Tag: hydroponic

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.

The census travel model can parameterize and weigh risk contribution of international travelers in various categories given there is a valid disease-associated concern. The census travel front-end allows users to run the model that best suits their needs without strong knowledge of background code and data. Risk maps have been generated for plant diseases and other human viruses . The model has been incorporated into risk-based Multi-Pest Survey programs to provide an augmentative risk factor for continued risk introduction from human-mediated disease spread. In addition, the model can be linked with survey scenario estimators to balance efforts and costs within agency constraints and among emergency programs. The underpinning for HLB control is early detection and early response, especially in Texas and California where incidence is low and Arizona where the disease has not been detected. Eventually Florida, which has been devastated by the disease, will likely replant large areas to reestablish the citrus industry. In all of these situations, the optimal control strategy is to inhibit HLB from entering and establishing in commercial plantings. Infected tree removal, to reduce inoculum in the early stage of the epidemic, remains the most effective deterrent to epidemic development. The earlier the detection of Candidatus Liberibacter asiaticus infections, especially when asymptomatic or better yet subclinical, the more efficacious infected-tree removal can be. A number of early detection technologies are being explored among which canine detection of CLas infected trees, shows considerable promise. Twenty dogs were trained for early detection via a USDA, APHIS HLB Mac grant. Using 10 dogs, each tested against 1000 trees in replicated randomized “field trials” in a gridded array with varying HLB-incidence,hydroponic gutter resulted in 99.17% overall detection accuracy. All dogs performed very well with statistically insignificant trends toward false negative or false positives.

Each dog has its own personality and interacts slightly differently depending upon the trainer-handler. However, there was no statistical difference in CLas detection by trainer-dog combination.When two or more dogs alert on the same tree, the tree statistically has a 100% probability of infection. Dogs were also capable of accurately detecting CLas-infected trees exclusively from 5-gm feeder root samples. Using 10 dogs in a time course experiment with ACP inoculated trees, dogs began to detect CLas infections within 2-3 weeks of inoculation, whereas, none of the trees became PCR-positive for CLas until three months post inoculation and the majority of CLas-infected trees required multiple months prior to PCR detection. This confirms that dogs are indeed a very early detection methodology; able to detect CLas in trees with subclinical infection, i.e., before symptom expression and considerably prior to the ability of PCR for detection/confirmation. In field trials of young and mature citrus plantations, canines trot along the rows with an average interrogation time of ~1 tree/sec, most rapid of all detection methodologies.Dogs are rewarded for detections by verbal praise and short duration play with handlers. Various deployment strategies will be discussed based on the known spatio temporal distribution of CLas infected trees. Using canines to detect HLB in citrus has shown extreme promise in the research environment. Tests on different aged infections, different cultivars, roots, and infections from both grafting and psyllids have all shown the dogs abilities to detect and differentiate an HLB infected tree from clean trees at accuracies exceeding 99%. The next logical step is the introduction of the canines to “real world” environments, i.e., commercial citrus plantings and residential or “dooryard” trees to determine if detection accuracy continued. The dogs were introduced to a young grove in Florida first, and trained over the course of several months. However, due to the high incidence of infection in Florida, finding a suitable low disease incidence training area for dogs in a mature grove is almost impossible. The dogs were therefore taken to other states, especially Texas, for training and testing.

This also allowed the dogs to work in different environments, different grove management practices, and different citrus species. Another aspect of early canine detection is the use of the dogs in residential environments. Again, using other states for a test bed, the dogs were run through various types of residential areas, including residential neighborhoods, mobile home parks, and even a Buddhist monastery. The dogs showed the adaptability to successfully work in these exceedingly diverse and sometimes environmentally hostile environments. Specificity includes the ability of dogs to accurately discriminate between Candidatus Liberibacter asiaticus infections and infections by other pathogens or extraneous scent profiles. To accomplish this, a group of the dogs were taken to Beltsville, Maryland and tested against trees held in the international pathogen collection composed of a number of different viral and bacterial citrus pathogen accessions from around the world. The canines consistently differentiated CLas-infected trees and did not alert on trees infected with other pathogens. One unique finding was that dogs trained on CLas were also intrinsically capable of detecting Liberibacter africancus and Liberibacter americanus infected trees as well, without additional sensitization or training. Asian citrus psyllid continues to spread in California both by natural means and human-assisted means. In areas of the state where the psyllid is well established, such as Southern California, growers are applying coordinated insecticide treatments to reduce populations. In these regions, urban areas receive parasitoid releases to establish Tamarixia radiata and Diaphorencyrtus aligarhensis. Insecticide treatments are only applied to residential citrus trees within 400 meters of commercial citrus. Studies are underway to determine the efficacy of these treatments and to improve the areawide programs in various regions of the state. In regions of the state where the psyllid is not well established, eradicative treatments of pyrethroids and neonicotinoids are applied to both commercial and residential sites around trap finds. These finds have a high correlation with traffic corridors, packinghouses and juice plants, indicating that humans are assisting psyllid spread by moving infested bulk citrus, equipment and plant material. Industry-led changes in how the commodity is handled are expected to reduce the impact of human assisted psyllid spread. Control of psyllid movement around the state is critical to prevent spread of huanglongbing. Early symptoms of HLB include fibrous root loss and leaf blotchy mottle, followed by premature fruit and leaf drop, and yield decline.

As a consequence of initial bacterial infection of fibrous roots, a 30-50% reduction in fibrous root density and elevated soil Phytophthora populations were detected in field surveys. Continued sampling of Hamlin and Valencia orange trees on Swingle citrumelo root stock in different stages of HLB decline revealed that root loss occurs in two stages. The second phase of root loss begins at the early stage of tree canopy thinning resulting from leaf drop and branch dieback. A more extensive survey of HLB-affected groves indicated that greater decline in fibrous root health and expression of HLB symptoms is observed where irrigation water is high in bicarbonates and/or soil pH > 6.5. HLB symptom expression of trees on different root stocks follows the known intolerance to bicarbonate . Acidification of irrigation water in central ridge and south central flat woods Valencia orange groves on Swingle citrumelo root stock for three seasons has maintained soil pH below 6.5 on the flatwoods and 6.0 on ridge. Over the last three seasons of survey, root density as an index of root heath has been sustained. Phytophthora populations remain below the damaging level in ridge groves and in flat woods increase to damaging levels coincident with the fall root flush but drop back to non-damaging levels for remainder of the season. Compared to the 2013-14 season,hydroponic nft channel yields in the ridge blocks have increased up to 4% and on the flat woods have increased up to 22%. Growers using acidification treatments with sulfuric and/or N-phuric acid for the last 3 seasons report an average cost of $60 per acre. This cost will analyzed in relation to yield response to provide a cost benefit of acidification.Our field and greenhouse data shows that secondary and micro-nutrient deficiencies are much greater in roots than in leaves of HLB-impacted trees. Not only is there severe feeder root loss, but the ability of the remaining roots to mine and translocate essential nutrients is compromised. Restoring root health is imperative for keeping trees productive in an HLB endemic environment. In an effort to improve tree health by focusing on the roots, we have been experimenting with polymer coated nutrients and more recently TigerSul micronutrients in the field and greenhouse. In a previous greenhouse study, HLB-infected Valencia sweet orange on UFR-3 root stock showed greater feeder root growth when grown with Harrell’s CRF supplemented with a 3x overdose of TigerSul manganese, or the Schumann blend of TigerSul manganese, iron and zinc . Results suggested that trees in the HLB world have higher specific micronutrient requirements than what are currently being recommended. PCR testing of the greenhouse trees suggested that an overdose of boron could suppress the bacterial titer in the midribs, whereas an overdose of manganese could suppress the bacterial titer in the roots. Several experiments are ongoing in the field to test hypotheses developed from the above information. We define ‘hybrid’ nutrition programs as any program that combines multiple sources of nutrient delivery with a goal of providing a constant supply of all required nutrients year round at an affordable cost. Successful field experiments with industry cooperators to be discussed include the St. Helena Project, where we are testing 100% CRF; and the Post Office Block, where we are testing a 50% CRF/50% traditional dry fertilizer program with micronutrient overdoses. Other successful ‘hybrid’ programs successfully developed by citrus growers will also be presented, including the Ed English program and the Duda program. Data from these trials will demonstrate that enhanced ground nutrition can restore health and sustainable profitable production to HLB-infected trees. Additional fine-tuning of fertilizer composition, type and delivery method could result in less need for psyllid control and thus lower production costs.

Genetic variability for HLB tolerance/resistance has been identified in existing experimental root stock germplasm planted throughout Florida, with both sweet orange and grapefruit scions. New root stocks are being identified in these trials that show a reduced infection frequency, and less severe symptoms once infected, as compared to commercial root stocks. Such root stocks are also showing a stronger recovery following infection in commercial trials. This information suggests that it should be possible to substantially increase the level of tolerance by conventional breeding at both the diploid and tetraploid levels, with focus on direct selection for HLB tolerance in progeny from carefully selected parental combinations. For the past 5 years, we have developed and utilized our ‘Gauntlet’ screening program described below. Each year, crosses of superior parents are made at both the diploid and tetraploid levels. Following a preliminary calcareous soil/Phytophthora screen, selected individual hybrid root stock candidates are transferred to citripots in commercial potting soil. Tops of these trees are propagated by rooted cuttings to produce seed trees on their own roots. The remaining individual liners are grafted with HLB-infected budsticks of Valencia sweet orange. The remaining root stock top is then removed, forcing flush from the HLB- infected Valencia bud stick. Trees are monitored for HLB symptoms, and healthy appearing trees are entered into a ‘hot psyllid’ house until psyllid feeding damage is observed on their leaves , followed by field planting at a challenging field site . Root stocks capable of growing off healthy sweet orange trees are identified for further study. To date, several thousand individual hybrids have been screened. The oldest ‘Gauntlet’ trees have now been in the field for approximately 4 years, and several promising new root stocks have been identified. Although PCR+, individual trees appear to have a reduced bacterial titer and are showing normal healthy growth and fruiting patterns. Seed trees of two of the most promising selections are producing abundant polyembryonic seed, and propagations for large-scale field trials are underway. Patterns are emerging regarding successful genetic combinations, and this will be discussed. Our goal is to develop root stocks that will facilitate sustainable and profitable citriculture in an HLB-endemic Florida, and possibly eliminate the need for psyllid control. Antibodies form the basis for various assays designed to detect plant pathogens in many pathosystems, but until recently have not been available to detect ‘Ca. Liberibacter asiaticus’ .

The oxygen output streams indicated in Figures 1 and 2 represent net oxygen production by the plants due to photosynthesis. However, oxygen production was not included as part of the model since it does not impact the economics of the process. Figure 3 shows the downstream processes for recovery and purification of the rBuChE, which was modeled after the rBuChE lab purification scheme from vacuum infiltrated N. benthamiana described by Hayward and the purification methods described by Lockridge et al.. Major operations include plant harvesting, shredding, screw press/disintegration, ammonium sulfate precipitation, centrifugation, tangential flow microfiltration, tangential flow ultrafiltration, ion exchange chromatography, affinity chromatography, and diafiltration.Table 3 shows the total capital investment and annual operating costs for the plant-made rBuChE facility at an expression level of 500 mg/kg FW plant biomass . The annual operating costs are shown with and without facility dependent costs to simulate a new facility and use of an existing facility, respectively. Table 4 shows the resulting rBuChE cost per dose for both cases. Table 3 shows the breakdown of the capital investment and operating costs for the plant-made rBuChE and indicates that the unit production costs are estimated to be about $234/dose if facility dependent costs are not included in the annual operating costs or about $474/dose if these costs are included. Most of the capital cost and a significant portion of the operating costs are associated with the recovery and purification of rBuChE. Our base case assumed rBuChE expression of 500 mg/kg FW because that is a target expression level in ongoing research at several institutions. If a currently achievable level of 100 mg/kg FW is used instead , the costs increase to $1,210/dose and $430/dose when including and excluding facility dependent costs, respectively.

In any scenario examined,growing lettuce hydroponically the production costs in plants are significantly lower than the estimated production costs for blood-derived BuChE . We recognize that additional modification or formulation of the plant-produced enzyme might be necessary or desirable prior to adoption for human use and that such additional modifications would increase the cost of the AI. For example, Geyer et al.reported improved pharmacokinetics of PEGylated plant-produced BuChE relative to the non-modified enzyme. However, because consensus on the preferred options for modification has not yet been reached, we omitted these additional steps from our calculations.The following premises and assumptions were used for evaluation of cellulase bio-manufacturing in open fields. Due to the fact that this process is specialized and due to the scale and input requirements of a modern bio-fuels operation, our analysis included the construction of a new, dedicated manufacturing facility to provide the required cellulase enzymes for a large scale cellulosic ethanol facility . Figure 4 shows the process operations required for cellulase enzyme production on a per-batch basis. The flow sheet on the top shows the blending tank needed for preparation of the ethanol induction solution to be applied in the field, and the flow sheet on the bottom shows the transport and storage operations following harvest of the transgenic tobacco.Table 6 shows the total capital investment and annual operating costs for the production of 2.87 million kg of cellulase enzymes per year at an expression level of 4 g cellulase/kg FW tobacco biomass and a plant density of 130 metric tons of biomass per hectare per year. The table also indicates the corresponding costs obtained from the JBEI model for fungal fermentation-based production of approximately the same amount of cellulase enzymes per year . For the base case study, the plant-based system results in a >30% reduction in unit production costs for the cellulases as well as an 85% reduction in the required capital investment. For the plant-based cellulase production system, the major contributors to the unit production cost were the costs associated with tobacco cultivation , the costs associated with ethanol spraying , followed by the costs associated with ethanol dilution, transporting and storage , and seed costs.

The differences in total capital investment and annual operating costs for the two cellulase production platforms are not surprising, since the fungal fermentation area alone requires twelve 288,000-L fermenters along with the seed train necessary to provide the inoculum for the production fermenters. The differences between the two systems would be expected to be even larger if the total capital investment included additional factors for associated piping, instrumentation, insulation, electrical facilities, buildings, yard improvements, and auxiliary facilities because these would be reflected in the facility dependent component of the annual production costs. Figure 5 shows the effect of biomass density on the unit production costs for cellulase enzyme using the ethanolinduced tobacco system and indicates, as expected, that the cost of goods decreases as tobacco biomass density increases. In agronomic studies with field-seeded tobacco cultivated at high density, biomass yields exceeding 150 mt/ha have been achieved; higher field densities may be possible with selected varieties and specialized agronomic practices.Comparison of responses of Cleopatra with the more tolerant root stock cultivar Carrizo citrange showed a similar down regulation for several pathways at 12 mai, most notably pathways associated with arginine and proline metabolism, galactose metabolism, and propanoate metabolism. The similarity of metabolic responses of Cleopatra and Carrizo is surprising, but may be associated with the foliar disease symptoms which were similar in manifestation in both cultivars at this stage of infection. It is important to note that except for galactose metabolism, other pathways of carbohydrate metabolism as well as TCA cycle reactions were not affected in Carrizo, which may explain the better performance of this root stock cultivar under HLB pressure. In contrast, the tolerant root stocks US-897 and US-942 which did not show any disease symptoms at 12 mai responded to infection by changes in the amounts of only three metabolites. This suggests that different and root stock specific mechanisms are associated with tolerant responses to HLB. Due to concern for pollinating bees in citrus trees in the spring, spraying for psyllid control with harsher, but often more effective pesticides is suspended from when 5 to 10 % of the citrus flowers are open until 95 % petal fall is reached. After use of dormant sprays at least one spray on emerging flush is desired before bee-friendly sprays are applied during the ‘flowering period’. Data was not available for how long a period existed from various stages of vegetative growth until 5 to 10 % open flowers is reached. Hamlin, Valencia,

Sunburst and Murcott blocks were monitored during the 2015, 2016 and 2017 bloom periods to determine vegetative bud break, leaf feather stage and unfolded leaves as well as pinhead, popcorn, open and petal fall stages of flowering. In the first two years the average time period from bud break to 10 % open flowers was 39 and 45 days, respectively, and the days from 10 % leaf feathers until 10 % open flowers was 26 and 20 days. Round oranges and mandarins differed in time from bud break to full bloom in 2015, 72 versus 57 days, but were similar in 2016, 57 and 56 days. Petal fall occurred 8 to 20 days after full bloom. Weather data will be evaluated to determine if any differences in time for vegetative development between the three years was due to daily temperatures, similar to the temperature response for flower development. More details including 2017 data will be discussed,4x8ft rolling benches particularly as pertaining to the best time to apply the first vegetative spray before the 10 % open flower stage is reached.This is most pronounced in early- to mid-winter under Florida conditions and has consistently occurred over the last four years. This flowering reduces the available buds for normal spring flowering and probably contributes to carry over of fungal inoculum for post bloom fruit drop . The role of fall tree water stress on the induction of this off-season flowering, and the association of this tree water stress with debilitation from HLB will be discussed. Huang long bing is a devastating bacterial disease of citrus and has been detected in most citrus producing areas worldwide. However, genetic sources of resistance/tolerance to this disease are extremely limited and little information is known about the inheritance of HLB tolerance. Several F1 populations derived from crosses between an advanced breeding selection and commercial germplasm are being evaluated under high HLB disease pressure conditions in Fort Pierce and Gainesville, Florida. Pedigree analysis of this trait originating from Poncirus trifoliata fits a Mendelian dominant inheritance pattern. These populations present an opportunity for identifying loci controlling HLB tolerance in citrus. Genetic variability in insect vectors can be a valuable tool to study vector competence determinants and to select or establish non-vector populations that may help reduce the spread of vector-borne pathogens. We collected and tested more than 20 isofemale lines of Asian citrus psyllid Diaphorina citri, vector of Candidatus Liberibacter asiaticus . CLas is the bacterial pathogen associated with huanglongbing, which is the most serious citrus disease worldwide. Individual ACP adult males and females were collected from Murraya plants in various parts of Florida and reared in pairs. After mating, females laid eggs on Murraya plants before being tested for CLas by qPCR. Nymphal progeny from non-infected parents were tested for CLas acquisition from infected citrus plants for three successive generations. So far, we identified one line as a ‘good acquirer’ with 26.8-48.6 % of CLas+ adults. We also identified several poor acquirer lines with 0-10.2 % CLas+ adults. The ‘good’ and ‘poor’ acquisition phenotypes were stable over the three successive generations, showing that variation in CLas acquisition has a genetic basis in the psyllid. ‘Good’ and ‘poor’ acquirer lines were selected and further tested for CLas transmission into healthy citrus, using our excised leaf assay method . Interestingly, CLas transmission assays indicated that Laurel 8 is also a good vector line and Laurel 16 is a poor vector line , which suggests that both acquisition and transmission of CLas are correlated at least in these ACP populations.

We are continuing these tests and studying the genetic composition of both good and poor vector lines using molecular biology methods to study vector competence determinants of CLas in ACP that could be valuable in developing new tools for combating this devastating citrus disease. As sequencing technologies rapidly advance, the successful application of other cutting edge molecular technologies has become increasingly reliant upon the availability of a complete and accurate genome sequence. In an effort to verify the accuracy of the Las bacterial genome while not being subject to the identical limitations encountered with the original sequencing method, a BAC library was constructed from the DNA of Asian citrus psyllids collected from Huanglongbing-affected citrus. Of the 61,440 clones contained within the BAC library, 27 clones specific for Candidatus Liberibacter asiaticus were sequenced in their entirety and used to reconstruct the genome. During the reassembly, a novel ~8.3 kb DNA fragment containing 9 putative ORFs was revealed. Although smaller segments of the 8.3 kb fragment were found dispersed amongst the other sequenced Liberibacter strains, 2 of the putative proteins did not share any homology with any previously identified Las protein. Moreover, 1 of these proteins is not homologous to any protein currently in the NCBI database. Further examination regarding the fragment demonstrated that it was absent in 20% of the Las-infected citrus samples and 5% of the Las-infected ACP samples collected from across the state of Florida, suggesting it is functionally important in the life cycle of the bacterium although not required for survival since the entire 8.3 kb fragment was absent in the genomic DNA used to sequence Ca. L. asiaticus strain psy62. Taken together, the data suggest that the fragment is involved in the generation of a heterogeneous population and may contribute to the virulence of the bacteria through a type of phase variation. It also demonstrates the plasticity of the Las genome and provides a possible marker for monitoring genetic shift in the bacterium.Huanglongbing -affected oranges are typically green or yellow in color, rather than orange, and the latter is more common in the Florida citrus groves. The yellow color is often associated with insufficient accumulation of carotenoids in the flavedo, lack of natural shine, and shriveled peel . The green color is an indicator of maturity due to HLB-associated phloem malfunction and resulting retarded growth and development of the fruit.

The Alameda County Public Health Department found that West Oakland adults are five times more likely to be hospitalized for diabetes, three times more likely to die of stroke, and two times more likely to be hospitalized for and to die of heart disease and cancer . Also similar to Bayview, West Oakland has been the focus of numerous revitalization efforts during the past decade. Plans are underway for the redevelopment of the Oakland Army Base, commercial revitalization along West Oakland’s historic main street, Seventh Street, and the development of artists lofts, condominiums, and affordable housing. Over the next decade, local planners and developers expect that West Oakland will be home to 6,000 new housing units, 200,000 square feet of neighborhood retail, 400,000 square feet of destination retail, and improved transit if plans progress as expected . Like Bayview, recent revitalization efforts have been received equally with enthusiasm and caution. Some argue that new housing has been prohibitively priced, perpetuating gentrification in the neighborhood . This dissertation uses an in depth, comparative case study approach to explore grocery store planning processes. In line with Yin’s criteria for case studies, this dissertation is focused on understanding processes over time and contemporary events where individual and organization behaviors cannot be manipulated .In some ways, this study could be understood as a comparison of two extreme cases—one top down, government-led process and a bottom-up activist driven process . Although one of these cases could be explored as a revelatory or critical case, this comparison of two extreme cases allows for a richer analysis of the factors that might explain their variation . While I am exploring Fresh and Easy and Mandela Foods as illustrative rather than representative cases, my analysis aims to draw out important lessons that might be generalizable to other contexts. As further discussed in the next section,hydroponic grow systems an important aspect of this comparative case study was “tracing and delineating a coherent story but keeping multiple voices .” Because my case studies involve past planning processes, I triangulated across multiple primary and secondary data sources. First, I relied heavily on a number of archival sources to tell the stories of both stores from multiple vantage points. This includes: 1) neighborhood and general plans, 2) neighborhood studies, 3) city council and government agency meeting minutes, 4) videos of public meetings and hearings relevant to both cases, 5) local and national newspaper articles, 6) press releases, and 7) websites and internet sources.

I supplemented these data with fifteen key informant interviews with urban grocery experts as well as government staff, community leaders, and residents associated with Fresh and Easy and Mandela Foods. Urban grocery retail experts were asked about national advocacy efforts around the Healthy Food Financing Initiative and the roles of Fresh and Easy and Mandela Foods. Government staff and community leaders were asked about their involvement with the Fresh and Easy and Mandela Foods projects and various facts about the planning processes, financing plans, and interactions between key actors. Residents were asked about their impressions of both stores.In many ways, the First Lady’s statement is not unique. As vital centers for everyday food and household shopping, grocery stores are cornerstones of neighborhood commercial districts. Therefore, it is perhaps no surprise that the phenomenon of food deserts has seized the attention of policymakers, politicians, and planners in recent years. In this chapter, I situate Fresh and Easy and Mandela Foods in a national policy context by tracing the rise of grocery stores as an urban policy and planning concern. Through a historical review and policy analysis, I find that the federal Healthy Food Financing Initiative reflects normative ideals about urban grocery stores as well as a long history of government interventions and private investment in urban poverty alleviation. At the same time, grocery stores are now part of this shifting federal policy context, where childhood obesity and health disparities are driving local decisions about community reinvestment. As I will discuss further in Chapters 4 and 5, this has implications for grocery development and tools, techniques, and governance patterns of urban revitalization more broadly. First, I show how the current rhetoric around urban grocery stores reflects longstanding normative ideals about the role of food markets in cities. Whether the Greek agora, the “Garden City,” or the neo-traditional neighborhood, food markets became the lifeblood of cities, neighborhoods, and their residents. Second, I show how current federal food access programs trace back to a long history of federal urban poverty programs beginning with the War on Poverty to the Clinton-era federal neighborhood revitalization policies. Community Development Corporations would play a vital role in early experiments in urban grocery development in line with the logic of Michael Porter’s classic campaign for “inner city revitalization.”

Growing evidence of the “urban retail gap” would cast grocery stores as a priority retail development in underserved urban neighborhoods— albeit with mixed outcomes. Third, I discuss how despite the shortcomings of these efforts, public health concerns placed grocery stores back on an urban policy and planning agenda. The community food security movement and growing body of ‘food desert research’ redefined the goals of grocery store development in line with the current Healthy Food Financing Initiative. Finally, I discuss the current context of federal food access policies and neighborhood revitalization, which represents a convergence of previously disparate neighborhood revitalization and public health agendas around grocery stores. The catalyst would become Michelle Obama’s Let’s Move childhood obesity campaign, which shifted national policy discourses around place-based determinants of health. This further elevated the status of grocery stores as a centerpiece of national public health and neighborhood revitalization goals. Be it the agora of Ancient Greece and or the forum of Ancient Rome, the marketplace was the center of social, economic and political life . With the industrialization of cities, central public markets became hubs for the sales of goods. Utopian visions of the city emphasized the role of the marketplace as an organizing element of cities and neighborhoods. For example, for 19th century British town planner, Ebenezer Howard, the “garden city” comprised variety of residential areas connected by rail and accessible to recreational green space, places of work, and basic retail services. Garden cities would be organized along swaths of central green space and the “Crystal Palace” where, “manufactured goods are exposed for sale, and here most of that class of shopping which requires the job of deliberation and selection is done .” Early 20th century urban planner/sociologist Clarence Perry saw a similar role for the marketplace in his vision of the “Neighborhood Unit.” As such, neighborhoods would consist of communities of 5000 to 6000 residents centered around schools and bound by basic commercial facilities on the periphery . Recalling pre-World War I urban neighborhoods, Perry’s concept emphasized physical accessibility and walk ability to institutions that supported the social fabric of neighborhoods, namely schools, churches, and marketplaces. While both the Garden City and Neighborhood Unit concepts would be criticized for prioritizing urban design over the social and economic organization of cities, they articulated particular ideals of neighborhood that maintained in 20th century planning practice. For example, despite being a staunch critic of top-down urban planning and the Garden City concept, Jane Jacobs captured elements of utopian planning ideals in her observations of the livable, mixed-use communities in New York, Boston, and Pittsburgh. Neo-traditional planning of the 1980s and 1990s, captured elements of Jane Jacobs’ vision of the ideal urban neighborhood as a solution to suburban sprawl,vertical grow table wherein cities were designed to be pedestrian-friendly and well serviced with amenities.

For New Urbanist planners, accessible grocery stores contribute to a “balanced mixed of activities-dwelling, shopping, working, and schooling” .For Peter Calthorpe and a related cohort of Transit-Oriented-Development planners, transit stops serve as hubs of dense mixed-use development. Regionally, TODs were to be distributed one-mile apart, a designation determined based on the “market area specifically to support a grocery store within a neighborhood retail center” . Since the late 1980s and early 1990s, New Urbanism, TOD, and related neo-traditional planning principles have been applied to the design of entirely new communities and for the enhancement of existing areas . Initially, New Urbanism was envisioned as a strategy to combat auto-centric, suburban greenfield development. And yet by the early 1990s, neo-traditional planning principles would explicitly inform the Clinton-era HOPE VI housing revitalization program and municipal form based codes, further elevating the role of urban design in land use planning . Overall, normative planning paradigms—be it the Garden City, Neighborhood Unit, New Urbanism and/or TOD—all considered grocery stores among neighborhood amenities essential to the creation of livable, walkable community. As such, grocery stores completed a certain vision of the ideal neighborhood, where the necessities of daily life could be readily accessible. Grocery stores entered more explicitly into urban agendas with the rise of federal urban policies and local institutional practices that developed in response to postwar urban decline. In the 1960s, controversial Urban Renewal plans, rampant social movements around civil rights and economic justice swept cities across the nation. Then President Lyndon Johnson instated a series of programs focused on workforce development or as put by Halpern , “enhancing opportunity and preparing poor children, youth, and to a lesser extent adults to take advantage of opportunities .” Through the enactment of the Economic Opportunity Act of 1964, the Office of Economic Opportunity funded “Community Action Agencies” primarily for the provision of social services spanning workforce development, early childhood education, and youth services—all considered “people-based” social services. In 1966, then Senator Robert F. Kennedy and Jacob Javits introduced an amendment to the Act, the Special Impact Program, to expand the scope of the War on Poverty to encompass economic development activities that addressed the focused on “place-based” neighborhood concerns. Prior to the instatement of this amendment, non-profit organizations and Settlement Houses dating back to the turn of the century pursued “place-based” revitalization activities spanning housing rehabilitation, infrastructure development, and small business incubation. Chicago’s Woodlawn Organization was among these early CDCs, which primarily focused on business and workforce development and significantly, the development of neighborhood grocery stores among other resident-led businesses . The Special Impact Program further expanded these activities, now reorienting federal monies specifically towards the creation of CDCs. The first of 39 CDCs included Brooklyn-based Bedford Stuyvesant Restoration Corporation and Cleveland-based Hough Development Corporation. Both organizations pursued housing assistance, rehabilitation, the provision of business loans for new enterprises, and the development of larger scale projects such as supermarket-anchored shopping center . By the late 1960s and early 1970s, the dismantling of OEO by the Nixon administration, as well as the mixed successes of commercial pursuits reoriented the focus of federally-funded CDCs from economic development to housing and social services primarily . Early evaluations found that despite growing numbers of CDCs nationally, they had limited successes with economic and business incubation strategies . This contributed to the dwindling of CDC funding, but instead, the creation of intermediary organizations who would serve as fiscal agents of federal CDC funds—namely Community Development Financial Institutions such as the national network of Local Initiatives Support Coalition . During the mid 1970s to early 1980s, more established CDCs such as BSRC continued to pursue supermarket-anchored shopping centers even as the large majority of CDCs shifted their focus to housing rehabilitation and social services. In the late 1970s and 1980s, cities across the U.S. experienced deindustrialization and as a result, unprecedented levels of unemployment, urban population decline, and mounting economic segregation. CDCs persisted during this time, even experiencing a net growth by the mid to late 1980s. On the heels of Bill Clinton’s election into office in 1992, grocery stores entered more squarely into neighborhood revitalization practice as a new administration sought new solutions to the old problem of urban poverty. The impacts of urban decline were well known but it was only in the early 1990s, that phenomenon of “supermarket redlining” and the resulting “grocery gap” commanded a national audience. In February 1992, amidst growing concerns about the economic conditions of urban areas across the nation, the U.S. Conference of Mayors issued a report examining urban retail gaps. Through a national survey of supermarket trends, the report revealed that the number of supermarkets in eight major cities experienced a net decrease in a phenomenon they described as “supermarket redlining” . That said, the mass exodus of supermarkets from urban areas well established in previous research.

However, at high substitution rates, substrate properties conducive to plant growth may be compromised. In particular, the high pH of many BCs could result in BC-substituted substrates with pH values unfavorable to plant growth. For example, pelleted wood BC substitution for peat required adjustment of pH due to the liming effect of the BC . The neutral to alkaline pH of BCs and their liming potential means that BC substitution for peat can increase pH beyond optimum for plant growth in potting media . Explicit evaluation of BC effects on substrate pH and plant performance provides a basis to improve design of BC-based substrates and inform trade-offs in this application of BC . The objective of this study was to determine the effects of BC substitution for peat and substrate pH on greenhouse production, using marigold as a model crop. In the United States, the wholesale value of marigolds plants was 30.3 million USD in 2015 . Softwood BC was substituted for peat in a typical 70:30 peat:perlite mixture at 10%v increments. Since many BCs are alkaline and will increase pH of substrates in proportion to the degree of substitution, the effect of adjusting pH of substrates to typical soil free substrate values was also evaluated. We hypothesized that under greenhouse conditions , marigold germination and growth would be more sensitive to BC substitution at higher rates and that this would be due to elevated substrate pH. Additionally, we hypothesized that pH adjustment of BC substrates would increase the extent to which this softwood BC could be substituted for peat without compromising plant growth.Multiple measurements of plant growth were evaluated in order to comprehensively assess the potential of BC as an alternative to peat in soil-free substrates . Germination rates were determined by daily counts for the first 10 days following sowing, after which seedlings were thinned to 1 per pot. Seedlings were transplanted into pots that had zero germination. Replacement seedlings were used from substrates with equivalent %BC but no pH adjustment and were the same age as seedlings in the experimental trial. Weekly measurements over 9 weeks were taken for plant height and for relative chlorophyll content as leaf greenness using a SPAD 502 Plus Chlorophyll Meter . SPAD meters measure the difference between red light and infrared light absorbance, and for a given species and cultivar under the same growing conditions SPAD values can be used as an indicator of relative chlorophyll content . To ensure accurate measurement of new leaf tissue,vertical farming in shipping container four separate points were consistently measured on the second fully extended leaf from the top of the plant . SPAD measurements were taken between the tip and apex of the leaf to better reflect chlorophyll content and reduce measurement variability . At early stage flowering in week 9 , above-ground biomass was harvested.

Fresh and dry biomass was measured individually for shoots, flowers, and buds. Total N was determined separately for non-flowering and flowering biomass by dry combustion using an elemental analyzer .Analyses of variance was used to analyze differences among the treatments for plant growth and substrate properties. Assumptions of normality and homoscedasticity of residuals were tested with the Shapiro-Wilk and Levene tests, respectively, using SAS Version 9.4 . Data were transformed when possible to meet these assumptions, including log transformation , square root transformation and Poisson transformation for variables with zero values . ANOVA was first performed using an exploratory model to test for potential interactions of substrate and pH adjustment for each response variable. If there was no interaction, simple mean differences of response variables were evaluated. If there was a significant interaction of BC substitution and pH adjustment, effects were analyzed separately for each factor. If transformations were not successful, non-parametric analysis was performed with JMP Version 11 using a Welch ANOVA, and significant differences in means for BC substitution treatments relative to the non-substituted control were evaluated using the Steel test. Relationships among post-harvest substrate properties were explored using linear correlation analysis with PROC CORR in SAS v9.4.By evaluating an alkaline BC at high volumetric rates in soil-free substrates, this study addresses a potential obstacle to the feasibility of BC-based substrates for plant production . The present data demonstrate that substituting a softwood BC with strongly alkaline pH for peat at high rates in soil-free substrates does not require pH adjustment under common greenhouse conditions because germination, shoot biomass and N content, and flowering of marigold did not significantly differ between substrates with and without initial adjustment to pH 5.8. BC substitution may even improve plant growth, as marigold plants with intermediate BC substitution exhibited higher relative chlorophyll content relative to 0% BC . These results are in mixed support of the stated hypothesis because BC substitution and pH adjustment effects on marigold depended on the stage of growth. As hypothesized, increasing BC substitution decreased plant height and chlorophyll content in the early stages of marigold growth. Though pH adjustment of BC substrates negatively affected germination and height, this may have been due to phytotoxicity of PLA used to decrease pH of high %BC substrates. By week 9, plant growth was similar regardless of BC substitution and initial pH adjustment, failing to support the hypotheses that high BC substitution rates would impair plant growth and that this would be alleviated by pH adjustment. However, since fertigation provided excess nutrients, pH was likely less important for nutrient availability.

Equivalent and slightly positive effects of BC substitution at high rates and without pH adjustment can be partially attributed to the convergence of pH over 9 weeks of fertigation and plant growth to pH 4.4–7.4. As this high-temperature softwood BC has a higher pH than most BCs and was used at high substitution rates , it represents a ‘worst-case scenario’ liming effect. BCs produced from other feed stocks and/or at lower temperatures may not have as pronounced liming effects. Decreases in substrate pH over time could reflect a number of processes: a residual liming effect of BC, which could also account for the slight upward pH drift of substrates initially corrected to pH 5.8; nitrification; rhizosphere acidification due to cation uptake. Though downward pH drift in peat-based substrates initially limed to a circumneutral pH has been found to be inverse to the base saturation of peat , the 0% BC substrates initially limed to pH 5.8 in this study did not exhibit significant pH changes.The availability and plant uptake of N may be impacted by substrate pH, as indicated by extractable inorganic N, relative differences in chlorophyll content, and above-ground plant N. This may explain initial decreased plant height and relative chlorophyll content in high BC substrates with high initial pH . Foliar chlorosis in ornamental plants,vertical grow racks including marigold, grown in high pH substrates has been induced by liming in peat substrates and could reflect non-N deficiencies such as iron and manganese . Similar above-ground biomass and total N despite greater relative chlorophyll content in high BC substrates by week 9 indicates that initial differences in chlorophyll content by BC substitution did not persist and that initial greater chlorophyll content for marigold in high BC substrates did not necessarily translate to greater biomass and N uptake. A lack of N deficiency under conditions of fertigation is further evidenced by overall high concentrations of available N in substrates at week 9 and by the absence of correlation between available N with marigold above-ground biomass and N content. Elevated chlorophyll content with BC substitution may therefore reflect enhanced plant access to non-N nutrients. Available N was inverse to SPAD values in week 9 and did not reflect similar above-ground plant N concentrations. The disparity between marked differences in substrate N availability under conditions of fertigation yet similar above-ground biomass N content could be explained by pH-dependent gaseous losses of N in pH unadjusted substrates and/or differences in extractability influenced by pH-dependent binding. That extractable inorganic P did not differ as much as inorganic N across pH gradient of pH unadjusted substrates could indicate similar anion exchange capacity of substrates. High available N and P in substrates challenges the hypothesis that BC substitution can influence marigold growth by affecting availability of nutrients added by fertigation. For example, post-harvest available P was positively correlated with marigold biomass but was two orders of magnitude higher than thresholds of deficiency . Though high C:N substrates such as peat can entail sufficient N immobilization so as to compromise plant growth , N fertilization as in this study would be expected to rapidly alleviate N deficiency. This time-dependent effect may have manifested as lower chlorophyll content in high BC substrates in week 1 but not week 9.

Similarly, N fertilization alleviated slightly lower biomass accumulation of marigolds grown in pine wood-based substrates compared to peat . Though the experimental design of this study removed water and nutrient limitations by daily fertigation, the present findings indicate a potential benefit of BC for water availability in soil-free substrates. The increase in WHC with BC substitution that peaked at 30% BC supports this hypothesized benefit of BC at high rates for soil-free substrates , as well as in inorganic matrices like soils .Marigold germination and growth response to BC substitution in pH adjusted substrates was likely due to the use of pyroligneous acid to decrease pH. An increasing amount of PLA was applied to reduce increasingly elevated pH at high rates of the alkaline BC used. Since pH adjusted substrates had the same target pH , the difference can be attributed to a non-pH effect of the almond shell PLA used in this study. PLAs are a complex mixture of organic compounds of varying biological and phytological activity, including toxicity. These include organic acids , phenols, ketones phenyl ethers, and furan and pyran derivatives . The survival and equivalent growth of marigold seedlings transplanted into pH adjusted substrates with no seed germination suggests greater sensitivity of seeds than seedlings to PLA effects and is consistent with previous findings of PLA inhibition of germination . Parallel in vitro experiments , revealed full inhibition of marigold and lettuce germination at PLA ≥ 2.50% and ≥ 1.25% , respectively, though a similiar response occurred for acetic acid, a major PLA component at the same concentration. Studies indicate mixed effects of PLA on biological activity, with both plant-growth promoting and toxic effects, and antimicrobial effects. For example, PLA improved in vitro rooting of pear , and at rates of up to 6% increased fruiting of edible mushrooms in sawdust-based substrates . On the other hand, germination of cress was inhibited by exposure to volatiles from pyrolysis, which are captured via condensation in the production of PLA . Similarly, cress germination was inhibited by BCs with high volatile contents . Like BC, feed stock and production conditions can significantly impact PLA composition and anti-biological activity , and thus the negative impacts of PLA observed in this study may be specific to the almond shell PLA used here.For example, Kono investigated the utility of charcoal to improve substrate physical properties such as water holding capacity and bulk density for orchid production . However, the rapidly expanding body of knowledge on BC, including the ability to design BCs based on feed stock and pyrolysis conditions, means that BCs can be engineered to target additional benefits for to soil-free substrates. Significant enrichment in available N and P over the course of 9 weeks of fertigation reflects high input conditions in greenhouse production systems. Compared to peat, the longer decomposition half-life of high-temperature BCs such as the one in this study, and the potential of nutrient ions to bind to BC and re-solubilize when applied to soils raises the possibility of re-using BC-based substrates as fertilizers. BC substitution may increase the longevity of peat-based substrates under conditions of high nutrient availability common in their use . Decomposition of peat during long grow periods, in particular under high N additions, can compromise physical and chemical properties . Partially replacing peat with less decomposable materials can decrease the overall decomposition rate of the remaining peat component of substrates even under N fertilization , raising the possibility of extending the lifetime of peat-based substrates with partial BC substitution.