change over time, reacting or adapting to the current situation. As ACP and HLB continue to spread, the need to assess and implement economically sustainable control options is paramount. We can investigate and compare the production benefits against the fiscal costs of various control strategies through mathematical modeling. We extend a spatially-explicit, stochastic, individual-based compartmental model to incorporate different combinations of control measures such as survey, insecticides/pest control, tree removal, and tree replacement. For surveying, we can implement different survey patterns and probabilities of disease detection. Insecticide spraying can be routine as well as reactive . Further, infected tree removal or localized culling can be invoked to reduce the local inoculum, and replanting can be initiated. Under different combinations of these control measures, we can calculate the costs, model the spread of HLB, and determine the yield/production benefit.