Horticulture – Page 33 – Naturehydro Horticulture Grow

Maximizing Yields, Minimizing Footprint: The Hydroponic Advantage

The observations suggest that OsZHD2 increases the amount of local auxin occurring in the dividing zone of the roots. We propose that OsZHD2 induces auxin biosynthesis in the RAM by increasing ethylene levels. However, we do not rule out the possibility that OsZHD2 directly increases auxin levelsby controlling other genes that we did not investigate in the present study . OsZHD2 induces root development by increasing ethylene biosynthesis and sequentially auxin biosynthesis. It determines meristem-specific homeobox protein functions as an activator for meristem activity by regulating the ethylene–auxin interaction. In the RAM region, ethylene–auxin crosstalk plays important roles .Root system architecture is a critical agronomic trait that influences crop productivity by altering soil mineral absorption and lodging . Deep rooting is a key trait that facilitates drought stress tolerance, since plants can absorb water from deeper soil layers . In addition, the introgression of the DEEPER ROOTING 1 allele from a deep-rooting rice cultivar into a shallow-rooting rice cultivar increases yield under drought conditions , while root-specific over expression of OsNAC5 enhances root diameter, resulting in greater drought tolerance and higher grain yield . Here, we demonstrated that the over expression of OsZHD2 increases the volume of the root system and overall yield, particularly under a poor nutritional status .

Therefore, our results suggest that OsZHD2 is a key trait that could be applied in the improvement of grain yield.We observed that the increased expression of OsZHD2 stimulated root growth. The effect was more significant for lateral roots. Although the total number of lateral roots increased due to the over expression of the gene,plastic pots plants their density did not change . Therefore, the function of OsZHD2 seems to be associated primarily with root growth rather than root initiation. In situ RNA hybridization analyses revealed the preferential and uniform expression of OsZHD2 in the lateral root meristem region, supporting the root growth function . The number of dividing cells in the RAM region was significantly higher in the OsZHD2-OX plants, further indicating that the gene stimulates root growth . The process of initiating lateral roots has been elucidated extensively using numerous mutants defective in that step. However, the molecular mechanisms of lateral root emergence and growth remain poorly understood . Mutations of orc3 in rice interrupt the cell cycle process and block lateral root emergence . The ORC is a critical element in DNA replication, cell cycle checkpoint regulation, heterochromatin assembly, and chromosome assembly. The expression levels of the genes of the D-type cyclin family are down-regulated significantly in orc3 mutants . In the present study, the expression levels of CYCD4;1 increased in OsZHD2-D, suggesting that OsZHD2 promotes cell cycle progression during lateral root growth .

Plants rely on an array of phytohormones to coordinate and finely regulate response networks to biotic and abiotic stresses . Studies of phytohormone regulation of defense responses in plant–microbe interactions generally have focused on salicylic acid , jasmonic acid , and ethylene . In Arabidopsis, SA-mediated defenses are considered deterrents to biotrophic pathogens, whereas JA/ET-mediated defenses deter necrotrophic pathogens . However, this dichotomy with respect to parasitic strategy may be less clear in other host species . While studies of SA and JA/ET signaling have shaped our current understanding of induced resistance mechanisms, consideration of other phytohormones is gaining traction in terms of how multiple stress response pathways overlap as non-linear networks to coordinate plant responses to diverse biotic challengers . These interactions can be synergistic or antagonistic, or phenotypically neutral if the disease assays cannot discern subtle differences. For example, SA and JA can be antagonistic in certain contexts leading to trade-offs in defense against different attackers . Similarly, there is substantial evidence that elevated levels of the phytohormone, abscisic acid , can diminish host resistance . Nonetheless, ABA enhances resistance in some biotrophic and other interactions . This parasitic context dependency of ABA action illustrates the challenge in identifying a unifying mechanism to explain ABA’s effects in plant–microbe interactions.

Various root stresses reduce plant water potential and induce ABA accumulation to trigger adaptive biochemical and physiological changes that enable plants to maintain water balance . However, episodic root stresses, even those from which plants fully recover, occur routinely in agricultural and natural systems, transiently elevating cellular ABA concentrations to levels that predispose plants to inoculum densities they would normally resist . Disease predisposition from abiotic stress has long been recognized in the plant pathology literature , and is particularly well-documented in classic studies of root and crown diseases caused by Phytophthora spp., where episodes of water logging, soil salinity, and drought are important factors in disease development . Although a role for ABA in conditioning the increased susceptibility during and following stress episodes is recognized, the underlying mechanisms and impacts on host defenses are unresolved . Furthermore, relatively little attention has been directed at defense-related phytohormone signaling in root–pathogen interactions where the predisposing stresses of water deficit, hypoxia and soil salinity are encountered most directly . Salicylic acid is involved in multiple physiological processes , but is perhaps most studied for its role in systemic acquired resistance and as a strong inducer of pathogenesis-related proteins . SA biosynthesis in plants occurs by two pathways, one via isochorismate synthase , and the other via phenylalanine ammonia lyase . Knockout mutants in the ICS pathway and transgenic plants carrying nahG encoding a bacterial salicylate hydroxylase have reduced SA levels, are highly susceptible to pathogens, have severely reduced levels of PRproteins, and fail to develop local and systemic resistance . ABA appears to antagonize SA action in defense . Jasmonic acid is an oxylipin involved in defense responses against necrotrophic pathogens and insect herbivores, and has been found to have positive or negative interactions with SA depending on the specific host-parasite/pest context . JA also acts synergistically with the phytohormone ET, and either synergistically or antagonistically with ABA . In soybean leaves, JA levels increase with ABA during dehydration, and a few studies have shown ABA signaling necessary for JA biosynthesis and elaboration of defense responses . In Arabidopsis, application of ABA suppresses some JA/ET activated genes such as PDF1.2, while JA/ET responsive genes are up-regulated in ABA-deficient mutants such as aba1 and aba2 . Several JA synthesis mutants are available in tomato, including defenseless-1 , blocked in the conversion of 13-hydroperoxylinoleic acid to 12-oxophytodienoic acid, and acx1, a mutant defective in acyl-CoA oxidase . These mutations result in reduced JA accumulation and pathogen-related transcripts . Phytophthora capsici is a broad host-range pathogen that can cause significant economic losses in vegetable crops in the Cucurbitaceae, Solanaceae, and Leguminosae families . Similar to other soilborne Phytophthora species, P. capsici causes extensive root and crown rots that are exacerbated by predisposing stresses such as water logging and salinity. In a previous study, we imposed acute levels of salt stress on hydroponically grown tomato seedlings prior to inoculation with P. capsici to show that predisposition in roots and stems occurred in an ABA-dependent and ET-independent manner . In a related study ,plastic nursery pots we showed that plant activators that engage SA-mediated defenses in tomato induce resistance to the bacterial speck pathogen, Pseudomonas syringae pv. tomato , both in non-stressed and salt-stressed plants, but not in the case of P. capsici where plants exposed to these same treatment regimes displayed similar severity of root and crown rot.

The objective of this study was to further assess the impact of salt stress on the infection and colonization of tomato roots by P. capsici and to determine if there is discernible interaction between ABA and SA or JA during salinity-induced predisposition. We examined the impact of salt stress on P. capsici zoospore attraction and early infection and colonization in tomato roots in wild-type and ABAdeficient mutants. Since ABA can alter the action of SA and JA , we evaluated SA- and JAdeficient tomato plants for altered predisposition phenotypes. In addition, we profiled the expression of hallmark genes for stress adaptation and defense during predisposition onset and recovery and P. capsici infection. Tomato plants of cultivars ‘New Yorker,’ ‘Rheinlands Ruhm,’ or ‘Castlemart’ and mutant or transgenic lines within these backgrounds were used in experiments. ‘New Yorker’ and ‘Rheinlands Ruhm’ are determinate and indeterminate cultivars, respectively, used primarily for fresh market consumption, and ‘Castlemart’ is a determinate, processing cultivar that was bred for the arid growing conditions of California and other regions. In our experimental format, all three cultivars are susceptible to P. capsici. ‘New Yorker’ seeds were obtained from a commercial source . The homozygous ABA-deficient mutants sitiens and flacca were compared with their isogenic, wild-type background, ‘Rheinlands Ruhm,’ and seeds for these were obtained from the C. M. Rick Tomato Genetics Resource Center, University of California, Davis. ‘Rheinlands Ruhm,’ sitiens, and flacca plants were grown for seed production in the greenhouse. NahG transgenic plants were generated in the ‘New Yorker’ background, similar to the method used by Gaffney et al. . The nahG construct containing the transgene salicylate hydroxylase under control of the CaMV 35S promoter in the binary vector pCIB200 was a gift of Syngenta Crop Protection, Inc. SA deficiency of our transgenic line was confirmed previously . The acx1 and def1 mutants in the cv. ‘Castlemart’ background were a gift of Gregg Howe, Michigan State University. Seeds of ‘Castlemart’ were obtained from the C. M. Rick Tomato Genetics Resource Center. Four-week-old plants with two or three true leaves were grown hydroponically as described previously . Experiments were conducted in a growth chamber . A pepper isolate of P. capsiciwas used for most experiments. A P. capsici isolate transformed with the green fluorescent protein was a gift of Christine Smart and William Fry, Cornell University . Wildtype and transformant P. capsici strains were maintained on V8 juice agar plates or V8 juice amended with 100 mg/L geneticin , respectively. Zoospore inoculum was prepared using methods described previously .The salt stress regime selected for these experiments was based on prior studies of root stress predisposition . The impact of salinity stress differs from other osmotic dehydration stresses primarily in that salt-stressed plants are additionally exposed to abnormally high extracellular concentration of ions such as sodium and chloride . The inclusion of calcium helps to mitigate the confounding toxicity caused by sodium and emphasizes the osmotic facet of salinity stress, which is likely applicable to other dehydration stresses . Plants were subjected to salt stress by replacing the 0.5X Hoagland’s solution with 0.2 M NaCl and 0.02 M CaCl2 for 18 h. Plants were returned to 0.5X Hoagland’s solution, allowed to regain turgor and recover for 2 h, and then inoculated with zoospores of P. capsici . To determine whether there was an effect on zoospore motility and chemotaxis, a microcapillary swim-in assay similar to that described by Morris and Ward was used with exudates collected from tomato roots. Following 18 h salt stress, tomato roots of uniform volume were rinsed in deionized H2O and transferred to tubes containing 2 ml of deionized water. Exudates were allowed to accumulate for 2 h, tomatoes were then removed, and the exudates were vortexed and immediately loaded into 1 µl microcapillary tubes . Exudate-loaded microcapillaries were placed into 15 cm petri dishes with one end submerged in a 500 µl droplet of 5 × 105 zoospores ml−1 . Microcapillaries were photographed under a dissecting microscope 15 min after being placed into the zoospore suspension. Zoospore attraction was determined as the proportion of the microcapillary’s inside diameter blocked by encysted zoospores and scored on a 0–5 rating scale .The P. capsici-GFP transformant was visualized 24 and 48 hours post inoculation in tomato roots using a Leica TCS SPE confocal system . Following infection and just prior to microscopy, roots were dipped into a 10 µg/ml solution of propidium iodide for 30 s and rinsed in sterile water before mounting on microscope slides . GFP was excited at 488 nm and emission was collected between 510 and 550 nm. PI was excited at 534 nm and emission was collected between 600 and 650 nm. Laser power was set to 50% with a gain of 800–900 for both the 488 nm and 534 nm channels. Final images were composites of five Z steps through root tissues approximately 40 µm in depth.To estimate the progression of P. capsici colonization in tomato seedlings by qPCR, nahG, def1, and acx1 plants and wild-type plants of their corresponding backgrounds were frozen in liquid N2 at 48 hpi, and stored at −80◦C until extraction and analysis.

Beyond Soil: Exploring the Wonders of Hydroponic Agriculture

Five replicate columns were prepared and analyzed for each treatment. Metal concentrations for all three ENMs are reported as ionic, although neither CeO2 nor TiO2 were expected to dissolve to a significant degree under the conditions used in this experiment. TiO2 is known to be highly insoluble in water and CeO2 is similarly insoluble at pHs similar to those found in the soils used here. However, Cu2 has been shown to undergo partial dissolution under acidic to neutral conditions, although at acidic pHs less dissolution occurs in media with high concentrations of dissolved organic matter.Based on this, dissolution of Cu2 is not expected to occur to a significant degree under the conditions and time scales used in this experiment. To measure size distribution of particles throughout the column, air-dried samples of contaminated soils were collected from the top and bottom 3 cm of columns and analyzed using environmental scanning electron microscopy with back scattering electron detection and energy-dispersive X-ray spectroscopy to confirm identification of CeO2, Cu2, or TiO2 ENMs. Beam voltage was set at 12 kV, spot size at 6.0, water vapor pressure was kept at 2.7 Torr,square plastic pot and working distance averaged around 10.5 cm. These settings were chosen in order to minimize X-ray subsurface penetration for EDS analysis. Elemental hypermap data was collected over a period of 6 min per image.

ImageJ image analysis software was used to determine particle or aggregate size. Soil solution extracts of potting, grassland, and farm soils were prepared following Rhoades, although no Na3PO4 was added in order to avoid influencing ENM physicochemical behavior. Soil solution extracts were stored at 4°C until use.Gravity-driven vertical transport of ENMs through unsaturated soil was found in general to follow the hypothesis that the majority of ENMs would be retained in the upper portion of the column, but as predicted was found to be highly dependent on soil type with increased retention occurring in the denser, less porous natural soils . However, ENMs coated with natural organic matter did not have increased vertical transport, and in fact were retained more in potting soil. TiO2 and CeO2 aggregate sizes were seen to decrease with column depth, suggesting physical straining to be the primary impediment to transport. Aggregate hydrodynamic diameters tended to be larger in soil solution extracts than Nanopure H2O and were also generally larger with NOM-coated particles, with several exceptions .All three ENMs largely passed through the entire length of potting soil columns, being present in lower concentrations than the hypothetical homogeneous concentrations at all points, although there was some retention in the upper 0-6 cm that was increased with NOM-coated particles . These trends can likely be explained by the primarily organic composition of the potting soil, which gave it very low density, high porosity, and high reactivity . The low density and high porosity prevented aggregates from being physically strained, which is shown for TiO2 and CeO2 by the similar aggregate sizes in the tops and bottoms of columns for both uncoated and NOM-coated particles .

If physical straining was strongly influencing particle transport in potting soil it is unlikely similar aggregate sizes would be observed throughout the length of the column, but rather would result in smaller aggregates or particles penetrating through the column while larger aggregates would be retained at the surface – as was seen in the two natural soils. All three ENMs had similar hydrodynamic diameters in potting soil solution , although NOM-coated aggregates were significantly smaller than uncoated aggregates . ζ-potentials for all three ENMs in soil solutions from all three soils were also similar , although again the presence of NOM coatings, as well as the ENM and soil types, had significant impacts on ζ-potentials . Coating particles with NOM appears to increase their affinity for the organic components of the potting soil. This resulted in the increased overall retention of NOM-coated CeO2 as well as the decreased vertical transport of NOM-coated TiO2 and Cu2 . Evidence for this can be found in Figures 2.3A-B, which show that both NOM-coated and uncoated aggregates have nearly identical hydrodynamic diameters in potting soil solution extract, so the additional retention of NOM-coated aggregates is unlikely to be due to increased physical straining. This was visually confirmed in micrographs of NOM-coated TiO2 in potting soil including Figure 2.4D, which revealed the formation of TiO2 encrustations occurring primarily on the organic components of potting soil over the Al/Si/Na/K perlite minerals. These encrustations may have been caused in part or whole to interactions between the NOM coating and the organic matter in the potting soil.

This finding is counter to several previous transport studies using TiO2, CeO2, and ZnO25 in quartz sand that found organic coatings decreased ENM retention by increasing electrostatic repulsion between coated aggregates and the sand grains, which further suggests interactions between the organic coating and organic soil components. In the grassland and agricultural soils CeO2 and Cu2 shared similar transport profiles , forming large aggregates in the soil solutions that were retained almost entirely in the upper 0-3 cm of the soil columns. However, the widely variable background concentrations of Ti in these natural soils prevented precise measurement of TiO2 ENM distribution throughout the soil columns by ICP-AES , the majority of TiO2 aggregates were confirmed to be retained immediately at the surface through both visual identification of white buildup on the column surfaces and through BSE/EDS analysis. As shown in Figure 2.4, both uncoated and NOM-coated TiO2 ENMs formed large encrustations on the surfaces of all three soils with the exception of uncoated TiO2 in potting soil. Despite having nearly identical surface charges in soil solution extracts , CeO2 formed large porous sponge-like aggregates instead of the more solid encrustations seen with TiO2. These differences in aggregate morphology may be due to differences between the primary particle shapes of these two ENMs, with TiO2 being nanospheres and CeO2 being nanorods. Afrooz, et al. found that spherical Au ENMs had higher attachment efficiencies and deposition rates than rod-like Au ENMs identical in composition, which they attributed to differences in electrosteric and physical packing characteristics. Similarly, Zhou, et al. found the critical coagulation concentration of TiO2 nanospheres was directly related to particle diameter while the CCC of TiO2 nanrods was better explained by particle surface area, which they postulated was a consequence of differences in exposed crystal faces. It has alsobeen shown that metal oxide nanospheres and nanorods interact differently with NOM,square plant pot which may also be a factor in explaining the differences in aggregate morphology seen here.Little research has been done on the effects of ENM exposure on soil properties. In one of the only available studies available on this subject, Ben-Moshe, et al. 1 observed that CuO and Fe3O4 ENMs did not change the total organic content or macroscopic properties of two types of soil but altered the humic substances in the soils. The authors also observed an effect on the soil microbial community, which has been reported in other studies , but did not attempt to link changes in important soil properties with these effects. VandeVoort, et al. found that silver ENMs could limit denitrification processes in soil, but that the effects were dependent on ENM concentration and coating. While previous studies in this area suggest that the effects of ENMs on soil properties are somewhat limited, there may be additional impacts not considered in these studies. For example, metal oxide surfaces are amphoteric, capable of producing both protons and hydroxide ions , but tend to be predominantly acidic in nature.

Due to this metal oxide ENMs may be able to alter the pH of soil pore water and consequently the overall pH of the soil. pH has been called one of the “master variables” for soil systems7 because it controls a number of critical physical and chemical properties, and if ENMs are able to alter soil pH when present above certain concentrations they may pose a hazard to organisms that rely on the soil for habitat or sustenance. However, soils are typically well-buffered, and may be able to withstand ENM accumulation without changing pH. Additionally, ENMs will likely aggregate as a result of the high ion content of soil solutions, thus decreasing total surface area and, potentially, proton/hydroxide production. Additionally, metal oxide ENMs bear many similarities to naturally occurring nano-scale poorly crystalline metal oxide minerals known as short-range order minerals. SRO minerals have been shown to influence nutrient availability in natural soils via sorptive processes, and metal oxide ENMs may also demonstrate this effect. In particular, metal oxides are well known for their ability to covalently adsorb phosphate ions and, depending on the strength of this interaction, may prevent organisms from accessing this important nutrient. Two hypotheses were addressed in these series of experiments. First, I hypothesized that none of the soils would experience a significant change in pH after spiking with ENMs due to the presence of buffering compounds in the soils. Second, I hypothesized that these ENMs would sorb soil nutrients, including phosphate, and reduce their mobility in the soil.The effect of ENM contamination on soil pH were tested over a range of ENM concentrations by adding potting, grass, or farm soil with 0, 0.1, 1, 10, and 100 µg g-1 TiO2, CeO2, or Cu2 ENMs with and without the addition of 10% NOM. Soil aliquots were then air dried and mixed with Nanopure water to make a 20% soil paste from which the pH was measured. All treatments were performed in triplicate. Changes in soil ion release due to the presence of ENMs was tested by mixing aliquots of potting, grass, or farm soil with 100 µg g-1 TiO2, CeO2, or Cu2 ENMs as suspensions, centrifuging at 8000 x g for 10 min, and analyzing the supernatant for ion concentrations. NO3 – was measured via colorimetric methods and Al, Ca, Fe, K, Mg, Na, P, and S were measured via ICP-AES after acidification to 10% HNO3. The influence of the three ENMs on the bio-availability and mobility of P was investigated further by contaminating agricultural, grassland, or potting soil samples with 100 µg g-1 ENMs and testing P content in three fractions: water extractable P, bio-available P, and immobile P. Soil aliquots were first mixed with water for 1 min, centrifuged at 8000 x g for 10 min, then the supernatant was removed and acidified to 10% HNO3. The same soil aliquots were then mixed with Bray extract11 for 1 min, centrifuged at 8000 x g for 10 min, then the supernatant was removed and acidified to 10% HNO3. The soil aliquots were then acid digested in 1:3 HNO3:HCl at 200°C for 1.5 hours in a microwave digestion system, and all samples were analyzed for P content via ICP-AES. Soil solution extracts of potting, grassland, and farm soils were prepared following Rhoades, although no Na3PO4 was added in order to avoid influencing ENM physicochemical behavior. Soil solution extracts were stored at 4°C until use. Despite varying ENM concentrations over four orders of magnitude, changes in soil pH due to ENM contamination were largely independent of both ENM type and concentration . Contrary to the first hypothesis, changes in soil pH due to ENM contamination did occur, but they were found to be highly dependent on soil type. All three ENMs increased grass soil pH , decreased farm soil pH , and had no effect on potting soil pH . Additionally, the presence of NOM had no effect on the influence of ENMs on soil pH except in the case of farm soil, where a slight buffering effect was seen . As nearly all changes in soil pH were independent of ENM concentration it is unlikely these ENMs directly influenced soil pH through the production of H+ /OH-due to their amphoteric properties. One possible alternate explanation is that the ENMs increased the release of ions that act as buffering or pH-altering agents, such as Al3+ , Ca2+, H+ , K+ , Mg2+, Na+ , and OH- , by replacing them on the mineral surfaces of the soil matrix. Since there is a limited pool of ions available for desorption in a unit of soil, changes in ion release due to ENM sorption would be relatively independent of ENM concentration beyond the point at which total sorption/desorption occurs.

Sustainable Solutions: Hydroponics as a Game-Changer in Agriculture

Using classical tax-onomic methods, the researchers were not able to differentially assign the isolate to either the B. thuringiensis or B. cereus species. The main objec-tives of this project are to develop molecular tools that can phylogenetically position GTN-degrading bacilli and to develop economical detoxification technologies. We have attempted the first route with mixed success. There are many degradation products and some may be strongly bound to the organic matrix of the wetland . This report considers the alternative approach using radio labelled 14C TNT. We used the same system of for other wetland experiments. A series of 1.5 L, replicated wetlands microcosms were set up using dead fragmented plant matter and a small soil inoculum. The microcosms were initially set up with nitrate to stimulate dentrifying bacteria. It is likely that bacteria that degrade nitrate will also be able to degrade TNT. The system was considered ready for TNT degradation when the nitrate concentration had fallen. The microcosms are anoxic, sparged with nitrogen gas and are slow flow-through systems. Incubations with added [U-14C] TNT were used to investigate mass balance and determine metabolite production including 14CO2. Any CO2 from the reaction solution was trapped in a series of three bottles containing 12-mL of scintillation cocktail. At the end of the experiment, the solution was acidified to pH 1 using concentration H2SO4 to volatilize any CO2 that remained in the solution. 14C in the final solution phase was also captured by scintillation cock-tail,blueberry plants in pots and then analyzed using a Beckman LS 6000 SC Scintillation Counter .

The projected costs of Department of Defense site restoration using existing technologies are staggering: the estimated cleanup cost is at least $24.5 billion for the 7,313 identified U.S. sites . The pollutants at these sites include chlorinated hydrocarbons, metals, petroleum products, explosives, mixed waste and other organics. There is clearly a need for new cost-effective treatment technologies. Bio-remediation, the use of microorganisms to detoxify hazardous waste, promises to provide economical and ecologically sound clean-up strategies. Tropical environments may have a large number of niches where unique microorganisms might have developed that can biodegrade organic contaminants and bio-transform heavy metals. This study is focused on the screening of new microorganisms in water samples from several sites in Puerto Rico and their potential use as agents for the transformation of wastes such as heavy metals, nitroaromatic compounds and other organic contaminants. The sites to be sampled include San Juan Bay in northern Puerto Rico and Yabucoa Bay to the southeast. An experiment has been designed that will use barrels holding soil contaminated with diesel fuel. The basic design is shown in Figure 1. Warm, humidified air will be pumped into the soil barrels. The warm, humidified air is meant to accelerate rates of bio-degradation within the barrels. A total of nine barrels will be used. Three barrels will have diesel contamination in the soil but will only be exposed to ambient dry air in order to provide a base line for monitored bio-degradation parameters. The diesel fuel will be mixed into the sand at a concentration of about 1,000 ppm. The sand, with a grain size of about 1 mm, will be spread out on a plastic liner. Diesel fuel will be dropped onto the sand while the sand is stirred to obtain a homogeneously contaminated sand. Then the contaminated sand will be put into the barrels.

The other three barrels with diesel-contaminated soil will be vented with warm, humidified air. The last three barrels will have a nitrogen-based fertilizer added to the diesel-contaminated soil to ensure that there is not a lack of nitrogen acting as a limiting factor to the rate of bio-degradation that can occur. Bio-degradation parameters will be monitored using the sampling ports as shown in Figure 1. Material and supplies needed for the project have been ordered and delivered to the project site, the University of Texas at El Paso Solar Pond facility. The barrels are currently assembled at the project site. The experiment started Jan. 10, 2000, with a six-month monitoring phase. Analysis and interpretation of results will start shortly after the first monitoring data are obtained.Most Department of Defense sites are contaminated with polycyclic aromatic hydrocarbons . PAHs contaminate military property as a result of open burning, incomplete fossil fuel combustion and accidental or deliberate hydrocarbon spills. PAHs rank among the most important environmental contaminants because of their persistence in the environment and their carcinogenic and mutagenic properties. Bio-remediation has been demonstrated to remove PAHs from contaminated soil. However there is evidence that the PAHs are not always mineralized during bio-remediation, but are transformed to stable intermediates that may remain in the soil after treatment. The identification of these intermediates is incomplete. Our task, the bio-degradation of PAHs, is an investigation of how bacteria transform high molecular weight PAHs in both laboratory culture and in the field. Our hypothe-sis is that we can use metabolite turnover to measure intrinsic PAH degradation. The scientific goals of the project are to develop a greater understanding of how bacteria that are able to grow on light PAHs can also oxidize heavier PAHs, such as pyrene and benz[a]pyrene. The technology or application goals of the project are to develop tools that allow us to study PAH degradation and PAH metabolite turnover in contaminated soils.

If we can link metabolite turnover and PAH degradation, we believe we can make a strong argument for an intrinsic degradation procedure based on monitoring metabolite turnover. The application of intrinsic remediation to fuel hydrocarbon contamination has proven a very cost effective approach to site clean-up and it would be beneficial if this technology could be applied to PAHs as well.A method for the extraction and analysis of PAH metabolites has been developed. This method uses an acetone extraction followed by a fluoracil column clean-up procedure that produces a methylene chloride fraction for GC analysis and an ethyl acetate fraction for HPLC analysis. This procedure was tested against known phenanthrene metabolites and found to be effective for their analysis in soil. Our current results suggest that this method is selective for carboxylic acids and excludes many alcohols. An HPLC method with fluorescence detection is used to selectively ana-lyze polynuclear compounds. Metabolite production was studied in a pure, well-characterized culture . As the bacteria grow on phenanthrene , we see a transitory accumulation of a metabolite. This metabolite is present only during active PAH turnover. Trinitrotoluene is chiefly used in military operations because it is a high explosive that is unaffected by shock or abrasion. TNT is also used in certain types of film processing. The compound has been manufactured and used for over a century with concomitant accumulation of TNT waste and unexploded ordinance. Although TNT is flammable, it is readily soluble in water and has accumulated in the groundwater of many military sites. In addition, TNT can also be found in small pellets,drainage plant pots which have become coated and are quite resistant to water. As with many hazardous wastes, TNT can be excavated from soil and incinerated, but costs are high and air pollution can be a problem. Thus, less costly alternatives are needed to remove TNT from a large number of sites covering a large area of the United States and elsewhere. It would prevent much trouble if the removal of TNT occurred on-site, since this would avoid shipping large volumes of contaminated soil or water on public highways. In the last two decades, wetlands have shown great promise for treatment of wastes, and continue to do so. In particular, there are now several large treatment wetlands for nitrate. These have operated for several years and are part of the water treatment system for several large public agencies. A number of investigators have already made important findings showing that TNT and other explosives could be taken up and transformed by living plants. Some of the plants tested may be amenable to full-scale wet-lands treatment systems. Wetlands are characterized as reducing systems but treatment wetlands are quite different from natural wetlands. In particular, treatment wetlands use plants grown in the wetland to provide carbon and habitat for bacteria that carry out the desired process. The soil underlying the dead leaf litter layer and the live plants play little role in the degradation or immobilization processes. It is not that these two components are not active, but that the flow rate and reaction rate in the leaf litter is engineered to dwarf that elsewhere.The main objective of this project is to study the ability of desert plants to adsorb toxic heavy metal ions from contaminated waters, thus reducing the threat to the public’s health. Metals such as lead , copper , nickel and chromium have been deter-mined to cause acute health effects in humans as well as ecological damage.

However, many desert plants have been found to survive in heavy metal contaminated soils. This resistance may be in part due to the development of chemical binding sites found on the exposed desert plant’s cell walls. Therefore, nature could hold the answer to removal of heavy metal ions from aqueous solutions. By utilizing the plant tissues that have evolved a natural affinity for heavy metal ions, we may be able to develop a method to remove these contaminants in a low-cost and highly effective manner. However, before a method can be developed to remove the heavy metal ions using desert plant tissues, the binding mechanism must be better understood.At the present time we are performing experiments with the desert plant Solanum ealeagnifolium . The plants were collected, dried and ground in preparation for the various metal binding studies. Batch laboratory methods are being employed to characterize the metal binding abilities of the desert plant. In addition, chemical modification experiments have been utilized to further study the metal binding mechanism of Solanum ealeagnifolium with the different metal ions being studied. Metal analysis for the batch experimentation has been performed utilizing flame atomic absorption spectroscopy and graphite atomic absorption spectroscopy with Zeeman background correction. In addition, samples have been analyzed utilizing synchrotron X-ray Absorption Spectroscopy at Stanford Synchrotron Radiation Laboratories at Stanford University in California. The XAS data are currently being examined to determine the X-ray Absorption Near Edge Structure of bound metals such as Cu and Cr. By using the computer software, Fourier transformation of the spectra can be performed, which provides the Extended X-ray Absorption Fine Structure of the metal bound on the biomass. These data can then be com-pared with model compounds to delineate the nearest neighbor atom bound to the metal ion on the biomass. The XAS data along with the chemical modification will help us pin down the actual chemical functional groups responsible for binding of the metal ions and help to find the most effective method to remove metal ions from contaminated waters. Thus far, we have determined that Solanum ealeagnifolium is able to bind appreciable amounts of metals tested. Figure 1 shows the percent of metal boundat various pHs. It is clear from Figure 1 that the binding of these metals is pH-dependent, with maximum binding occurring at or near pH 5.0. Since carboxyl lig-ands are known to have pKa values in the 3-4 region, we believe that carboxylic acids found on the cell walls may be involved in the binding of these metal ions. However, the binding of Cr did not follow the same trend. This indicates that Cr may be binding through a different mechanism. In order to better understand how carboxyl ligands on the cell walls of Solanum ealeagnifolium are involved in the metal binding process, we chemically modified the plant tissues and performed batch experiments with modified biomass. The results of the esterification and hydrolyzation capacity experiments are shown in Table 1. From the table, it is clear that the esterification of the cell walls of Solanum ealeagnifolium blocked the majority of the binding for these metals. These data support the finding from the pH profile, which indicates that carboxyl ligands are involved in the metal binding process. In addition, it can be seen form the table that by creating additional carboxyl ligands, there is an increase in the binding of these metals, which further supports that carboxyl groups found on the cell walls of Solanum ealeagni-folium are involved in the binding of these metal ions.

Revolutionizing Crop Growth: The Rise of Hydroponic Farming Techniques

We identify key the data gaps that need to be filled in order to proceed with meaningful ecological risk assessments, whether they are more global/ regional in nature, or for site specific assessments. Finally, we attempt to draw conclusions from the literature about the relative sensitivity of different model organisms, as well as the importance of particle properties on fate, transport and effects.The European Commission estimates the global production of nanoceria to be around 10 000 tons.Similarly, a comprehensive market study provides an estimate of 7500 to 10 000 tons for the year 2011.According to the US Geological Survey , over 80% of the global CeO2 supply originates from China.The major nanoceria producers are located in Asia, Australia, and Europe. It is estimated that only 35–700 tons are produced per year in the US.Nanoceria is used in electronic and optical devices, polishing agents for glass and of silicon wafers, exterior paints, metal-lurgy, and diesel fuel additives.Additionally, nanoceria is used in automotive catalytic converters.It is also used in catalysts in petroleum refining, in the fluid catalytic cracking process . Based on the amount of total global CeO2 annual production and global nanoceria production rates,package of blueberries 10 roughly 15–25% of total CeO2 production is nano . Cerium oxide is used in these applications in both nano and non-nano form and quantitative estimates of cerium oxide use within specific applications do not distinguish between nanoceria and its bulk counterpart.

Nanoceria is used to improve catalytic reactions in catalytic converters.However, studies of CeO2 use in catalytic converters do not distinguish between nanoceria and its bulk counterpart. According to the USGS, approximately 80 g of CeO2 are contained in an average catalytic converter, and roughly 85% of cars and light-duty trucks are equipped with catalytic converters.11 By combining these estimates with global automotive sales reports13 the global demand for CeO2 for use in catalytic converted was estimated to be roughly 4900 tons per year.There are few studies that quantify the release of engineered nanomaterials during use, and even less nanoceria specific studies. One of the few studies by Park et al., indicates that 6–100% of CeO2 will be released during the use phase of diesel fuel additives.This has not yet been validated by other researchers. In laboratory conditions, particles filters from diesel cars removed 99.9% of Ce present in fuel additives.However the manuscript does not specify whether the Ce additive was in the nanoscale. Considering the applications and the likelihood that the nanomaterials are released to the environment, the following assumptions were made. For example, nanoceria in batteries is enclosed within a protective casing, which is likely to minimize release during use. If the batteries are disposed of improperly, the most likely environmental compartment would be soil, with negligible release to air, water or wastewater treatment plants . Similar assumptions were made for metallurgical products, catalysts in FCC, polishing powders used in industry , and other applications. Experimental studies have been conducted to measure the release of various manufactured nanoparticles from surface paints on exterior facades. Kaegi et al. measured concentrations as high as 600 μg L−1 of nano-TiO2 in runoff from newly-painted building facades,and estimated that as much as 30% of nano-Ag is released from surface paints within a year of paint application.

However, no data exist on nanoceria released from paints. Based on similar information, estimated nanoceria concentrations in treated WWTP effluent discharged to water bodies are expected to be in the range of 0.003–1.17 μg L−1 . In bio-solids, nanoceria concentrations are expected to be around 0.53–9.10 mg kg−1 . These estimated concentrations are expected to increase as nanoceria is used more widely, and there will likely be accumulation of CeO2 in soils and sediments, further increasing exposure concentrations in these media.The detection and characterization of nanoceria under conditions relevant to environmental, toxicological and biological systems remains a challenging, and frequently impossible, task. However, there is little or nothing that is ceria specific, but applies to all nanomaterials. However, aspects of charac-terization are included here since it is fundamental to under-standing of all nanomaterials, including nanoceria. Essential general aspects are listed below: i) In environmental systems, the specific and accurate detection and characterization of manufactured nanoceria remains essentially impossible,20,21 due to the gap between metrology and analysis and the complexity of the system . Total Ce detection is useful as it acts an upper limit of nanoceria concentrations for risk assessment, but is not synonymous with manufactured nanoceria. The discussion below applies primarily to spiked materials, mainly in the laboratory or mesocosm. ii) As with other nanomaterials, nanoceria should be fully characterized using suitable preparation methods and a multi-method metrological approach. In a multi-method approach, independent techniques operating on independent measuring principles provide cross-validation of measured properties. The source of the nanomaterial also needs to be fully reported, given the likely effects on properties. Fuller discussion is given elsewhere. iii) A number of properties require characterization which can be grouped as size, shape, morphology, aggregation/ agglomeration, surface charge and dissolution . These groups, or classes, contain several individual properties.

For instance, for size, an average size should be reported, along with some measure of spread .iv) Given the changes that are well known to occur upon storage or changing media, it is essential to perform appropriate measurement over temporal and spatial scales which adequately capture the dynamics of the nanomaterial system. Although, none of the points above are ceria specific, nanoceria is capable of oxygen storage, which is size and shape dependent.Nanoceria is generally thought to have low solubility in water,although this is size and oxidation state dependent. Where dissolution and solubility are low, study is rendered simpler because dissolved ions should have little impact on toxicity. However, recent work has shown potential effects of even low level dissolution.Nano-ceria has two stable oxidation states ĲCeĲIII and Ce under environmental conditions and cerium has the ability to transi-tion readily between these two states.This redox activity gives nanoceria some of its key properties.However, oxidation state and morphology are usually poorly controlled or defined and spatially variable within an individual particle,giving rise to poorly reproducible data and uncertainties in understanding toxicity or exposure. These uncertainties, along with dynamic changes that occur in complex media, could explain the variable environmental and toxicological results that are seen in the literature for nanoceria.Table 1 shows a non-definitive selection of studies of nanoceria in a variety of different environmental, toxicological and standard complex media. These studies are examples of some of the most complete characterization in the literature, although there is still little consistency between studies and it is often not clear which nanomaterial properties require analysis because it is not well understood how each affects biological or environmental processes. Lastly,plastic plant pot because of logistical or other constraints, characterization is often not performed as fully as necessary to interpret such processes. Some of the most powerful techniques for the visualization of nanoparticles are transmission electron microscopy , atomic force microscopy and scanning electron microscopy . These techniques not only provide direct visual images but can be used to quantify other properties such aggregation, dispersion, sorption, size, structure and shape of the nanoparticles,although the sample prepara-tion may alter considerably the sample. These techniques have been extensively applied to nanoceria, occasionally in complex media. Van Hoecke et al.and Rodea-Palomares et al.used TEM to visualize the interaction between the nanoceria and algal cells in order to test whether the nanoparticles are taken up or adsorbed by the algal cell wall. Zhang et al.used TEM to further investigate the needle like clusters on the epidermis and in the inter-cellular spaces of cucumber roots after treatment with nano-ceria over 21 days. In some cases, TEM has been coupled with spectroscopy, for instance TEM coupled with EDS was used to determine the elemental composition of ceria clusters on both the root epidermis and in the intercellular regions of the cucumber plant.Merrifield et al.used AFM to image and quantify the size of PVP-coated nanoceria while compared them using TEM and DLS in toxicology exposure media.

TEM confirmed that the larger particles are aggregates composed of smaller individual particles , but that nanoceria properties did not measurably change in the exposure media tested. In the same study, EELS was used to quantify the oxidation states showing that the smallest and the largest samples were composed of entirely CeĲIII), with only small amounts of Ce present in the largest sample. Such spectroscopy is essential to microscopy imaging in complex media and is required to unambiguously identify the nano-particles of interest in the presence of materials with similar sizes, shapes and electron densities/tip interactions. Micros-copy, although a powerful single particle method, remains challenging when attempting to provide statistically meaningful measurements. Much data reported in the literature is pictorial and non-quantitative; careful design and time consuming analysis are required to be able to determine representative parameters with confidence. Nanoparticle tracking analysis is another widely used characterization technique which utilises microscopy to determine size distributions and number concentration of nanoparticles in liquid samples. NTA has been infrequently used for nanoceria, for instance to determine the mean size of nanoceria in green alga and crustaceans and to better understand the effect of natural organic matter on the particle-size distribution of nanoceria settling in model fresh water as a function of time.However, the methodology has some limitations in complex and realistic media.X-ray photoelectron spectroscopy has been used in only one relevant study, to our knowledge, in this case to understand the antioxidant capacity of nanoceria to DNA. The calculation of CeĲIII) : CeĲIV) ratios was performed,in an analogous manner to EELS, within a multi-method approach. Similarly, synchrotronbased X-ray spectroscopy has been used in several studies to assess Ce speciation. Studies using micro X-ray fluorescence coupled with X-ray absorption near edge structure in natural matrices have been conducted concluding that nanoceria can undergo bio-transformations within a matrix, so the modifications, the mechanism and extent of these transformations should be fully addressed.Scanning transmission X-ray microscopy is an analytical microscopy which, with extended X-ray absorption fine structure spectroscopy, provided 2D quantitative maps of chemical species at concentrations which are environmentally relevant.X-ray microscopy can in principle provide a spatial resolution down to ~30 nm while imaging the specimen in the aqueous state without the need for sample preparation.Synchrotron-based techniques provide direct structural information regarding the nanoparticles and their interaction with the environment. It is clear that X-ray spectroscopy, XPS and EELS are complementary methods for oxidation state analysis and combination may prove fruitful. Field flow fractionation has also been used on nanoceria to measure the size distribution of nanoceria in synthe-sized samples30 as well as to understand the aggregation behavior of other nanoparticles in the presence and absence of humic substances.ICP-MS can be used as a detector for FFF, but has not been for environmental or toxicological studies of nano-ceria, to ourknowledge. Preliminary studies56 have shown the feasibility of ICP-MS for nanoceria analysis in single particle mode, although its further application in real systems has yet to be demonstrated. Infrared spectroscopy has also been used40 to study bio-transformations in plants by comparing the molecular environment of the sample before and after exposure hence concluding that cerium speciation changes after incubation of nanoceria in different exposure media over 21 days. Ultraviolet-visible spectroscopy has been used43 to monitor the dynamic aggregation process of nanoceria in various waters with time along with DLS and TEM.Nanoparticles properties are altered by the water chemistry such as pH, ionic strength, nature of electrolytes or presence of NOM. One of the most important changes may be aggrega-tion of nanoparticles: between the same nanoparticle, homo-aggregation, or between nanoparticles and an environmental particle, heteroaggregation. The increase in size of the aggregates affects their transport, behavior, reactivity, uptake by organisms, and toxicity. In pure water, the stability of non-coated nanoparticles in solution depends on their surface charge. Nanoparticles brought into close contact via Brownian diffusion processes will repel each other if the charge is strong enough to overcome attractive forces. Nano-ceria surface charge is dependent of the pH; nanoceria are positively charged at low pH, negatively charged at high pH and have an isoelectric point at approximately pH 8.The methods of synthesis and the cleanup of nanoceria have been shown to play a role in affecting the experimental point of zero net charge for nanoceria suspensions, which range from 6.5 to 8.1.

Two light sources were used in this study including UV-A and light-emitting diode arrays

Our low environmental impact assessment for plant-based manufacturing should compare favorably with fermentation based approaches to producing Griffithsin . In the latter, the complexities of purification suggest less efficient utilization of materials and higher disposal volumes, although a side-by-side environmental analysis between the two platforms was not conducted in this study.Upstream, Griffithsin expression rates were based on empirical findings using TMV whole virion as the expression vector, which can achieve typically 0.5–1.0 g Griffithsin/kg plant biomass . An average pilot-scale expression rate of0.52 g/kg was used in our model . Although this expression level is quite good for TMV, higher Griffithsin expression levels can be achieved with different technology. For example, Nomad Bioscience GmbH has achieved Griffithsin expression in N. benthamiana exceeding 2.5 g Griffithsin/kg FW biomass using NomadicTM agrobacterial vectors applied to plants either through vacuum infiltration or agrospray , albeit these results were obtained in small-scale studies. The utilization of such an induction process instead of TMV virions could further improve process economics. For example, even with the same recovery efficiency of 70% assumed in the current model,tomato grow bags the output of Griffithsin at the higher expression level would be 1.75 g API/kg plant material, instead of the current 0.37 g/kg; this represents more than 4.7- times the modeled output of protein per kg biomass.

Under such conditions, the costliest parts of the current process, namely biomass production and upstream procedures, would be lowered by the reduced biomass needs to produce the required 20 kg/year of API. Although a full analysis of the cost of agrobacterial inoculation for Griffithsin production needs to be conducted, we know from similar analyses that economics can be favorably impacted by higher expression efficiencies. We can therefore envision that by using a more efficient induction process the per-dose production cost could be less than the current $0.32. Still other gene expression methods can be considered, including using transgenic plants expressing Griffithsin either in constitutive or inducible systems , which could also lead to higher API accumulation in host plant biomass and potentially lower COGS . Increasing expression yield upstream might shift costs to downstream operations to handle process streams with higher concentrations of API. Definition of the comparative cost benefits of these improvements relative to the current process modeled awaits a subsequent evaluation. From a process standpoint, improvements in the efficiency of lighting technologies and/or incorporating solar panels would reduce upstream utilities costs, one of the major contributors to the upstream operating costs. Improving hydroponic nutrient utilization through recycling and minimizing runoff in the simulation model will reduce raw material costs as well as aqueous waste disposal costs, thereby reducing the COGS. In the downstream portion of the process consumables play a major role, particularly dead-end filters and plate-and frame filters; if these could be replaced with tangential flow filtration systems that utilize reusable, cleanable ceramic filters, downstream operating costs could be further reduced. At the time of this writing, such systems were being considered and their impact on Griffithsin COGS will be the subject of a future analysis.

Fisheries and aquaculture are a growing industry, and seafood consumption has been increased from an average of 9.9 kg per capita in the 1960s, to 20.3 kg per capita in 2017. Pathogenic Vibrio spp., specifically, Vibrio parahaemolyticus, are the leading causes of seafood-associated disease in U.S., and 45% of the seafood-borne outbreaks are related to molluscan shellfish. Vibrio spp. are natural inhabitants of estuaries and coastal marine environments. They can be found in water, sediments, and all flora and fauna in coastal environments, including freshly harvested seafood. Another aquatic important microorganism is Aeromonas hydrophila which can cause disease in both fish and humans, affecting seafood safety, quality, and causing severe losses for production and marketing. The importance of controlling these pathogenic bacteria is highlighted by the fact that the aquaponics industry isgrowing globally, and the number of aquaponics producers in the U.S. has continued to grow. Since most fresh produce is consumed raw, the potential for cross-contamination and transfer of pathogenic bacteria from aquaculture water to edible parts of the plants represents a serious risk to public health. Controlling zoonotic fish diseases and food borne pathogens in recirculating aquaculture systems and aquaponics using antibiotics, chemical sanitizers, and pesticides, is challenging due to the sensitivity of the microbial community in bio-filters which oxidize ammonia to nontoxic nitrate, chemical residue concerns in both fish and plants, and regulatory stand points. These challenges motivated researchers to develop novel chemical-free, or bio-based antimicrobial approaches. For example, the application of light-based interventions has emerged recently as an alternative approach to inactivate bacteria.

The photo sensitizer curcumin, in combination with light, has been used for inactivation of various pathogenic and spoilage microorganisms. Exposure of curcumin to light radiation results in photo oxidative generation of reactive oxygen species , which have strong antimicrobial properties. Recent reports have documented curcumin-mediated photo oxidative DNA damage as a mechanism for bacterial inactivation. Light emitting diodes and UV-A are two main safe sources of lights for photo dynamic inactivation which have been used to improve sanitation of food products. Nanobubble technology represents another promising antimicrobial approach that has been recently described. Cavitative collapse of nanobubbles generates reactive oxygen species, as well as a physical insult to microbial cell structures. Due to nanobubbles’ unique properties, this technology has been applied in various areas of advanced science and technology including engineering, medical, agricultural, and food sectors; for cleaning surfaces; dental hygiene; wound cleaning; removing bacteria from fresh produce ; inactivation of norovirus; and removing microbial biofilms. Nanobubbles can exist in both bulk solution and at liquid–solid interfaces, and due to their unique physical properties , nanobubbles can remain stable for up to 24 h, resulting in a supersaturated bubble phase. However, only a few studies have investigated the antimicrobial properties of nanobubbles alone or in combination with chemicals and other nonthermal processes such as ultrasound. Thus, to address the potential challenges in water sanitation in Recirculating Aquaculture Systems and aquaponics, we propose the use of two technologies including curcumin-mediated photo sensitization and nanobubbles with ultrasound to inactivate V. parahaemolyticus and A. hydrophila. The efficacy of light -activated curcumin at different concentrations and exposure time and temperature against these bacteria was evaluated. Antimicrobial properties of synergistic approaches of nanobubbles–ultrasound were also determined at different exposure times at room temperature. This study illustrates the potential of light activated food grade antimicrobial materials, such as curcumin,grow bags garden and a novel chemical-free approach combining nanobubbles and ultrasound for water sanitation in intensive RAS and aquaponics systems to reduce reliance on chemical-based approaches.The efficacy of curcumin activated by UV-A and LED lights was studied against V. parahaemolyticus and A. hydrophila. Samples with curcumin were prepared by adding 5 mL fresh V. parahaemolyticus and A. hydrophila cell suspensions to 5 mL curcumin solution . The final concentration of the cells was 106 cfu/mL. Samples were placed into sterile 6-well clear polystyrene microplates and were treated by UV-A for 5 and 15 min, and LED for 15 and 30 min at 4 and 22 ◦C. After exposure, V. parahaemolyticus and A. hydrophila were cultured on TCBS and TSA , and incubated at 35 and 37 ◦C for 48 h, respectively. Bacteria with curcumin without exposure to lights, and bacteria without curcumin exposed to lights were used as control groups. All the experiments were repeated at least two times in triplicate . The pH of the water samples was adjusted to 5.6 using 1 N citric acid. RAS-aquaponics water sanitation was simulated using water from a seventy liter pilot-scale RAS-aquaponics system . Before conducting the experiment, to ensure that there were no Vibrio sp. and Aeromonas in the RAS-aquaponics water, 100 mL RAS water was filtered using 0.45 µm, and a filter was placed on TCBS and selective Aeromonas medium , and no bacterial colonies were observed. The pH of the water samples was adjusted to 5.6 using 1 N citric acid.

Then, samples were prepared as mentioned previously, by mixing 5 mL of the pH-adjusted aquaponics water with bacterial cell suspensions to obtain 106 cfu/mL and 10 mg/L curcumin concentrations, which were treated with light sources for 5, 10, and 20 min. The synergistic antimicrobial activity of nanobubbles and ultrasound was studied against V. parahaemolyticus and A. hydrophila. A nanobubble solution was provided using deionized water and pure oxygen gas using a Moleaer 25 L nanobubble generator . Our preliminary experiments showed that nanobubbles produced by pure oxygen showed stronger anti-biofilm and antimicrobial properties as compared to nanobubbles generated by pure carbon dioxide or air. A total of 1 mL of the previously washed bacterial cell suspensions was added to 9 mL deionized water containing nanobubbles. The solutions were exposed to ultrasound for 5, 10, and 15 min. Bacteria in PBS with ultrasound and bacteria in nanobubbles without ultrasound were used as control. Bacteria were also exposed to LED light for 15 and 30 min with curcumin . The results indicated that increasing the curcumin concentration from 1 to 10 mg/L resulted in increasing the efficacy of the combination approach to inactivate bacteria. Curcumin concentration demonstrated a strong impact on the reduction in V. parahaemolyticus and A. hydrophila regardless of the light source. Overall, the results indicated that V. parahaemolyticus was less resistant to light-activated curcumin compared to A. hydrophila. A higher reduction was also observed for V. harveyi compared to A. salmonicida, and V. parahaemolyticus exhibited a faster rate of inactivation compared to Staphylococcus aureus and Lactobacillus plantarum in the presence of 405 as well as 470 nm LED illumination. Gram-negative bacteria are more resistant to light-activated curcumin, compared to Gram-positive bacteria, which is mainly because of the Gram-positive bacteria outer wall structure permeability for curcumin. The outer-wall structure of Gram-positive bacteria contains up to 100 peptidoglycan layers, which display a relatively high degree of porosity, which is permeable to molecules such as curcumin. However, on the contrary, a higher sensitivity of Gram-negative bacteria compared to Gram-positive bacteria has also been reported. Furthermore, our results indicated that UV-A 400 nm caused a higher bacterial reduction compared to LED 470 nm. After 5 min of light treatment, a greater than 5 log cfu/mL of V. parahaemolyticus and a greater than 4 log cfu/mL reduction in A. hydrophila were achieved by UV-A light. However, LED 470 nm only reduced 3 and 3.5 log cfu/mL of V. parahaemolyticus and A. hydrophila, respectively. Similar results have been previously observed by other researchers, where at an equal radiant energy dosage of 1100 J/cm2 , the 395 nm LED treatment was more effective with a 2.48 log cfu/g reduction, than the 455 nm with a 1.6 log cfu/g reduction in Salmonella in wheat flour. Kumar et al. also reported low antimicrobial properties of 460 nm compared to 405 nm, regardless of bacterial species and illumination temperature. This can be explained by the fact that UV-A can generate ROS and directly cause microbial cell damage compared to visible light, and UV-A light may excite curcumin molecules compared to LED 470 nm. UV-A has been used to activate curcumin for treating E. coli and L. innocua in water and fresh produce. Moreover, mainly LED or visible light were used in combination with photosensitizers for inactivating bacteria including Vibrio sp. and Aeromonas sp.. The results of these experiments illustrate the potential of using the photo dynamic inactivation approach for food production systems for which using antibiotics, therapeutics, and pesticides is restricted such as intensive indoor aquaculture systems, aquaponics, and the oyster industry.Similar to the room temperature experiment, curcumin and UV-A light together caused a more than 5 log cfu/mL reduction in V. parahaemolyticus and a more than 4 log cfu/mL reduction in A. hydrophila at 4 ◦C . Compared to UV-A treatment, LED 470 nm only caused a 4.5 and 2.5 log cfu/mL reduction in V. parahaemolyticus and A. hydrophila, respectively at 4 ◦C . Temperature control is a critical food safety and quality step during seafood harvesting and processing. It has been reported that antimicrobial properties of UV-A light-activated bio-based compounds were inhibited at refrigerated temperatures against E. coli O157:H7 and L. innocua, which is due to the lower bacterial metabolism rates at a lower temperature.

All the supernatant was discarded and the pellet was dried briefly in a vacuum desiccator

To generate the sog1-7 and suv2-3 mutations, als3-1 seeds were treated with EMS. M2 seedlings were screened for mutants that suppressed the hypersensitivity of als3-1 to Al in a 0.75 mM AlCl3 gel soaked environment. EMS treated seeds were planted around the periphery of the plates and were grown for 7 days at 20°C with a 24 hr continuous light cycle. After 7 days, seedlings with roots that grew to the bottom of the plate were rescued on PNS media. After 2 weeks, putative mutants were transferred to soil and allowed to self-pollinate. M3 seeds were collected from their M2 mutant parent and were screened using the same system. Two of these lines that were able to restore growth on the AlCl3 media, were then further characterized, and after mutation identification named sog1-7 and suv2-3.o generate sog1-7 without als3-1, sog1-7;als3-1 was crossed with Col-0 wild type. F1 lines were allowed to self-pollinate and F2 lines were screened for wild type ALS3 by PCR. The als3-1 mutation can be followed by amplification with CAPS markers as listed in Table 1 followed by restriction enzyme digest with ClaI . The wild type ALS3 contains a ClaI site, while als3-1 mutation has lost this site. Digested amplicons were analyzed by gel electrophoresis,plastic garden container resolving wild type or mutant genotype results in a 2% agarose gel. Lines that were homozygous for ALS3 were then screened for the sog1-7 mutation.

The sog1-7mutation can be followed by the amplification with CAPS markers as listed in Table 1 followed by a restriction enzyme digest with DdeI . The wild type SOG1 contains a DdeI site, while sog1-7 has lost this site. Digested amplicons were analyzed by gel electrophoresis, resolving wild type or mutant genotype results in a 3% agarose gel. To generate suv2-3 without als3-1, suv2-3;als3-1 was crossed with Col-0 wild type. F1 lines were allowed to self-pollinate and F2 lines were screened for wild type ALS3 by PCR. The als3-1 mutation can be followed by amplification with CAPS markers as listed in Table 1 followed by restriction enzyme digest with ClaI . The wild type ALS3 contains a ClaI site, while als3-1 mutation has lost this site. Digested amplicons were analyzed by gel electrophoresis, resolving wild type or mutant genotype results in a 2% agarose gel. Lines that were homozygous for ALS3 were then screened for the suv2-3 mutation. The suv2-3 mutation did not result in the loss of a restriction enzyme recognition site, therefore this mutation was followed by the amplification with markers as listed in Table 1 followed by sequence comparisons to the SUV2 published gene sequence of the Arabidopsis thaliana genome archived on The Arabidopsis Information Resource to identify the suv2-3 mutation. To generate sog1-7;atr-4, sog1-7 was crossed with atr-4. F1 lines were allowed to self-pollinate and F2 lines were screened for sog1-7 by PCR and restriction enzyme digest.

The sog1-7 mutation can be followed by amplification with CAPS markers as listed in Table 1 followed by a restriction enzyme digest with DdeI . The wild type SOG1 contains a DdeI site, while sog1-7 has lost this site. Digested amplicons were analyzed by gel electrophoresis, resolving wild type or mutant genotype results in a 3% agarose gel. Lines that were homozygous for sog1-7 were then screened for the atr-4 mutation. The atr-4 mutation can be followed by amplification with CAPS markers as listed in Table 1 followed by a restriction enzyme digest with BstXI . The wild type ATR contains a BstXI site, while atr-4 has lost this site. Digested amplicons were analyzed by gel electrophoresis, resolving wild type or mutant results in a 2% agarose gel. To generate suv2-3;atr-4, suv2-3 was crossed with atr-4. F1 lines were allowed to self-pollinate and F2 lines were screened atr-4 by PCR and subsequent restriction enzyme digest. The atr-4 mutation can be followed by amplification with CAPS markers as listed in Table 1 followed by a restriction enzyme digest with BstXI . The wild type ATR contains a BstXI site, while atr-4 has lost this site. Digested amplicons were analyzed by gel electrophoresis, resolving wild type or mutant results in a 2% agarose gel. Lines that were homozygous for atr-4 were then screened for the suv2-3 mutation. The suv2-3 mutation did not result in the loss of a restriction enzyme recognition site, therefore this mutation was followed by the amplification with markers as listed in Table 1 followed by sequence comparisons to the SUV2 published gene sequence of the Arabidopsis thaliana genome archived on The Arabidopsis Information Resource to identify the suv2-3 mutation.

To generate the sog1-7 and suv2-3 lines carrying either the CycB1;1::GUS or QC46 reporters, parent lines of sog1-7 and suv2-3 were crossed and F1 seeds allowed to self-pollinate. Genomic DNA was extracted from F2 plants for genotyping. For sog1-7; CyclinB1;1 and sog1-7;QC46 plants were screened to select for plants homozygous for the sog1-7 by PCR and restriction enzyme digest. The sog1-7 mutation can be followed by amplification with CAPS markers as listed in Table 1 followed by a restriction enzyme digest with DdeI . The wild type SOG1 contains a DdeI site, while sog1-7 has lost this site. Digested amplicons were analyzed by gel electrophoresis, resolving wild type or mutant genotype results in a 3% agarose gel. Plants that were identified as sog1-7 homozygotes were GUS stained to identify lines that were homozygous for the reporter line, either CycB1;1 or QC46. For suv2-3;CyclinB1;1 and suv2-3QC46 plants were first screened to select for GUS activity to identify lines that contained the reporter constructs. Plants that stained positive for GUS were then screened to select for plants homozygous for the suv2-3 mutation. The suv2-3 mutation did not result in the loss of a restriction enzyme recognition site, therefore this mutation was followed by the amplification with markers as listed in Table 1 followed by sequence comparisons to the SUV2 published gene sequence of the Arabidopsis thaliana genome archived on The Arabidopsis Information Resource to identify the suv2-3 mutation.To generate an atr-4 mutant line carrying either the SOG1:GUS or SOG1:GFP reporters, parent lines of atr-4 were crossed to parents of the respective reporter and F1 seeds allowed to self-pollinate. Genomic DNA was extracted from F2 plants.For atr-4;SOG1:GUS the atr-4 mutation was first screened for to select atr-4 homozygous mutants. This mutation can be followed by amplification with CAPS markers as listed in Table 1 followed by a restriction enzyme digest with BstXI . The wild type ATR contains a BstXI site, while atr-4 has lost this site. Digested amplicons were analyzed by gel electrophoresis,plastic pot resolving wild type or mutant results in a 2% agarose gel. Plants that were identified as atr-4 homozygotes were allowed to mature and seeds from the F3 were harvested for GUS staining to identify lines that were homozygous for the reporter line SOG1:GUS. 30 seeds from the F3 were planted on PNS and stained again for GUS activity. Lines with staining in all 30 seedlings proved to be homozygous for and SOG1:GUS. For atr-4;SOG1:GFP F2 plants were grown on PNS medium containing kanamycin for 10 days and seedlings were selected for resistance to the antibiotic. Selected individuals were allowed to recover on PNS and transplanted into soil until maturity. The F3 seeds were then harvested, and 30 seeds were planted again on PNS media containing kanamycin, and lines with 100% germination proved to be homozygous for kanamycin resistance, and therefore SOG1:GFP homozygotes.

Plants that were identified as SOG1:GFP homozygotes were then screened for the atr-4 mutation. This mutation can be followed by amplification with CAPS markers as listed in Table 1 followed by a restriction enzyme digest with BstXI . The wild type ATR contains a BstXI site, while atr-4 has lost this site. Digested amplicons were analyzed by gel electrophoresis, resolving wild type or mutant results in a 2% agarose gel.All GUS staining experiments were conducted as previously described , with microscopy performed using a Leica DMR differential interference contrast light microscope. Seedlings were grown on soaked gel media for 7 days, unless otherwise specified, collected, and subsequently fixed in 5 mL 90% acetone on ice for 20 to 30 minutes. Acetone was removed and seedlings were rinsed in 5 mL of rinse solution [50 mM NaPO4, 0.5 mM K3Fe6, and 0.5 mM K4Fe6]. Rinse solution was removed and seedlings were treated with 5 mL GUS stain [50 mM NaPO4, 0.5 mM K3Fe6, 0.5 mM K4Fe6, and 2 mM X-Gluc ], vacuum infiltrated for 5 minutes at room temperature, and incubated at 37°C for noted times. Stain solution was removed and seedlings were stored in 70% ethanol until analyzed using differential interference contrast microscopy. For CYCB1;1 GUS analyses, 60 total seedlings from each line and each treatment were scored for level of blue color at the root tip after 1 hour staining for GUS activity. To generate the SUV2:GUS line, the full length At1g45610 including the promoter was amplified from genomic DNA. PCR was performed using a high fidelity Taq polymerase, Phusion following the manufacturer’s instructions. This was assembled in the pGEMT EASY vector and then cloned into pBI101 containing GUS to create the SUV2:GUS fusion. The construct was electroporated into Agrobacterium tumefaciens strain Aglo, which was then used to transform Col-0 wt. F0 lines were screened on PNS with kanamycin for resistance. Kanamycin resistant lines were allowed to self and F1 lines were then screened again on kanamycin to identify homozygous lines. To generate the SUV2:GFP line, the full length At1g45610 including the promoter was amplified from genomic DNA. PCR was performed using a high fidelity Taq polymerase, Phusion following the manufacturer’s instructions. This was assembled in the pGEMT EASY vector and then cloned into pBI101 containing GFP to create the SUV2:GFP fusion. The construct was electroporated into Agrobacterium tumefaciens strain Aglo, which was then used to transform Col-0 wt. F0 lines were screened on PNS with kanamycin for resistance. Kanamycin resistant lines were allowed to self and F1 lines were then screened again on kanamycin to identify homozygous lines. Images were taken with a Lecia SP2 confocal microscope and nuclei were counter stained with Hoescht 33342. All pBI101 constructs were electroporated into the Algo strain of Agrobacterium tumefaciens using a Bio-Rad MicroPulser following the manufacturer’s instructions. Colonies were screened for transformants on LB medium containing Kanamycin and Streptomycin and colonies that appeared to contain the plasmid were grown and tested via PCR for the insert. Genomic DNA from the Agrobacterium line was grown overnight in 1mL LB medium containing Kanamycin and Streptomycin and at 28°C. Cells were pelleted and resuspened in 0.1 mL of ice-cold Solution I . Cells were incubated for 5 minutes at room temperature. To the cell suspension, 30 μL of phenol equilibrated with two volumes of solution II was added and vortexed gently for a few seconds. To this mixture, 150 μL of 3 M sodium acetate, pH 4.8 was added and the tube was shaken briefly. The tube was incubated at -20°C for 15 minutes, centrifuged for 3 minutes and then supernatant was poured into a new 1.5 mL tube. The tube was filled with ice-cold 95% ethanol, mixed by inversion and stored at – 80°C for 15 minutes. The tube was centrifuged for 3 minutes and the supernatant was discarded. To the pellet, 0.5 mL of 0.3 M sodium acetate, pH 7.0 was added and the pellet was resuspended. The tube was filled with ice-cold 95% ethanol and mixed well by inversion. The tube was then stored at -80°C for 15 minutes. The tube was centrifuged for 3 minutes, the supernatant was decanted and the tube was inverted on a paper towel to drain remaining supernatant. To the tube, 1 mL of icecold 70% ethanol was added, the tube was vortexed briefly and centrifuged for 1 minute. The pellet was resuspended in 50 μL of TE . DNA was then screened for by PCR using original cloning primers to detect complete SUV2:GUS or SUV2:GFP constructs. Flowering Col-0 wild type Arabidopsis thaliana was transformed with Agrobacterium lines that were confirmed to contain the appropriate plasmid. Agrobacterium lines carrying the appropriate plasmid were grown over night at 30°C in 500 mL of LB with appropriate antibiotics from a 5 mL starter culture. The overnight culture was pelleted at 5,000 rpm in a GSA rotor for 10 minutes.

Phosphorylation patterns were visualized by autoradiography

Root tips were subsequently visualized using confocal microscopy at the same magnification for each. Al treatment results in substantial increases in both cell and nuclear size for als3-1 roots, which is consistent with terminal differentiation in conjunction with endore duplication . In contrast, atr-4;als3-1, alt2-1;als3-1, and sog1- 7;als3-1 roots did not show the dramatic Al-dependent increases in cell and nuclear size as seen for Al-treated als3-1 roots . This shows that all three suppressor mutants block the Al hypersensitivity of als3-1 in conjunction with prevention of terminal differentiation and endore duplication.Since loss-of-function mutants for SOG1 and ATR are phenotypically similar with regard to Al tolerance , it might be expected that these two cell cycle checkpoint factors act together to trigger Al-dependent terminal differentiation of the root. In order to test whether there is a relationship between these two factors in Al-dependent stoppage of root growth, a sog1-7;atr-4 mutant was generated and tested for its capability to grow in the presence of Al. For this experiment, Col-0 wild type, sog1-7, atr-4, and sog1-7;atr-4 were grown for 7 days in the absence or presence of 1.50 mM AlCl3 in a soaked gel environment. Root lengths were measured and the sog1-7;atr-4 double mutant was comparable to sog1-7 and atr-4 for Al tolerance . This result suggests that SOG1 and ATR are part of the same pathway that halts root growth following exposure to Al.

Al toxicity is most pronounced at the root tip ,square flower bucket and since SOG1 works in conjunction with other factors to shut down root grown in the presence of Al, it was of interest to determine the tissue localization pattern for SOG1. For this analysis, a previously reported transgenic Arabidopsis line carrying a SOG1:GUS fusion construct was grown in the absence or presence of 1.50 mM AlCl3 in a soaked gel environment. After 7 days of growth, GUS activity in seedlings was assessed. GUS activity was clearly observed throughout the root tip, which is consistent with the role of SOG1 in promoting terminal differentiation of the root tip following Al exposure . In contrast, root tips treated with Al for 7 days showed no GUS activity. This indicates that SOG1 does not persist after a root has terminally differentiated following Al treatment. Since it was determined that ATR and SOG1 act within the same pathway, SOG1:GUS was introgressed into the atr-4 mutant. Loss of SOG1 expression in the presence of inhibitory levels of Al is apparently ATR-dependent since SOG1:GUS is maintained in root tips of the loss-of-function atr-4 mutant even following Al treatment . Even though effects of Al toxicity severely compromise root growth, the terminal differentiation seen in Al-treated roots is not associated with tissue death as shown by Evan’s blue staining in previous studies .In order to determine the subcellular localization of SOG1, examination of a previously published SOG1:GFP translational fusion allowed for visual localization via confocal microscopy. Transgenic seedlings of Col- 0 wild type carrying native SOG1 promoter controlled SOG1:GFP fusion construct were grown in 0µM AlCl3 hydroponic growth media for 6 days, after which they were exposed to either 0 or 100µM AlCl3 for 48 hours.

Root tips were subsequently examined using confocal microscopy and treated with the nuclear specific stain, Hoechst 33342. In the absence of Al, SOG1:GFP is found at the perimeter of nuclei of root tip cells . Exposure of roots to a highly inhibitory concentration of Al in a hydroponic system for 48 hours results in either a redistribution of SOG1:GFP to the interior of the nucleus or a morphological change to the nuclear architecture . While it is unclear what is occurring to affect the visualization of nuclear aggregation of SOG1 following Al treatment, analysis of SOG1:GFP localization in the atr-4 loss-of-function mutant shows that in both the absence and presence of 100µM AlCl3 in hydroponics, SOG1:GFP is maintained in a comparable fashion to SOG1:GFP in Col-0 wild type without exposure to Al . Despite the uncertainty of the nature of the change in SOG1:GFP visualization in response to Al, this result does indicate that this change is dependent on ATR.ATR is a member of the phosphoinositide 3-kinase-like kinase family and major regulator in the DNA damage response in mammalian systems . Since there is an apparent functional relationship between ATR and SOG1 with regard to terminal differentiation of the root tip following Al exposure, it was of interest to determine whether SOG1 is a phosphorylation target of ATR. For this analysis, the entire coding sequence of Arabidopsis ATR representing 2702 amino acids was produced as a GST fusion protein in a baculovirus protein expression system. In conjunction with this, the entire CDS of Arabidopsis SOG1, representing 449 amino acids, was produced as a Maltose Binding Protein fusion in an Escherichia coli BL21-DE3 pLysS protein expression system. Approximately 50 ng of GST-ATR was subsequently incubated with 1 mg of either MBP or MBP-SOG1 in the presence of [γ- 32P] ATP, after which samples were separated on an SDS-PAGE gel.

While incubation of MBP with GST-ATR did not result in measurable phosphorylation of MBP, MBP-SOG1 incubated with GST-ATR resulted in a distinct radiolabeled band that was the same size as that predicted for MBP-SOG1 . At least in vitro, SOG1 is a phosphorylation target of the Arabidopsis ATR kinase. While Al treatment has been associated with upregulation of a large group of genes in multiple plant model systems , it has been difficult to identify which members of these Al-inducible groups are of primary relevance to Al toxicity and response. Therefore, demonstration that SOG1 is responsible at least in part for stoppage of root growth following chronic exposure to Al is expected to allow for determination of which Al-inducible genes are central to Al-dependent terminal differentiation. With this in mind, it was of interest to determine whether Al results in similar SOG1-dependent changes in gene expression as seen with γ-radiation . Several genes have been found to be substantially upregulated following exposure to γ-radiation, including many that are involved in response to and repair of damaged DNA. Examples include BRCA1, PARP2, XRI1, RAD50, and RAD51, along with a number of genes whose functions in relation to DNA damage response have yet to be elucidated . In order to determine whether Al causes changes in expression of these SOG1-regulated genes, it was first necessary to determine the conditions that would allow for the best capture of these changes. This was particularly problematic since SOG1 does not persist after Al-dependent terminal differentiation , making it necessary to determine at which point damage had accumulated to a high enough level to promote entrance into endore duplication but not late enough to where the transition had already been initiated. For this work, SOG1:GUS expression in the root tip was followed over a time course of Al exposure. Col-0 wild- type transgenic plants expressing SOG1:GUS were grown in the presence of 1.50 mM AlCl3 in a soaked gel environment, and samples were taken on successive days for visualization of GUS activity. GUS activity persisted in the root tip throughout the course of the experiment for untreated samples,black flower bucket whereas growth of roots in the presence of Al resulted in a progressive loss of GUS activity starting 3 days after planting . It was also necessary to assess the status of the root QC on a daily basis through the use of the QC46:GUS reporter line. Consistent with the loss of SOG1:GUS activity, the root QC disappeared by day 5 of growth in the presence of 1.50 mM AlCl3 in a soaked gel environment with a substantial decrease in GUS activity occurring between days 3 and 4 . When considered in conjunction with the results from the SOG1:GUS time course, the transition from an actively growing root tip to one that has transitioned to endore duplication occurs between days 3 and 4 of chronic Al exposure, indicating that SOG1-dependent increases in gene expression in response to Al would be most likely observed within this 3 to 4 day window. Because of this, seedling tissue was collected after 3 days of exposure to Al to assess whether Al causes upregulation of genes in a similar SOG1-dependent manner as seen for γ-radiation. For this experiment, Col-0 wild-type, als3-1, sog1-7, and sog1-7;als3-1 seedlings were grown in the absence or presence of 1.50 mM AlCl3 in a soaked gel environment for 3 days.

Subsequently, whole seedlings were harvested for isolation of total RNA, cDNA synthesis, and subsequent RT-PCR analysis. For this experimental approach, a normalization gene needed to be selected that acts as a house keeping gene, which is expressed consistently in different genetic lines in the absence and presence of Al. Generally, genes encoding cytoskeletal elements are often used; however, as discussed previously, Al disrupts micro-tubule and actin dynamics so this was not an option . Based on a previous study, At5g60390 was selected due to its consistent expression under other heavy metal toxicities such as nickel and copper . To ensure that EF-1 α would be an ideal normalization gene candidate, seedlings of Col-0 wild-type, als3-1, sog1-7, and sog1-7;als3-1 were grown in the absence or presence of 1.50 mM AlCl3 in a soaked gel environment for 3 days. Subsequently, whole seedlings were harvested for isolation of total RNA, cDNA synthesis, and subsequent RT-PCR analysis. All genetic lines under all test conditions showed equivalent expression . Several genes that have been found in a previous study to be highly induced by γ-radiation in a SOG1-dependent manner were used to perform a survey with regard to Al response . Genes tested included those encoding a Zn finger of unknown function , a protein with an unknown role in DNA damage repair , a putative ubiquitin conjugating enzyme , a putative telomere maintenance protein , an ortholog of the human breast cancer susceptibility gene , and a key component of microhomology-mediated DNA repair . Primers for these genes were generated and tested for parameters essential to accurate RT-PCR analysis including single amplicon replication verified via melts curves and amplification efficiencies . Treatment with Al resulted in a measurable increase in expression of this subset of genes in Col-0 wild type compared with no Al . In Al-treated als3-1 seedlings, the expression of these genes also increased significantly, even more so in comparison to Col-0 wild type , consistent with the extreme Al hypersensitivity seen for this mutant. In contrast, an increase in expression of these genes was not observed for sog1-7 and sog1-7;als3-1 roots in comparison to the respective controls . This result indicates that Al triggers a SOG1-dependent transcriptional program that is similar to that observed following treatment with γ-radiation. SOG1 has been demonstrated previously to function downstream of ATM in response to γ-radiation . ATR and ATM are both members of the PIKK family of kinases, and now that ATR has been established to work in conjunction with SOG1 to inhibit root growth in the presence of Al, it was of interest to determine whether Al-responsive stoppage of root growth also required ATM. An atm-2;als3-1 double mutant was generated . The capability of the atm-2 loss-of-function mutation to suppress the Al hypersensitivity of als3-1 was compared with that of atr-4. Seedlings of Col-0 wild type, als3-1, atr-4;als3-1, and atm-2;als3-1 were grown for 7 days in the absence or presence of 0.75 mM AlCl3 in a soaked gel environment . Exposure to Al resulted in severe Al hypersensitivity in als3-1 roots compared with Col-0 wild type, whereas atr-4;als3-1 mutant roots were indistinguishable from Col-0 wild type in the presence of Al . In contrast, Al-treated roots of atm-2;als3-1 were only marginally longer than those of als3-1, with both displaying the same terminal differentiation phenotype following Al exposure . Because there is a clear discrepancy regarding the roles of ATR and ATM in mediating stoppage of root growth following exposure to Al, it was determined whether loss-of-function mutations for each had an impact on SOG1-dependent expression of genes following Al exposure. For this analysis, Col-0 wild type, als3-1, sog1-7;als3-1, alt2-1;als3-1, atr-4;als3-1, and atm-2;als3-1 were grown in the absence or presence of 1.50 mM AlCl3 in a soaked gel environment for 3 days.

Even harvesting methods can affect the rate of acidification of the soil solution

We first assessed if the 23 genes up-regulated in elf18 treated EFR:XA21:GFP could be validated by qPCR analysis. Eleven out of 23 DRGs were up-regulated in EFR:XA21:GFP rice leaves after elf18 treatment. Transcripts of the remaining 12 candidate genes were detectable by qPCR amplification but were not up-regulated in elf18 treated EFR:XA21:GFP leaves . We established a detached leaf infection assay to test if genes identified in the EFR:XA21:GFP experiments are representative of genes differentially regulated in Xoo infected Myc:XA21 rice. We observed bacterial ooze from the detached rice leaves three days after inoculation with Xoo strain PXO99A . To further assess if Xoo infects rice leaves in our detached leaf infection assay, we measured the expression level of Os8N3 , which was previously shown to be up-regulated in rice upon Xoo infection and is thus a useful marker of successful infection . For these experiments, we also included a mutant PXO99A strain that is unable to infect rice as a control. The hrpA1 gene encodes a pilus protein that is essential for type IIIsecretion of effectors required for host infection . We observed that the PXO99A1 hrpA1 Xoo mutant is unable to infect Kitaake and Myc:XA21 rice plants . Both WT Kitaake and Myc:XA21 detached leaves express Os8N3 at higher levels compared to mock treatments 24 hpi with WT PXO99A, but not with PXO99A1 hrpA1 .

These results demonstrate that Xoo infects detached rice leaves. We next employed the detached leaf infection assay to examine the expression of the stress-related marker PR10b in Xoo infected Myc:XA21 rice leaves. Compared with mock treated controls,flower bucket PR10b is up-regulated in flg22 treated rice, elf18 treated EFR:XA21:GFP rice and Myc:XA21 rice treated with the RaxX21-sY . Using qPCR, we detected significant up-regulation of PR10b expression in Myc:XA21 rice leaves 24 hpi with PXO99A and PXO99A1 hrpA1. PR10bup-regulation was not observed in infected Kitaake leaves . These results show that the detached leaf infection assay can be used to assess XA21-mediated marker gene expression and also indicate that RaxX expression or secretion is not affected by the 1 hrpA1 mutation. In this study we identified 8 genes that are specifically up-regulated in both elf18 treated EFR:XA21:GFP and Xoo infected detached Myc:XA21 rice leaves. At the time of these experiments, the activator of XA21, RaxX, had not yet been identified . We therefore treated rice plants expressing the EFR:XA21:GFP chimera with elf18 to identify candidate marker genes because EFR:XA21:GFP rice are partially resistant to Xoo and respond to elf18 treatments as described above in the introduction. Our results show that even though the EFR:XA21:GFP-mediated response does not confer robust resistance to Xoo , similar genes are up-regulated during both EFR:XA21:GFP- and Myc:XA21-mediated responses .

Further studies are necessary to determine why the expression of EFR:XA21:GFP in rice does not confer robust resistance to Xoo. We show that stress-related gene induction of PR10b in Myc:XA21 rice leaves is maintained in plants inoculated with PXO99A1 hrpA1 mutant strains. These results suggest that RaxX expression, modification and secretion is not compromised by the 1 hrpA1 mutation. These results indicate that RaxX function is independent of type-III secretion mediated by hrpA1. It was previously reported that the raxSTAB operon, which encoded predicted components of a type-I secretion system, was required for the processing and secretion of the XA21 elicitor . Our finding that RaxX function is independent of hrpA1-mediated type-III secretion is consistent with the hypothesis that RaxX is a type I-secreted molecule and may provide insight into the largely unknown biological function of RaxX. The discovery of RaxX and the establishment of the detached leaf infection assay described here provide useful tools for studying XA21-mediated immunity. XA21 activation can be measured through ROS production and marker gene expression in detached leaves treated with the RaxX21-sY peptide . One advantage of this approach is that researchers can study XA21-mediated immunity without working with Xoo. Instead, researchers can activate XA21-mediated immunity by treating leaves with RaxX21-sY peptide rather than Xoo. This strategy eliminates the need for select agent permits, which are costly and time-consuming.

The assay described in this study now allows researchers to use Xoo infected plants to monitor XA21 activation by gene expression, which was previously only possible using peptide treatments. This provides the benefit of monitoring bacterial induced genes, such as Os8N3 . While we are not able to definitively assess resistance versus susceptibility to Xoo using this assay, we demonstrate that we can use gene expression to monitor an immune response specifically mediated by XA21. The detached leaf infection assay can also be used for other studies of bacterial-rice interactions. For example, this system can be used to study rice immune responses conferred by different resistance genes or induced by different bacterial strains. For example, the detached leaf infection assay can be used to study the immune response conferred by other rice Xa genes that confer resistance to Xoo such as Xa3/Xa26, which also encodes a cell surface receptor kinase . The detached leaf infection assay can also be adapted to study immune responses to other races of Xooor other Xanthomonas pathovars such as Xanthomonas oryzae pv. oryzicola .Throughout every organism’s lifecycle, exposure to a variety of biotic and abiotic stresses can threaten their survival. Unlike motile animals that can relocate to avoid certain threats, plants are sessile organisms that must cope with external stresses to a much greater extent than mobile organisms. During the course of their evolution, the development of complex mechanisms has allowed plants to grow under unfavorable and even hostile conditions. The majority of plant species are rooted in the soil; thus the quality of the soil greatly influences the virility of plants. From the soil, plant roots must absorb sufficient water and mineral nutrients to sustain both their underground and above ground organs. While plants are dependent on the nutrients provided in soils in order to grow, they are adversely affected by undesirable compounds in the soil such as salinity,plastic flower bucket extremes of pH, and toxic substances. Aluminum is a detrimental metal ion in soils around the globe that drastically reduces root growth with a concomitant reduction of shoot growth. This negatively impacts global crop growth due to this failure of the roots to support the above ground organs with the water and nutrients needed in order for the whole plant to thrive. The phytotoxicity of Al is dependent on soil pH, and while Al is abundant in soils all around the globe, Al toxicity is confined to acidic soils. The mechanisms of Al resistance are of great interest to better understand how plants, especially crop species, can grow on Al toxic soils in order to feed a growing global population.Al toxicity is a global agricultural problem and is one of the predominant factors that limit crop productivity in acidic soil regions. Acidic soils have a pH < 5.5 at the surface layer and comprise greater than 30% of the land in the world , with varying estimates having 50-70% of the potentially arable lands in the acidic range . While plant growth inhibition in acidic soils is due to a combination of mineral toxicities as well as nutrient deficiencies, Al toxicity is the single most important factor inhibiting plant growth and the major constraint for crop production . Al is the most abundant metal in the earth’s crust and the third most prevalent element on earth behind oxygen and silicon .

The majority of Al is contained within aluminosilicate compounds in the primary mineral, or as Al oxides and aluminosilicates in secondary minerals, which are formed during weathering . However, Al in these forms is not phytotoxic and only a small percentage of Al in the soil solution contributes to Al toxicity in plants . It is the soluble Al ion, Al3+, that is primarily responsible for plant growth inhibition due to Al toxicity. Al solubility is dependent on the pH of the soil solution; therefore Al toxic soils are confined to acid soil environments. Al can become dissociated from the primary and secondary minerals in acidic conditions, which can add to soil acidity. In acid soils, soluble Al speciates into the toxic trivalent cation, Al3+, at concentrations that rapidly inhibit root growth. As the pH decreases further, the concentration of Al in the soil solution increases. Between pH of 4.0 to 4.5 in the soil solution, a small change in the acidity can cause large changes in the solubility of Al . For example, at pH <4.2, Al3+ can be found at extremely high concentrations ranging from 0.1 to 1.0 mmol/L . Even though Al exists in many forms such as monovalent, divalent and trivalent Al ions, Al oxides, Al sulfates, Al fluorides and the complex Al13, only Al3+ and the Al13 complex have been proven to be toxic to plants . Of these forms, Al3+ is the most prevalent species in soils that are pH < 4.7, and is therefore the most significant form for Al phytotoxicity. As such, from here out the elemental abbreviation Al will represent Al3+ in regards to phytotoxicity unless otherwise specified. Soil acidity is determined by the concentration of hydrogen ions in the soil solution. A variety of factors both natural and manmade affect soil acidity including the innate characteristic of the soil, farming practices, as well as biological and environmental changes. Decay of organic material that forms carbonic acid and other weak acids can add to the acidity of the soil solution . The amount of rainfall can affect the rate of soil acidification depending on the rate of rain water flow through the soil solution . Also, soils that develop from granite materials acidify faster than soils that have developed from calcareous materials. Sandy soils also acidify more rapidly due to higher leaching of alkalinizing agents and reduced buffering capacity . Utilization of high-input farming practices is common in industrialized nations that include the overuse of ammonia-based fertilizers, which cause further acidification of agricultural soils . Over cropping can also lead to the depletion of essential nutrient cations from the soil solution such as calcium, potassium, magnesium and sodium .For example, removal of the straw from wheat cropping depletes basic cations to a great extent and further enhances acidification by nitrification . While the cumulative impact of these factors may be difficult to quantify over the course of a few years, it is important to acknowledge that other possible factors could additionally be contributing to the rapid acidification of the soil solutions. Originally Al toxicity was considered to be restricted to tropical agricultural regions, or areas of high rainfall with highly weathered soils; but soil acidity has now attracted global attention. Acid soils occur primarily in two geographic humidity belts: a cool-climate northern belt spanning North America, Europe, and Northern Asia, and a warm-climate southern tropical belt spanning Central and South America, Sub-Saharan Africa, South East Asia including its islands and Australia . In addition, two thirds of the acid soils are found primarily in forested areas and 17% are covered by prairie, savanna, or steppe vegetation while only 6% of the world’s acid soils are used for agriculture, indicating that acid soils severely compromise agricultural production . Because acid soils are detrimental to crop production, especially several cereal species, the widespread occurrence of acid soils serves as a severe limitation on agricultural production in these geographic belts. Often the practice of liming surface soils is used in industrialized countries to ameliorate soil acidity, and therefore Al toxicity; however this practice is neither affordable nor effective as a long-term practice. The bulk of agricultural lime is derived from limestone but is also sourced from marl, slag from iron and steel making, flue dust from cement plants, and refuse from sugar beet factories, paper mills, calcium carbide plants, rock wool plants, and water softening plants . However total production and use of lime is relatively low and is generally only applied to farming areas near the source, ie industrialized areas. Lime is either sprinkled onto soils to be mixed in during tillage operations or dissolved in water where it hydrolyzes to form –OH ions to raise soil pH, but only at the surface of soils .

The energy of the incident X-ray beam was adjusted at 30 keV to excite the K-edge of Cd

A mix of accumulation and suppression of terpenoids and an increase of methoxyphenols was observed in the leaves of ANE-root-treated plants. Chemical enrichment analyses broadly revealed classes of metabolites that were induced or suppressed in AA- or ANE-treated plants. To examine which specific variables provide the strongest discriminatory power between the two treatment groups, a two-group comparative supervised multivariate analysis, orthogonal projections to latent structures discriminant analysis , was utilized. OPLS-DA score plots show strong between group variability discrimination between AA and ANE treatment groups compared to the H2O control across all tested time points with the x-axis describing the inter-treatment variability, and the y-axis showing the intra-treatment variability . S-plots derived from OPLS-DA were examined for both AA and ANE treatments in pairwise comparison with H2O control. S-plots of OPLS-DA revealed that treatment with AA or ANE induced shared changes in the levels of several defense-related metabolites in roots . Variables with the most negative and positive correlation and covariance values are the most influential in the model. These metabolites are located on either tail of the S-plot and contribute most greatly to the separation between treatment groups Bar charts depicting mean LC-MS signals for top OPLS-DA S-plot metabolites visualized across all time points illustrate that AA and ANE have similar effects on plant metabolic response .

Treatment of tomato roots with AA and ANE resulted in a sharp increase in metabolic intermediates in ligno-suberin biosynthesis. This includes AA-induced accumulation of moupinamide and significant increases in coniferyl alcohol in the roots of ANE-treated plants across all tested time points. In roots, AA and ANE treatments also induced increased levels of N-ethyl phydroxycinnamide and N1-trans-feruloylagmatine compared to H2O treatment,wholesale plant containers reflecting strong upregulation of the shikimate pathway and phenolic compound synthesis. Reduced levels of tomatine and dehydrotomatine were observed in the roots of AA- and ANE-treated plants indicating suppression of steroid glycoalkaloid biosynthesis. Treated plants also showed lower levels of lyso-phosphatidyl ethanolamine that could reflect enhanced membrane lipid turnover. AA and ANE can induce disease resistance locally and systemically, alter the accumulation of key phytohormones, and change the transcriptional profile of tomato with a striking level of overlap between the two treatments . The current study examined and characterized the AA- and ANE-induced metabolomes of tomato. AA and ANE locally and systemically induce metabolome remodeling toward defense-associated metabolic features. Early studies investigating transcriptional and metabolic changes in potato revealed selective partitioning and shifting of terpenoid biosynthesis from steroidal glycoalkaloids to sesquiterpenes following treatment with AA or EPA or challenged with P. infestans . Similarly, our work here with AA- and ANE-treated tomato seedlings has shown a marked decrease in the levels of two abundant glycoalkaloids, tomatine and dehydrotomatine .

Our data also show strong enrichment of sesquiterpenes in leaves of AA-treated plants at 96 hours post treatment, although the identity of these sesquiterpenes is unresolved . This work further supports evidence for differential regulation and sub-functionalization of sterol/ glycoalkaloid and sesquiterpene biosynthetic pathways in solanaceous plants in different stress contexts . AA and EPA are strong elicitors that are abundant in structural and storage lipids of oomycete pathogens, but absent from higher plants. Although their initial perception by the plant is likely different from that of canonical MAMPs , there is some convergence in downstream defenses induced by these various MAMPs. Work to characterize the effect of canonical MAMP treatment on the metabolomes of various plant species has implicated common metabolic changes that prime for defense. Cells and leaf tissue of A. thaliana treated with lipopolysaccharide showed enrichment of phenylpropanoid pathway metabolites, including cinnamic acid derivatives and glycosides . In the same study, SA and JA were also positively correlated with LPS treatment, as we also observed in tomato following treatment with AA . Recent work in A. thaliana wild-type and receptor mutants treated with two chemotypes of LPS showed increases in hydroxycinnamic acid and derivatives and enrichment of the associated phenylpropanoid pathway . Work in tobacco similarly found treatment with LPS, chitosan, and flg22 all induced accumulation of hydroxycinnamic acid and derivatives, and that defense responses elicited by these MAMPs were modulated by both SA and JA . More recent work in the cells of Sorghum bicolor treated with LPS showed enrichment of hydroxycinnamic acids and other phenylpropanoids in coordination with accumulation of both SA and JA . Treatment of tomato with flg22 and flgII-28 also enriched hydroxycinnamic acids, and tomato treatment with cps22 revealed a metabolic shift toward the phenylpropanoid pathway with hydroxycinnamic acid, conjugates and derivatives as key biomarkers .

Similar to traditional MAMPs, AA and the AA/EPA-containing complex mixture, ANE, both induce enrichment of cinnamic acid and derivatives in tomato seedlings . This supports the hypothesis that MAMPs broadly induce similar metabolic changes to enrich pools of specialized secondary metabolites that contribute to plant immunity. AA- and ANE-treated roots showed strong enrichment of metabolic features classified as fatty acyl glycosides of mono- and disaccharides . Fatty acyl glycosides have been studied in several plant families and are most extensively characterized in members of Solanaceae . Investigations into the function of fatty acyl glycosides in plants suggest they may act to protect against insect herbivory through various mechanisms and provide protection against fungal pathogens . A recent study isolated and identified fatty acyl glycosides from strawberry capable of inducing immune responses in A. thaliana, including ROS burst, callose deposition, increased expression of defense-related genes, and induced resistance to bacterial and fungal challenge . This same work also demonstrated that the strawberry-derived fatty acyl glycosides induced resistance in soybean and, due to their antimicrobial activity, also protected lemon fruits post harvest from fungal infection . AA- and ANE-root treatments locally elicit accumulation of the same class of defense associated metabolites that Grellet et al. illustrated to have direct antimicrobial activity and protect against disease . Cell wall fortification is an important plant defense often initiated upon pathogen infection. Cell wall lignification is a well-studied mechanism with localized accumulation of phenolic intermediates and lignin at attempted penetration sites . Lignification reinforces and rigidifies the cell wall to create an impervious barrier to microbial ingress . In our study, AA treatment of tomato roots induced accumulation of a phenylcoumaran intermediate in lignin biosynthesis, while ANE treatment induced accumulation of coniferyl alcohol, an important monomer unit of lignin. Interestingly, coniferyl alcohol has recently been shown to act in a signaling capacity in a regulatory feedback mechanism to intricately control lignin biosynthesis, an irreversible process that is energetically costly . The findings of our study coincide with the well-characterized role of lignin and its intermediates in plant defense. This work characterizes local and systemic metabolic profiles of AA- and ANE-treated tomato with the oomycete-derived MAMP, AA,plastic pot manufacturers and the AA-containing biostimulant, ANE. AA and ANE profoundly alter the tomato metabolome toward defense-associated secondary metabolites with notable overlap in enriched metabolite classes compared to H2O control. Further investigation is required to elucidate the functional contribution of these metabolic features in AA- and ANE induced resistance and, more broadly, plant immunity. Our studyadds to the understanding of MAMP-induced metabolomes with implications for further development of seaweed-derived biostimulants for crop improvement.Although the major N forms available to crop plants in most agricultural soils are ammonium and nitrate, urea is found ubiquitously in the soil and additionally represents the most widespread form of nitrogen fertilizer used in agricultural plant production. Due to its fast hydrolysis by urease the concentration of urea in agricultural soils is in general below 70 µM and most of the urea derived nitrogen is thought to be taken up from the soil solution in the form of ammonium .

However, biphasic uptake kinetics of labelled urea in Chara cells indicated that membrane transport systems might facilitate the uptake of urea into plant cells . By heterologous expression of plant genes in yeast and frog oocytes, two different transport systems were identified, that allow the membrane permeation of urea. Several aquaporins of the TIP, NIP and PIP families have been identified as low affinity transport systems , while AtDUR3 encodes a H+ /urea cotransporter , that facilitates high affinity urea transport in heterologous systems. In addition to externally supplied urea, internal production of urea represents an important intermediate of the N metabolism in higher plants. Urea is amongst others increasingly generated during protein degradation, a process of particular importance for the retranslocation of nitrogen during plant senescence. In senescence the earliest and most drastic change in plant cellular structures is the breakdown of chloroplasts, which hold the majority of the leaf protein . The degradation of these photosynthetic proteins results in the generation of mostly free amino acids, in particular arginine. This is assimilated in the mitochondria within the ornithine cycle by degradation into ornithine and urea . Urease is thereby the only enzyme known in plants that is able to recapture nitrogen from urea, which otherwise would remain an unavailable N source. It has been localized to the cytoplasm and was detected in particular in generative tissues of plants , although it seems to be synthesized in almost all organs . The product of the urease reaction, ammonia, is then assimilated via the GS-GOGAT pathway into glutamine . To date it is unclear if urea also has a meaning for short-term storage and long-distance transport of N. Cadmium is one of the most widespread hazardous metals found in agricultural soils. In a market-basket study conducted in Japan, 7% of 381 eggplant samples were found to exceed the maximum Cd concentration for fruiting vegetables recommended by codex Alimentarius Commissions. These results indicate the urgent requirement for cultivation systems that reduce the Cd concentration in eggplant. Grafting eggplant Solanum melongena onto Solanum torvum has been reported to be an effective method for reducing the Cd concentration in the fruits of eggplant by up to one-fourth.1). The Cd concentration in the shoot and xylem sap of S. melongena was higher than that of S. torvum; however, the concentration in the roots of both species was almost identical.2). It was likely that the limited translocation of Cd into the shoot of S. torvum was partly due to the presence of a barrier function in root tissues. In this study, we compared the microscale distribution pattern of Cd in root tissues of S. melongena and S. torvum by using the synchrotron micro-X-ray fluorescence technique and thus elucidated the role of the roots of S. torvum in restricting Cd transport to the shoot. Hydroponically cultivated S. melongena cv. Senryo 2 and S. torvum cv. Torubamubiga were exposed to CdCl2 for 24 h with fresh nutrient solution at pH 5.5 in a controlled environment chamber. From 5-mm root segments obtained at a distance of 10 and 40 mm from the root apex, 200-μm thick transverse sections were prepared using a microtome. The freshly prepared sections were frozen immediately by contacting them on an aluminum block with a mirrored surface that was pre-cooled by liquid nitrogen; the sections were subsequently freeze-dried. The freeze-dried sections were then mounted on an acrylic plate for SR m-XRF analysis. A few of the sections were stained with berberine hemisulfate/aniline blue.3) to identify the presence of Casparian bands by fluorescence microscopy. The distribution of Cd, Zn, and Fe in the transverse sections of the root was mapped using SR -XRF at BL37XU, SPring-8, Japan Synchrotron Radiation Research Institute. The beam spot size was 2.0 m wide and 1.0 m in height, and the scanning step size was 3 m. The spectral acquisition time was set to 2 s. Although copper-based engineered nanomaterials currently comprise a relatively small fraction of global ENM production ,1 their toxicity and life cycle characteristics raise concerns regarding their environmental risk. For example, a common use for Cu-based ENMs is as the active ingredient in marine antifouling paints or agricultural biocides,2 where they are directly introduced into the environment as intentionally toxic substances. Copper based ENMs are somewhat unique among the most widely used ENMs in that they can participate in redox reactions to form three oxidation states: Cu0 , Cu1+, and Cu2+. Copper can also participate in a number of inorganic complexes with compounds found in natural waters, such as sulfate, sulfide, phosphate, chloride, and carbonate.

Neither the developer nor the local government had this sort of financial capacity

For example, Jinhu villagers consider the Guoqiang company’s waste of arable land to be incomprehensible. Thus, a few peasants tried to grow rice on fallow land they had previously plowed. However, they were forced to stop. The “subsistence ethic” is evident in Mrs. Tang’s husband and father-in-law, both of whom believe that a peasant has a natural right to cultivate fallow land not being exploited by another villager. The Guoqiang company, by contrast, has argued that, in accordance with the legally-binding contracts signed by the peasants when they handed over their land use rights, the company had the authority to decide what to do with the land, even if this meant leaving it unused for a long period of time. Finally, we should consider the middle class urbanites who have visited Jinhu Rural World as tourists. In general, they are looking for adventure and entertainment. Although there are some complaints about overcrowding,macetas de 30 litros disorderliness, and unprofessional behavior among staff members, most on-line reviews and comments about Jinhu Rural World are positive.

Many urban clients—often young couples with one child and a car—were excited that Jinhu Rural World theme park combined entertainment with a concept of agricultural education, thus affirming the Guoqiang Conglomerate’s own advertisements. Since many Chinese are recent urbanites who only recently left the rural world in order to enjoy an urban modern life, they have apparently not yet developed a sense of nostalgia. Though Timothy Oak describes rural tourism as a “playground” for rich and middle-class urbanites, it seems to me that there is something else to be said about the relationship between urbanites and the countryside. The above quote reveals that a rural nostalgia ideology has a practical side that concerns food security in today’s China, daily concerns of ordinary urban Chinese following several toxic food and poison scandals in recent years.16 Thus, for example, a national brand of infant milk formula caused babies to die due to toxic additives in its formula. This incident aroused national skepticism toward food items purchased from the market in general. Health has now become a pertinent day-to-day issue. In sum, although the “New Countryside” campaign is a national policy imposed throughout China by the central government, it is too simplistic to see its implementation as merely a manifestation of top-down authoritarian rule. In reality, multiple actors, each with their own concerns and agendas, have participated in rural development. Needless to say, each group’s vision of rural development does not necessarily accord with the central government’s conception and overarching goals.

The process of envisioning and reenvisioning the “New Countryside” by the various actors inevitably leads to conflicts and contradictions. For example, one finds inconsistencies between the central government and local government on issues of implementation, between the central government’s concern for “national food security” and urban consumers’ concern for food safety and hygiene, and between the real estate developer and the peasants regarding the logic of land use. As one might imagine, there are also power differentials between the different actors, with the peasants on the ground probably having the least say in the process. With all these different actors and agendas, “Building a Socialist New Countryside” is by no means a straightforward process. Here, I describe the process of implementation in the Jinhu case, demonstrating how this process involves some form of negotiation between the different actors, and how this negotiation has manifested itself. Much of the initiative at Jinhu came originally from a well-connected local entrepreneur. According to the young assistant of this man, whom I interviewed in 2012, the boss in question was the executive president of the Guoqiang Conglomerate, which had initially been founded as a government-owned Township and Village Enterprise . This man was a provincial representative to the National People’s Congress.

Although representatives to the National People’s Congress generally do not in fact have political authority,he was clearly well connected and, moreover, would have been one of the first individuals in the county to hear about the “New Countryside” campaign and to learn how to secure state funding for relevant projects. According to the young assistant, when the eleventh five-year plan was first promulgated by the central government in 2005, the boss almost immediately sensed a business opportunity and spent two years mobilizing resources to plan the Jinhu Rural World. In 2007, after taking over a local “dragon head”agribusiness firm that produced agricultural and food products, he established a new subsidiary under the Guoqiang Conglomerate called the New Countryside Conglomerate . Although the young assistant provided a fairly coherent account of how Jinhu Rural World cameabout, it turns out that this is not the whole story. According to one of my peasant informants, there were actually two rounds of contracts signed between villagers and the developer, suggesting that the process was, in fact, more convoluted. Online information provides more details, indicating that the Jinhu project did indeed pass through two phases—an initial failure, followed by later success. According to the well-known virtual community forum called Tianya—which frequently exposes injustice and official corruption—even before getting involved in the New Countryside campaign, the Guoqiang Conglomerate initially rented 2000 mu for 18 years from the peasants, hoping to take advantage of a rumor that the area that is now Jinhu Rural World would be formally absorbed into the Wuhu metropolitan region, a thriving economic zone just to the north. Guoqiang was apparently gambling on a more than ten-fold increase in land value in the immediate future. According to the Tianya article, in order to evade paying large state land taxes, Guoqiang avoided getting the proper land conversion permits. The company was eventually accused of leasing land illegally, and was ordered by the county Department of State Land to cease their activities immediately. At this point, Guoqiang risked losing its entire initial investment. Seeking to turn the situation around,macetas de 5 litros the entrepreneur took advantage of political connections and spent large sums of money bribing local officials to allow Guoqiang to transform the Jinhu development project into one officially responding to the central government’s call for a New Countryside. Once the company convinced the local government to get involved in the project, it expanded the scope of the project site to transform it into a national showcase. Henceforth, what had been a simple act of real estate land speculation turned into the “Jinhu New Countryside Construction Experimental Site” . As we can see, the initial development plan at Jinhu failed when the entrepreneur and the company acted on their own. It became a much more successful project only after the entrepreneur’s interests were coordinated with those of local state officials. This sort of coordination is a critical element in understanding rural development in today’s China. More in depth coordination was also required between the entrepreneur, the local government, and the central government. According to the master plan of the Jinhu New Countryside Experimental Project, the project required an intensive capital investment of around 700 million to 900 million RMB. At the time, Guoqiang had less than 300 million on hand in total assets, and, thus, needed bank loans and government subsidies. Financing of such large amounts inevitably required central government not local government money.

Central government funding is distributed through “proposed projects” , managed and monitored by the Development and Reform Commission . In recent years, the central government has allocated large amounts of funds directed towards the modernization of agriculture, as well as to rural education, health care, social welfare, and other concerns of the New Countryside campaign. Any sponsor of a proposed project can fill out a detailed application form, subsequently endorsed by local government officials. Once a project is selected by the central government, the sponsors receive large government grants, as well as opportunities to take out special loans from state banks. It is clear that, in order to get state funding, local government support and coordination were required. As a corollary, once the local government had approved the project, the local government shared with Guoqiang all responsibility for the project’s failures or successes. In addition, the local government played another important role in the project. Not only did it facilitate the process of getting state funds, but also it played an equally important role as a broker between the real estate developer and local peasants. As the young assistant of the Guoqiang boss put it, there were many instances that “required the government to show its face” , for example, when it was necessary to persuade farmers to adhere to the company’s master plan in a timely fashion. It was always local officials and village cadres who went to each household to persuade residents to sign contracts. He further mentioned that the New Countryside Conglomerate included a “Jinhu Experimental Site Management Committee,” whose salaried members were simultaneously county government officials. Their most challenging task was to make sure the rural theme park project went smoothly, confronting peasants with the “face” of government when necessary. Whereas the local government at this point had a direct interest in Jinhu’s success, the central government did not have the same stake in the project. It is quite clear that, as a consequence, Guoqiang continued to work hard to portray the Jinhu development site as a project aligned with the Chinese central government’s macroeconomic concerns regarding rural development, including the issues of “national food security” and ecological sustainability. In some cases, this has involved changes with concrete effects on the ground. For example, I was told by both peasants and staff members of the Guoqiang Conglomerate that the first resettlement housing style consisted of relatively attractive townhouses designed to lure peasants to want to move there. However, Guoqiang was later ordered by the central government—the Ministry of State Land Resources specifically—to stop wasting land. Consequently, for the sake of saving land to help achieve “national food security,” Guoqiang began to build apartment buildings six to seven floors in height in all new constructions in Jinhu New Village. The Jinhu case is not unusual. In fact, I have witnessed other high-rise apartment buildings in new villages across the countryside.Another strategy did not involve actual changes to plans, but rather the deployment of the central government’s rhetoric to describe preexisting projects. Thus, Guoqiang in conjunction with local state officials developed a strategy that adopted key terminologies relevant to central government policies, while at the same time achieving their own goals of company profits and local economic growth. The most common approach was simply to exploit the campaign slogan “Building a New Socialist Countryside” when seeking to justify a particular local development project. Another popular term is “ecological,” as improvements to the environment now constitute an important concern of the government. This phenomenon helps explain why references to the concept of “eco-friendly,” “low carbon,” and “sustainability” are encountered on publicity billboards all over the Rural World theme park. For example, inside the theme park, an “eco-restaurant” proudly advertises in the park’s brochure as one of the core attractions. The “eco-restaurant” is walled and roofed with transparent materials, thus in principle saving energy by using natural light. Yet, inside the “eco-restaurant” —which is filled with plastic cherry trees covered in pink and white blossoms, as well as plastic green banyan trees—the use of the “eco” language is primarily a rhetorical game. There is nothing ecological about fake trees. As in the West, the word “eco” is a buzzword to be exploited in the political and economic arena. In fact, although Jinhu Rural World is said to be the first “low-carbon” “eco-tourist” site, tourists in on-line comments have complained that they found garbage strewn about everywhere, especially inside the crowded tsunami water dome. Even after Guoqiang managed to work things out to the satisfaction of both local and central government authorities, numerous issues remained to be negotiated with local peasants. It is quite clear that the final arrangement reached with the peasants was economically disadvantageous to the peasants. For instance, according to my informants, residents were given 100 RMB per square meter for their old houses. The houses in the New Village, however, were more expensive, at 500 RMB per square meter. In addition, there were numerous additional costs associated with the relocation.