Tag: hydroponic

Distributing the risk, responsibilities, and knowledge-intensive labor among partners is a yet-to-be-thoroughly-tested strategy for overcoming some of the land access challenges facing farmers in the San Juan Island region.Agroecology rests upon an essential foundation of building healthy soil, through ecological cultivation of plants, insects, and food webs governed by the “Law of Return” creating a rich network of life on the farm. On Lopez, land clearing for farming, homesteading, and haying posed a threat to the island’s biological and pedologic resource base starting in the late 19th century with the arrival of European-Americans. Today, there is growing attention around restoring and revitalizing soils, forestland, and ecosystem services. Farms such as Midnight’s Farm are managing land for three purposes: healthy food production, economic viability, and soil carbon storage . Other farms are following suit, seeking to build soil and revitalize land that has been degraded especially from repeated haying. The soils on the island vary across short distances, from sandy and well-drained hilltops to heavy clay and moisture-retaining wetlands. The island geology is mostly rock, with a thin soil layer, not considered ideal for farming activities. In the words of one farmer, “we don’t have much rich farmland for row crops on Lopez, so most of us are in a constant dance to balance income-producing crops with inputs to improve the soil and, therefore, the harvest” . Farmers and ranchers are involved in a suite of soil-building practices out of necessity for maintaining productive small-scale operations year after year. These practices include compost production and application, cover cropping,vertical grow rack system bio-char production and co-composting, crop rotations, intercropping , managed rotational grazing, minimal- or no-till cultivation, and combinations of perennial and annual plantings with animals to create a diverse ecological farming system that takes less than it gives back to the ultimate life-source: the soil.

Farmers receive support, training, and information from researchers at WSU SJC Extension, SJICD, and through annual farmer to farmer workshops. Several farmers collaborated in 2015 to host a visit from the Soil Carbon Coalition’s Peter Donovan in order to sample local soils as a baseline and collect additional samples in later years to measure carbon storage, an important component of soil health. WSU researchers offer regular guidance and workshops around crop rotations and pasture management to improve island soils. Recently, WSU partnered with local farmers and the local bakery to host a Field Day on small scale grain production, part of a soil-building rotation that can enhance fertility in concert with legumes and other crops. Other WSU researchers collaborated on a successfully funded Western Sustainable Agriculture Research and Education grant proposal with five local producers to explore the use of bio-char co-composted with cattle bedding and other woody biomass materials as a soil amendment, which will be applied in experimental trial plots beginning in summer 2020 . The SJICD received a WA State Department of Ecology grant to purchase a no-till seed drill that is shared among islands, and recently used in a sequence of liming and seeding Lopez pastures with diverse seed mixes to restore grassland soil health. While the support and education provided by local agriculture and conservation organizations is essential, there is a constant need for further financial resources to extend and improve educational initiatives and technology pilots.Related to efforts of building healthy soil through crop rotations and appropriate farming decisions, farmers on Lopez are taking steps to provide their own inputs for crop production that do not need to be imported or purchased from off island.At a Department of Ecology-approved facility, the farm produces compost from forest and agricultural debris dropped off from across the island, grinding, composting, and screening materials in an aerated static pile system to create a finished product that is widely applied to local croplands.

Manure and bedding material from the farm’s cattle, pigs, and chickens are valuable feed stocks to the composting process as well. Midnight’s produces over 600 yards of compost annually, which is all applied to Lopez agricultural lands and gardens. Farms also self-compost, recycling waste products in smaller decentralized systems and supplementing with purchased composts. Animals also play a role: “Our pigs really close the loop for us on the farm,” one farmer stated, referring to food and plant scraps she was feeding to her American Guinea hogs who were in the midst of transforming it into high quality meat . More recently, due to wildfire risk mitigation efforts, the island has begun to selectively remove and burn some trees in a controlled, limited oxygen environment to create local bio-char, a potentially valuable soil amendment with implications for increased soil carbon sequestration. Current production is happening at a very small scale, but regional interest in larger-scale bio-char production abounds. Midnight’s Farm initiated a research collaboration between WSU extension, U.C. Berkeley, and five local producers from across Western Washington to address the question: can bio-char be a multi-use farm product that improves farm-based co-composted products and vegetable production, and promotes soil C sequestration? Two regionally sourced bio-chars will be applied to cattle bedding at Midnight’s Farm, and then the bio-char-bedding will be co-composted with other on-farm feed stocks to produce a bio-char-enhanced compost product. Through absorbing Nitrogen and other nutrients from the cattle bedding, the “charged” bio-char is intended to provide valuable fertilizer-like qualities to the compost, reducing the need for other amendments to cropping fields. The research hypotheses are: 1) blending bio-char into cow bedding will result in greater N retention, reducing the potential for environmental loss, 2) adding the bio-char bedding blend to compost will increase nutrient content, thereby adding value to the compost product, and that 3) compost with bio-char as a feed stock will lead to increased soil carbon, cation exchange capacity, and pH when applied to soil . The research team will measure impacts on manure handling, composting, soil quality and crop yields, following field application trials on two local farms . Data will be collected in Spring 2020 on soil profiles before amendment, and again in Fall 2020 on soils and crop yields.

The research underway is based on prior work from local bio-char researcher Kai Hoffman-Krull and others, who have worked with universities in Washington and Montana over the past five years investigating on-farm bio-char soil amendments. They have found through field trials on nearby Waldron Island, WA, that in addition to improving soil C storage, locally produced bio-chars have potential to “significantly improve soil fertility and crop productivity in organic farming systems on sandy soils” . However, there remains controversy around the impacts of bio-char in disparate contexts, evidenced by several meta-analyses pointing out varied outcomes based on pyrolysis and feed stock conditions,mobile vertical grow racks and differential effects of in temperate vs. tropical soils .Pending outcomes of the local study on Lopez and across Western Washington, best practices for creating a locally sourced “complete” soil amendment could be scaled regionally, minimizing “external inputs” on a growing number of small-scale organic farms. The goal of minimizing external inputs extends from farmers to others in the food supply chain, including island bakers of Barn Owl Bakery. Rather than purchase bulk inputs like sugar and wheat for their baked goods, Sage and Nathan are actively pursuing the local cultivation of grains and sugar beets to create their own 100% organic island grown products– sprouted Lopez wheat locally milled into flour for wild leavened breads, fruit scones, flat breads, and weekly specialties incorporating other island grown ingredients. Their work is also supported by local researchers from WSU Extension and a Western SARE grant to understand the impact of seeding rate and fertilizer application on the agricultural performance and baking quality of land race wheat. The goal of local input sourcing is also local waste management and reuse of waste as inputs into other ecological processes. Outputs from some farms become inputs for others, in a cost-minimizing closed-loop cycle for those involved.Transitioning food systems to agroecological practices will not be possible without investing in the “re-skilling” of an agroecological workforce.

Lopez has a series of educational offerings in place to reach a variety of audiences from K-12 students to beginning and current. At the farmer-to-farmer level, Lopez farmers engage in regular meet ups and events, including the monthly farmer coffee. On the second Wednesday of each month, Lopez farmers gather at the Lopez Grange for an hour of information and resource sharing. Organized in 2019 by Faith Van de Putte, the forum is a meeting of the minds and transactional space for connecting problems with solutions, questions with answers. Where do people get good, affordable organic chicken feed? Who has straw for goat bedding? How do you get rid of persistent weeds like thistle and morning glory? Do deer get into the grain fields through the electric fence? How can we arrange for annual small animal vet clinics to provide appropriate care for our sheep, goats, and pigs? Disease and pest identification and management topics swirl around room, some finding mostly empathy, and others finding a speedier resolution. At a September 2019 coffee, several farmers shared positive results from experimenting with a new Organic Materials Review Institute -approved herbicide called “Weed Slayer,” said to be effective against the pernicious thistles. Underlying these informational exchanges is the challenge of continuing to run the iconic, diversified small farms of Lopez, lauded as beacons of sustainable agriculture and agritourism, yet requiring countless hours of hard work, determination and passion. Lopez farmers recognize that they cannot “go it alone” on their small farms and rely on the support of other farmers as well as researchers from WSU Extension. Two county extension agents were present at a recent coffee gathering to generate a list of future workshop and clinic topics to offer for farmers, as well as to gauge interest in collaborating on planned future research experiments, grants, and educational demonstrations. In addition to educating each other, Lopez farmers educate aspiring farmers primarily through the Lopez Community Land Trust Sustainable Agriculture internship program. Each year on average five interns live and work on one of the islands six main educational farms, learning from the farmer how to seed, transplant, weed, water, and regeneratively farm diverse vegetable varieties and care for animals such as chickens, sheep, pigs, and cows. These interns select several readings and a documentary to discuss with other interns under supervision from Land Trust staff. Interns complement the practical and hands-on skills of farming with bigger picture reflection and dialogue about ideal vs. real food systems, connecting the production element to all the other moving parts of the system. According to the internship program director, the three biggest takeaways for participants are 1) importance of good local food, 2) basic life skills and 3) the experience of living in community. It is an “empowering experience;” however, it is not a formal or comprehensive beginning farmer training program and has thus far not led to the transition of farmland from an aging farmer to a former agricultural intern. There are additional opportunities for young farmer mentor ship through a Beginning Farmer and Rancher Development grant where more experienced farmers receive funds to support and mentor younger farmers as they begin their own operations. This is geared towards new farmers who have already taken steps to start up operations on Lopez or other islands. All farmers, new and old, have a recurring opportunity to learn more about evolving farm practices at the annual San Juan Agriculture Summit , which rotates between Lopez, San Juan, and Orcas Island, and is held in February each year. The Ag Summit began nine years ago at the impetus of the Agricultural Resource Committee and now WSU Extension has taken on the primary organizing role. Topics presented are wide ranging, from soil health to business and marketing to climate change, and feature speakers from all over the Western United States. The Ag Summit is a social as well as educational event, bringing farmers together for dining, dancing, and community building. On Lopez Island, farm to school programming is run through the Lopez Island Farm Education program. It began as a collaboration between LCLT, the Lopez Island School District, Lopez Island Education Foundation, the Family Resource Center, S & S Center for Sustainable Agriculture, the SJI Conservation District, WSU SJC Extension and the Heller Family.

Total soil C and N were strongly associated with EOC and EON, the soil C:N ratio, and POXC. These variables had negative values along axis 2 and thus contrasted with the pattern of soil inorganic N. Weak loading of AMT1.1, NRT2.1, Nii, and GS2 on the first two principal components reflects the lack of association of expression levels of these genes with biogeochemical and plant variables. Non-overlapping confidence ellipses for seven out of 13 fields on the PCA biplot indicated distinct N cycling patterns . Fields 1 and 2, with the highest values along axis 1, had low values of all variables included in the analysis. Field 4 had the highest values along axis 2 corresponding with higher soil NH4 + and NO3 – . Fields 10, 11, 12, and 13 were associated with high values of labile and total soil C and N. Overlapping confidence ellipses of fields 3, 5, 6, 7, 8, and 9 close to the origin indicate similar, moderate values of this suite of variables for these fields. Three groups of fields were identified by k-means cluster analysis of the same 28 variables included in the PCA . Group 1 included fields 1 and 2, which had low mean values for yield , the lowest mean soil C and N and soil inorganic N pools , and the lowest mean value of GS1 relative expression in roots. Groups 2 and 3 had similarly higher mean yield , shoot N, and petiole NO3 – than group 1, but these two groups differed substantially in their soil C and N pools. Group 2 had higher soil NH4 + and NO3 – pools as well as root expression of AMT1.2 while group 3 had higher total and labile soil C pools. Expression of GS1 was similar in both groups. Based on the relative magnitude of F-statistics calculated for each variable,vertical indoor hydroponic system soil C and N, EOC, EON, shoot N, and soil NO3 – at transplant and anthesis were most strongly differentiated across the three groups.

The high F-statistics of AMT1.2 and GS1 relative to other N metabolism genes indicate that root expression of these genes are most responsive to soil N cycling.This study confirms that working organic farms can produce high yields with tightly-coupled N cycling that minimizes the potential for N losses. Such farms had the highest soil C and N and used high C:N organic matter inputs coupled with labile N inputs that resulted in high soil biological activity, low soil inorganic N pools, high expression for a root N assimilation gene, adequate plant N, and high yields. Organic systems trials have previously shown crop N deficiencies that lead to less-than-ideal crop productivity; losses of N when Navail ability is poorly synchronized with crop N demand; or alternatively, that organic production can reduce N losses. But how working organic farms achieve yields competitive with high-input conventional production with low potential for N losses has not been demonstrated. Elevated expression of a key gene involved in root N assimilation, cytosolic glutamine synthetase GS1, in fields with tightly coupled N cycling confirmed that plant N assimilation was high when plant-soil-microbe N cycling was rapid and inorganic N pools were low, thus showing potential as a novel indicator of N availability to plants. Improving biologically-based farming systems will benefit from research that uses novel tools to uncover innovations happening on farms, especially if the research process helps facilitate knowledge exchange among farmers and researchers.To characterize the substantial variation in crop yield, plant-soil N cycling, and root gene expression across 13 fields growing the same crop on similar soil types, we propose three N cycling scenarios: “tightly-coupled N cycling”, “N surplus”, and “N deficient”.

Values of indicator variables suggest differing levels of provisioning, regulating, and supporting ecosystem services in each scenario . Fields in group 3 show evidence of tightly-coupled plant-soil N cycling, a desirable scenario in which crop productivity is supported by adequate N availability but low potential for N loss. Despite consistently low soil NO3 – pools in these fields, well below the critical mid-season level for conventional processing tomatoes in California, total above ground N concentrations were very close to or only slightly below the critical N concentration for processing tomatoes. Tomato yields were also above the county average . This discrepancy between low soil inorganic N pool sizes and adequate tomato N status is due to N pools that were turning over rapidly as a result of efficient N management, high soil microbial activity, and rapid plant N uptake. Composted yard waste inputs with relatively high C:N ratios in concert with limited use of labile organic fertilizers applied during peak plant N demand provided organic matter inputs with a range of N availability. A companion study showed how high potential activities of N-cycling soil enzymes but lower activities of C-cycling enzymes in this set of fields reflect an abundant supply of C but N limitation for the microbial community, thus stimulating production of microbial enzymes to mineralize N. Plant roots can effectively compete with microbes for this mineralized N, especially over time and when plant N demand is high. High root expression of GS1 in these fields indicates that root N assimilation was elevated and thus actual plant N availability and uptake was higher than low inorganic N pools would suggest . Fields from group 2 demonstrated N surplus, showing similar yields to group 3 but with lower total and labile soil C and N and a higher potential for N losses, given much higher soil inorganic N . While actual N losses depend on a host of factors , high soil NO3 – is considered an indicator for N loss potential. Results from a companion study support the idea that soil microbes were C rather than N-limited in these fields, showing higher potential activities of C-cycling soil enzymes but low activities of N-cycling soil enzymes, the inverse of group 3 .

An alternative multivariate clustering approach based on an artificial neural network suggests multiple potential drivers of higher inorganic N pools in these fields, including both management factors and soil characteristics . For instance, field 4 had strong indications of surplus N driven at least in part by a large application of seabird guano , a readily-mineralizable organic N fertilizer, at tomato transplanting when plant N demand is low. In contrast, higher inorganic N in field 8 was likely driven by low plant N demand based on very low soil P availability, which resulted in plant P limitation. These site-specific problems were identifiable due to the focus on variability across similar organic fields and illustrate the need for site-specific approaches to reduce N losses. Finally, the two fields included in group 1 were exemplary of N deficiency, in which low N availability compromises crop productivity but also likely limits N losses within the growing season. While low soil NH4 + and NO3 – concentrations were similar to group 3,vertical farming tower for sale low total and labile soil organic matter and poorly-timed organic matter inputs compromised microbial activity and likely limited N mineralization.Cytosolic glutamine synthetase GS1 encodes for the enzyme that catalyzes the addition of NH4 + to glutamate, the former resulting from either direct uptake of NH4 + from soil or reduction of NO3 – in roots. GS1 is thus the gateway for N assimilation in roots and is upregulated to increase root N assimilation capacity. Similar levels of GS1 expression in groups 2 and 3, in spite of large differences in soil NH4 + and NO3 – concentrations at the anthesis sampling, suggests that plant N availability is indeed higher in group 3 fields than would be expected based on measurement of inorganic N pools alone. The low levels of GS1 expression found in fields with clear N deficiency supports this idea. These results complement recent experimental approaches that showed rapidly increased expression of GS1 in tomato roots in response to a pulse of 15NH4 + -N on an organic farm soil, which was linked to subsequent increases in root and shoot 15N content, even when this pulse did not significantly change soil inorganic N pools. GS1 transcripts and glutamine synthetase enzyme activity also increased with increasing NH4 + and NO3 – availability in sorghum roots, suggesting this response may be widespread among plant species.

Interestingly, inclusion of soil GWC in multiple linear regression models increased the proportion of GS1 expression variability explained to nearly 30% ; soil water content increases microbial activity as well as the mass flow and diffusion of inorganic N to roots. Further research will undoubtedly show how other factors like crop physiological N demand relative to C fixation and P availability increase the interpretability of N uptake and assimilation gene expression in roots.The N cycling scenarios identified on this set of organic fields corresponded at least in part with landscape clusters based on landscape and soil characteristics . Fields that balanced high yields with low potential for N loss and high internal N cycling capacity were part of PAM cluster 1, which had the highest productive capacity rating . Landscape clusters encompassing more marginal soils included both low-yielding fields exhibiting N deficiency or high-yielding fields that used inputs of highly available N like seabird guano to alleviate N deficiency . But these inputs led to the highest soil NO3 – levels and thus came at the cost of higher potential for N loss. Long-term efforts to increase internal soil N cycling capacity would help alleviate both N deficiency and the need for such large inputs of labile N. Whether farmers are willing to invest in management to increase soil N cycling capacity depends in part on how likely they perceive the benefits to be, especially on marginal soils. The discussions that we had with each farmer in this study indicated genuine interest in adaptive management to further tighten plant-soil N cycling, but this may not always be the case. Indeed, the proportion of management vs. inherent soil characteristics responsible for driving differences in N cycling is challenging to untangle. Farmers may allocate more resources to more productive land and likewise fewer resources to more marginal land, or may selectively transition more marginal land to organic management. Documenting the multiple services provided by increases in soil quality and facilitating information exchange among organic growers such as through the landscape approach used here may help build momentum for efforts to improve soil quality and plant-soil-microbe N cycling.The so-called chelation strategy , which is mainly found in graminaceous plants, is based on the excretion of phytosideropores to the rhizosphere. Phytosideropores rapidly chelate Fe, to form Fe-PS chelates that are subsequently transported into the root cells through a specific transporter. The socalled reduction strategy relies on the coordinated action of a membrane bound Fe reductase, that reduces Fe to Fe, an Fe uptake transporter and an H+ -ATPase that lowers the pH of the rhizosphere, is mainly used by non graminaceous plants, including Beta vulgaris. The reduction strategy includes root morphological, physiological and biochemical changes that lead to an increased capacity for Fe uptake. Morphological changes include root tip swelling, development of transfer cells and an increase in the number of lateral roots, leading to an increase in the root surface in contact with the medium. Some plants are able to accumulate and/or release both reducing and chelating substances, such as phenolics and flavins, which may have a role in Fe acquisition. Iron has been shown to down-regulate riboflavin synthesis in flavinogenic yeast strains and some bacteria. In plants, Rbfl and derivatives are accumulated and/ or excreted in Fe-deficient roots and could act as a redox bridge for electron transport to the Fe reductase. Moreover, FRO2 belongs to a super family of flavocytochrome oxidoreductases, and a recent study confirmed that the FRO2 protein contains FAD sequence motifs on the inside of the membrane. Also, a connection between Fe deficiency perception and Rbfl excretion has been described to occur through basic helixloop-helix transcription factors in Arabidopsis thaliana. At the metabolic level, increases in the activity of phosphoenolpyruvate carboxylase and several enzymes of the glycolytic pathway and the tricarboxylic acid cycle have been found in different plant species grown under Fe deficiency.

Through mechanical weathering processes, these clay fragments become incorporated into the soil and may provide a long-term source of PAH contamination in the environment . Polycyclic aromatic hydrocarbons are ubiquitous environmental contaminants and 16 PAHs are considered priority pollutants by the U.S. .There have been a few ecotoxicological evaluations concerning the large PAH concentrations from clay target fragments, but these studies have reported that the PAHs elicit low toxicity in aquatic organisms . This low toxicity was determined to be primarily due to the low bio-availability of PAHs resulting from the process of making clay targets in which the PAHs in the binding agent are bound under heat and pressure with dolomitic limestone . In addition, due to their aromatic nature and hydrophobicity, PAHs typically bind to nonpolar soil domains such as organic matter, further decreasing their bio-availability . However, a recent clay target ecotoxicity study using Eisenia andrei showed that the content of clay fragments in soils was correlated with PAH bio-accumulation in the terrestrial soil organism, suggesting that direct ingestion can be a more important route of exposure and potentially explain the lack of toxicity in exposed aquatic organisms . Clay-target contaminated site evaluations have also concluded that the elevated PAH concentrations in the soil from the clay target fragments pose an unacceptable level of risk to future potential residents and current site workers.

There are several PAH remediation strategies involving physical, chemical, biological,stackable flower pots and thermal technologies; however, conventional PAH removal methods such as incineration, excavation, and land filling are expensive and inefficient . Because of these issues, biological remediation practices such as bio-augmentation and phytoremediation have become preferred in situ treatment technologies as they are considered to be cost-effective and more environmentally friendly for the cleanup of PAH-contaminated soils . However, biological remediation operations can also be ineffective due to the limited PAH soil bio-availability that is a consequence of the clay target manufacturing process and the physicochemical properties of these compounds, which can be further exacerbated by the aging effect in field-contaminated soils . These PAH bio-availability limitations can be overcome through the use of surfactants that increase the desorption of PAHs from the soil to the aqueous phase, thus increasing their bio-availability to the degrading soil microbes . Bio-surfactants such as rhamnolipids or glycolipids offer an environmentally-friendly alternative to synthetic surfactants and are becoming more economically-feasible through the use of low-cost substrates and offer distinct advantages to synthetic surfactants such as reduced toxicity, high biodegradability, and greater stability under different temperature, pH, or salinity conditions . In practical surfactant-enhanced PAH contaminated soil remediation applications, mixtures of surfactants are commonly used to take advantage of the potential synergistic effects that can result in increased solubilization at a reduced effective surfactant concentrations . The bioaugmentation of biosurfactant-producing soil microbes has also been shown to be an effective strategy for the remediation of PAH-contaminated soils. For example, M. vanbaalenii PYR-1, a glycolipid-producing microorganism isolated from an oilcontaminated estuary near the Gulf of Mexico, has been shown to enhance PAHsolubility and degradation in PAH-contaminated soils .

Another in situ biological remediation treatment commonly used to increase PAH bio-availability is phytoremediation, or the use of plants and the associated rhizosphere to restore contaminated sites . Phytoremediation is considered to be an effective, low-cost alternative to cleanup large contaminated sites . The PAH bio-availability is enhanced in the plant rhizosphere, as plant roots secrete root exudates that promote PAH desorption from the soil matrix . In addition, plant roots may release enzymes that play a key role in the degradation of PAHs including oxygenases, dehydrogenases, phosphatases, and lignolytic enzymes . Finally, plant roots also provide easily degradable carbon sources and other nutrients that increases microbial biomass, diversity, and activity, contributing to enhanced PAH degradation through direct metabolism or co-metabolism . Because of the numerous benefits provided by the rhizosphere, grass species are recommended for phytoremediation treatments due to their extensive fibrous root systems and large root surface area, and hence more extensive interactions between PAHs and the rhizosphere microbial community . Typically, the primary contaminants of concern during the remediation of outdoor shooting range soils are heavy metals from ammunition; however, large concentrations of PAHs from the clay target fragments remain in the contaminated soil and could possibly become more bioavailable during the remediation of the metals.Brij-35 nonionic surfactant and sodium dodecyl sulfate anionic surfactant were purchased from Sigma-Aldrich. Rhamnolipid biosurfactant isolated from P. aeruginosa NY3 was purchased from AGAE Technologies .Diatomaceous earth, Ottawa sand, and all GC-MS grade solvents used in this study were purchased from Thermo Fisher Scientific . All substrates utilized for soil enzymatic analyses were purchased from Tokyo Chemical Industry Co., .

Bermudagrass, switch grass, and lettuce [Lactuca sativa] seeds were purchased from Lowe’s.A Vista coarse sandy loam was collected manually using a shovel from the 0-15 cm soil depth of an abandoned shooting range located near Lake Elsinore, California that was littered with clay target fragments with no prior soil remediation or waste removal from the site. The collected soil was air-dried for 5 d at approximately 23 °C and sieved through a 2-mm stainless-steel mesh screen.The soil pH and electrical conductivity were determined potentiometrically in a 1:2 soil-to-water suspension . Total metal analysis was carried out using an Optima 7300 DV inductively coupled, argon-plasma optical emission spectrometer following U.S. EPA Method 3050B after a 6-h digestion in a mixture of nitric acid, hydrogen peroxide, and hydrochloric acid at 95 °C .Mycobacterium vanbaalenii PYR-1 was stored at -80 °C in a 30% glycerol stock and the inoculum was prepared according to a previous method in MBS solution amended with pyrene as a carbon source . The CMC of Brij-35 and rhamnolipid biosurfactant was determined previously . The CMC of the Brij-35/SDS surfactant mixture was determined by measuring the surface tension of surfactant solutions over a concentration range using a Du Noüy ring-tensiometer and using the inflection in the plot of surface tension against surfactant concentration. The CMC was determined to be 0.099 mM at 0.5/0.5 molar fraction, which was similar to a previous study .After the soil was thoroughly mixed, 1 kg soil was placed in a stainless-steel bowl and 150 mL of distilled water was added and mixed to achieve a soil water potential of approximately -33 kPa determined by a soil tensiometer. For the M. vanbaalenii PYR-1 bioaugmented treatments, 15 mL M. vanbaalenii PYR-1-MBS solution was added to yield approximately 106 CFU/g soil and thoroughly mixed . The same procedure using only the MBS solution was added to the non-inoculated, or native, soil as the control. The PYR-1-MBS solution was reapplied every 2 months by adding the inoculum solution into the soil rhizosphere 5 cm below the soil surface . Once the soil treatments were prepared, the soil was added to the phytoremediation sample containers,flower pots for sale which consisted of 800-mL glass jars that were first painted on the outside with black paint, followed by aluminum enamel to prevent exposure to light . The pots contained approximately 50 g of 2-cm diameter gravel at the bottom to allow for accumulation of any excess soil water . Bermudagrass and switch grass seeds were surface-sterilized by three sequential washings in 0.1% sodium hypochlorite, followed by two rinses with sterile distilled water . Bermudagrass and switch grass seeds were planted at a rate of 20 seeds/pot and sealed with plastic wrap for 1 week for optimal seedling emergence conditions. After 2 weeks, plants were thinned to 8 plants/pot and amended with a commercial fertilizer for bermuda grass establishment. Treatments were then fertilized monthly with 100 mg/kg-N as urea, and 12.5 mg/kg-P as monobasic potassium phosphate . Due to the potential toxicity of surfactants to emerging plant seedling , surfactant addition at 50 mg/kg was initiated 1 week after plant thinning and initial fertilizer application. Since rhamnolipid biosurfactants have been previously shown to be degraded by the soil microbial community and are considered more biodegradable than the synthetic surfactants used in this study, surfactants at the initial rate were reapplied to the soil surface every 40 d .

Each pot was placed randomly in one of four blocks, each containing one replication of all treatment combinations in a climate-controlled growth chamber . The PAH phytoremediation experiment was continued for 8 months in the growth chamber under a 12/12 hour day/night period at 23±1/19±1 °C and 40% relative humidity. The average light intensity was obtained through fluorescent and incandescent lighting in the growth camber . Each pot was weighed daily for 8 months and the soil moisture was gravimetrically adjusted to 20% by application of distilled water . The quantity of distilled water added to the soil to achieve proper soil moisture was not adjusted for vegetation biomass produced during the study. Plant shoots were trimmed to a height of 5 cm every 3 months in order to stimulate continuous plant growth . At the end of the 8-month phytoremediation experiment, plant shoots and roots were separated from the soil as described in section 2.7. Once the vegetation was removed, the soil was sieved to pass through a 2-mm sieve and separated into two subsamples. The first soil subsample was air-dried for 7 d at approximately 23 °C in the dark and used for PAH analysis and toxicity assay . The second soil subsample was used for soil enzyme analysis and kept at fieldmoist conditions and analyzed within 1 week after the termination of the experiment. set at 60 °C and then raised at 5°C/min to 280°C . Quantification of PAHs was performed using an internal standard-normalized calibration curve and coefficients of determination for all calibration curves fulfilled the requirement of R2 ≥ 0.99. Soil dehydrogenase soil activity was analyzed by the use of 2–3- -5-phenyl tetrazoliumchloride as a substrate . A 1.0-g soil aliquot was mixed with Tris buffer and INT substrate in a stoppered 100-mL Erlenmeyer flask, and the mixture was incubated for 2 h at 40 °C in the dark. After incubation, the mixture was extracted using 10 mL N,Ndimethylformamide:ethanol mixture for 1 h at 23 °C in the dark and shaken every 20 min. Immediately after filtration, iodonitrotetrazolium formazan formation was measured colorimetrically at 464 nm against the reagent blank using a UV-Visible spectrophotometer . Soil dehydrogenase activity was expressed as µg INTF produced/g dry soil 2h. Soil polyphenol oxidase activity was measured by the utilization of pyrogallic acid as a substrate to form purpurogallin . Ten mL of 1.0% pyrogallol was added to 1.0 g soil sample and incubated at 30 °C for 2 h at 200 rpm. Afterwards, 5 mL of citrate-phosphate buffer was added to the treatment to stop the reaction, followed by the addition of 35 mL ether and shaking for 30 min at 200 rpm. The colored ether with dissolved purple gallic prime was measured colorimetrically at 430 nm on a UV-Visible spectrophotometer.The polyphenol oxidase activity was expressed as mg purpurogallin produced/g dry soil 2h. Control assays for each soil enzyme activity included autoclaved soil treatments, assays without soil, and assays without substrate addition during incubation . Results of soil enzyme activities are reported on an oven-dry-weight basis.At the end of the phytoremediation experiment, plant shoots were cut at the soil surface and rinsed with distilled water to remove any adhering soil. Approximately 4 g shoot subsample was taken and freeze-dried for PAH extraction and the remaining shoots were dried to a constant weight at 55 °C and weighed to determine total shoot biomass. The freeze-dried plant shoots were ground to pass a 2-mm, stainless-steel mesh screen using a Wiley Mill Grinder and 2 g was used to determine PAH shoot concentrations using procedures similar to those for soil PAH extraction . Plant roots were manually collected from the soil using forceps, placed on a 500-µm stainless-steel sieve, and thoroughly rinsed with distilled water to remove any adhering soil particles. Approximately 2 g root subsample was taken and freeze-dried and 1 g was used to determine PAH root concentrations similar to shoot analysis. The remaining plant roots were dried to a constant weight at 55 °C and weighed to determine total root biomass. The lettuce seed toxicity assay was performed to evaluate changes in phytotoxicity before and after remediation treatments by following a method in Cofield et al. . Briefly, 100 g soil at 85% water-holding capacity was placed in a 150 mm ´ 15 mm Petri dish and 40 lettuce seeds were evenly distributed and pressed into the soil.

The respiration rate of shade grown segments that were shifted to high light became significantly more negative four days after transplant . The shade grown segments exposed continuously to low light showed small changes in Afull sun and Rd relative to the SH-SU segments. Rd acclimation in SU-SH segments took about 13 days, with an initial rapid change followed by a more gradual change .The intercellular CO2 concentration remained constant among the treatments , and the changes in Afull sun were attributable to shifts in photosynthetic capacity rather than CO2 supply. Stomatal conductance paralleled the changes in Afull sun . We did not find evidence of changing patterns of stomatal control, and the simplest explanation is that conductance simply responded to Afull sun acclimation. Leaf acclimation was highly localized; individual leaf segments acclimated to local light autonomously from the rest of the leaf. Previous studies of leaf acclimation have focused on entire leaves, and acclimation by segments of mature leaves has received less attention . The ability for mature leaf acclimation varies among species, and has been reported in several herbaceous and a few woody species . Our findings of segmented acclimation are most closely related to those of Prioul et al. , who found that Afull sun, chlorophyll content,fodder growing system and Rubisco activity changed markedly from the base to tip of Lolium multiflorum leaves. Likewise, reciprocal transplants to contrasting light conditions inL. multiflorum showed the capability for rapid photosynthetic reacclimation to high and low light along the leaf, even in fully expanded leaves .

Detailed investigations of leaf anatomy and biochemistry are beyond the scope of this study, but our observations provide evidence of the mechanisms responsible for acclimation. Photosynthetic acclimation in Typha appears to result from biochemical or cellular changes, and a general up or down regulation of metabolic activity within individual leaf segments.Acclimation did not involve a significant change in nitrogen content on either a mass or area basis. We did not find evidence that a morphological change in leaf thickness, or a net movement of nitrogen into or out of a leaf segment, were required for photosynthetic acclimation.Our results confirm previous reports that species from highly variable light environments have a strong capacity for photosynthetic acclimation. In the case of T. latifolia, light heterogeneity is created by the combination of a basal meristem and a dense canopy of live leaves and litter . Typha leaves are exposed to markedly different light environments as they grow and individual segments are pushed upward . The upper segments of leaves in the field, which occurred in a brighter environment, had higher rates of CO2 uptake . Previous field studies on T. latifolia have also reported large CO2 assimilation and gs gradients along leaves . We hypothesize that the patterns of leaf photosynthesis and conductance in Typha reflect four properties. Mature Typha leaf segments are morphologically preformed to function in high light and allow high rates of Afull sun, regardless of the current or growth environment. Mature Typha leaf segments contain sufficient amounts of nitrogen to support high rates of Afull sun, regardless of the current or growth environment. Mature Typha leaf segments rapidly reallocate nitrogen between active and inactive pools in response to local light availability; acclimation occurs at a local level and does not require nitrogen translocation into or out of a leaf segment.

The controls on stomatal conductance remain constant over time; the patterns of conductance can be explained based on simple, short-term adjustments that act to maintain a nearly constant Ci concentration despite the changes in Afull sun and the physical environment. We interpret these patterns as a highly plastic strategy that maximizes carbon gain by a monocot growing in a vertically heterogeneous light environment. The construction of leaves that are morphologically capable of high rates of Afull sun is a simple consequence of the spatial decoupling of the growth environment fromdegradation of cellular components, such as Rubisco, cytochrome f, and chloroplast ATPase . The amount of nitrogen in leaf segments remained nearly constant over time, leading us to hypothesize a fraction of the nitrogen in shaded segments is stored in inactive pools and is rapidly activated following transfer to high light. These changes may include adjustments in partitioning among carboxylation, electron transport and light harvesting, chloroplast ultrastructure, volume, and orientation . The high N content of shaded segments should not be viewed as wasteful. These nutrients can be reabsorbed and reallocated to the rhizome during senescence; a high reabsorption efficiency of P and N has been reported for Typha dominguensis . Moreover, this strategy allows a leaf segment to rapidly and autonomously respond to a change in light availability, without importing or exporting nitrogen to or from other leaf segments or organs.Polycyclic aromatic hydrocarbons are a group of persistent organic pollutants that are composed of two or more fused aromatic rings in linear, angular, or cluster arrangements . Depending upon the structure of the rings, PAHs are classified as either alternant or non-alternant.

Alternant PAHs contain only fused six membered rings , while non-alternant PAHs contain four- or five-membered rings in addition to the six-membered rings . The aromatic structure of PAHs results in increased thermodynamic and chemical stability due to electron delocalization in the π orbitals, which plays a critical role in the environmental fate and toxicity of these contaminants . There are 16 PAHs designated as priority pollutants by the United States Environmental Protection Agency due to their occurrence in the environment and toxicity. The physicochemical properties of the 16 priority PAHs are detailed in Table 1.1 . These compounds are all hydrophobic, as demonstrated by their relatively high octanol-water partition coefficients and low solubility in water . The impact of the PAH structure on its chemical behavior is primarily dependent upon molecular size and angularity. Typically, an increase in the number of rings and angularity results in increased electrochemical stability and hydrophobicity . For example, low-molecular-weight PAHs are considerably more water soluble and volatile than high-molecular-weight PAHs . In addition to increases in hydrophobicity and environmental persistence with increasing PAH molecular size, PAH genotoxicity generally increases and toxicological concern shifts towards chronic toxicity, primarily carcinogenesis . Numerous studies have indicated that LMW PAHs exhibit acute toxicity to humans, whereas HMW PAHs exhibit chronic effects such as genotoxicity . The acute effects of PAHs on human health such as nausea, vomiting, and respiratory and skin irritation depend primarily on the extent of exposure, the route of exposure , and the concentration and toxicity of the individual PAHs . Polycyclic aromatic hydrocarbons can be widely distributed throughout the human body and have been detected in almost all internal organs, especially adipose tissues due to their lipophilicity . Once they enter the body, PAHs undergo metabolism primarily through the cytochrome P450 mixed-function oxidase system. This metabolic pathway transforms PAHs into polar epoxide intermediates that are further converted to dihydrodiol derivatives and phenols, which then form glucuronide and sulfate conjugates that are finally excreted in the bile and urine . However, this metabolic transformation can also result in the formation of electrophiles that elicit deleterious human health effects . Because of this, PAHs are considered procarcinogens because they do not directly induce DNA damage, but require metabolic activation to exert their genotoxic, mutagenic, or carcinogenic effects . There are three major pathways for PAH carcinogenic activation: the bay region dihydrodiol epoxide pathway, the radical cation pathway, and the o-quinone pathway,which result in the formation of radical cations, diol epoxides, and electrophilic and redox-active o-quinones, respectively, all of which may react with DNA to produce DNA adducts . Following extensive and systemic studies on the toxic effects of individual PAH metabolites in animals, it has been determined that the vicinal or bay-region diol epoxides are considered the ultimate mutagenic and carcinogenic species of PAHs .The International Agency for Research on Cancer classifies numerous PAHs as known, probably,chicken fodder system or possibly carcinogenic to humans . The IARC has determined that benzo[a]pyrene is one of the most potent carcinogenic PAHs, benzo[a,h]anthracene is a probable human carcinogen, and naphthalene, benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, and indeno[1,2,3-c,d]pyrene are possible human carcinogens . Altogether, PAHs rank as #9 on the Agency for Toxic Substances and Disease Registry’s Substance Priority List , which ranks contaminants based on a combination of their toxicity, frequency, and potential for human exposure at National Priority List sites . Of the approximately 1,400 NPL sites that are targeted for remediation by the U.S. EPA, more than 700 sites are contaminated with PAHs.

Polycyclic aromatic hydrocarbons are formed primarily during the incomplete thermal decomposition of organic substances and their subsequent recombination . Thus, the composition of the PAHs formed is dependent upon the temperature and the starting organic material . Polycyclic aromatic hydrocarbons occur as complex mixtures in the environment instead of single compounds due to their differing physicochemical properties during the incomplete combustion process . There are three primary sources of PAHs in the environment: pyrogenic, petrogenic, and diagenetic/biogenic. Pyrogenic PAHs areproduced from the rapid oxygen-depleted, high-temperature incomplete combustion of fossil fuels and organic materials . These pyrogenic PAHs are formed from the breakdown of organic matter to LMW radicals during pyrolysis, which is then followed by rapid reassembly into PAH structures . Pyrogenic PAHs are typically found at greater concentrations in urban areas because the major sources of pyrogenic PAHs are the incomplete combustion of gasoline and diesel in vehicles, the production and use of coal tar and asphalt, heat and power generation, and discharges from aluminum smelters and manufactured gas plants . The most abundant pyrogenic PAHs are typically fluoranthene and pyrene . Petrogenic PAHs originate from diagenetic processes at relatively low temperatures over a long duration, leading to the formation of petroleum and other fossil fuels containing PAHs . Petrogenic PAHs are introduced into the environment through accidental oil spills, discharge from tanker operations, and underground and aboveground storage tank leakage . Diagenetic/biogenic PAHs are produced from biogenic precursors by plants, algae/phytoplankton, and microorganisms . For example, concentrations of perylene, naphthalene, and phenanthrene concentrations have been found in hydromorphic soils, Magnolia flowers, and Coptotermes formosanus termite nests . While diagenetic/biogenic PAHs are often found at background levels in recent sediments, they are frequently the primary PAHs in older sediments deposited before increased industrial activity . The environmental ubiquity of PAHs is due to their chemical stability and numerous natural and anthropogenic sources. Natural sources of PAHs include volcanic eruptions, forest and prairie fires, and seeps of crude oil deposits . Anthropogenic sources, which contribute the vast majority of PAH contamination in the environment, include the production and use of fossil fuels such as coal, oil, and natural gas, refinement of crude oil, heat and power generation, wood treatment preservation processes, landfills, residential wood burning, and improper industrial waste disposal or spillage . Over the past century, there has been a substantial increase in environmental concentrations of PAHs following increased anthropogenic sources from industrialization, which can be demonstrated by PAH levels being the greatest in urban areas followed by agricultural and rural environments . Even the lowest PAH concentrations in temperate soils are approximately 10 times greater than PAH concentrations assumed to have been present before global industrialization . Once PAHs are emitted to the environment primarily through the combustion of fossil fuels, they are distributed atmospherically and deposited onto terrestrial, lacustrine, and marine surfaces . However, unlike most persistent organic pollutants like polychlorinated biphenyls that follow the global distillation transportation effect, PAH concentrations generally decrease as the distance from the initial source increases . Atmospheric PAHs are generally more abundant at night than daytime, and during the winter months compared to the summer months due to greater deposition at lower temperatures and increased coal combustion for heating . Polycyclic aromatic hydrocarbons are semi-volatile organic compounds; therefore, PAHs can be found in both vapor and particle phases depending on the vapor pressure of the PAH, temperature, and size and surface area of suspended particles . However, given the physicochemical properties of PAHs, they tend to more readily sorb to atmospheric particulates than be present in the gas phase . Because PAHs are commonly adsorbed onto atmospheric particulates, PAH transformation and degradation by thermal or photodecomposition and reactions with O3, SO2, NOx, or OH radicals are reduced and can even be completely inhibited .

Up or down changes in carbohydrate and lipid metabolism and protein handling pathways are also evident, although these are more difficult to interpret. Aquaporins, which affect membrane water permeability, were found to be upregulated in two studies . When drought-stressed seedlings are re-watered, most gene expression quickly returns to normal. In Pinus taeda, only 76 of the 2445 genes with altered expression during drought were still different after 48 h of recovery . Lorenz et al. found 11 genes upregulated in ‘recovered’ P. taeda seedlings relative to either drought-stressed or well-watered seedlings, including probable cell wall proteins, an aquaporin and a gene involved in vacuole function. These may reflect recovery or repair processes that occur once drought stress is removed.Pinus pinaster has at least eight dehydrin genes, based on expressed sequence tag analyses . Three of five were downregulated during drought, whereas the other two were upregulated . Most dehydrin induction occurred after 20 d of drought , which may be why a similar but shorter study did not reveal the upregulation of dehydrins. Expression can also vary by tissue. Of seven dehydrins examined in P. abies, drought stress upregulated four in needles, but only two in bark, with one being down regulated in bark . To investigate the link between drought and defense gene expression, Fossdal et al. exposed P. abies seedlings to a pathogen , drought stress or both,grow strawberry in containers and examined the transcription of 14 candidate defense genes. Genes were upregulated more slowly in drought-stressed seedlings than in pathogeninoculated seedlings.

The combined treatment led to more rapid and/or higher expression of many defense genes than either alone, which may be related to the synergistic mortality risks posed by biotic and abiotic stressors. Pleiotropic effects for some drought/ defense-related genes are also possible, but none have been identified to date.Multiple provenance studies have identified patterns consistent with local adaptation to drought. Trees from drier climates often exhibit conservative growth strategies , such as slower height or needle growth , less above ground biomass or a shorter growing season . Seedlings from dry environments often also exhibit more root growth and higher drought survival . Provenance trials of Pinus halepensis have shown mixed responses, with low growth and high water use efficiency in dry-sourced populations , but high growth in populations from intermediate-aridity areas , which may be related to growth plasticity. Because of the importance of carbon resources for plants, WUE – the ratio of carbon fixed to water lost – has long been considered to be closely tied to drought tolerance . Measures that integrate over longer time periods, such as the carbon isotope ratio d13C , are most frequently used to represent changes in WUE in trees. However, although different measures of WUE are often correlated , they are not interchangeable. For example, carbon discrimination is sensitive to chloroplast carbon concentrations and mesophyll conductance, whereas WUE itself is heavily influenced by evaporative demand, which does not directly affect D . In addition, nitrogen fertilization can increase WUE and decrease D, but does not affect gs or transpiration . Thus, WUE and D do not always co-vary, and caution is needed in the interpretation of d13C as a measure of WUE. Additional caution is warranted when using WUE as an indication of drought tolerance. High WUE may not be adaptive in some dry environments if the use of less water per unit carbon fixed does not result in slower depletion of soil water , or if plants with higher WUE grow faster and thus use more total water. Although a few studies have shown higher d13C for populations from dry sites , others have shown the opposite .

There was no difference between three populations of Pinus ponderosa seedlings from varying climates in d13C or instantaneousWUE; the drought adapted populations exhibited greater plasticity in water use . In P. halepensis, however, individuals from more mesic sources showed higher plasticity of WUE than those from drier sources , but dry sources may show higher average WUE . Highly plastic growth and water usage reduce apparent WUE over the whole season compared with consistently moderate to low water usage . Instantaneous measures of WUE can change over a day, whereas integrated measures can differ significantly for a source population grown under different conditions or for the same tree across years . Changes in WUE may thus be a useful indication of drought stress, but, in conifers, radial growth and WUE are often weakly or negatively correlated . In pines, higher WUE usually results from reduced gs and/or reduced leaf area , which can limit photosynthesis and growth . Low gs can also result in higher tissue temperatures, which can be damaging, particularly in seedlings . Drought length and severity can influence measures of relative drought tolerance between populations. In P. ponderosa seedlings,the relative growth rate under moist conditions was positively correlated with previously measured tolerance to severe drought, whereas, under 4-wk drought, the intermediate-drought-tolerant population grew faster . When Silim et al. examined Picea sitchensis, P. glauca and their hybrids, they found that P. sitchensis and the hybrids had the highest WUE and growth in well-watered conditions, but P. glauca and the hybrids had higher WUE and growth in drought conditions. Similarly, the relative transpiration and photosynthetic rates, WUE and growth of P. halepensis tree provenances differed between near-desert and Mediterranean planting sites . Such shifts in ranking are often a result of plasticity differences between populations. Provenances of P. pinaster from across the species’ range did not vary in cavitation resistance, suggesting uniform selection or lack of genetic variation . In P. halepensis, however, the percentage loss of conductivity differed significantly between provenances, but not between environments .

Although plasticity has been observed in xylem wall thickening, time to thickening and number of cells in Picea mariana in drought experiments , cell anatomy studies often focus on only one population, so that the extent of local adaptation is unknown.Genome scans have identified loci in conifers that may be under differential selection across environments . Of 13 candidate genes for drought response in P. pinaster, two showed signs of divergent selection, although only one exhibited a pattern correlated with a climatic gradient; three, including two dehydrins, showed evidence of balancing selection . Prunier et al. examined SNPs from 313 candidate genes in P. mariana and found 16 that exhibited differentiation correlated with precipitation, including a LEA protein and genes in the ubiquitin protein handling pathway. However, differentiation between populations can be driven by processes unrelated to climatic gradients. Conifer populations are likely to violate the assumptions of such tests because they rarely form discrete isolated populations and are often far from demographic equilibrium; mis-specification of population hierarchical structure can lead to high false positive rates . However, newer methods are being developed that avoid frequently violated assumptions and reduce false positives . The approach preferred by recent studies is to directly assess the association of loci with environmental gradients ,hydroponic nft channel whilst controlling for population structure . JaramilloCorrea et al. examined the correlation of P. pinaster candidate gene SNP allele frequencies with climate principal component axes, using transcriptome-wide SNPs to control for population structure and demographic history. They identified 18 environmentally associated SNPs, many of which were in genes relating to carbohydrate transport, cell wall construction and photosynthesis. Two surveys of P. taeda examined associations between candidate gene SNPs and environmental gradients. One examined the association of these loci with five climatic PC axes , whereas the other used an aridity index for each county . There was some overlap in function between the loci identified. However, the studies disagreed on whether SNPs associated with climate also tended to be Fst outliers. G2E associations have been detected even over short geographical distances, suggesting that selection can drive local adaptation in the presence of high gene flow. Eckert et al. examined Pinus lambertiana populations around Lake Tahoe, an area of c. 35 9 65 km2 , and found 11 genes associated with environmental PCs reflecting differences in water availability. These included genes involved in carbohydrate metabolism and transport and response to biotic stress . A cross-species comparison of environmental associations suggests some similarities in the genetic mechanisms involved in climatic tolerances across conifer genera. For each of four European conifer species in the Italian Alps, 6–18 SNPs were associated with precipitation/temperature PC axes . There was some overlap between species in the genes represented, including heat shock proteins, and cell wall construction and carbohydrate metabolism genes .Gene expression studies have identified a range of genes that may be involved in drought responses, but these results are not easily connected to the results of physiological or provenance response studies.

First, RNA transcripts reflect the genes being expressed at a particular instant, whereas morphological or physiological traits are the result of processes acting over a longer time. Second, most gene expression studies do not examine differences between populations. Although some evidence suggests that stronger gene expression changes during stress are associated with greater growth or survival, different genotypes and demographic stages can show significant differences in gene expression changes . A few studies have begun to address this. Provenances of P. pinaster differed in the expression response of two dehydrin genes, as well as in physiology and mortality rates . Similarly, three genotypes of P. taeda differed in their gene expression responses to drought and re-watering . More such studies are needed, but care must be taken to distinguish between drivers of expression differences. For instance, a more drought-sensitive tree might express higher levels of dehydrins at a given drought stage because the leaf water potential has dropped faster than in a drought-resistant tree, whereas the resistant tree might express higher levels of dehydrins than the sensitive tree at a given leaf water potential. Genome scan and G2E association studies can be useful tools in the search for genes responsible for local adaptation. Although such studies can identify loci at which allele frequencies differ between environments, it is not always clear how these differences are connected to phenotypic differences, and thus what traits are under selection in a given environment. This is where QTL and G2P association studies are useful.Most conifer QTL studies have focused on wood traits, growth or yield. Of the two that have examined drought tolerance, the first identified four significant and four suggestive QTLs for d13C in P. pinaster, none of which co-located with QTLs for ring width . The second examined a wider range of traits – photosynthesis , chlorophyll fluorescence, gs, d13C, intrinsic WUE and specific leaf area – in F1 cross seedlings of P. pinaster when well watered or after 1 or 2 wk without water, and identified 28 significant and 27 suggestive QTLs . Locations of the QTLs for each trait varied by time point. Candidate genes within the QTLs were identified : those for gs and WUEi included stomatal regulation, ABA signaling and cell wall construction genes; those for d13C included an aquaporin; and those for chlorophyll fluorescence included transcription factors and a histone chaperone. G2P studies focusing on quantitative traits have generally been successful in identifying associated loci . However, only a few studies have investigated drought tolerance in conifers , with less success. All such studies used d13C as the focal trait. As we argue in Section VI, other traits would probably yield results that are more helpful for the understanding of drought responses. Gonzalez-Martinez et al. examined 41 candidate stress response genes of P. taeda, using 61 tree families planted at two sites. However, drought stress was probably mild, and they only identified one strongly associated gene and one weakly associated gene at each site. A later study on the same species examining 3938 SNPs identified seven new associations with d13C . Four of the associations were with unknown proteins, with only a transcription factor probably involved in the ABA-mediated stress response having an obvious connection to drought responses. G2P and G2E association studies complement one another, with the first identifying loci linked to targeted traits, but not whether these loci are under selection in nature, and the second doing the opposite.

A better understanding of the transmission of HLB between the psyllids and citrus hosts in natural landscape is crucial to formulating effective control strategies. A spatially-explicit agent-based model, which simulates the actions and interactions of autonomous agents within the epidemiological system, has been developed to investigate how ACP and HLB spread in the Central Valley of California, an intermixed landscape of residential and commercial citrus. This study is a practical extension of the mathematical model with purposes to quantify the influence of input epidemiologic parameters on disease progress under sensitivity analysis, and investigate the efficiency of ACP/HLB management strategies by running scenario-based simulations. Although there are numerous hypothetical management strategies for testing, we focus more on survey design , and biological and chemical control strategies . In addition, we incorporated these management strategies into disease modelling with consideration of the social-economic perspective of citrus growers. Growers’ awareness of ACP/HLB and their attitude toward control strategies will change along with disease development, and we evaluate the set-points that yield optimal operation cost and sustainable control using a cost-benefit analysis. Our results indicate that climate change can have a large effect on the performance and spatiotemporal distribution of ACP and biological control agent populations in central California. ACP spread occurs more frequently and faster within commercial citrus clusters, but comparatively slower for low density or well separated residential areas. For different spray strategy scenarios,vertical rack system a comparison between simulation outputs confirms that the synchronize rate for coordinated spray plays an important role in slowing ACP epidemic development.

The Asian citrus psyllid vectors causal pathogens of huanglongbing or citrus greening disease. Its control is critical in all habitats including organic citrus grown in the United States and other regions of the world. Three separate organic programs, organic insecticides applied alone or with horticultural mineral oil and insecticidal soap were compared with one conventional program for impact on ACP and beneficial insects including release of nymphal parasitoid Tamarixia radiata in bearing citrus in southwest Florida. During the dormant winter season, Pyganic applied alone or with 435 oil or M-pede applied in November, December and January and Danitol applied in November and January all provided significant reduction in ACP with residual lasting longer for Danitol.Organic programs 2 and 3 rotated organic insecticides with 435 oil or M-pede resulting in 50% reduction in use of insecticides while providing better ACP control than program 1 with organic insecticides only. However, ACP was reduced more in the conventional program. Tamarixia radiata, ectoparasitoid of ACP nymphs was released in all programs but recovered more from ACP nymphs in the organic program compared to the conventional program. Lacewings, spiders, ants and ladybeetles were observed in all programs that also may have contributed to ACP reduction. Best yields were obtained in programs using organic insecticides with 435 oil or conventional insecticides. Monthly applications of Pyganic with 435 oil during dormant winter period and rotation of organic insecticides with oil during growing season appear to be reasonable options for ACP management in organic citrus. Organic insecticides will also be suitable for conventional citrus growers as selective options to avoid excessive use of non-selective insecticides, to limit pesticide resistance and harm to beneficial insects.

Candidatus Liberibacter asiaticus colonizes the citrus phloem and causes the most destructive citrus disease. Understanding how the bacterium interacts with citrus is important to improve or devise new methods of disease control. The objective of this work was to assess Las movement in citrus plants, from the initial infection site to the root, which is the part of the tree that apparently first suffers after pathogen infection. Experiments were carried out using 208 three-year-old, 60-cm high, potted Valencia/Volkamer lemon. The plants were grafted at the stem 60 cm above the substrate surface with 3- to 4-cm-long buds from Las-positive branches, and assessed over time at different distances from the inoculation site to determine the presence of the bacterium. Two evaluation procedures were used in three experiments carried out in two time periods. In the first, the stem of the inoculated plants were pruned sequentially at 10, 20, 30, and 40 cm below the inoculation site at 14, 21, 28, 35, and 42 DPI. The new leaves that developed at the top of the remaining stems were assessed regularly for HLB symptoms and through qPCR at six months post inoculation. This procedure was used in two experiments carried out from August 2015 to January 2016 and from March to August 2016 . In the second procedure, fibrous roots and one-centimeter long bark rings were removed just below the inoculation site and, at 10-cm intervals from it, up to near the soil level. The evaluations were made at 7, 14, 21, 28, 35, 42, and 49 days post inoculation , with the samples processed and analyzed through qPCR. This procedure was used in just one experiment carried out from March to April 2016. The fastest Las speed were 1.14, 1.43, and 2.02 cm per day for E1, E2, and E3, respectively. The frequencies of the evaluated sites positive for Las, at all evaluation dates, were then subjected to regression analysis. The logistic model better described Las distribution in the plants over time.

The probability of detecting Las at any portion of the plant was a function of time and distance from the inoculum. The longer the time between the inoculation and sampling dates and the shorter the distance from the inoculum, at any given time, the higher the probability of testing positive. Extrapolations made using the regression model allowed estimations of minimum 51 and 131 days for Las to reach the root of 2- and 7-m high citrus trees from a single inoculation site at the top of the tree. This fast movement explains the difficulties of curing an HLB affected tree and reinforces the importance of the implementation of areawide preventive actions to control the disease in citrus orchards. One of the long-term solutions for cultivation of citrus in presence of HLB may be development of disease resistant cultivars. In the genus Citrus, resistance to HLB is not known although some tolerant varieties have been reported. We have identified resistance and significant field tolerance in many citrus relative genera native to Australia. Field and greenhouse studies indicated that many naturally occurring hybrids of citrus and Eremocitrus/Microcitrus are resistant to HLB. Since the HLB resistance in the Australian types appears to be heritable, we have generated hundreds of citrus hybrids by crossing Citrus and Poncirus with disease resistant/tolerant, sexually compatible Australian citrus relatives. The hybrid plants are challenged by exposure to psyllids carrying HLB-associated Liberibacter pathogen. Preliminary evaluations of the hybrids in green house experiments conducted in Fort Pierce, FL indicate that a significant number of the novel hybrids appear to be disease resistant. Further testing to confirm HLB resistance is in progress. The promising hybrids will be useful in understanding the basis of HLB resistance and in developing methods to impart resistance to commercial citrus cultivars. The Asian citrus psyllid is the insect vector responsible for the worldwide spread of ‘Candidatus Liberibacter asiaticus’ , the bacterial pathogen associated with citrus greening disease. Developmental changes in the insect vector impact pathogen transmission,mobile grow rack such that D. citri transmission of CLas is more efficient when bacteria are acquired by nymphs as compared to adults. We hypothesize that expression changes in the D. citri immune system, including the insect’s commensal microbiota, occur during development and regulate vector competency. In support of this hypothesis, more proteins, with greater fold changes, were differentially expressed in response to CLas in adults as compared to nymphs, including insect proteins involved in bacterial adhesion and immunity. Discovery of protein interaction networks has broad applicability in the study of hostmicrobe relationships. Using Protein Interaction Reporter technology, we show how protein interaction networks in D. citri are regulated during development and in response to CLas-infected citrus trees. Notably, a hemocyanin protein highly upregulated in response to CLas was found to physically interact with the CLas coenzyme A biosynthesis enzyme phosphopantothenoylcysteine synthetase/decarboxylase. In addition, hemocyanin was found to physically interact with several other D. citri signaling and stress response proteins.

Co-evolved protein interaction networks at the host-microbe interface are highly specific targets for controlling the insect vector responsible for the spread of citrus greening.Huanglongbing , also known as citrus greening, is one of the most destructive citrus diseases worldwide and is seen as a major threat to the multimillion dollar citrus industry in California. The vector of two of the bacterial species associated with this disease, Candidatus Liberibacter asiaticus and Ca. L. americanus , is the Asian citrus psyllid , Diaphorina citri. In August 2008, the first ACP in California was found in a trap located in San Diego County. The detection triggered an ongoing state wide risk-based survey and HLB testing program for ACP and HLB host plants in California. Since 2008, a total of 135,000 citrus trees and 252,000 ACP samples have been tested so far by the California Department of Food and Agriculture using the United States Department of Agriculture HLB Work Instruction which utilizes a TaqMan real-time PCR assay based on 16S rDNA primers and probe developed by Li et al. . After eight years of challenging Li’s 16S HLB primers with hundreds of thousands of environmental samples, we have found that Li’s primers are highly specific but on rare occasions will cross amplify other closely related bacteria such as Ca. L. crescens . Lcr has never been reported in citrus before, and has only been reported in Babaco papaya during a survey for Papaya Bunchytop Disease in Puerto Rico. Currently there are six recognized species of Candidatus Liberibacter, four of which are pathogenic and two that have not been shown to cause symptoms in plants. Lcr falls in the latter category and is also the only member of this group that has successfully been cultured in artificial media. To date, Lcr has been confirmed in six citrus samples collected in three different counties in California and one from Hawaii. The detections were made during routine HLB testing in which the samples produced Fam Ct values between 33-36. A 1,149 bp fragment of the 16S ribosomal RNA gene was amplified from the DNA extracts using conventional PCR with primers Ol1 and OI2c. The sequence showed 99% identity with the corresponding regions of Ca. L. crescens strain BT-1. All samples had the same two single nucleotide polymorphisms differing with positions characteristic to Lcr. This is the first report of Lcr in citrus. Follow up sampling and testing of the CA trees confirmed all were negative for the HLB associated bacterium. Only one tree was retested for Lcr and was confirmed positive. Plans to retest the other trees for Lcr are underway. We evaluated the percentage of psyllid population with Las and its correlation with insect population captured in sticky cards and the HLB management in the property over a year. Assessment was carried out in four citrus regions in São Paulo state according to the Fundecitrus Phytosanitary Alert System. Cards were installed, read and replaced every two weeks on the period of February 2014 to February 2015 in the regions of Avaré, Santa Cruz do Rio Pardo , Araraquara and Bebedouro . In each reading, up to 50 psyllids per regions were collected and detection of insects Las+ was made by qPCR. Bi-monthly averages of the percentage of psyllids Las+ were compared. Santa Cruz do Rio Pardo region had 71% of Las+ samples and differed from Bebedouro region that had 56%. Avaré and Araraquara regions had 68 and 66%, respectively. The number of psyllids increased gradually between July and August in Avaré and Santa Cruz regions, from September to October in Araraquara region and intermittently from October in Bebedouro region. Potentially infective psyllid was higher in early spring in Avaré and Santa Cruz, with the largest population; Araraquara were higher starting in October, peaked in December ; Bebedouro region reaches the highest values in January and February . Percentage of Las+ psyllids collected in properties without HLB management were higher and statistically different from the averages on the properties that adopted psyllid control. The relationship between the percentage of psyllids Las+ and the number of captured psyllids in the region in a given time denotes the most critical time of year that psyllids Las+ intake in the orchards can occur.

We have used genome sequence data to identify epitopes on the surface of CaLas. Genes that encode these epitopes have been cloned in E. coli, and the proteins expressed, purified and used to immunize rabbits. The major outer membrane protein A has been used as the basis of a immune tissue print assay for CaLas. A problem for assays of CaLas isthe well known erratic distribution of the pathogen in infected citrus trees. DNA based detection methods require extraction and purification of the DNA prior to the assay. The immune tissue print assays are done directly on plant material and preserve and record the distribution of the pathogen with resolution of single infected phloem cells. Using this method, the pathogen has been readily detected in all tissues of infected sweet orange and lemon trees, a portion of the leaf midrib has been identified as ideal site for sampling. The tissue print assay also scales well to many samples. The rabbit polyclonal antibody has also been used to purify CaLas cells from plant extracts prior to PCR. Immune tissue prints and standard qPCR assays have been carried out on the same samples and are complementary. When both immune tissue prints and qPCR assays are used, the combined data show instances where each assay records a false negative, but together nearly all infected samples are detected.Huanglongbing is a devastating bacterial disease of citrus which causes yield loss, tree decline and reduces fruit and juice quality. HLB is caused by Candidatus Liberibacter asiaticus and transmitted by the Asian citrus psyllid.Many horticultural management practices have evolved to manage HLB infection such as pest management practices,nft growing system the establishment of Citrus Health Management Areas and addition of bactericides. There has been much investment by many funding agencies in many research topics to find solutions to HLB, including plant breeding and biotechnology.

Plant improvement to develop HLB resistant or tolerant root stocks and scions through conventional or biotechnology is the most likely to provide sustainable solutions to HLB. The goal of Citrus Research Development Foundation is to support research and to deliver HLB solutions to growers as quickly as possible. There are several plant improvement research programs in the US using conventional and biotech methods. In Florida, there are researchers at the University of Florida and USDA-ARS research departments comprised of several programs and many researchers. Due to the diversity of programs, program objectives, approaches to research and evaluation, it is difficult to understand the scope and progress of post HLB research and the current status of projects. CRDF is investing in efforts to work with researchers to develop a common development, evaluation, and deployment platform to accelerate commercial release of economically viable HLB resistant or tolerant root stocks and scions.The Asian citrus psyllid Diaphorina citri Kuwayama is the insect vector of the fastidious plant pathogen bacterium Candidatus Liberibacter asiaticus , the causal agent of citrus greening disease, also known as Huanglongbing which is the most significant and widespread threat to the citrus industry. To investigate gene expression profiles that associate with ACP- CLas interactions and identify genes in response to CLas infection, we constructed RNA-seq libraries from CLas-infected and CLas-free ACP samples of three different developmental stages . With 150 bp paired-end sequencing on the Illumina Hiseq2500, we generated 152 Gb of sequence data from 56 million reads per library/replicate, which was assembled into 34,122 contigs with 18,827 being annotated, which were then further analyzed for potential functional classification and potential roles in infection. The results suggested that gene expression in different developmental stages did not respond in the same manner to CLas infection. With more contigs being up or down- regulated, nymphal instars 4-5 showed a more sensitive response to CLas infection than nymphal instars 1-3 and adults. A comprehensive analysis of the transcriptomes revealed vector life stage differences and differential gene expression in response to CLas infection, and identified specific genes with roles in nutrition, development, immune response and transmission pathways.

In Florida approval was granted for field application to citrus trees of formulations of streptomycin and oxytetracycline to control infections by ‘Ca. Liberibacter asiaticus’ and the subsequent development of the associated disease, huanglongbing . We developed a procedure for infecting individual trees by grafting a single symptomatic leaf to the tree. We wanted to determine if this procedure could serve as a small scale assay system for testing conventional antibiotics and other compounds for antimicrobial activity effective against ‘Ca. L. asiaticus’. The working hypothesis behind the assay was that the application of the candidate compound to the inoculum leaf before or after grafting would impede or prevent bacteria in the inoculum leaf from infecting the receptor tree. Aqueous solutions of oxytetracycline or streptomycin, containing either glycerol or the commercial compound Pentra-Bark to aid penetration were applied under 20 psi of pressure to both the adaxial and abaxial surfaces of individual symptomatic leaves before or after grafting the leaf to a receptor tree. Receptor trees were inspected periodically for symptoms and after five to seven months leaf samples were evaluated for infection using real time PCR to detect pathogen DNA. The cumulative results from six trials found 44/60 , 46/75 and 53/80 of trees infected by grafted leaves treated with oxytetracycline, streptomycin or no treatment, respectively. The data suggest that under the conditions of this study, these two compounds failed to impede infection of the receptor trees relative to non-treated controls. The reasons for the lack of success in preventing infection are unknown but could be related to either the effectiveness of the compounds or the method of application, or both. The Asian citrus psyllid is the insect vector of the bacterium Candidatus Liberibacter asiaticus , the causal agent for the citrus greening or Huanglongbing disease which threatens citrus species worldwide. This vector is the primary target of approaches to stop the spread of the pathogen. Accurate structural and functional annotation of the psyllid’s gene models and understanding its interactions with the pathogenic bacterium, CLas, is required for precise targeting using molecular methods. The draft genome was annotated with automated pipelines.

Knowledge transfer from well-curated reference genomes like Drosophila to a newly sequenced insect is challenging due to the diversity and complexity among all insect genomes. We opted for manual curation of gene families that have key functional roles in D. citri biology and pathology. The community effort produced more than 450 manually curated gene models across developmental, RNAi regulatory, and immune-related pathways. Curators included undergraduate and graduate students from multiple institutions as well as experienced expert annotators from the i5k community. Here we report the official gene set for the Asian citrus psyllid genome that includes manually curated genes involved in pathways of experimental interest for vector control strategies. The Asian citrus psyllid, Diaphorina citri is the insect vector of ‘Candidatus Liberibacter asiaticus’ ,nft hydroponic system the causal agent of HLB. Proteomic characterization of D. citri identified hemocyanin as central to the insect’s response to CLas-infected trees. Hemocyanin expression is highly induced psyllids reared on CLas-infected Citrus medica Linn. and has been found to physically interact with a CLas vitamin A biosynthetic enzyme. Hemocyanins function as respiratory proteins of mollusks and arthropods with conserved histidine residues forming a coordination complex with copper ions binding oxygen for transport in the hemolymph. Hemocyanins have evolved from oxygen-binding tyrosinase enzymes, which participate in melanin biosynthesis and have documented roles in insect defense, including parasite encapsulation. This study evaluated the role that hemocyanin plays in the insect response to CLas infection. Specific dsRNA to hemocyanin in D. citri were designed and delivered via artificial diet feeding. Effects of hemocyanin suppression on CLas transmission were evaluated using detached leaf transmission assays. The levels of hemocyanin mRNA expression were highly correlated with the different D. citri color morphs and thus, we hypothesized that the blue abdominal color commonly observed in D. citri may be attributed to the oxygen coordinating copper associated with hemocyanin in the insect. Insight into the function of D. citri hemocyanin may reveal novel strategies to specifically disrupt CLas transmission by the insect vector. The commonly employed technique for monitoring the Asian citrus psyllid utilizes yellow cards covered with glue. These have several disadvantages: the sticky surface is messy and indiscriminate for other insects or debris trapped, it is difficult to remove intact specimens for complete identification, and insects are not well preserved for tissue analysis. An alternative design that can preferentially attract ACP while avoiding the sticky substance, while also preserving the specimens for complete analysis, is therefore desirable. This project enhances the mitigation of citrus greening through early detection of huanglongbing through improved trapping technologies.

Trapping of ACP, with “smart” traps that make it possible to accurately test for the presence and spread of the HLB pathogen, Candidatus liberibacter spp., facilitates the rapid site management response to enable delimitation and potential containment of citrus greening in regions where the disease is not widespread. The first attempt at psyllid trap design was a simple, color-attractive, yellow cylinder that eliminated the sticky glue, but the catch yield was consistently lower than that of the sticky trap, even when tested inside a psyllid rearing facility where the target population was high. Since making manual tweaks to our design quickly became both labor and time intensive, we decided to make the various physical modifications to our prototype by utilizing computer modeling software coupled to a 3D printer. This approach allows us to virtually model the design before testing, and then we can print viable traps within a single day, without the time needed to purchase and assemble the components derived from various sources; this removes a major impediment to rapid innovation. The modeling software can be used to make iterative tweaks to successive designs or to immediately create entirely new models, and the designs are limited only by the capabilities of the 3D printer. The printers deposit heated plastic into intricate designs, a method that is not possible using injection molding techniques, so the shapes of our traps can include projections and voids in strategic places to exploit the natural behaviors of citrus psyllids. Trap designs can easily be developed within a single day, allowing modifications and adjustments to be made in real time as new field data is acquired. Our improved “SmartTrap” designs can favor the capture of certain insects only, such that >50% of the insects collected would be psyllids. The trap base also contains a preservative liquid that allows those captured psyllids to be analyzed by standard molecular techniques. This enables researchers to better study the vector of citrus greening, therefore leading to improvements in control and prevention methods that may be more effective than the expensive, broad-spectrum pesticide sprays. The yellow trap color may be an adequate psyllid attractant for sticky traps, but our group is experimenting with slight color variations of different types of plastics and LED lights, incorporating the UV wavelengths that have been identified as psyllid-specific attractants. The concept of our latest design is to attract ACP to colored plastics and LEDs, where they will land on the primary cylindrical body of the trap. As they naturally crawl upward on rubberized plastic projections, designed to resemble the tender new growth of citrus stems, they will pass under the shade of an opaque overhang, where they will then crawl toward a view of sunlight that appears to be coming through the holes in the trap body, protected from the elements by a clear umbrella dome. Once inside the trap they will remain inside, preferring not to reenter the holes which appear dark from the interior perspective. They then fall down a slippery, fluon-lined funnel and into a preservative-filled test tube, which can be removed for inspection without dismantling the trap itself. Other researchers have identified potential chemical and auditory attractants for psyllids, and these features can easily be incorporated into a single trap design, made possible by 3D print technology.This presentation will show our latest designs and provide commentary on our observations.

HLB-affected Valencia fruit together with healthy fruit, were harvested in the middle of the harvest season, and peel oil extracted from flavedo tissues using methylene chloride/pentane solvents. Volatile compounds were analyzed by GC-MS. All HLB-affected fruit produced a lower volume of peel oil compared to healthy fruit, however, only green fruit showed substantially different volatile profiles from healthy fruit. The green fruit contained low concentrations of most of sesquiterpene hydrocarbons and derivatives, such as, nootkatone, valencene, α-selinene, 7-epi-α-selinene and caryophyllene oxide, some monoterpene hydrocarbons and derivatives with orange/citrus characteristics, such as, limonene, myrcene, α-pinene, neral, geranial, and α-terpineol, and straight-chain aldehydes, such as, octanal, nonanal and decanal. Eight compounds were determined in the green fruit peel which were not present, or present in very minor quantities, in the healthy fruit. Their retention indices on a DB-5 column were RI 1137, 1144, 1149, 1189, 1320, 1334, 1346 and 1387. The preliminary identifications based on MS and the NIST database RI comparisons were -p-mentha-2,8-dien-1-ol, 1,8-p-menthadien-2-ol, 3-[-3- methyl-1-butenyl]-cyclohexene, 6-methyl-bicyclo[3.3.0]oct-2-en-7-one, p-mentha-2,8-diene-1-hydroperoxide, an unknown, p-menth-8-en-2-ol and p-mentha-[1,8]-diene 2-hydroperoxide, respectively. Other compounds in HLB-affected green fruit were cis-3-hexenol, cispinocarveol, carvone, and α-cubebene. The large scale surveys for detecting Asian citrus psyllids and HLB in Texas and California are on-going. We continue to analyze the spatial pattern of the diagnostic Ct-values from psyllid samples and our goal is to help focus the survey effort for HLB infected citrus trees. The realtime polymerase chain reaction diagnostic methods used to detect HLB are set to run for 40 cycles. The reaction must surpass a threshold of ≤ 32 prior to the completion of the run to be considered positive for HLB. Currently,growing hydroponically reactions that surpass the threshold at 33 or more cycles have proven impossible to acquire a confirmatory PCR bands and DNA sequence data and are considered inconclusive.

The question we are trying to answer is whether there is additional information in the spatial pattern of inconclusive psyllid samples for predicting locations with HLB infected trees. Based on analysis of the spatial pattern of psyllid Ct-values proximate to HLB positive tree detections in both Texas and California, there appears to be a biological process underlying indeterminate Ct-values in the range of 32 – 38. Analyses indicate significant clustering of indeterminate Ct-values of psyllid samples, and some clusters in both Texas and California were around known positive HLB trees. The San Gabriel area infections in California were found after more intensive survey around an inconclusive psyllid sample. Analyses have also revealed significant space-time scales of pattern in the California psyllid data, such that inconclusive samples tend to cluster within 1.5 miles and 15 months of each other. A secondary cluster was found for inconclusive samples > 4.5 miles and > 3 years from the first detection in Hacienda Heights. These analyses may serve as early warning indicators for HLB positive trees, as well as informing dynamic spread models in development. A long period of data about the occurrence of HLB in commercial citrus farms located in São Paulo state, Brazil, was used to evaluate the disease progress and its management. Approximately 260 Sweet orange plots from five farms , naturally affected by HLB and under strict disease management , were evaluated considering the proportion of HLB-affected trees, occurrence of Diaphorina citri, plot location, and presence of neighboring commercial and non-commercial citrus plants. The logistic model better explained, than Gompertz, the yearly proportion of HLB-affected trees. The cumulative disease incidence varied from <4.0% to 80% and highly correlated with area under disease progress curve, independently of plant age, but not with the disease rate of progress . Adults of D. citri were poorly detected by shoot evaluation conducted 2 to 5 times/month in 1% of trees in each plot. In contrast, higher frequency of D. citri identified in yellow stick cards occurred in the most HLB-affected plots.

However, the majority of yellow cards presented no adults for most of the time. Spatial heterogeneity was observed for both HLB-tree incidence and D. citri detection . In all farms, a strong concentration of HLB-affected trees occurred in some plots located in their borders.In the majority of plots, especially those presenting higher disease incidences, >95% of HLB-trees occurred up to 200 meters from their borders. The disease incidence in each farm varied from 7.1% to 25.9%, though the similar disease management adopted for HLB . Recently, surveys around those farms have been started to identify neighboring citrus and Murraya sp. plants that could may act as HLB-sources of inoculum. Their location, represented by commercial and non-commercial citrus plants, strongly suggests that non-commercial trees are very effective sources of HLB, even when represented by a few plants. The Asian citrus psyllid Diaphorina citri Kuwayama is the insect vector of Candidatus Liberibacter asiaticus , the causative agent of the citrus greening disease. Understanding the interactions between the insect vector and the bacterial pathogen is essential for the development of efficient strategies aimed at limiting the spread of the infection and inhibition of bacterial growth. Furthermore, better knowledge of insect-pathogen interaction at the metabolite level will help develop in vitro culture of CLas, a much needed tool for studying its metabolism and designing means to combat it. In this study, we compared the primary metabolite and lipid profiles between CLasinfected and healthy psyllids using GC-TOF-MS and LC-TOF-MS, respectively. We found that both feeding on infected trees and the actual infection with CLas cause dramatic changes in monitored compounds’ accumulation. The results of metabolomic analyses and the possible implications for the pathogen-vector interactions will be presented and discussed. Huanglongbing is a devastating disease of citrus caused by the Candidatus Liberibacter asiaticus . The inability to sustainably culture and reinoculate Las into any host has prevented functional genomics and impeded research and development of disease control methods.

Phosphatidylcholine has recently been established as an important membrane component of those few bacterial species that associate with eukaryotic membranes, such as Legionella pneumophila, Agrobacterium tumefaciens and Rhizobium leguminosarum. For many of these species, PC is required for virulence. Las colonizes living citrus phloem cells, and infects psyllid hosts systemically, moving both intraand inter-cellularly, crossing through multiple membrane layers. We hypothesize that Las PC is similarly required for colonization of psyllids. Liberibacter crescens , the only cultured Liberibacter to date, is a useful and genetically tractable proxy for functional genomics of Las. It carries two predicted pathways for synthesis of PC: 1) import of exogenous choline and subsequent conversion of choline to PC utilizing phosphatidylcholine synthase , and 2) de novo synthesis of PC utilizing phospholipid N-methyltransferase . Notably, Las is missing the de novo pathway, and must import choline,flood and drain table which is abundantly present in psyllid hemolymph and phloem sap, to make PC. We created individual marker interruption mutations of both Pcs and Pmt in Lcr. Rescue of the Pmt mutant in Lcr required 0.1% choline supplementation in the medium. The standard culture medium for Lcr is BM7 which contains 6X10-6 % choline, an amount that appears to be insufficient for culturing Las. Other genes found in Lcr but not found in Las and that should significantly limit Las culturability are: glyoxylase I and the ATP/ADP translocase. Phage lytic cycle genes found in Las but missing from Lcr also likely significantly limit Las culturability. Despite the availability of multiple complete genomic DNA sequences of Candidatus Liberibacter asiaticus , inability to sustainably culture, manipulate or re-inoculate Las has seriously impeded the identification of virulence factors and subsequent development of disease control methods. Liberibacter crescens , is a culturable proxy for Las, but is not pathogenic. Given that the Las genome is even more reduced than Lcr and several Lcr genes are suspected of contributing to its culturability, one long term goal of our lab is to sequentially delete multiple Lcr genes not found in Las and to make its morphology and metabolism more comparable to Las, and make informed incremental adjustments in the growth medium to help compensate for the loss of the Lcr functions. To date, the best growth of Lcr has been obtained in the complex growth medium BM7, containing fetal bovine serum , ACES, α-ketoglutarate, and TNM-FH insect medium. We have found that replacing FBS with 1 mg/mL of methyl-β- cyclodextrin reduced growth, even though such replacement has been reported to improve culture of the fastidious pathogenicmicrobes Coxiella burnetii. In addition, both BSA and glucose, major components of FBS, inhibited growth of Lcr. Both MβCD and BSA are hypothesized to inhibit Lcr growth by sequestering fatty acids and other small hydrophobic molecules that are important to Lcr growth. Lcr growth was unaffected by the presence of cholesterol in the medium.

Replacing TNM-FH with Schneider’s insect medium resulted in removal of sucrose and marginally improved growth of Lcr. Eliminating sucrose from the culture medium allows use of conditionally lethal DNA cloning vectors such as pUFR080 carrying sacB that can be forcibly and reliably evicted by adding sucrose to growth media. Such a system is important to make sequential mutations. We report here that Lcr is able to import isotopic sucrose in a time- and dose-dependent manner, enabling use of the sacB eviction system. We have successfully created 2 marker interruption mutations in Lcr that make Lcr more Las-like and that will likely inform culture media components. Other genes found in Lcr but not found in Las and that should significantly limit Las culturability are: glyoxylase I; ATP/ADP translocase and the sucrose transporter. We are currently attempting a series of conditional knockout mutations involving a sacB vector with genes suspected of enabling Lcr growth in media, each time evaluating different rescue strategies to keep the resulting mutant strains alive. Fruit from trees with severe symptoms of Huanglongbing produce juice that is described as less sweet, more sour with some bitterness and harshness together with typical off-flavor in comparison with juice made with fruit from healthy trees. Commercial juice processing includes separation and recovery of peel oil from the fruit, which is then used in flavor manufacturing including add-back to orange juice to standardize flavor. To this date, there is no published data on the aroma quality of peel oil from fruit affected by HLB. Hamlin and Valencia fruit from healthy and HLB-infected trees were harvested in December 2013 and April 2014, respectively. Fruit were washed and juiced using a JBT industrial extractor. Peel was separated and the oil was expressed using several cycles of manual cold pressing followed with centrifugation. Peel oil was then analyzed by gas chromatography mass spectrometry and olfactometry , as well as presented to a trained taste panel for aroma differences. Juice from the same fruit were also presented to the trained taste panel for sensory descriptive analysis. For peel oil from Hamlin, 14 compounds had higher peaks by GC-MS in healthy samples, including 1-octanol which was perceived by GC-O. Further, three unidentified peaks with mint , citrus and pungent odors were perceived only in the oil from healthy fruit by GC-O. For Valencia oil, differences by GC-O were for peaks with odors of -cadinene and metallic which were only perceived in HLB samples. Other differences were for RI=955 , α-phellandrene , and terpinolene , which had higher intensities in Valencia healthy samples. Overall, those differences detected by GC-O were not translated to aroma differences between oil samples by a trained panel. The same trained panel found differences when tasting the juice of Hamlin, with HLB juice having more ‘typical HLB flavor’, ’bitter’, ‘metallic’, ‘tingling’ and ‘astringent’, likely due to differences in nonvolatile compounds. There were no difference between HLB and healthy samples for Valencia juice. Some new hybrid root stocks grafted with commercial citrus scion cultivars produce trees with reduced huanglongbing symptoms and higher fruit production over a period of many years, following infection by Candidatus Liberibacter asiaticus , as compared with trees on other common commercial root stocks in the same trials. In one replicated trial with ‘Valencia’ scion severely affected by HLB, highest yields over five consecutive harvests were with ‘US-942’ and ‘US-1516’ root stocks. Other components of tree health, including fruit quality, tree growth, and tree survival, differed greatly among the root stocks, and provided additional evidence of improved field tolerance to HLB for some root stocks.

Previous research had found that “supermarket flight” in fact traced back to the 1950s and 1960s, when retail suburbanized with housing and the grocery industry began a gradual process of consolidation to remain competitive . By the 1980s, Reagan-era deregulation relaxed previous corporate antitrust policies. This would encourage the trend towards larger, fewer supermarkets, each of which gained larger shares of grocery sales over time—an benefit to stockholders but not necessarily consumers . Between 1979 and 1989, 16 of the nation’s top 20 grocery retailers experienced mergers and leveraged buyouts, resulting in a complete reorganization of the grocery industry . Furthermore, between 1970 and 1990, half of the supermarkets in the three largest U.S. cities closed . The U.S. Mayor’s Conference report reinforced this findings emphasizing the implications for these industry wide changes on local communities and vouching for the creation of new policies to ‘end the redlining of urban America by the supermarket industry’ . As a result, the report drew a strong response from the national media, trade associations, grocery retailers, and the federal government. The news media featured the findings of the report as well as the beginnings of coordinated local responses by local entrepreneurs. Despite disputing the rhetoric of the report, the Food Marketing Institute created an Urban Initiatives Task Force designed to revitalize communities through the development of grocery stores and job creation.Regional retailers Lucky’s, Von’s,hydroponic growing system and Path mark Supermarket made new commitments to open stores in underserved areas mostly in the Pacific West and Northeast respectively .

The hearing featured statements from urban grocery experts, Ronald Cotterill and Mark Winne, residents of grocery-deficient communities, and notably, the work of the Monsignor William Lindor, executive director/founder of Newark’s New Community Corporation . A faith based CDC operating in Newark’s poor urban areas since 1968, NCC garnered widespread acclaim for its comprehensive scope of work including retail, housing, health centers, and social service programs initiatives for poor urban residents. Amongst its most notable achievements was its joint venture partnership with Path mark supermarket, among the first of larger supermarket chains to reenter urban areas by way of CDC partnerships. Together, these speakers highlighted several key trends among urban grocery stores that were beginning to be explored but had not yet been widely publicized. This included new evidence showing that poorer urban residents pay between 8 to 30% more for food each year than more affluent residents and spend an extra $400-$1000 per year to commute to supermarkets outside of their own communities due to limited vehicle ownership . Several ways forward were identified including: 1) more stringent corporate anti-trust policies that promote more competition within the grocery industry and Electronic Benefits Transfer systems ; 2) grocery shuttles for resident of low income communities ; 3) regulatory and tax incentives for urban grocery development ; 4) public private partnerships among entrepreneurs and community organizations ; and 5) municipal and federal policies to support the financing, development, and operations of new grocery stores, new funding streams to support inner city economic development . The hearing did not lead to immediate federal policy actions per se, but it generated political awareness of the visceral nature of disinvestment in poorer urban communities as exemplified by access to grocery stores. Additionally, it reinforced the critical role of CDCs as key drivers of economic development, even in spite of dwindling federal funding channels and limited organizational capacities for retail-oriented initiatives. Finally, while the hearing did not result in immediate federal policy actions around grocery stores, it would pave the way for a new era of federal revitalization policies advantageous to urban grocery development.

As such, the “urban grocery gap” was situated within broader discussions on how best to leverage the private sector, the burgeoning “community development industry” , and government to systemically address urban poverty and disinvestment. In line with the scope and intent of Clinton-era federal urban revitalization policies, scholars of business, economic development, and community planning looked to these CDC experiments as further confirmation of the need for modes of “inner city revitalization.” For example, Bendick and Egan argued for mutual integration of community development and business development to more systemically address the symptoms of urban poverty. Namely, community development activities can become a boon to business by expanding the pool of potential employees, improving service delivery, rehabilitating real estate. And yet, business alone may not impart the trickle down social impacts, which may be best provided in the form of community development activities including social services, housing rehabilitation, and workforce development. Ultimately, business that serves community goals and vice versa, may be more effective in revitalizing poor communities than one singular approach. Bendick and Egan’s view reflects a longstanding divide between social and economic goals in community development practice, despite integrated efforts through the Economic Opportunity Act of 1964 and subsequent practices by CDCs. And yet the gradual diminishing of funding for business-oriented activities by CDCs reflected the continued challenge to truly marry these largely disparate approaches to neighborhood revitalization. Bendick and Egan begin to suggest the business benefits brought on by community development, however pre-eminent business scholar, Michael Porter, perhaps made the most influential argument for business development in the “inner city.” In his seminal article, “The Competitive Advantage of the Inner City,” Porter argued that entitlements and social investments alone may not mitigate urban poverty. Rather, inner cities must create wealth in order to redistribute wealth through “private, for profit-initiatives and investments based on economic self-interest and competitive advantage” . Furthermore, contrary to perceptions, inner cities are poised for successful economic development given their locational benefits, untapped market demands, an available workforce, and potential for regional economic development.

In a follow-up piece, Porter highlighted CDC-supermarket partnerships as models of how “the private sector is already waking up to the potential of inner cities” and how “entrepreneurs are creating businesses that cater to the distinct needs of inner city consumers” . Critics have argued that Porter’s model oversimplifies the solution to urban poverty , understates the role of minority-owned and locally-owned businesses in economic revitalization , and discounts the role of government and community development corporations in encouraging private investment .Yet Porter has been credited for widening ongoing discussions about inner city revitalization to encompass the question of retail. Porter put forth his proposal amidst growing research on the magnitude of urban grocery and retail gaps and continued suburban retail expansion due to available land, lower development costs, looser zoning laws, and higher purchasing power . Still Porter elicited a strong response from grocery retailers in particular,mobile vertical grow tables who began reentering poorer under served communities in the early 1990s in part due to new federal incentive programs . Although federal programs specifically focused on grocery stores would emerge in the 2010s, the Clinton Administration initiated a string of federal urban revitalization programs that would provide preliminary solutions to the urban grocery gap. The first of these programs was Empowerment Zone program, which aimed to combine the economic incentive structure of the statewide Enterprise Zones with workforce development and social services. Administered by U.S. Department of Housing and Urban Development since 1993, the program provides competitive block grants to local governments for collaborative economic and workforce development activities. Food security was part of a national policy concern since 1930s and 1940s with the instatement of the first Food Stamp program and the National School Lunch Act of 1946 . Although the initial goals of the Food Stamp program was primarily focused on hunger alleviation, some argue that it was focused more on assisting farmers by purchasing food surpluses and feed the poor . The School Lunch Act was premised in improving childhood health, when almost half of potential draftees were rejected due to physical ailments traceable to malnutrition. Essentially, access to nutritious and healthy food was part of national security agendas—whether keeping the U.S. self-sufficient in terms of its food needs or as a way to ensure the health of future citizens. And yet by the 1960s, hunger became a central national policy issue with growing medical research and journalistic accounts of childhood hunger in poor urban communities.The Kennedy administration reinstated the Food Stamp Program, which was later expanded to include a School Breakfast Program and what is now known as Women, Infants, and Children . In cities across the U.S., anti-hunger movements proliferated against the backdrop of the civil rights, free speech, and anti-war movements. Between 1969 and 1970, the Office of Economic Opportunity funded the first anti-hunger voluntary associations, including the Food Research and Action Center, based in Washington D.C. . Amidst continued local advocacy around childhood malnutrition and hunger, in December 1969, Nixon initiated his own “war on hunger” at a White House conference on Food, Nutrition, and Health noting, “the moment is at hand to put an end to hunger in America itself. For all time” . For the decade that followed, federal food programs expanded its focus on cash subsidies and vouchers—all premised in a “medical model” of hunger and malnutrition . However, the anti-hunger movement would shift more broadly towards “food security,” with growing research and evidence of the connections between socioeconomic status and hunger. During the early years of Reagan administration, social assistance programs experienced significant reductions amidst an economic recession . As a result, existing emergency food providers reported an abrupt rise in the use of their services by ‘the new poor,’ suddenly under-resourced children, youth, and families . Evaluations of food assistance recipients at the time further confirmed that the risk of hunger multiplied with increasing poverty and reduced food assistance benefits .

Amidst this crisis, the Reagan administration looked to private sector food banks, food pantries, and soup kitchens to meet increasing food demands. These circumstances elicited a strong response from anti-hunger, anti-poverty, and social justice activists who began promoting “community food security” policies in the late 1980s and 1990s. The culmination of these early efforts was the Community Food Security Coalition, a national network of activists promoting new provisions in the 1995 U.S. Farm Bill ensuring community food security: that ‘all persons obtaining at all times a cultural acceptable, nutritionally adequate diet through local non-emergency sources.” The proposed 1995 Community Food Security Empowerment Act called for reductions in traditional food assistance programs and instead the creation of a new competitive grants program for community food projects35 in low income, under served communities. Already prior to the Community Food Security Act, the U.S. had a long history of community-based and local government community garden programs , counterculture food movements involving food buying clubs and cooperatives , and community-driven food assistance programs . However, the Act was distinct in its focus integrating social equity, economic justice, environmental sustainability through community-based means . As of 1996, the Act was included in the Farm Bill, authorizing the USDA Community Food Projects Competitive Grants Program, which allocates $16 million over the course of seven years with guidance from the Community Food Security Coalition. Through a competitive application process, the USDA administers one to three-year matching grants ranging from $10,000-300,000. The community food security movement distinguished itself from the anti-hunger movement by focusing on community food needs through community food planning, direct marketing, community gardening, food assistance, farmland protection, food retail strategies, and associated community and economic development . Domestically, this initial scholarship inspired hundreds of ‘food desert’ studies since 2000, ranging from academic studies, government-sponsored assessments, health assessments, and GIS analyses . These studies tend to use different definitions of what food deserts are and entail- some tend to define food deserts in terms of the number of grocery stores or lack thereof, while others focus on the type of quality of foods . The most widely cited early study on food deserts emphasized the connections between food access, wealth, and racial make-up of neighborhoods, noting that supermarkets abound in higher income areas while convenience and fast food stores dominate in low-income areas . Subsequent studies found similar trends regarding the distribution of stores and their impacts: poorer residents tend to pay 3-37% more for food items at small stores compared to residents shopping at suburban supermarkets and available food is often processed and of poor quality . Residents living in areas without a grocery store or supermarket tend to be of ethnic minority groups and with higher rates of obesity, diabetes, and diet-related diseases . In light of these findings, researchers continued to suggest that increase access to supermarkets might lower prevalence of obesity, improved fruit and vegetable consumption, and better diet quality among African Americans, low income households, and pregnant women .

The availability of BC as a secondary product of bio-energy production and/or waste stream management , as well as lower transportation costs made possible by regional or on-site BC production, could further leverage economic advantages over peat and peat alternatives . Recent studies support the unique ability of BC to mediate biological interactions with benefits for greenhouse production such as enhanced pathogen and pest suppression. For example, 1–5% additions of citrus wood BC to peat-based substrates increased expression of pathogen defense genes in strawberry and as a result suppressed fungal disease ; for tomato and pepper , such additions delayed and reduced disease from fungal pathogens and mites . However, lower susceptibility of plants to pathogens in soil-free substrates with a BC component may be muted by fertilization , and therefore may not be possible under intensive greenhouse production. On the other hand, substrates with a high proportion of BC such as in this study could have detrimental effects on biological processes that support plant productivity , largely due to interference of chemical signals between beneficial microorganisms and host plants . As a result, BC could lessen establishment of rhizobial and mychorrizal associations and reduce nodulation in leguminous species . Strong sorption by BC could afford horticultural advantages, however. For example, bulbet organogenesis of grape hyacinth was enhanced with the use of BC-like material in substrates due to its sorption of inhibitory compounds . Potential plant health benefits of BC-based substrates are relatively under-investigated in evaluations of peat alternatives, despite one of the main uses of soil-free substrates being the avoidance of plant exposure to pathogens . Finally, the ability to replace peat with BC offers potential economic and environmental benefits. The expense of peat is expected to increase in the coming decades due to production costs, competing uses for peat, and its perception as being unsustainable . Such a perception in part stems from the negative impacts on wetland ecosystems of some peat mining operations , though the sustainability of peat harvesting is a subject of debate . Peat mining operations and the eventual decomposition of peat after its use in substrates represents a transformation of a terrestrial C sink of global importance into a net C source, with climate change forcing effects . Assuming a conservative aerobic decomposition rate for peat in substrates of 5% per annum ,vertical hydroponics within one century of mining and use in soil-free substrates 95% of mined peat would be expected to revert from a C sink to source .

In contrast, high-temperature BCs are thought to generally exhibit lower decomposition rates than undecomposed or humified biomass such as compost and peat and exhibit centennial to millennial residence times . The molar O:C = 0.36 for the BC in this study corresponds to a half-life of 100–1000 years , suggesting that one century after production and use in soil-free substrates, at least 50% of C in the softwood BC in this study would be converted to CO2. The use of non-peat biomass or even waste in the form of BC in soil-free substrates is an additional strategy for ‘sustainable bio-char to mitigate global climate change’ due to its greater stability and ability to preserve a key global C sink.In landscape, we form meaning through placefulness; ‘‘place’ places man in that dimension which reveals the revealing meaning of being’. Gardens imply a more accelerated and amplified rendition of this process, while the gardens that we personally make and dwell in further magnify this condition. The garden is in effect the most permanent communion we can make with a piece of the world, whether that patch is on traditional earth or elevated in an artificially constructed environment. Those who have been faced with moving from somewhere they have resided for a long time may confer that vacating the house is one issue, but leaving the associated garden is an altogether more fraught separation. The most fundamental biological fact that plants are rooted and sedentary—while we are not—is laid bare during this process. Accordingly, John Brinkerh off Jackson defines the landscape of place as ‘a space on the surface of the earth … with a degree of permanence’.Nonetheless, we are also remarkably adept and manufacturing meaning on the run. There are countless accounts of how travelers, explorers and refugees have rearranged their immediate surroundings to assemble meaning from the background void. Furthermore, this phenomenon is not restricted to those who move long distances by choice of profession or byproduct of circumstance, since modern urban dwellers also possess this capacity. As Ian Nairn notes, ‘people need to put down roots in a terribly short time’, himself taking ‘about forty-eight hours’. Nairn concludes that movement paradoxically amplifies the sense of place, observing that ports—while being highly fluid—are nevertheless very well defined places. The gardens in a garden show can be considered within this context. Such exhibits are not gardens with which the visitor grows and coinhabits with its meaning, but rather passes through en mass in a matter of minutes.

And not long after they are experienced, the gardens are either wholly deleted or at the very least downgraded to mere residual features in the landscape-park that typically inherits the site once the spectacle of the expo has concluded. Under these circumstances meaning is absorbed and place manufactured on the run. To apply a vegetal analogy, this is less a process of terrestrial rootedness than the ‘continuous-flow solution culture’ associated with hydroponics. This interplay between rootedness and mobility in the place making and meaning-construction of the individual in the garden prefigures a society-scale condition found within Modernity as a whole. That is, the tension between the rapidity of globalism and the romantic yeaning to resist-and-return as Paul Ricoeur describes: ‘how to become modern and return to the sources, how to revive an old dormant civilization and take part in a universal civilization’. While visible in the landscape generally, the tension of this ‘paradox of place’ is manifest most acutely in the garden. The ‘sense’ or ‘spirit of place’ is both a manifestation of this paradox and an attempt to resist or realign it.In the midst of a fast-tracked industrial-to-consumption revolution, this tension is patently visible in the rapidly urbanizing cultural landscape of China. Framed by these dialectics of transience versus groundedness and tradition versus modernity, I focus in this essay on a particular example of a phenomenon that has persevered throughout the West’s Modernity and has found new vigor in China’s; that of the garden show or horticultural exposition. Both an expression of the yearning for otherness within the totalizing fabric of Modernity and a product of the very global reach of Modernity, international garden shows are increasingly commandeered into the mega-events that are used to influence the fortunes of cities. Typically, in the vein of the World’s Fairs and indeed Olympic Games , installments of the World Horticultural Exposition have fulfilled this transformative role, involving themed extravaganzas underpinned by massive quantities of construction far beyond that which is required for the simple promulgation of horticulture. Positioned at the northeastern periphery of the ancient capital, the 2011 Xi’an International Horticultural Exposition continues this bootstrapping city-building logic by leveraging the adjacent development of alluvial farmland and traditional villages into a regional financial centre.

The site preparation for the Expo involved remodeling a clay quarry into a simulacrum of the ancient Guangyun Lake, which was once an important port on the Chan-Ba River. Reinterpreted as a constellation of lined lakes interconnected with weirs,hydroponic vertical farming systems the shorelines inform the necklace structure of the exhibits . While most displays represented other provinces and countries, two areas moved beyond kitsch regional simulacra; the first being the collection of gardens by selected ‘Masters’ of landscape architecture, and the second a collection of University gardens by invited international academic teams. To investigate the state-of-the-art of current garden expo design, I explore these two collections of gardens with several objectives: to position the gardens in relation to contemporary landscape architecture design paradigms; to examine the role of the frame in the contained context of the expo garden with the implicit hypothesis that these tactics have agency in the wider contemporary metropolis; to understand why one set of gardens appeared to function as intended within the Expo, while the other appeared to be dysfunctional; and to create a record of Expo Gardens themselves, since despite pretences of being ‘permanent’ installations, it is highly unlikely that any of the gardens will survive physically or semiotically intact beyond the short extravagance of the Expo event.In the first part of the essay I describe, interpret and theoretically and poetically position a number of exhibits from both the Masters and University collections. In the second part of the essay I explore the issue of framing that so vividly distinguishes the Masters’ from the University Gardens. I develop the argument that dissolving the frame—while relevant in contemporary landscape praxis—does not necessarily translate into the context of individual gardens; rather it is the boundary between the Expo and the city that is the most potent threshold. Extending this argument to the city itself, I conclude the essay with discussion regarding the fate of the horticultural expo and its devolution into the urban fabric. In terms of methodology, I draw on my involvement with the Expo from several perspectives; participation in the design workshop for the University Gardens; observations on site during the construction process; and my experiences at the opened Expo as a member of the public.In regards the latter, when on site I was sensitive to Bernard St-Denis’s critique of the tendency for contemporary garden scholarship to place semantic interpretation over ‘the gratification of spending time in a garden’.To be sure, whereas St-Denis was undoubtedly referring primarily to established gardens, the transient nature of the Horticultural Expo gardens tested this challenge to its practical limits. I made repeat visits to the gardens and loitered insofar as was practical, but never attained a contemplative communion with any one garden. However, the impracticality of experiencing ‘time in a garden’ in the Expo context was offset by the heightened experience of the ‘first encounter-reality’ of the perception that results from the initial visit to each garden. Far from being superficial, first impressions are a potent mechanism in our ongoing formation of a sense of place. They are also rarely isolated as purely experiential events; as Donald Apple yard notes, ‘prior indirect information supplied through social contacts or media are also influential’. Like over-the-horizon radar that allows us to cognitively image un-experienced places, these preconceptions are legitimate component of the construction of an environmental image.In addition to my own experiences—keenly honed but biased as a gardenphile and designer of gardens—I also observed the behavioral tendencies of Chinese visitors in each garden. When visiting other more thematic representative gardens of local provinces, it was clear that on the whole the Chinese knew how to act in each garden, and seemed to be far more in tune with the living cultural narrative of garden history than Westerners in equivalent situations in the West, who as Robert Riley intimated, have lost connectivity with and hence knowledge of what to do in a garden.That said, although the gardens in question are located in China, an apparent deficiency of this essay may appear as a lack of attention to traditional Chinese garden landscape themes and narratives. This is perhaps partially a consequence of my limited command of this material, but most importantly it is a product of the Expo being very much a condition of modern China. Indeed, the modern history of the botanic garden / horticultural expo in China is transplanted from the West rather than emerging from Daoism or Confucianism.That Westerners designed all but one of the Masters Gardens and the majority of University Gardens, but that virtually all Expo visitors were domestic in origin, illuminates this complex condition.In raw form, the 10 000 sq ft plots allocated to each designer are typically flat in profile and trapezial in plan, buffered by thick stands of bamboo with controlled access on two sides. These manufactured site circumstances present a strong case for utilizing the timeless phenomenology of the walled garden as an other world decisively withdrawn from the surrounding landscape.