Tag: vertical farming

Human pathogen contamination of produce was the leading cause of food borne illnesses and outbreaks associated with a single-ingredient commodity between 2004 and 2013 . Lack of visual evidence that indicates the presence of contamination on produce contributes to the estimated 9.4 million cases of food borne illness in the United States annually . Various pathogen groups and toxins can be causal agents of food borne illness associated with produce; however, non-typhoidal Salmonella ranks as the second leading cause of all illnesses associated with consumption of produce . In a pre-harvest setting, enteric pathogenic bacteria are introduced to fresh produce through many routes, including low quality irrigation water, use of contaminated organic fertilizers, close proximity to livestock operations, wildlife intrusions, improper worker hygiene, or contaminated equipment . Once on the leaf surface, bacteria are faced with harsh conditions, such as UV irradiation, low nutrient and water availability, and unfavorable weather . Bacteria may escape these conditions by attaching to the leaf surface and forming bio-films or by transitioning to an endophytic lifestyle through internalization into the leaf extracellular space via natural pores or wounds . While leaf internalization is likely to confer some protection to the bacteria, it is not without some disadvantages. Plants can detect endophytic bacteria in the apoplast through pattern recognition receptors localized at the cell membrane that perceive conserved microbial molecules known as pathogen- or microbe-associated molecular patterns . PRR-PAMP binding leads to initiation of PAMP-triggered immunity , which functions to prevent further internalization of bacteria and to eradicate those that have already entered the apoplast . This suggests that internalization trades one challenge for another ,black plastic planting pots and only bacteria that can cope with these challenges will be able to colonize leaves successfully.

Previous studies have shown that Salmonella spp. interact with plants in a sophisticated manner, although the exact mechanisms are not fully understood . For instance, similar to some plant pathogens, Salmonella enterica serovar Typhimurium SL1344 can modulate stomatal movement in Arabidopsis leaves, where it induces a transient stomatal closure and re-opening at 4 h post incubation . Stomatal closure can diminish bacterial internalization and subsequent contamination of internal leaf tissues. The mechanism for stomatal re-opening by the phytopathogen Pseudomonas syringae pv. tomato is through the action of coronatine , a polyketide phytotoxin . However, the genomes of STm strains LT2 and 14028s do not encode genes for coronatine synthesis . Furthermore, stomatal re-opening is not a ubiquitous response to human pathogens. For instance, Escherichia coli O157:H7 induces a lasting stomatal closure in lettuce and Arabidopsis for at least 4 and 8 h, respectively . Beyond the ability to modulate stomatal movement, STm SL1344 can survive at a higher titer within the apoplast of Arabidopsis leaves than O157:H7 after surface-inoculation and S. enterica serovar Thompson strain RM1987 can survive at high titers on the surface of romaine lettuce leaves . Therefore, S. enterica may either induce a weak plant immune response or can counteract plant immunity and consequently persist on and in leaves . Internalization and persistence within the apoplast are arguably the most important targets for managing contamination of produce by Salmonella, as endophytic populations cannot be removed through typical washing tactics . Here, we provide details of multiple genomic regions required for internalization and persistence of STm 14028s into lettuce leaves. These genomic regions were identified with a genetic screen of multi-gene deletion mutants of STm 14028s to pinpoint proteins and metabolic pathways responsible for stomatal re-opening and apoplastic persistence. Selected MGD mutants were further characterized regarding their ability to survive in the apoplast, induce hallmark plant defenses, and replicate in apoplastic wash fluid. While all mutants induced a prolonged stomatal closure when applied to the leaf surface, the mutants were found to vary in other aspects of phyllosphere survival. Five-mL of LSLB bacterial cultures were grown in 14.0 mL culture tubes on a rotary shaker to an OD600 of 0.8–1.0. Cells were harvested by centrifugation and resuspended in water to an OD600 of 0.002 . Inoculum was infiltrated with a needleless syringe into leaves of 5-week-old lettuce plants that were kept under the same environmental conditions used for plant growth. Stomatal bioassay was conducted as previously described at 2 and 4 hpi. Mean stomatal aperture widths from three independent leaves and SE were calculated. The difference between the means was compared by Student’s t-test to determine statistical significance.

Apoplastic wash fluid was extracted from 5-week-old lettuce leaves, omitting the cotyledons, using an infiltration centrifugation method as previously described . To ensure that plant cellular contamination did not occur during extraction, AWF was evaluated for cellular contaminants using the Sigma-Aldrich RGlucose-6-Phosphate Dehydrogenase Assay Kit . None of the AWF used exhibited detectable levels of G6PDH . AWF was saved in aliquots to limit freeze-thaw cycles and stored at −20◦C and filter sterilized at the time of use. Bacterial cultures were grown in LSLB liquid medium on an orbital shaker to an OD600 of 0.8–0.1. Cells were harvested by centrifugation and resuspended in water to an OD600 of 0.2 . An aliquot of this inoculum was added to each medium to achieve an initial bacterial culture concentration of 5 × 106 CFU/mL in a 96-well plate format. Growth curves were obtained by growing cultures stationary, except for a 30-s rotation prior to each OD600 reading using a BioTek EPOCH 2 Microplate Spectrophotometer . OD600 readings were obtained every 30 min throughout a 24-h period and blanks were included as a control. This experiment was performed three times with three technical replicates each time. Mean OD600 for each time point of the growth curve was calculated after subtracting the mean blank value and subsequently converted to bacterial cell number per mL of culture. Growth rates in the log-phase of growth were determined using the formula N0 x 2 n = Nf where N0 is the number of bacteria at the first time point of interest, Nf is the number of bacteria at the final time point of interest, and n is the number of generations.We utilized a collection of MGD bacterial mutants derived from STm strain 14028s . We first confirmed that this strain induces a similar stomatal response to that of STm strain SL1344 . We evaluated changes in the stomatal aperture width in leaves of young lettuce plants by floating leaf pieces onto bacterial inoculum as previously reported . Both STm strains induced an initial stomatal closure at 2 h post inoculation followed by re-opening at 4 hpi , suggesting that the MGD library could be useful to identify STm genomic regions required for successful stomatal re-opening at 4 hpi. Second, to ensure that lack of re-opening was due to deletion of genes required for stomatal re-opening by STm 14028s rather than temporal factors, the circadian movement of lettuce stomata was determined. This analysis indicated that the stomatal aperture was widest at 6 h after first light . We therefore, chose to start the stomatal bioassay at 2 hafl to ensure that the 4 hpi time point corresponded to a time with maximum expected stomatal aperture width. A primary screen of 303 MGD strains with a single biological replicate indicated that 177 mutants were unable to re-open stomata, suggesting a high rate of false-positives. Thus, we functionally annotated the predicted deleted genes in these 177 mutants .

Considering the current knowledge of STm epiphytic behavior , we reasoned that genes involved in secretion,black plastic pots for plants perception of environmental signals, signaling, and regulatory functions could be involved in opening of the stomatal pore. Thus, we selected 51 MGD mutants based on their functional annotation for retesting with at least three biological replicates. The primary functional units missing in these 51 mutants are described in Supplementary Table S2. From this confirmation screen, only eight mutants were unable to reopen lettuce stomata consistently and they were selected for further characterization. Furthermore, previous results indicated that mutants for the Salmonella Pathogenicity Island 1 and 2 were unable to open lettuce stomatal pores . Thus, we also analyzed two MGD strains from our collection that have a predicted deletion of these regions in addition to a few adjacent genes . To confirm that the lack of stomatal re-opening using leaf pieces floating on bacterial inoculum was a reproducible response that can also be observed in leaves still attached to the plant, we designed a stomatal bio-assay that included infiltration of mature lettuce leaves with STm 14028s, Mut3, or Mut9 bacterium suspensions. In this assay, bacteria are placed in the leaf apoplast, including the sub-stomatal chamber, where they can be in contact with the guard cells. All three strains induced a strong stomatal closure at 2 hpi , similar to observations made using surface inoculation of mature, whole plants . Furthermore, the wild type strain STm 14028s, but not the mutant strains, induced stomatal reopening at 4 hpi , suggesting that this response is robust. To rule out the possibility that the infiltration procedure induced an unpredictable stomatal movement, we assessed the circadian stomatal movement in untreated lettuce leaves as well as leaves infiltrated with water , STm 14028s,or Mut9. Mock-treated and untreated leaves showed an almost identical movement pattern, stomata of Mut9-infifiltrated leaves remained closed throughout the daylight period, and STm 14028s-infiltrated leaves showed a transient reduction in stomatal aperture width at 2 hpi that corresponded to 4 hafl. Upon completion of this screening procedure, the genome position of the deleted region for each mutant was identified at the nucleotide level by whole genome sequencing of the mutant strains. This procedure, which was readily available in a time and cost-effective matter, allowed us to predict the genotype and the functional units missing in each mutant using the available STm 14028s and LT2 genome annotations . Furthermore, each mutant, except Mut5, was able to swim and swarm , confirming the predicted genotype of Mut5 is missing genes involved in flagellar biosynthesis and chemotaxis . Movement and chemotaxis have previously been associated with STm SL1344 internalization through the stomatal pore . Thus, the identification of Mut5 during the Salmonella genetic screening validates our procedure, which identified known and novel features associated with bacterial epiphytic behavior. The 10MGD strains were further tested for phenotypic traits required for colonization of leaves as described below. As all selected mutants were unable to stimulate stomatal re-opening at 4 hpi , we sought to determine whether each mutation also affected the population dynamics in the lettuce leaf apoplast. To characterize each mutant’s ability to survive within the apoplast, leaves were infiltrated with bacterial inoculum. This allowed for direct analysis of population titer changes due to apoplastic interactions and eliminated confounding factors, such as failure to survive on the leaf surface and/or lack of internalization through stomata.The wild type bacterium STm 14028s population declined significantly in lettuce leaves , while variable population titers were observed among the mutants . For instance, seven mutants, Mut1/2/4/5/7/8/10, had significantly greater population titers than that of the wild type bacterium at 21 dpi, whereas Mut3 and Mut6 apoplastic persistence did not differ from that of the wild type at 21 dpi . Interestingly, only Mut9 showed significantly impaired endophytic survival . This finding indicates that genes missing in this mutant, including the SPI-2 and the suf, ynh, lpp, and ttr operons , may be required for the bacterium to cope with or overcome plant defenses and/or the ability of the bacterium to obtain nutrients from the apoplastic environment necessary to maintain its population. To test for these possibilities, we performed a callose deposit assay and a bacterial growth rate assay using lettuce apoplastic wash fluid . Callose deposition is a hallmark plant defense response that is induced upon biotic stress . Thus, we determined the average number of callose deposits in lettuce leaves inoculated with STm 14028s, Mut3, and Mut9. We observed that all three bacteria induced similar numbers of callose deposits that were significantly higher than those seen in the water control . Because all three STm strains induced a relatively low number of callose deposits , we also inoculated Arabidopsis with the virulent phytopathogen Pst DC3000 for comparison with this well-established system.

A corollary goal is to provide the nursery industry with a source of new and interesting, economically advantageous and environmentally sound plant revenue. With increasing pressure from state and regional waterquality control boards for zero runoff in the nursery industry , plants requiring fewer inputs will be a welcome addition.A field was prepared to test 240 plants on a UC Davis research farm in USDA Zone 9 . Plants were placed 2 yards apart along rows that were 2 yards apart, with 20 plants per row in each of 12 rows. This allowed the simultaneous testing of six individual plants on each of four different water treatments for each of the 10 species. The rows were covered with 3 to 4 inches of bark mulch, and two 2-gallon-perhour drippers were buried beneath the mulch in the root zone of each plant. The plants were placed according to a randomized complete block pattern in three blocks throughout the field. Each row was furnished with four water lines to deliver one of the water treatments to each plant after they were established. It is important that even drought-tolerant plants be given supplemental water until well established, because the development of an adequate root system is a key component of drought-tolerance . The 10 species were planted in fall 2004, and frost-killed specimens of creeping sage were replaced in spring 2005. Likewise, the plants were also watered during long, rain-free periods in the winter of 2005 to 2006. Experimental irrigation treatments were carried out during the 2006 growing season. The four irrigation levels were based on percentages of reference evapotranspiration as described in Water Use Classification of Landscape Species III . ETo was defined as the amount of water evaporated from a 4- to 7-inch-tall, cool-season grass in open field conditions. WUCOLS classifies landscape plants according to how much water they need compared to cool-season turfgrass,drainage planter pot which is high water-use and needs 80% of ETo to look green and healthy in the summer growing season.

In our trial, we used the following percentages of ETo: 20% , 40% , 60% and 80% . We wanted to assess not only if these plants were truly drought-tolerant, but also if they could survive under garden conditions where they might be combined with higher water-use species or adjacent to a high water-use lawn. The average water-holding capacity of the soil was determined from soil samples collected at field capacity along a transect across the field. Irrigation was measured to replace half of the soil’s water-holding capacity in the root zone of each treatment to a depth of 1.5 feet. Since some of the moisture in the soil is held too tightly to soil particles for plant uptake, plant water stress is usually avoided by providing an irrigation when 50% of field capacity has been depleted. This amounted to 21.2 gallons of water per plant delivered over a period of approximately 5.25 hours. We used ETo values calculated by the California Irrigation Management Information System , which comprises data collection stations in various locations throughout the state that measure precipitation, relative humidity, solar radiation, temperature and wind speed. The California Department of Water Resources provides values daily for ETo online for the public . During the May to October 2006 irrigated growing season, the Davis CIMIS station was accessed daily via the Internet, and the ETo values were placed into a water budget worksheet to calculate the four percentages of accumulated water deficit. From this data, the subsequent need for irrigation in any one of the water-use treatments could be determined. In brief, all the plants received the same amount of water at each irrigation, but how often they received it was determined by their water-use percentage of ETo treatment . This low water-tolerance screening is somewhat unique to the needs of a California introduction program, since most states do not deal with complete drought from May to November each year.After the first summer of regular irrigation followed by wintering over in open field conditions, four species had suffered 50% or greater mortality, leaving six species in sufficient numbers to collect data . The species that did not survive the first year were coast gum plant, California beach aster, seaside daisy and creeping sage. The species that did survive were Apache plume, California lilac ‘Valley Violet’, serpentine columbine, rosy coral bells, eyelash grass and San Diego sedge. The first three species that did not survive are native to warm coastal areas, as reflected by their common names. Although they had grown well in the UC Davis Arboretum for years, the unmitigated summer heat and cooler winter temperatures of our field-trial site proved too inhospitable for them.

The fourth species that did not survive, creeping sage, was bitten back by frost in winter 2005 and did not transplant well into the clay-loam field soil in spring. However, the few creeping sage plants that did survive spread up to 9 feet in two directions across bare paths where the soil did not stay moist. It is native to well-drained slopes and is probably a good choice for restoration in its native range in the coastal and Sierra foothills, but was not deemed a good selection for most Central Valley gardens with space restrictions and heavier soils.One of the six species that survived in the UC Davis open field, Apache plume, did not advance to the next stage of zone garden trials. It is a woody shrub with small, dissected leaves and a profusion of pink staminate flowers that lend it a fuzzy appearance when in bloom. While the September 2006 plant growth index was higher with moderate levels of irrigation than with either low or high levels , this difference was statistically insignificant . This species bloomed heavily over a long period of time, and showed no signs of disease or pest damage. However, Apache plume also had some undesirable characteristics. Large branches tended to flop over, yielding an untidy, open habit as the season progressed, and the abundant seeds selfsowed rather freely in dry paths and mulched beds.The second woody shrub was a UC Davis Arboretum selection of California lilac that has become our banner species, ‘Valley Violet’. This California lilac performed beautifully at any watering level,plant pot with drainage which was unexpected since so many other species of this genus will not tolerate summer water. It should be noted that July 2006 was exceptionally hot, even for Davis , and yet the lilac’s appearance was unaffected even at the lowest level of summer water. Steady increases in relative plant growth index over the season from 1.15 to 1.45 were observed for all irrigation levels, with no significant difference between the treatments . In the spring, this plant bloomed in profusion from the base of its branches to the tips and was unbothered by pests or disease. This California lilac, with its yearlong deep green color and staggering spring floral display, was eagerly accepted by all the demonstration gardens involved in the next phase of the trial.Two of our herbaceous species, serpentine columbine and rosy coral bells, are naturally found in shady woodland locations. Consequently, they all showed a loss in plant growth index at all irrigation levels during the hottest part of the growing season in our exposed site, with values between 0.7 and 0.9 .

However, there were no statistically significant differences between the irrigation treatments, leading us to conclude that during the hottest months, protection from the sun was more critical to the success of these species than the availability of water. Interestingly, under the highest watering regimen, two of the six columbines died by the end of July and two more died by the end of August, possibly showing an intolerance of wet soil during the hot season. However, the remaining two columbines were already beginning to recover by September when temperatures began to drop, and all irrigation levels for both of these woodland herbaceous species showed dramatic recovery by the following June. Noteworthy in both species was the prolific flower display, far beyond what was observable with specimens in shady locations in the nearby Arboretum during the same year. While both the coral bells and columbine leaves showed signs of sunburn and necrosis during the summer, their flowering seemed to benefit from the availability of light during the winter and spring months. Both were attractive to bees and syrphid flies, but were unbothered by pests or diseases. So, even though our test site’s exposure was damaging to foliage, their mere survival under these conditions, combined with their spring beauty and attraction of beneficial insects, caused us to advance them to the next phase of the trial with a recommendation for planting sites with at least afternoon shade during the summer. Plants suited to dry shade are sorely lacking in the nursery trade, making serpentine columbine and rosy coral bells good introduction candidates.Also called blue grama grass, eyelash grass is a bunchtype grass with a wide native range in prairies throughout North America. The amount of water it received in our trial made no significant difference in the amount of summer growth, with a September relative plant growth index of 1.9 to 2.0 . Regardless of the amount of water, this species maintained a neat, fountain-form habit desirable for an ornamental grass, and had no pest or disease problems. For these reasons, we advanced eyelash grass to the zone garden trials.San Diego sedge showed an unexpected toughness and drought tolerance for a plant that grows along streams in the wild. It sent up handsome flower spikes that matured to an attractive, buff-colored seed head held above sword-shaped leaves. None of these seeds has been observed to self-sow in the field, making it unlikely to be invasive in dry areas. At all irrigation levels, the plants showed consistent, positive changes in plant growth indices until the end of August, when growth leveled off, presumably in favor of seed production . San Diego sedge plants irrigated at the two lowest water levels did show slightly lower relative growth indices as the season progressed, with those given the second lowest water level inexplicably displaying the lowest relative plant growth index. However, the only statistically significant differences were between the low-medium and high-medium treatments in September . Plants at all irrigation levels became more attractive as the season progressed, and they were pest-free and disease-free. There was no consistent pattern to which watering level the plants preferred, making San Diego sedge a good candidate for a strong structural element in a variety of garden situations.The key to the next stage of this endeavor was the Master Gardener Program, which is coordinated by UC Cooperative Extension. Because these programs are located in most counties throughout the state, they are uniquely situated to grow and collect data on the plants that are advanced from the first phase of the trials. Many counties have demonstration gardens, which make perfect sites for both data collection and exposure to the public. The counties currently participating in the second phase of the native plant trials are Shasta , Placer/Nevada , Alameda , Santa Clara , Mariposa , Fresno and San Diego . The sites include coastal, inland valley and low mountain gardens, but all are within the boundaries of the climate zones recognized as “Mediterranean.” As plants became available beginning in fall 2006 through fall 2007, each site was provided with six plants each of several prospective species advanced from the irrigation trial. Master Gardeners in these areas have planted, tended and collected data on the plants provided. They are taking monthly measurements using the same protocol as the plant growth index used in the field trial. Soil types have been noted, irrigation frequency is being tracked and any unusual weather events noted. In addition, each garden is supplied with data sheets that allow them to rate each plant on a scale of 1 to 5 each month infive categories: foliage, flowering, pest resistance, disease resistance and overall vigor. Table 3 and figure 4 provide examples of the first year’s compiled observations for rosy coral bells.

However, as pH decreased, Cd removal efficiency in tests without nitrate addition was relatively higher than in tests with nitrate addition, especially at low pH conditions. This indicates that the removal capacity of iron hydroxide or iron oxide derived from the reaction between nZVI and nitrate is much lower than that of pristine nZVI. As seen in Fig. 3, the removal efficiency in both reaction systems increased by more than 20% when pH increased from 7.5 to 8.0, and 15% as pH increased from 8.0 to 8.5. Meanwhile, in a control test a significant increase in Cd removal efficiency was observed at pH 8.5 , although Cd started to precipitate out from water at pH
8.0 . This meant the increase in Cd removal efficiency was mainly due to the presence of nZVI. The sharp increase in removal efficiency and capacity may be due to three main reasons: the critical pH value for Cd hydrolysis þ and Cd23 þ) and precipitation 2) is 8.0 , which may have contributed to the increased removal rate through electrostatic interaction and deposition; the isoelectric point of nZVI is around 8.1 , below which nZVI particles are positively charged and above which they are negatively charged. The negatively charged surface at pH > IEP favors Cd adsorption due to strong electrostatic attraction. Thus, the change of surface charge of nZVI may also have contributed to the increased removal of Cd; and high pH, especially above 7, could also improve Cd adsorption on iron oxide . Comparing the results of these two series of experiments, the enhanced Cd removal capacity in the presence of nitrate most likely resulted from elevated solution pH. Specific sorption may have also contributed to Cd removal because of the relatively high removal efficiency observed around pH 6 .Under global warming and climate change,25 liter pot cultivated plants are encountering increased biotic and abiotic stresses, which lead to reductions of plant growth and reproduction and consequently economic losses. The use of plant endophytic bacteria to promote plant growth and increase tolerance of environmental stresses has provided an alternative to standard agricultural practices that has fewer safety concerns.

Endophytic bacteria can be defined as non-pathogenic bacteria that colonize the interior of host plants and can be isolated from surface-sterilized plant tissues. These bacteria can obtain a constant nutrient supply from host plants by living inside the plants and having close contact with plant cells. The endophytic bacteria colonization process is usually initiated at wounds and cracks in the roots by a rhizospheric population of the bacteria in the soil. After entering the plant roots, endophytic bacteria can systemically colonize the above ground parts of plants, including stems and leaves.Endophytic bacteria communities include five main phyla. Proteobacteria is the most dominant phylum isolated from host plants, which includes α-, β-, and γ-Proteobacteria. Actinobacteria, Planctomycetes, Verrucomicrobia, and Acidobacteria are also commonly identified. The most frequently isolated bacteria genera are Bacillus, Burkholderia, Microbacterium, Micrococcus, Pantoea, Pseudomonas, and Stenotrophomonas, with the two major genera being Bacillus and Pseudomonas. Several factors affect the composition of endophytic bacteria populations, including plant growth conditions, plant age, types of analyzed plant tissues, soil contents, and other environmental factors. Endophytic bacteria can have several beneficial effects on host plants, such as promotion of plant growth and yield, increased resistance to plant pathogens , enhanced tolerance to abiotic stresses, elimination of soil pollutants through the facilitation of phytoremediation, and production of various metabolites with potential applications in agriculture, medicine, and industry. Some endophytic bacteria help host plants acquire increased amounts of limited resources from the environment. This can include enhancing the uptake of nitrogen, phosphorous, or iron by expressing nitrogenase, solubilizing precipitated phosphates, or producing iron-chelating agents in bacteria, respectively. Some endophytic plant-growth-promoting bacteria can increase host plants’ metabolism and nutrient accumulation by providing or regulating various plant hormones, including auxin, cytokinin, gibberellins, or ethylene.

Auxin and ethylene are the two major hormones that affect plant growth and development and that are involved in plant-endophytic bacteria interactions. In addition to these four hormones, several endophytes can utilize signaling pathways mediated by salicylic acid, jasmonic acid, and ethylene to initiate induced systemic resistance and protect host plants from phytopathogen infection. A number of endophytic bacteria can also produce various antibiotics, toxins, hydrolytic enzymes, and antimicrobial volatile organic compounds to limit pathogen infection. We previously isolated a plant endophytic bacterium, Burkholderia sp. strain 869T2, from surface-sterilized root tissues of vetiver grass. Strain 869T2 can also live within banana plants, in which it promoted growth and reduced Fusarium wilt disease occurrence. Genomic sequences of the strain 869T2 contain the gene for 1-aminocyclopropane-1-carboxylate deaminase, which may modulate host plant ethylene levels. Strain 869T2 also has genes related to the synthesis of pyrrolnitrin, which may function as a broad-spectrum anti-fungal agent, as well as dioxin-degradation-related genes. Furthermore, strain 869T2 can degrade the toxic dioxin congener 2,3,7,8-tetrachlorinated dibenzo-p-dioxin , mainly via its 2-haloacid dehalogenase. A recent study compared the genome sequences of 31 Burkholderia spp. and reclassified Burkholderia cenocepacia strain 869T2 as Burkholderia seminalis. We also compared the genome sequences of the strain 869T2 with those of five published B. seminalis strains: FL-5-4-10-S1-D7, FL-5-5-10-S1-D0, Bp9022, Bp8988, and TC3.4.2R3. The strain 869T2 shared 93–95% of its genome with the other five B. seminalis strains. Furthermore, strain 869T2 lacked several genetic loci that are important for human virulence. Based on the results of our analysis of the core genome phylogeny and whole-genome average nucleotide identity , strain 869T2 was classified as B. seminalis. B. seminalis is a member of the Burkholderia cepacia complex , which is a group of Gram-negative, aerobic, non-sporulating, rod-shaped bacteria. Bcc consists of opportunistic human pathogens that exist in patients suffering from cystic fibrosis as well as pathogens of many vegetables and fruits,25 liter plant pot such as onion and banana . Contrary to the pathogenic traits that led to their original discovery, some Bcc bacteria have ecologically beneficial interactions with host plants.

The plant endophytic bacterium B. seminalis strain TC3.4.2R3, isolated from sugarcane, can serve as a bio-control agent to reduce infections with Fusarium oxysporum and the cacao pathogens Moniliophthora perniciosa , Phytophthora citrophtora, P. capsici, and P. palmivoraas well as orchid necrosiscaused by Burkholderia gladioli through the production of pyochelin, a rhamnolipid, and other unidentified diffusible metabolites. Another strain of Burkholderia seminalis, strain R456 isolated from rice rhizosphere soils, decreased the occurrence of rice sheath blight disease caused by Rhizoctonia solani. Furthermore, Burkholderia seminalis strain ASB21 was found to be able to produce the plant hormone auxin, promote rice seedling growth, and reduce aluminum toxicity symptoms in host plants. Similarly, a Burkholderia seminalis strain isolated from Bangalore, India can produce indole acetic acid and enhance tomato seedling growth. Although it is known that Burkholderia seminalis belongs to the plant-growth-promoting rhizobacteria , only limited strains and their promoting abilities are well characterized. In this study, we examined the amounts of IAA produced by B. seminalis strain 869T2 in various growth conditions, detected the strain’s siderophore synthesis and phosphate solubilization abilities, and demonstrated its growth-promoting abilities in several leafy vegetables, including pak choi, lettuce, and amaranth. Indole acetic acid production was determined as described previously, with minor modifications. Bacterial cultures were grown on LB media containing 100 µg mL−1 of tryptophan with different pH levels and appropriate antibiotic for 48 h at selected temperatures . Bacteria were also cultured on M9 salt media at 30 ◦C for 48 h with 100 µg mL−1 of tryptophan and 2% of different kinds of sugar: glucose, fructose, or sucrose. Fully grown bacteria cultures were then centrifuged at 5000 rpm for 10 min, and the supernatant was passed through a syringe filter with a pore size of 0.2 µm to remove bacteria. The 500 µL of supernatant was mixed with 1 mL of the Salkowski reagent and incubated at room temperature for 25 min. Finally, the concentrations of IAA in the supernatants were determined by comparison of the absorbance measured at 530 nm with a standard curve of 0–100 µg mL−1 IAA.Various growth parameters of different plant species were measured at selected days, ranging from 14 to 80 days, after inoculation with strain 869T2. The fresh weight, dry weight, and length of leaves and roots as well as the width, number, and surface area of leaves were measured in harvested pak choi, lettuce, and Chinese amaranth as described previously. The fresh weight, length, number, and color of fruits of hot pepper and okra were recorded following previously described methods. The chlorophyll content of the lettuce leaves was measured using a previously published protocol. Chlorophyll was extracted from the leaves with N, N-Dimethylformamide for 1 hour in the dark, and chlorophyll a and b concentrations were calculated from the absorbance of the crude extract at 647 and 664 nm. Anthocyanin concentrations were determined using a published acidified methanol method. Hot pepper fruits were first ground with liquid nitrogen. Acidified methanol was then mixed with the ground materials for 10 min in darkness with shaking. These crude extracts were subsequently mixed with an extraction solvent containing 1:1 chloroform:water to isolate anthocyanins. After centrifugation, the absorbance of the supernatant was read at 530 and 657 nm by the spectrophotometer, and anthocyanin contents were calculated from these values. The effects of pH were also examined by culturing strain 869T2 in LB media at 30 ◦C over a pH range of 4 to 9. Strain 869T2 was able to grow over this entire pH range . The results shown in Figure 1D demonstrate that IAA production was at a similar level when bacteria were grown at pH 6 to 9, whereas the IAA amount decreased 44.0% when bacteria were grown at pH 4. Additionally, three different sugars, glucose, fructose, and sucrose, were used in the minimal medium to examine the effects of different carbon sources on IAA production.

Strain 869T2 grew similarly in the M9 salt media with different kinds of sugars . The results shown in Figure 1F indicate that when strain 869T2 was grown in the media with two kinds of monosaccharide, glucose and fructose, the IAA amounts were higher than for the bacteria grown in the media with sucrose. We further investigated whether strain 869T2 had other plant-growth-promoting traits, including siderophore production and phosphate solubilization abilities, with agar plate assays. Supplementary Materials Figure S1A shows that the strain 869T2 colonies exposed to CAS agarose turned yellow, indicating the siderophore production ability of strain 869T2. Furthermore, Figure S1B reveals that the formation of halos around the strain 869T2 colonies grown in Pikovskaya’s agar medium with 0.5% tricalcium phosphate suggests that strain 869T2 may have the ability to solubilize phosphate.A previous study by Ho et al. demonstrated that strain 869T2 promoted plant growth in banana, a monocot. Here, the growth promotion ability of strain 869T2 was tested in three different eudicot plants from the Brassicaceae family, namely Arabidopsis thaliana, ching chiang pak choi, and pak choi. Because strain 869T2 produced relatively higher amounts of IAA at 25 ◦C to 37 ◦C , we cultured strain 869T2 at three different temperatures, 25 ◦C, 30 ◦C, and 37 ◦C. Subsequently Arabidopsis thaliana ecotype Columbia was inoculated with these strains to determine which strain had the best plant growth promotion ability. We confirmed the endophytic colonization of the Arabidopsis plants by strain 869T2 by reisolating the bacteria from surface-sterilized inoculated plant tissues. The identities of the isolated bacteria were determined via sequencing and phylogenetic analysis of the 16S ribosomal RNA gene. Subsequently, different plant growth parameters were examined in Arabidopsis plants inoculated with strain 869T2 and in mockinoculated controls. Two weeks after inoculation, the presence of strain 869T2 increased the average fresh weight , rosette diameter , root length , number of leaves , total leaf area per plant , leaf area per leaf , number of inflorescences , and number of siliques of Arabidopsis plants more than 1.5- to 2.1-fold compared with mock-inoculated controls. As shown in Figure 2I–K, the overall size and number of leaves of plants inoculated with strain 869T2 were greater than those of control plants, indicating that strain 869T2 promoted Arabidopsis plant growth. Furthermore, when the plants were inoculated with strain 869T2 grown at 30 ◦C, the average root length and average total leaf area per plant were slightly higher than for the strains grown at 25 ◦C and 37 ◦C.

This branch of the immune system known as pathogen-triggered immunity is the first line of active defense against infection. Human pathogen on plants is an emerging field that only recently has caught the attention of plant biologists and phytopathologists. A few studies have been reported in the last 5–10 years, which focused on the most well studied PAMPs, flagellin and lipopolysaccharide , in the interaction of human pathogens with plants. Table 1 lists the plants, bacterial strains, and method details for such studies.Flagellin, the structural component of flagellum in bacteria, is involved in bacterial attachment and motility on the plant , is recognized by plant through the FLS2 receptor , and induces plant defenses . Similar to the well-studied PTI elicitor flg22 , the flg22 epitope of S. enterica serovar Typhimurium 14028 is also an effective PAMP and elicitor of downstream immune responses in Arabidopsis , tobacco, and tomato plants . Flagellum-deficient mutants of S. enterica serovar Typhimurium 14028 are better colonizers of wheat, alfalfa, and Arabidopsis roots as compared to the wild type bacterium further suggesting that the Salmonella flagellum induces plant defenses that may restrict bacterial colonization of several plant organs. However, the Salmonella flg22 peptide is not the only PAMP for elicitation of plant immune response as fls2mutant of Arabidopsis still shows a low level of PTI activation in response to this PAMP . Purified flagellin or derived epitopes of E. coli O157:H7 has not been used to induce plant defenses. However, flagellum-deficient mutant of this strain does not activate the SA-dependent BGL2 gene promoter as much as the wild type strain and shows larger population in Arabidopsis than the wild type strain further suggesting that surface structures in the bacterial cell are perceived by plants. The differences in responses observed could be attributed to the presence of other microbial signatures eliciting plant defense.Although flagellin sequences from S. enterica strains and other bacteria are highly conserved,best vertical garden system even a minor change of five amino acids in the flg22 epitope leads to reduced activation of PTI in Arabidopsis, tobacco, and tomato plants .

Adding to the specificity, it has also been shown that Brassicaceae and Solanocecae plants recognize specific flagellin . Hence, evolving variations in flagellin sequences could be a strategy employed by the pathogens to avoid plant recognition, which in turn leads to the development of pathogen- specific immune responses in the plant. Flagella also play an important role in bacterial behavior on the plant. Several studies have pointed out to the usefulness of flagella for attachment to leaf surfaces and movement on plant surfaces .Lipopolysaccharide is a component of the cell wall of Gram-negative bacterial pathogens of animals and plants. In the animal host, LPS is a well-characterized PAMP that is recognized by host Toll-like receptor 4 . In plants however, receptors for LPS have not been discovered yet. Nonetheless, current evidence suggests that human pathogen-derived LPS can be perceived by plants resulting in PTI activation. For instance, on the leaf surface, purified LPS from Pseudomonas aeruginosa, S. Minnesota R595, and E. coli O55:B5 induces strong stomatal closure in Arabidopsis . Purified LPS from Salmonella triggers of ROS production and extracellular alkalinization in tobacco cell suspension but not on tomato leaves suggesting that LPS recognition may be either dependent on experimental conditions or variable among plant species. Genetic evidence suggests that the high activity of SA dependent BGL2 gene promoter in Arabidopsis is dependent on the presence of LPS in E. coli O157:H7 as higher activity of this promoter was observed in the wild type bacterial as compared to its LPS mutant . However, LPS-dependent responses seem not to be sufficient to restrict bacterial survival on plants as the population titer of E. coli O157:H7 LPS mutant or wild type in plant is essentially the same . Additionally, live S. Typhimurium cells do not induce ROS in epidermal tissue of tobacco suggesting that, at least Salmonella, can suppress LPS-induced ROS and extracellular alkalinization. Similar to flagellin, the O-antigen moiety of LPS is not only important for plant perception of bacterial cells, but also for bacterial attachment, fitness, and survival on plants .

One of the earliest PTI responses in plants is stomatal closure that greatly decreases the rate of pathogen entry into plant’s internal tissues. This response requires molecular components of PTI including such as flagellin and LPS perception and hormone perception and signaling . Stomatal immunity is also triggered by the presence of human pathogens S. enterica serovar Typhimurium SL1344 and E. coli O157:H7 , albeit at various levels. For instance, E. coli O157:H7 induces a strong stomatal immunity and Salmonella SL1344 elicits only a transient stomatal closure in both Arabidopsis and lettuce suggesting that the bacterial strain SL1344 can either induce weaker or subvert stomata-based defense. Active suppression of stomatal closure by SL1344 may be unlikely because it cannot re-open dark-closed stomata . However, it is possible that signaling pathways underlying bacterium-triggered and dark-induced stomatal closure are not entirely overlapping and SL1344 acts on immunity- specific signaling to subvert stomatal closure.Recognition of PAMPs by PRRs leads to several hallmark cellular defense responses that are categorized based on the timing of response. Zipfel and Robatzek have discussed that early responses occur within seconds to minutes of recognition including ion fluxes, extracellular alkalinization, and oxidative burst. Intermediate responses occur within minutes to hours including stomatal closure, ethylene production, mitogen-activated protein kinase signaling, and transcriptional reprogramming. Late responses occur from hours to days and involve callose deposition, salicylic acid accumulation, and defense gene transcription. These hallmark plant cellular defenses have also been tested for both E. coli and S. enterica . In particular, S. enterica infection results in the induction of MPK3/MPK6 kinase activity and plant defense-associated genes PDF1.2, PR1, and PR2 in Arabidopsis leaves as well as PR1, PR4, and PR5 in lettuce . MPK6 activation in Arabidopsis is independent of FLS2 , indicating that flagellin is not the only active PAMP of Salmonella and plant response to other PAMPs may converge at MAPK signaling.

Direct comparison of the PR1 gene expression in Arabidopsis indicated that both E. coli O157:H7 and Salmonella SL1344 are able to induce this defense marker gene, however at difference levels . The PR1 gene induction is low in SL1344-infected plants indicating that immune responses are either weaker or are suppressed by Salmonella.The ethylene-insensitive mutant of Arabidopsis, ein2, supports higher Salmonella 14028 inside whole seedlings as compared to the wild type Col-0 plants . Furthermore, addition of a specific inhibitor of ethylene mediated signaling, 1- methylcyclopropene , to the growth medium resulted in increased S. enterica 14028 endophytic colonization of Medicago truncatula, but not M. sativum, roots and hypocotyls suggesting that the role of endogenous ethylene signaling maybe be specific to each plant-bacterium interaction. However, ethylene signaling may play a contrasting role during fruit contamination. Tomato mutants with defects in ethylene synthesis, perception, and signal transduction show significantly reduced Salmonella proliferation within their fruits as compared to the wild type control .Similar to the ein2 mutant, the coronatine-insensitive mutant of Arabidopsis, coi1-16, also supports high Salmonella 14028 inside whole seedlings . Along with the induction of the jasmonate-responsive gene PDF1.2 addressed in the same study and mentioned above, it seems that jasmonate signaling isalso an important component to restrict Salmonella infection in, at least, Arabidopsis. These results are surprising as coi1 mutants are well known to have increased resistant to various bacterial pathogen of plants, such as P. syringae, but not to fungal or viral pathogens .Two genetic lines of Arabidopsis has been extensively used to determine the role of salicylic acid in plant defenses against phytopathogens,frambuesa cultivo the transgenic nahG plant that cannot accumulate SA and the null mutant npr1 that is disrupted in both SA-dependent and -independent defense responses . Both of these plant lines support higher populations of Salmonella 14028 inside their roots and seedlings as compared to the wild type plant. NPR1-dependent signaling is important reduce the population of the curli-negative strain of E. coli O157:H7 43895 but not for the curli-positive strain 86-24 in Arabidopsis leaves . Although only a few strains of Salmonella and E. coli have been used, there is an emerging patterns suggesting that SA itself and activation of SA-signaling can potentially restrict HPOP. In attempts to understand the overall cellular transcriptional response to human bacterial pathogens, global transcriptomic analyses have been used. Thilmony et al. showed that E. coli O157:H7 regulates PTI-associated genes in Arabidopsis leaves, albeit in a flagellin-independent manner. A similar transcriptomic analysis with medium-grown Arabidopsis seedlings 2h after inoculation with S. enterica serovar Typhimurium 14028, E. coli K-12, and P. syringae pv. tomato DC3000 showed a strong overlap among genes responsive to each bacterial infection suggesting a common mechanism of plant basal response toward bacteria . Gene expression analysis of Medicago truncatula seedlings root-inoculated with only two bacterial cells per plant indicated that 83 gene probes were commonly regulated in response to S. enterica and E. coli O157:H7 . All together, these studies indicate that each human pathogenic bacterium can modulate specific plant genes beyond a basal defense response; however the mechanisms for plant-bacterium specificity are largely unknown.Successful virulent pathogens of plants are able to defeat this army plant defense by employing its own set of artillery and cause disease in the host plant .

In incompatible interactions , the host plant already has pre-evolved molecules that recognize these effectors and cause a specific defense response to this pathogen. This specific response is called effector-triggered immunity . Because the type 3-secretion system is important for the virulence of both animal and plant pathogenic bacteria on their natural hosts as evidenced by the use of bacterial mutants, it is reasonable to expect that T3SS would be important for HPOP as well. However, animal and plant cell surfaces are structurally different; the plant cells wall seems to be impenetrable by the secretion needle of the extracellular animal pathogens as discussed by He et al. raising the question of how these effectors can reach the plant cytoplasm and interfere with plant defenses. To date, there is no evidence for the ability of human pathogens to inject T3SS effectors inside plant cells. It is possible that the T3SS is still active on the plant cell surface and the effectors are secreted into the plant apoplast. If that is the case, however, plant membrane receptors would be necessary to recognize the effectors and trigger plant cellular responses. Nevertheless, it has been observed that the T3SS mutant of E. coli O157:H7, escN, has reduced ability to attach to and colonize baby spinach leaves similar to the fliC mutant . Furthermore, apoplastic population of T3SS structural mutants of S. enterica serovar Typhimurium 14028 is smaller than that of the wild type bacterium in Arabidopsis leaves and plant defense-associated genes are up-regulated for longer time by the prgH mutant than wild type Salmonella in Arabidopsis seedlings . Contrary to these findings, Iniguez et al. reported that two Salmonella 14028 T3SS-SPI1, the structural mutant spaS and the effector mutant sipB, hypercolonize roots and hypocotyls of M. sativum and fail to induce SA-dependent PR1 promoter in Arabidopsis leaves. More studies need to be conducted to conclude whether T3SS of Salmonella acts as “recognizable” surface structure similar to flagellum and/or as a conduit to deliver effectors in plant tissues and trigger ETI. It is worth mentioning that T3SS and effectors of the phytopathogen P. syringae pv. syringae have functions on ETI as well as bacterial fitness on plant surface and the filamentous T3SS protein EspA is required for E. coli O157:H7 attachment to arugula leaves . The invA structural mutant, that is defective in all T3SS-1 system-associated phenotypes, induces high ROS and extracellular alkalinizing in tobacco BY-2 cell suspension and hypersensitive reaction in tobacco leaves as compared to the wild type strain suggesting that T3SS is important for this suppression of immunity. However, Shirron and Yaron also reported that plant response to the regulatory mutant phoP that modulates the expression of many effector proteins and membrane components , is no different to that of the wild type bacterium. These findings raised the question whether the phenotypes observed are due to the T3SS structure itself or due to the translocated effectors.

More deeply rooted species can access existing and newly thawed deep soilnitrogen [Keuper, 2012]. In addition, roots acclimated to low temperatures in deep soil may have higher nutrient uptake capacity than roots in warmer surface soils [Chapin, 1974]. However, nitrogen in deeper soil is available for plant acquisition for a relatively shorter period than nitrogen in near-surface soil because the active layer thaws and increases in thickness throughout the growing season. Shallow-rooting species access soil nitrogen nearer the surface, and do so in the context of stronger microbial competition, but with more abundant soil nitrogen and over longer periods during the growing season. Therefore, different tundra species may respond dramatically differently to climate warming-induced soil nitrogen availability changes. The trade offs and ecological significance of plant carbon investments to compete for nitrogen in relatively warm shallow soils with high microbial competition, or to access nitrogen in relatively cold deeper soils with less microbial competition warrant further investigation. Second, root nitrogen uptake capacity is also an important trait for nutrient competitiveness. Species with low nitrogen uptake capacity must develop dense or long-lived roots in order to acquire enough soil nitrogen. For example, Carex aquatilis’s fine roots live for multiple years, and the fine root to leaf biomass ratio can be as large as 16 [Iversen et al., 2015b]. In contrast, species with high nitrogen uptake capacity invest less carbon for the growth of relatively short-lived roots [Eissenstat et al., 2000]. Third, tundra species with different carbon allocation strategies may contribute differently to carbon-climate interactions. For example, Carex aquatilis may fix more carbon per unit additional nitrogen uptake than Eriophorum angustifolium,vertical growing towers because the former allocate more carbon to grow roots and root C:N ratios are much higher than leaves .

Carbon costs of constructing roots are commonly lower than above ground tissues [Poorter, 1994]. In addition, tissue lifespan [Withington et al., 2006], decomposability [Hobbie et al., 2010], maintenance respiration [Segal and Sullivan, 2014], and contribution to soil carbon accumulation [Hu et al., 2016] differ among leaves and roots. Integration of these essential root traits into ESMs will improve understanding of how arctic tundra plants will respond to climate warming, through informing the magnitude of warming-induced increases in nitrogen availability on tundra carbon production.Current ESM land models have rudimentary representations of plant traits because of a lack of mechanistic understanding of how those traits control plant and ecosystem bio-geochemical processes and a lack of trait data to structure and parameterize large-scale simulations. We have recommended several key traits, which should improve predictions of root nitrogen uptake and how arctic tundra plants may respond to warming-induced elevated nitrogen availability. Some knowledge of the global spatial distributions of several of the aforementioned root traits is available. For root biomass profiles, the first global database was presented by Jackson et al. [1996]. Zeng [2001] further analyzed those biomass profile data according to Plant Functional Types and derived PFT-based root distribution data needed for large-scale land models. Schenk and Jackson [2002] expanded the Jackson et al. [1996] data set to include 475 root biomass profiles. However, most of those profile data are from temperate regions .Moreover, the PFT-based root distributions have not been updated accordingly. A global-scale maximum rooting depth data set was synthesized by Canadell et al. [1996] and included 253 plant species.

They also aggregated maximum rooting depth data based on PFTs, which is readily applicable to large-scale land models. The rooting depth followed the order: forest > shrub > herbaceous plants > – crops. However, within-PFT variation was quite large. For example, the maximum rooting depth of tropical species was 68 m, while the mean of tropical evergreen plant maximum rooting depth was about 15 m. Particularly for arctic tundra, a more detailed rooting depth data set was developed by Iversen et al. [2015a]. Tundra maximum rooting depth ranged from 0.7 cm for a deciduous shrub species to 100 cm for a forb species . In general, evergreen shrub tundra has the shallowest rooting depth . Grass, forb, and deciduous shrub tundra have deeper root systems , and sedge tundra has the deepest roots . This data set casts doubt on land model PFT classifications for arctic tundra. For example, CLM and ALM represent arctic tundra with only two PFTs , which substantially under represents root traits across the wide range of dominant tundra species, including arctic grasses, sedges, forbs, deciduous shrubs, and evergreen shrubs [Chapin et al., 1996].Travel to space is limited by the expense of transporting resources beyond Earth’s gravity well. As a result, early metrics of usability for space systems, especially life support, favored mass as the primary decision factor. Following a request to “provide the designers of future missions with mature technologies and hardware designs, as well as extensive performance data justifying confidence that highly reliable Advanced Life Support Systems that meet mission constraints can be developed” by the 1997 NASA Research Council, the scope of the Equivalent System Mass framework was broadened to account for differences in the cost of resources. The general principle behind this early metric was to calculate the mass of all of the resources required to make the system work. ESM was expanded from theory to the practice of accounting for processes ranging from controls, agriculture, and recycling. Currently, ESM remains the standard metric for evaluating ALS technology development and systems.

It has been adopted for use in trade studies, as the metric for life support sizing, and has been incorporated into several tools.Previous efforts to quantify the cost in problems of mission planning/space logistics have relied on metrics based solely on the Initial Mass to Low Earth Orbit for constant commodity supply and demand or on carry along mass. In such logistics frameworks like Space Net and HabNet, the cost is kept simple to allow for the analysis of complex mission architectures with multiple mission segments. Comparatively, ESM has been most fully developed for ECLSS where the costs of capital equipment, power, operations, transport, and other things have been captured on a common unit scale of mass. While it provides a method for summing the weighted terms of many subsystems, there is no explicit ESM equation that captures total mission costs across systems in various stages of a complex mission. Thus the standard ESM approach faces limitations in that there exists no explicit language for capturing the set of all segments and there exists interdependent relationships between the decision variables within separate segments. Here, we see a trade-off in the complexity of the cost function for the complexity of the mission architecture. As plans for human exploration continue to be made in anticipation of returning to the moon and traveling to Mars, an added emphasis will be required for the optimization of mission architecture. As of now, the current instance of the ESM framework does not lend itself to use as an objective function in optimization over a mission—although this ESM has been proposed as the metric for mission optimization. The result is that this standard framework remains fixed for multi-stage missions and generally faces challenges in providing design or planning information based on subsystem risk. Thus,container vertical farming the ESM metric is not always helpful when comparing missions with differential reliability for systems in their proper context. That is, given two possible technologies for meeting a mission objective, the one that is less likely to fail might be a better choice. To demonstrate how to formally add reliability metrics to the ESM framework, we take the case of a new technology platform, bio-manufacturing, for which there are known and quantifiable reliability concerns and for which there are little in situ testing for space missions. In the following work, we propose an extended ESM framework to account for the proposed multi-stage missions and critical mission features, such as reliability. As the scope of human exploration missions has expanded, the need for new technology platforms has grown, and it has been proposed that these features best capture the potential of bio-manufacturing systems. We do not claim completion of xESM, but rather, we demonstrate progress along this trajectory in the form of a more generalized framework to account for multi-staged mission segments ; account for reliability; and feed into downstream optimization problems. We also note that this later progress is less developed in more in line with a discussion rather than a ready-to-use operational strategy.Figure 1 depicts three profiles with varied transit architectures. Profile 1 uses a single journey from Earth to Mars, and although it has been proposed in some forms, it is unlikely this architecture will be adopted due to the substantial mass demands of the transit ship and the ascent propellant required to leave Mars. In the case of Profile 2 , cargo can be predeployed to Mars through some number of predeployment missions.

Profile 2 introduces segments to a crewed mission to Mars which are not actually crewed, but instead are either purely cargo-based in which case only the M and V terms factor into the ESM cost, or autonomous where M, V, P, and C for uncrewed operations matter. Since cargo missions do not require life support systems, the M cost is reduced greatly, leading to a reduction in overall mission cost, especially for missions that require a great number of goods that can be pre-deployed. In the most likely Profile 3, crew transportation can be further broken down such that smaller crewed vehicles make the jump from planet to surface and viceversa, but the interplanetary transit is made on a larger craft to reduce the mass required for egress from planetary gravity wells. Previous ESM literature allows for varied equivalency factors based on mission staging, and in such cases, the ESM of distinct segments of a mission are calculated separately, then normalized through the use of location factors. However, ESM M for any set of systems is calculated using a single location factor Leq term as a multiplier. In this form, it is assumed that each subsystem is transported in a uniform fashion or that all parts of a subsystem would correspond to a single Leq term. The profile expansion in Fig. 1 shows that inventory can be transported in different segments using different crafts which changes the value of Leq. This is supported by non-ESM logistics methods.The three Cases in Fig. 4 consider the food system and the potential impact of agricultural biotechnology to supply astronauts with their caloric and nutritional needs. We assume that each of six CMs has a daily dry mass food requirement of 0.617kg/ CM-d. We use this requirement to calculate the prepackaged food requirements of the two transit legs of each mission scenario, as well as the extra 70 or 500 days of food for surface operations in Cases 2s and 2b respectively. Given the recently updated infrastructure costs associated with a Mars Surface Habitat Vehicle, we calculate ESM through consideration of the food subsystem including food, packaging, refrigeration, and processing. In Case 2s, we consider only the stored food requirements from Case 2 from Fig. 3. In Case 2b, we consider the stored food requirements during surface operations decreased from 500d to 70d and the remaining food was produced via agriculture. In a long-duration mission scenario in which food is grown during surface operations, and where literature suggests that a sizable initial hardware set would be required. This set could include hydroponic growth chambers, water filtration, refrigeration, etc. along with additional support hardware like pumps, filters, etc. In Case 3, we consider the transportation of the bio-manufacturing system during predeployment rather than with the crew. During initial transit as well as the return transit, the crew relies on prepackaged food—crop growth begins on the first day of surface operations, necessitating another ~70 days of predeployed food while the surface hardware grows the first crop. Variations in crop selection and growth conditions during surface operations have been proposed, butthis bounding assumption is consistent with crops such as lettuce and wheat. Like Cases 0–3, xESM costs for Cases 2s, 2b, and 3b are larger than their ESM alternative, however, in Case 2s and Case 2b , the xESM option is significantly larger than the ESM option for calculation. The difference between the xESM and ESM calculation results is an increased mass on the transit to Mars and reduced mass for surface operations and return transit.

While the creation of transgenic plants may be relatively straightforward for a number of species, the strategy has its own substantial time requirements. The greatest advantage of transgenic technology is its ability to overcome fertility barriers for the dissemination of genes originating from a different species; two examples from the Solanaceae family highlight this advance. Bs2, as mentioned above, was identified originally in pepper and its resistance has been durable in the field against isolates of X. campestris . Due to the fitness requirement associated with avrBs2 locus, the incorporation of the resistance locus Bs2 via transgenic technology may offer durable resistance in a number of plant systems affected by X. campetris. To assess this hypothesis, tomato was transformed with the Bs2 gene from pepper. Inoculations of X. c. pv vesicatoria isolates onto Bs2– containing transgenic tomato plants failed to cause disease therefore Bs2 function was conserved in tomato . Tomato and pepper when crossed cannot form a fertile hybrid and this resistance could not have been utilized with standard breeding protocols. In another example the N gene from tobacco, conferring resistance to the tobacco mosaic virus , was transferred into tomato. The resulting transgenic tomato plants, expressing the N resistance gene, were inoculated with TMV and complete resistance was observed. While TMV is not as devastating economically to tomato as is X. campestris, the conceptual notion that resistance loci can be transferred among species while retaining their function points illustrates a key advance for engineering resistance using transgenic technology. These examples demonstrate conservation in disease signaling pathways that can be exploited for cultivar improvement.Disease resistance research has largely focused on understanding the specific pathogen–host interactions mediated by R and avr loci.

Recently, studies have revealed signaling components that function downstream of R genes or other pathogen sensors. Studies on broad-spectrum resistance pathways, such as the rhizobacteria-mediated,vertical plant rack induced systemic resistance pathway and the insect-responsive pathway involving jasmonic acid are rapidly gaining momentum . However, research on the pathway transducing a broad-spectrum defense response termed the systemic acquired resistance response has progressed most rapidly. Chemical and abiotic inducers of SAR, along with inherent signaling components of this pathway identified by basic research in model plant systems, are among the initial targets being used to engineer multi-pathogen disease resistance in important crop plants. The SAR defense response is manifested when a plant host is inoculated with a pathogen that results in a localized infection. This primary infection subsequently primes the host to resist secondary infections by viral, oomycete and bacterial pathogens . In the model plant Arabidopsis, SAR is associated with a rise of internal levels of the plant hormone salicylic acid , and is correlated with the increased expression of a set of genes termed pathogenesis related genes . Several PR genes encode proteins with antimicrobial activity and thus contribute to an overall defense response directly . Research aimed at modulating this pathway and generating broad-spectrum resistance has largely targeted three parts of this response for further study: the ability of SA to trigger the response, the increased expression of PR genes and the identification and modulation of other signaling components.SA is both necessary and sufficient to induce SAR in Arabidopsis and SA added exogenously initiates the SAR response . Conversely, transgenic plants constitutively expressing the bacterial nahG gene, which encodes a salicylate hydroxylase, do not accumulate SA and do not mount an SAR response . Other potent chemical inducers of SAR for higher plants are the synthetic chemicals benzo–thiadiazole-7-carbothioc acid and isonicotinic acid , both functional analogs of SA . BTH in particular, is associated with low phytotoxicity and has demonstrated efficacy against multiple pathogens when applied in field trials.

Notably, resistance is increased against the wheat powdery mildew fungus and the rice blast fungus, as well as against the late blight pathogen of tomatoes .These plants have low SA levels when not induced by pathogen attack. In contrast, high levels of endogenous SA are constitutively detected in some crop plants such as potatoes and rice . Therefore, the role of SA in rice and potato SAR-like responses is not clear and these examples may indicate points of divergence in the composition of defense pathways in different plants. It will be important to understand the individual differences before applying similar engineering strategies for all plant types. For example, BTH treatment of wheat enhances resistance to the pathogens Erysiphe graminus and Puccinia recondita, and induces the expression of five novel WCIgenes . BTH does not induce the full subset of wheat PR genes and the expression of the WCI genes after induction by BTH is not sufficient to provide resistance to disease caused by another pathogen, the wheat head blight fungal pathogen Fusarium graminarium . Infection of wheat by this fungus will induce the expression of wheat PR-like genes but not the WCI genes . Thus, at least in wheat, biotic and chemical inducers promote the expression of different genes sets suggesting roles for multiple defense response pathways. Studies in rice and barley yield similar results . Evidence for the conservation of a SAR-like response in cereals and other economically important crop plants arises from studies indicating that defenses against multiple pathogens can be induced after treatment with the SAR-inducing chemicals SA and BTH. Biotic induction of systemic resistance has also been described. Systemic resistance to the rice blast pathogen, Magnaporthe grisea, is established after an initial infection with the non-host pathogen Pseudomonas fluorescens . In wheat and barley, an initial infection by E. graminus heightens a plant’s ability to respond to a subsequent infection by the same pathogen . Hence, several studies indicate that in multiple plants, including cereals, SA and similar chemicals can induce defense responses and that pathogens can prime secondary resistance, two key SAR features.

It is then reasonable to postulate that at least some of these responses may be mediated via a SAR signaling pathway. Therefore,growing strawberries vertical system identifying and characterizing the components of such a pathway will likely provide targets that can be used to engineer resistance in plants.Genetic screens were initiated in model plant systems such as in Arabidopsis to identify genes involved in SAR. Many genes encoding components of the SAR signaling pathway have been identified through these screens . Below, a few examples which may provide targets for engineering resistance are discussed in detail. Mutants identified by their enhanced disease resistance, and thus likely to function as negative regulators of the SAR pathway include the edr1and mpk4mutants . Plants carrying an edr1 mutation have enhanced resistance to the fungal pathogen Erisphye cicoracearum. In addition, the phenotypic effects of this mutation can be suppressed by removal of endogenous SA from the mutant plants . The EDR1 locus has been cloned and was shown to encode a protein with high sequence similarity to a MAPKK kinase . The mpk4 mutant plants show strong expression of the PR genes 1, 2 and 5. When mpk4 mutants carry the nahG transgene the resistance phenotype is lost and these plants no longer constitutively express PR-1 indicating that the mpk4-mediated negative regulation of SAR requires SA accumulation . Other mutants showing constitutive PR gene expression and enhanced pathogen resistance are the cpr mutants . The SA levels in these mutants are also increased. Several members of the cpr mutants and the mpk4 mutant have growth defects, indicating metabolic problems with constitutively expressing defense responses . A contrasting class of mutants contains members that can no longer express PR genes and fail to mount a defense response . The NPR1gene was isolated from this second class of mutants. NPR1 was cloned and shown to encode a protein with limited overall homology to the mammalian immune response signaling protein IkB . Subsequently, studies of NPR1 have indicated that it is a key positive regulator of the SAR response in Arabidopsis that functions downstream of the SA signal. NPR1 may help induce PR gene expression through interaction with a class of transcription factors of the basic leucine-zipper type . Recently, genomic-analysis technologies have identified novel defense-associated genes that may be additional targets for genetic engineering. In two examples, the expression of Arabidopsis genes under several SAR-inducing conditions was analyzed using microarray technology . Along with the transcripts for the well characterized PR genes the mRNAs upregulated by defense stressors include those that encode for proteins such as a zinc-copper, superoxide dismutase, and a WRKY transcription factor, AtWRKY7 . WRKY transcription factors are novel, plant transcription factors that can bind to promoters containing a ‘W’ box; these motifs can be found in high abundance in the promoters of defense-related genes , including NPR1 . While these technologies for global gene analysis are starting to reveal additional components of defense pathways, work using these components in crop plants has so far focused on the transgenic expression of the PR genes and NPR1.Attempts to alter the expression of individual PR genes and their encoded proteins were among the first examples of experiments manipulating SAR components to engineer crop plants to display broad-spectrum pathogen resistance. PR proteins were originally classified as plant host proteins that were induced only in pathological or related situations . The function of PR-1 is not clear but PR-2 and PR-3 have anti-fungal properties in vitro. This suggests they may play a direct role in defense by attacking and degrading pathogen cell wall components . Many PR and PR-like proteins have been identified in crop species including, rice, wheat, barley, sorghum and maize . In crop plants each PR gene class has multiple family members. For example, there are at least 17 unique members of the PR-3 gene family, and seven PR- 2 family members in barley . Increasing the expression of individual and multiple PR proteins in various crops has demonstrated some success in enhancing disease resistance to particular pathogens. Examples of enhanced protection conferred by altering PR gene expression are mainly derived from studies manipulating levels of the PR-2 and PR-3 gene family members.

For example, over-expression of a rice PR-3 gene, Chi11, slightly reduces the extent of infection by the rice sheath blight pathogen, R. solani, in rice and the extent of the disease reduction correlates with the expression level of the transgene . Additionally, over-expression of another ricechitinase, Cht-2, reduces the severity of the disease conferred by the rice fungal pathogen M. grisea, although the extent of this resistance was decreased in subsequent generations . This protective ability is not limited to rice; over expression of a barley chitinase in wheat confers resistance, in isolated leaf assays, to the powdery mildew pathogen E. graminus . Furthermore, in sorghum, over-expression of the rice chitinase, Chi11, enhances resistance to the fungal pathogen Fusarium thapsinum . The effects of altering the expression of the PR-5gene family members have also been studied in wheat and maize . Constitutive over-expression of a rice PR-5 gene in wheat delays the onset of the symptoms caused by the wheat scab pathogen, Fusarium graminearusin, in a stable and heritable manner . Hence, at least for a subset of PR genes, manipulation of their expression levels may offer some enhanced protection to crop plants, even if the protection is not the broad-spectrum resistance originally desired. It appears that over-expression of any single PR gene, similar to the results seen employing single R genes, will not be sufficient to generate broad, durable resistance. An alternate approach to engineering resistance using PR genes is to over-express regulatory genes, such as NPR1, that are found upstream of defense genes in signaling pathways.NPR1 plays an important role in stimulating many downstream components of the SAR pathway; it is therefore a natural target with which to engineer disease resistance in crop plants. Over-expression of NPR1 in Arabidopsis leads to enhanced disease resistance to both bacterial and fungal pathogens in a dose-dependent manner . Plants containing the NPR1 transgene display no obvious pleiotropic effects. Once induced by pathogen attack or by chemical induction, NPR1 localizes to the cell nucleus where it can interact with a class of basic-leucine zipper transcription factors that are predicted to modulate PR gene expression, thus mediating the SAR response .

Root chemical concentrations were expressed per segment water volume based on root radius measurements at each location.The current study is one of the few that has determined both endogenous and exogenous patterns of nutrient supply and the only one that contrasts the two major inorganic N forms, NH4 + vs. NO3 – . A previous report estimated root net influx of exogenous N from the disappearance of N ions from the bathing solution; the current study calculated the endogenous rate at which the N ions are deposited locally with a continuity equation. Comparison of net influx to deposition rate indicates the extent to which the tissue is either retaining or exporting the N taken up from the bathing solution . If the deposition rate exceeds the exogenous net influx, then the difference shows the rate at which the tissue is importing the ion from older tissue or generating it metabolically.For roots receiving an exogenous NH4NO3, net NH4 + influx was fast enough to support the local deposition only in tissue basal to 5 mm . Because NH4 + deposition rate exceeded the exogenous net influx through the apical 5 mm, the meristem and apical half of the growth zone must retain most of the exogenous supply as well as import NH4 + or NH4 + precursors from more mature tissue. This conclusion is supported by the observation that similar NH4 + deposition rates occurred in the apical 6 mm when the source was Ca2 ; some combination of chemicaltransformation of NO3 – , import of NH4 + from more mature tissue, or deamination of amino acids must have occurred at these locations. With the exogenous Ca2 supply, the NH4 + deposition rate becomes small or even negative in the region basal to 6 mm. These conclusions confirm and, by showing the spatial profiles, extend the conclusions of Walter et al. , who found that as a whole,vertical rack the growth zone receives from non-growing tissue 31 nmol h 1 NH4 + while it exports 45 nmol h 1 NO3 – .

The excess of net influx over deposition of NH4 + and the co-occurring decline in tissue NH4 + in regions basal to 7 mm suggest that NH4 + absorbed near the apex remained unassimilated, whereas NH4 + absorbed in the region 7–60 mm from the apex was assimilated . Profiles of net NH4 + and H+ fluxes in the maize seminal root also support this interpretation . Assimilation of NH4 + produces H+ that roots rapidly excrete . The current study found that net NH4 + influx was greater at the root apex than at the regions 4–10 mm from the apex, but that net H+ efflux was greater 4–10 mm from the apex than at the apex, suggesting that NH4 + assimilation was more rapid in the more basal regions . This seems reasonable given the carbon/nitrogen balance in the root apex. The assimilation of NH4 + into glutamine is highly carbohydrate dependent, requiring carbon skeletons from 2-oxoglutarate and a respiratory expenditure of about 2 ATP equivalents per NH4 + . The root apex, however, lacks mature vascular tissue to facilitate carbohydrate translocation from more basal tissues, and nonvascular, symplastic diffusion of carbohydrates appears to be inadequate to meet the energy requirements of this tissue . Thus, the apex probably suffers from carbohydrate limitations. Maize seminal roots most likely store some of the NH4 + absorbed at the apex in vacuoles to avoid toxicity. Indeed, the location of maximum deposition of NH4 + coincides with the location where vacuoles in root cells are enlarging. Assimilation of NH4 + is greater in more basal regions where the phloem is more fully developed and capable of supplying sufficient carbohydrates. Although the NH4 + concentrations in the N-free and Ca2 treatments were significantly lower than those in the NH4NO3 and NH4H2PO4 treatments, the N-free and Ca2 treatments still showed small amounts of NH4 + in their root growth zones. Several explanations come to mind. First, the extraction and analysis protocols may have resulted in some deamination of amino acids. Second, some of the signal may have come from free amino acids in the root sample because free amino acids interfere with the NH4 + analysis technique, although the sensitivity of this analysis to amino acids is less than 10% of its sensitivity to free NH4 + . It is concluded that the apical 4 mm imported or produced free NH4 + through deamination during N-cycling even when NH4 + was not present in the bathing solution. When NH4 + was present exogenously, the presence of NO3 – in the medium had only a small effect upon NH4 + concentrations in the root tissue or in the xylem sap . This fits with earlier studies that demonstrated root NH4 + acquisition to be relatively independent of NO3 – or other anions .

The profile of NO3 – concentration in the growth zone was the same with Ca2 as with NH4NO3 , but the way in which the patterns were produced varied with the form of nitrogen supplied. With the Ca2 treatment,NO3 – influx exceeded the net deposition rate at all locations, but especially in the parts of the root basal to the growth zone where the influx was 4- to 5-fold greater than the deposition rate. The patterns with the NH4NO3 treatment were quite different: in the meristem and beyond 12 mm NO3 – influx slightly exceeded the deposition rate, but throughout the rapid growth zone the deposition rate exceeded influx. Thus, with the Ca2 treatment, the entire root tip absorbed more NO3 – than it deposited; indeed, the NO3 – influx greatly exceeded the amount that remained in the tissue. In contrast, with NH4NO3 treatment, most of the growth zone was importing NO3 – from mature tissue and NO3 – influx greatly exceeded that which remained in the tissues only in the more basal regions . Thus when NH4 + was present in the medium, NO3 – absorbed near the root apex was stored in the tissue and negligible amounts were assimilated or translocated. It is concluded that the more mature tissues were assimilating and exporting much of the NO3 – absorbed. This is a reasonable conclusion because NO3 – influx exceeded the deposition rate in these tissues and NO3 – in the xylem sap doubled .During its development, an individual tissue element is displaced from the root meristem through and then beyond the growth zone. The position of the basal and apical ends of the segment can be tracked to find the location and length of the segment over time. The growth trajectory gives the time course of the element position and can be calculated by integrating the displacement velocity over time or by counting the number of cells to the position of interest and multiplying by the ratio of mature cell length to root elongation rate . This paper is interested in calculating the total uptake of NO3 – into the tissue element as it expands and moves farther from the apex. To model the ‘potential uptake’ of NO3 – that results from influx,vertical farming hydroponic this study assumes no assimilation or translocation and then considers what the NO3 – content would be in the moving tissue element. Before calculating the total uptake in the developing tissue element, two extreme cases are considered: localized influx only in the apical 3 mm; and uniform influx along the growth zone. In the first case, with influx occurring only near the apex , then the NO3 – content would first increase and then decrease .

Where growth continues after influx ceases, then NO3 – would decrease more rapidly with position. Where influx and growth have both stopped, NO3 – would remain uniform in the absence of assimilation and translocation. Therefore, if influx is restricted to the apical 3 mm, the potential uptake into the older root segment would be quite small. In the second case, with influx occurring throughout the root, then the potential uptake would increase slowly as the element moves through the growth zone and more rapidly after growth has ceased in the tissue element . NO3 – is in fact taken up throughout the root . Fig. 9B shows that the total NO3 – uptake slightly exceeds the observed content while the tissue element is moving through in the growth zone, and the total uptake vastly exceeds the content when the tissue element is in the 10–20 mm region. Comparisons of the influx and deposition rates are most useful to analyse the physiology and biochemistry of the local nitrogen transformations and to determine the source–sink relations. It is also instructive, however, to compare the total uptake to the content , to appreciate the amount of the influx that has been retained in the tissue element over time.Glucose and fructose contributed about half of the osmolarity in the zone of elongation . Sucrose was undetectable . Other studies estimated sucrose in the maize root apex from tissues extracted with 80% ethanol at 80  C and estimated sucrose after its chemical or biochemical conversion to glucose , procedures that can overestimate sucrose and underestimate glucose and fructose . The current study directly measured glucose, fructose, and sucrose via HPLC immediately after boiling water extraction to inactivate any enzymes that might hydrolyse sucrose. K+ and its counter-ions contributed the other half of the osmolarity in the zone of elongation . Previous studies on maize seminal roots have not addressed the issue of counter-ions for K+ . This study found that the counter-ions for K+ included malate and NO3 – , but these could balance less than half of the K+ . The nutrient solution also contained H2PO4 – and SO4 2–. Walter et al. measured H2PO4 – and SO4 2– along the apical 10 mm of maize seminal roots receiving NH4NO3 and found their concentrations to be less than a third of the NO3 – concentrations. Most likely a combination of these anions, organic anions other than malate and citrate, and an increase in cellular pH accounted for the remainder of the counter-ions . Osmolarity remained high in the more basal zones of the root despite a substantial decline in glucose, fructose, and K+ concentrations . NO3 – accumulated in these more basal regions, as discussed above, but NO3 – and its counter-ions such as K+ contributed less than half of the observed osmolarity . Unfortunately, previous studies have not analysed solute concentrations in these more basal regions or have analysed only the soluble sugars .These results indicate that when both NH4 + and NO3 – were available in the rhizosphere, maize roots absorbed both forms, but preferentially assimilated NH4 + and stored NO3 – . Assimilation of NO3 – to glutamine expends 12 ATP equivalents versus only 2 ATP equivalents for NH4 + to glutamine . For the root apex, which may be carbohydrate-limited, a 6-fold difference in energy requirements was obviously critical. When NO3 – was the sole N-source, the root stored about the same amount of NO3 – in its tissues, while apparently importing or assimilating some NO3 – to support the rapid protein synthesis in the meristem and translocating a large portion of the NO3 – from the young mature tissues to the shoot. Shoots can use surplus light to assimilate NO3 – so that the large energy demands of this process do not detract from growth . The storage of substantial quantities of NO3 – at the base of the growth zone and in the young mature root tissues argues that NO3 – may serve as a metabolically benign osmoticant to balance other ions in plant tissues . Zhen et al. , using intracellular NO3 – -selective microelectrodes, found that most of the NO3 – in the epidermal and cortical cells of barley roots was stored in the vacuole and at levels that varied between 50 and 100 mol m 3 . Here, accumulation of hexoses and K+ in root cells of the elongation zone sustained root expansion, and malate served as counter-ions to K+ , as it does in other tissues .

A central aim of the original proposal was the concept of a circulating low flow regimen above the reconstituted streambed: “The park design would be centered about a small creek to be reestablished within the right of way by pumping Temescal Creek water to ground level. The reconstituted creek would harmonize with and accentuate the linear nature of the proposed park, providing a focus for lay out of trails, resting spots, and landscaping. The design of park trails would be integrated with the creek and the narrow right or way to achieve a corridor effect inviting the user to walk and experience what the park has to offer. The reconstituted small creek will also serve as a local storm drain by receiving runoff from adjoining properties which have historically drained to the creek”. The other project studied is located in northeastern Ventura County, near the Santa Susanna Pass. Regionally, White Oak Creek is tributary to the Arroyo Simi, which drains the Simi Valley over a distance of approximately 36 miles in a southeasterly direction towards the Oxnard flood plain and Ventura Harbor. The White Oak Creek restoration program was part of a coordinated restoration effort involving a number of developers who have participating in building out housing and other uses within the Douglas Ranch Specific Plan Area. The segment of White Oak Creek comparison study was implemented by Standard Pacific Homes under the regulatory authority of the Corps of Engineers, Department of Fish and Game, and the City of Simi Valley. The restoration program was designed to satisfy two purposes: first, to compensate for the major disruptions to White Oak Creek which resulted from the construction of access roads and flood control measures,indoor growers and to create a common area that functions as both a habitat amenity and as a density buffer and urban design feature within the neighborhoods situated on either side of the Creek.

The Standard Pacific Homes White Oak Creek restoration is immediately linked upstream to a major flood control facility that was built within the Creek’s active streambed boundary in the newly constructed Mount Sinai Memorial Park. To comply with the Clean Water Act, horticultural riparian restoration programs were required for both the Memorial Park and the Standard Pacific residential development. As in the case of the Temescal Creek project, the scope of these restoration projects was clearly flood control, not process driven. The Standard Pacific Homes restoration included a subdivision of land which set aside about three acres of riparian restoration which is served by conducting stored detention waters beneath a manufactured, at grade, “re-constructed” streambed alignment. This reconstructed streambed carries a perennial flow of up to 195 c.f.s. through a diversion from the immediately upstream detention structure . The upstream detention basin gathers both neighborhood street discharge, as well as potential irrigation run-off from the Memorial Park as well as storm water flows.The gradients between the cemetery and residential projects were carefully adjusted and matched. In contrast to the Temescal Creek program, this artificial streambed was designed, disclosed in detail, and is considered a success by both the regulatory agency and the lead agency . Although flood control issues were primary considerations, in this case riparian horticulture restoration and habitat planning were also considered to be of primary importance—unlike Temescal Creek. This is clearly expressed in both the planning efforts and the design. The adjacent development changed ‘Faux’ White Oak Creek from a natural ephemeral channel to a manufactured perennial flow channel. This is a common outcome in southern California restoration projects because most of the time, such projects are designed to incorporate increased continual flows from upstream residential developments.As an amenity, creating a protected common area with habitat functions in a contemporary subdivision is unusual. The reconstructed reach of White Oak Creek now has the character of a young riparian greenbelt. Based on my review of the monitoring records , over the period of monitoring, perennial flow has been provided without interruption into the Standard Pacific Homes restoration area. This site has been under regulatory monitoring for four years.

The monitoring period has been extended beyond the established five-year time frame because of a series of early failures which more frequent monitoring would have revealed. The relative success of each project is compared in Table 1 through a series of defined success criteria. Unlike the outcome at Temescal Creek, for this project, there is a close correlation between original concept and implementation. The low flow diversion has been carefully designed so that it is reliable and operates within the parameters of the source detention waters. The low flow device is calibrated not to rainfall data but to neighborhood runoff flow rates, the actual reliable source of water for the restoration. In this respect, “natural” hydrologic conditions were determined not to be the governing design requirement for long-term success—flow rates out of the upstream neighborhoods were the determinant. The establishment of a firm legal basis for the protection of the restored area, , and the creation of an enclosure around the restoration site have ensured that the surrounding community does not encroach upon the project.Two different strategies of Fe uptake have been described in plants. The so-called chelation strategy , which is mainly found in graminaceous plants, is based on the excretion of phytosideropores to the rhizosphere. Phytosideropores rapidly chelate Fe, to form Fe-PS chelates that are subsequently transported into the root cells through a specific transporter. The so called reduction strategy relies on the coordinated action of a membrane bound Fe reductase, that reduces Fe to Fe, an Fe uptake transporter and an H+ -ATPase that lowers the pH of the rhizosphere, is mainly used by non graminaceous plants, including Beta vulgaris. The reduction strategy includes root morphological, physiological and biochemical changes that lead to an increased capacity for Fe uptake. Morphological changes include root tip swelling, development of transfer cells and an increase in the number of lateral roots, leading to an increase in the root surface in contact with the medium. Some plants are able to accumulate and/or release both reducing and chelating substances, such as phenolics and flavins, which may have a role in Fe acquisition.

Iron has been shown to down-regulate riboflavin synthesis in flavinogenic yeast strains and some bacteria. In plants, Rbfl and derivatives are accumulated and/ or excreted in Fe-deficient roots and could act as a redox bridge for electron transport to the Fe reductase. Moreover, FRO2 belongs to a super family of flavocytochrome oxidoreductases, and a recent study confirmed that the FRO2 protein contains FAD sequence motifs on the inside of the membrane. Also, a connection between Fe deficiency perception and Rbfl excretion has been described to occur through basic helixloop-helix transcription factors in Arabidopsis thaliana. At the metabolic level, increases in the activity of phosphoenolpyruvate carboxylase and several enzymes of the glycolytic pathway and the tricarboxylic acid cycle have been found in different plant species grown under Fe deficiency. Transcriptomic and proteomic studies in Fe deficient plants have also reported increases in root transcript and protein abundances, respectively, of enzymes related to the glycolytic and TCA cycle pathways, among others. Iron deficiency also induces an accumulation of organic acids,danish trolley mainly malate and citrate, in roots. The induction of C metabolism in roots of Fe-deficient plants would not only provide a source of reducing power, protons and ATP for the Fe reductase and H+-ATPase enzymes, but also lead to an anaplerotic root C fixation. Carbon accumulated in roots is exported in the form of organic acids via xylem to leaves, which have otherwise drastically reduced photosynthetic rates when Fe-deficient. The higher energy requirements in Fedeficient root cells are tackled by increasing mitochondrial oxidative processes, and roots from Fe-deficient plants show enhanced respiratory activities and higher O2 consumption rates. On the other hand, the mitochondrial respiratory chain is strongly affected under Fe-deficient conditions, since some of its components are Fe-containing enzymes. Iron deficiency leads to an enhancement of different ROS detoxification strategies. Furthermore, an increase in anaerobic metabolism has also been described in Fe-deficient roots, probably as an strategy to oxidize all the reducing power generated by glycolysis and TCA cycle that can not be easily oxidized in the respiratory chain. When resupplied with Fe, Fe-deficient plants reorganize its metabolism by readjusting metabolic cycles and changing root morphology towards those typical of Fe-sufficient plants. The most common approach used to study Fe deficiency in roots is to analyze only a small number of genes, proteins and/or metabolites. A more comprehensive knowledge of the processes taking place in Fe-deficient roots has been recently provided by the application of modern techniques allowing for the simultaneous and untargeted analysis of multiple genes or proteins. The aim of this work was to characterize the changes induced in the root tip proteome and metabolome of sugar beet plants in response to Fe deficiency and resupply, in order to provide a holistic view of the metabolic processes occurring in plants under different Fe status.The polypeptide pattern of root tip extracts was obtained by 2-D IEF-SDS PAGE electrophoresis. Real scans of typical 2-D gels are shown in Figure 1; an average number of 141 and 148 polypeptides were detected in Fe-sufficient and Fe-deficient plants, respectively .Several causes may account for this discrepancy, including i) protein extraction method and amount of protein loaded in the gels, ii) gel size, iii) pI range and iv) sensitivity of the staining method.

Averaged 2-D polypeptide maps were obtained using gels of three independent preparations, each from a different batch of plants . To better describe polypeptide changes we built a composite averaged virtual map containing all spots present in both Fe-deficient and control root tip extracts . Iron deficiency caused 2-fold increases in 29 spots and 2-fold decreases in signal intensity in 13 spots . Furthermore, 6 spots were only detected in Fe-sufficient plant samples and 13 spots were only detected in Fe-deficient plants . All polypeptides in the composite averaged map are depicted again in Figure 1D, to permit annotation of those polypeptides where identification was achieved by matrix assisted laser desorption ionization – time of flight MS . These polypeptides were labeled from a to v as described in Figure 1D, and homologies found are described in detail in Table 1. From the 29 spots that showed increases in signal in root tip extracts of Fe-deficient as compared to Fe-sufficient controls, the 20 more abundant were excised and analyzed by MALDI-MS. Since the sugar beet genome has not been sequenced yet and few sequences are avail-able in the databases, identification was performed by homology searches with proteins from other plant species. From the 20 spots analyzed, 14 proteins were identified . These include proteins related to glycolysis such as fructose 1,6-bisphosphate aldolase , triose-phosphate isomerase , 3-phosphoglycerate kinase and enolase . Three spots gave significant matches to malate dehydrogenase , and two more polypeptides presented homology with α and β subunits from F1 ATP synthase . Other proteins increasing in root tip extracts from Fe-deficient sugar beet plants as compared to the controls were fructokinase and formate dehydrogenase . Also, one spot gave significant matches to a cytosolic peptidase At1g79210/YUP8H12R_1 . Spot n gave significant match to a glycine rich protein, which possibly has a role in RNA transcription or processing during stress conditions. From the 13 spots detected de novo in proteome maps from root tip extracts of Fe-deficient plants , the 6 more abundant were excised and analyzed by MALDI-MS, resulting in only 2 positive matches . These significant matches were found for glyceraldehyde 3-phosphate dehydrogenase and DMRL .Changes in the amount of DMRL as well as DMRL gene expression and flavin analysis were further studied using root tip extracts of Fe-sufficient, Fe-deficient and Fere supplied sugar beet plants . From the 13 spots showing a decrease in signal intensity in root tip extracts from Fe-deficient plants as compared to controls , 3 were identified by MALDI-MS. Spots q and rgave a significant match to nucleoside diphosphate kinase I and to oxalate oxidase-like germin, respectively.

All vehicles are described by attributes that are common to all of the study vehicles, e.g., range. The attribute levels are varied over several trials to elicit different choices. With these data, econometric models are run to estimate the partial utility values for consumer preferences of each attribute Turrentine and Kurani argue that the underlying assumptions of consumer behavior in many EV Stated preference studies are flawed. These studies assume that the survey respondents have well-formed preferences for driving range, for example. Second, they assume that these preferences remain stable to forecast changes in preferences . Finally, these studies evaluate several vehicle attributes, which study participants have not yet experienced. Consequently, it is very difficult to explore the market for unknown technologies. Demonstration projects can provide a useful platform for examining early reactions and traveler responses to new transportation technologies. In the absence of such field studies, many researchers rely upon Delphi techniques–repeated queries of a panel of respondents–to explore the potential market for ITS technologies. In this evaluation, researchers conducted a modified Delphi evaluation of the market for various ITS technologies to construct the ITS scenarios, which include market penetration estimates, that will be used in the modeling exercise in this study. The modeling results will be used to identify and prioritize a range of ITS technologies on the basis of a set of energy and environmental criteria.Results from ITS operational tests underscore the importance of user acceptance in estimating the environmental impacts of ITS. For example, the SmarTraveler project in Boston found that nearly half of those who used the services changed their travel behavior in ways that could reduce traffic congestion. Nevertheless, system usage remained “too low by any measure to provide noticeable impacts on congestion” . As cited above, Van Aerde and Rakha showed for Orlando’s TravTek that emission impacts were a function of market penetration, 25 liter pot and could even switch from negative to positive depending on market penetration. This section presents an overview of various market penetration estimates that have been made for the ITS technologies examined in this study.

The market penetration results presented here serve as context and comparative points for the estimates we produced in our modified Delphi study. As with the emission studies reviewed earlier, each of these market studies was conducted using differing methods and a variety of ITS descriptions. As mentioned above, One common methodological approach has been the use of Delphi techniques. This technique is designed to facilitate the convergence of the estimates provided by experts.In 1992 the Volpe National Transportation Systems Center was commissioned by the Federal Highway Administration ITS Joint Program Office to develop an analytical framework to predict ITS impacts and assess the potential benefits of ATMS user services. The study focused on ATMS under the premise that it provides the foundation for other types of ITS services. Within the bundle of ATMS services, the study concentrated on ramp metering, signal coordination, integrated traffic management systems, and HOV lanes and ramp meter HOV bypass lanes. The framework integrates a regional planning model with freeway and arterial simulation models, which provide input to emission models, a fuel consumption model, and a safety model. The emission modeling component of the framework uses MOBILE5a and EMFAC7F to provide emission rates for input to EMISSION and EMIS . The EMIS model also provides the structure for applying fuel consumption rates to link-based data. The fuel consumption rate model used for this study was developed by the Caltrans Office of the Transportation Laboratory in cooperation with the US DOT and the FHWA . A new safety model was developed specifically for the framework, incorporating safety factors that were determined from historical accident data for the study corridor. The US DOT framework generates a set of MOEs that can be used to evaluate the impact of implementing ATMS user services. The MOEs are categorized under four major impact areas: congestion or operational measures; emissions; fuel consumption; and safety. No attempt was made to distinguish between supply- and demand-related impacts as in Brand . The framework was applied to model five alternative ATMS scenarios and the results were compared to those for a baseline configuration. The scenarios were made up of various combinations of fixed time and demand-based signal coordination with fixed time and synchronized freeway ramp metering.

A summary of the findings of these analyses are presented in the section of this report titled “Quantitative Assessment: Modeling Studies.”Brand describes an evaluation framework that is designed to be sensitive to the differences between ITS and traditional transportation improvements. An extensive set of evaluation criteria is presented, distinguishing between the supply and demand impacts of ITS deployment. The criteria are categorized into five main impact types. The first two criteria types, increased operational efficiency and increased output , are separated to avoid the possibility of dramatically underestimating the benefits of a given technology. These criteria are also structured in a second dimension explicitly to address the time frame of the impacts . The additional criteria types are safety, energy and environmental, and implementation impacts. Brand shows how the full set of criteria can be grouped to avoid double-counting of benefits because of the correlations that exist between certain criteria. An example also demonstrates how evaluation criteria can be weighted to allow the evaluator to produce an overall weighted measure of merit for each project. Finally, Brand provides default values to evaluate ITS improvements for inclusion in transportation system plans.Lo et al. developed a framework for comparison of dynamic traffic models, emphasizing the dimensions of functionality, traffic and route choice dynamics, and overall network performance. The framework is set up to compare the models against a check-list of functions and also to compare the ability of each tool to model twelve scenarios developed for five different test networks. The report provides a list of performance measures to be determined for each modeling tool and a discussion of how the results are to be interpreted. The comparison framework is designed to be generic and permit comparison of many traffic models. However, four specific models were selected for comparison in this study: INTEGRATION, DYNASMART, DINOSAUR, and METS. The comparison results and the impact of perturbations to O-D data will be presented in Part II and III of the report, which are almost complete. Some of the traffic simulation tools mentioned above are capable of estimating the energy and environmental impacts of the simulated traffic conditions . Others can provide output suitable for input to some fuel consumption and emission models. However, raspberry cultivation pot modeling frameworks that provide output suitable for average speed based emission models such as the US EPA MOBILE and the California Air Resources Board EMFAC models are not capable of providing accurate assessments of the environmental impacts of the ITS scenarios being considered.

The MOBILE and EMFAC emission factors predict vehicle emissions based in part on average trip speeds and a large number of FTP bag emission measurements. These models are intended to predict regional emission inventories, and hence they are not adequate for evaluating microscopic-level operational improvements, such as those achieved by ITS strategies. To evaluate the emission benefits of such systems, it is necessary to employ an emission model that considers the modal operation of a vehicle . The INTEGRATION and DYNASMART models make use of modal emission models that account for microscopic changes in vehicle speed profiles and Ramachandran , respectively. A brief summary of these models is presented in the sections below .Much has been written about the capabilities of the INTEGRATION traffic simulation model . This section presents some of the aspects of this model that are relevant to our research. For more detailed information about the model development, capabilities, limitations, and applications, the reader is referred to the reports just mentioned and the INTEGRATION User’s Guide . The INTEGRATION traffic simulation model was developed by Michel Van Aerde of Queen’s University in Ontario, Canada . This microscopic simulation model was developed with the purpose of simulating integrated networks composed of freeways and arterial roads, with a particular emphasis on modeling ITS scenarios. INTEGRATION 2.0 simulates the behavior of individual vehicles on signalized arterial and mainline freeway links, with the ability to model merges, diverges, and weaving sections. The model contains algorithms to simulate many aspects of traffic behavior including: lane changing; link-to-link lane transitions; car following; route selection and traffic assignment; signal cycles,including turning movements, shock waves, over saturation delay, and gap acceptance at traffic signals; signal coordination; stop and yield signs; and incidents and diversions. INTEGRATION also provides estimates of effectiveness measures for individual vehicles; links; O-D pairs; and complete networks, including link travel time, fuel consumption, and vehicle emissions.The revised ozone standard is intended to replace the current one-hour standard with an eight-hour standard. However, the one-hour standard will continue to apply to areas not attaining it for an interim period to ensure an effective transition to the new eight-hour standard. Title I of the CAA addresses the requirements for different classifications of non attainment areas that do not meet the current one-hour standard . These requirements include such items as: 1) mandatory control measures, 2) annual rate of progress requirements for emission reductions, and 3) offset ratios for the emissions from new or modified stationary sources. These requirements have contributed significantly to the improvements in air quality since 1990. Based on the US EPA’s legal review, the Agency has concluded that Title I should continue to apply as a matter of law for the purpose of achieving attainment of the current one-hour standard. Once an area attains the one-hour standard, those provisions will no longer apply and the area’s implementation of the new eight-hour standard would be governed only by the provisions of Title I. To streamline the process and minimize the burden on existing non attainment areas, the one-hour standard will cease to apply to an area upon a determination by the US EPA that an area has attained air quality that meets the one-hour standard.

In light of the implementation of the new eight-hour standard, which is more stringent than the existing one-hour standard, States will not have to prepare maintenance plans for those areas that attain the one-hour standard. For areas where the air quality does not currently attain the one-hour standard, the one-hour standard will continue in effect. The provisions of Title I would also apply to designated non attainment areas until the time each area has met the one-hour air quality standard. At that time, the US EPA will take action so that the one-hour standard no longer applies to such areas. In any event, the “bump-up” provisions of Subpart 2, of Part D of Title I, which require that areas not attaining the standard by the applicable attainment date be reclassified to the next higher classification, will not be triggered by the failure of any area to meet the new eight-hour standard. The purpose of retaining the current standard is to ensure a smooth legal and practical transition to the new standard .For areas that attain the one-hour standard but not the new eight-hour standard, the US EPA will follow a flexible implementation approach that encourages cleaner air sooner, responds to the fact that ozone is a regional as well as local problem, and eliminates unnecessary planning and regulatory burdens for State and local governments. A primary element of the plan will be the establishment under Section 172 of the CAA of a special “transitional” classification for areas that participate in a regional strategy and/or that opt to submit early plans addressing the new eight-hour standard. Because many areas will need little or no additional new local emission reductions to reach attainment, beyond those reductions that will be achieved through the regional control strategy, and will come into attainment earlier than otherwise required, the US EPA will exercise its discretion under the law to eliminate unnecessary local planning requirements for such areas. The US EPA will revise its rules for new source review and conformity so that States will be able to comply with only minor revisions to their existing programs in areas classified as transitional.

High root expression of GS1 in these fields indicates that root N assimilation was elevated and thus actual plant N availability and uptake was higher than low inorganic N pools would suggest . Fields from group 2 demonstrated N surplus, showing similar yields to group 3 but with lower total and labile soil C and N and a higher potential for N losses, given much higher soil inorganic N . While actual N losses depend on a host of factors , high soil NO3 – is considered an indicator for N loss potential. Results from a companion study support the idea that soil microbes were C rather than N-limited in these fields, showing higher potential activities of C-cycling soil enzymes but low activities of N-cycling soil enzymes, the inverse of group 3 . An alternative multivariate clustering approach based on an artificial neural network suggests multiple potential drivers of higher inorganic N pools in these fields, including both management factors and soil characteristics . For instance, field 4 had strong indications of surplus N driven at least in part by a large application of seabird guano , a readily-mineralizable organic N fertilizer, at tomato transplanting when plant N demand is low. In contrast, higher inorganic N in field 8 was likely driven by low plant N demand based on very low soil P availability, which resulted in plant P limitation. These site-specific problems were identifiable due to the focus on variability across similar organic fields and illustrate the need for site-specific approaches to reduce N losses. Finally, the two fields included in group 1 were exemplary of N deficiency, in which low N availability compromises crop productivity but also likely limits N losses within the growing season. While low soil NH4 + and NO3 – concentrations were similar to group 3, low total and labile soil organic matter and poorly-timed organic matter inputs compromised microbial activity and likely limited N mineralization.Cytosolic glutamine synthetase GS1 encodes for the enzyme that catalyzes the addition of NH4 + to glutamate,fodder systems for cattle the former resulting from either direct uptake of NH4 + from soil or reduction of NO3 – in roots.

GS1 is thus the gateway for N assimilation in roots and is upregulated to increase root N assimilation capacity. Similar levels of GS1 expression in groups 2 and 3, in spite of large differences in soil NH4 + and NO3 – concentrations at the anthesis sampling, suggests that plant N availability is indeed higher in group 3 fields than would be expected based on measurement of inorganic N pools alone. The low levels of GS1 expression found in fields with clear N deficiency supports this idea. These results complement recent experimental approaches that showed rapidly increased expression of GS1 in tomato roots in response to a pulse of 15NH4 + -N on an organic farm soil, which was linked to subsequent increases in root and shoot 15N content, even when this pulse did not significantly change soil inorganic N pools. GS1 transcripts and glutamine synthetase enzyme activity also increased with increasing NH4 + and NO3 – availability in sorghum roots, suggesting this response may be widespread among plant species. Interestingly, inclusion of soil GWC in multiple linear regression models increased the proportion of GS1 expression variability explained to nearly 30% ; soil water content increases microbial activity as well as the mass flow and diffusion of inorganic N to roots. Further research will undoubtedly show how other factors like crop physiological N demand relative to C fixation and P availability increase the interpretability of N uptake and assimilation gene expression in roots.The N cycling scenarios identified on this set of organic fields corresponded at least in part with landscape clusters based on landscape and soil characteristics . Fields that balanced high yields with low potential for N loss and high internal N cycling capacity were part of PAM cluster 1, which had the highest productive capacity rating . Landscape clusters encompassing more marginal soils included both low-yielding fields exhibiting N deficiency or high-yielding fields that used inputs of highly available N like seabird guano to alleviate N deficiency . But these inputs led to the highest soil NO3 – levels and thus came at the cost of higher potential for N loss. Long-term efforts to increase internal soil N cycling capacity would help alleviate both N deficiency and the need for such large inputs of labile N. Whether farmers are willing to invest in management to increase soil N cycling capacity depends in part on how likely they perceive the benefits to be, especially on marginal soils.

The discussions that we had with each farmer in this study indicated genuine interest in adaptive management to further tighten plant-soil N cycling, but this may not always be the case. Indeed, the proportion of management vs. inherent soil characteristics responsible for driving differences in N cycling is challenging to untangle. Farmers may allocate more resources to more productive land and likewise fewer resources to more marginal land, or may selectively transition more marginal land to organic management. Documenting the multiple services provided by increases in soil quality and facilitating information exchange among organic growers such as through the landscape approach used here may help build momentum for efforts to improve soil quality and plant-soil-microbe N cycling.Net tropical forest loss of 7 million hectares per year occurred between 2000 and 2010, with conversion to agriculture accounting for 86% of deforestation . Annual deforestation in tropical Asia during the 1990s reached up to 5.6 million ha yr−1 , resulting in the emission of 1.0 Pg C yr−1 to the atmosphere . In Indonesia, the total forest area of 117 million ha in 1990 dropped to 89 million ha in 2011–2012 with primary, secondary and plantation forests occupying 45.2, 40.8 and 3.0 million ha, respectively . The average forest loss of 1.3 million ha yr−1 from 1990 to 2012 resulted from burning and conversion to agriculture, mining and infrastructure with Indonesia contributing to ∼10% of total global forest loss each year. Short-term changes in soil properties following conversion of tropical forests to agricultural land use are often pronounced and in most cases detrimental to sustainable agricultural production. In contrast to the Amazon rainforests supported by Oxisols and Ultisols , Indonesia’s rainforests are largely supported by volcanic soils, primarily Andisols. These Andisols support high agricultural productivity with some of the world’s highest human-carrying capacity being found on volcanic soils in Indonesia . With respect to greenhouse gases, fodder sprouting system Andisols are notable for having the highest soil carbon storage capacity among the mineral soil orders in temperate and tropical climatic regimes with an average carbon stock of 25.4 kg C m−2 . Matus et al reviewed soil carbon storage and stabilisation in andic soils and concluded that the most important mechanism of sorption of soil organic matter by short range ordered amorphous minerals is the ligand exchange.

While short-term changes in properties of tropical rainforest soils have been extensively studied, there is a paucity of information concerning long-term changes in soil properties resulting from changing land use and management practices, especially with respect to Andisols. Greenhouse gas emissions from agriculture are reported to contribute up to 30% of anthropogenic emissions . Soils can be a major source or sink of GHG from terrestrial ecosystems depending on the ecosystem disturbance regime and soil management practices. Soil carbon storage is dependent on soil mineral constituents, with volcanic ash soils typically having exceptionally high potential C stocks owing to their high content of active Al and Fe constituents . In Andisols, Chevallier et al. showed organic matter transformation to CO2 via microbial respiration was lower as allophane content increased. In addition, changes in land use/land cover alter organic matter quantity and quality, which are major factors controlling soil microbial biomass and activity . Given the high C stocks in Andisols, it is important to assess the fate of soil C following land-use conversion from forest to intensive agricultural production, especially with regard to rapid deforestation in the tropics. Andisols have several unique properties that affect agricultural productivity, such as high P fixation, high organic matter concentrations, a clay-size fraction dominated by pH dependent variable charge, low bulk density, high porosity, high water retention capacity and high mesopore content . In particular, high P retentionin Andisols can limit agricultural productivity by limiting plant availability of P. Currently, there is little information on how P retention and availability in tropical Andisols change with different land use and agricultural practices. Nitrate leaching characteristics in Andisols are also strongly affected by variable charged constituents as positive charges can retain nitrate enabling higher plantutilization efficiency. In southern Chile, Huygens et al. reported NH4 + and NO3 − retention of 84 and 69% of N fertilizer additions, respectively, after one year based on 15N pool-dilution and 15N tracer studies of forested Andisols. In Japan, the maximum nitrate adsorption by Andisols ranged from 0.4 to 7.0 cmolc kg−1 with the highest values occurring in soil horizons with high allophane content and low organic carbon content . Furthermore, Deng et al. evaluated the denitrification rates from eight Andisols under three different cropping systems in an intensive livestock catchment of central Japan and reported that N loss via denitrification from upland fields was almost negligible in spite of substantial N inputs . In addition to retention of NO3 − by positively charged colloids, a laboratory study by Matus et al. reported high retention of NO3 − in Andisols through transformation of NO3 − to dissolved organic nitrogen . In Indonesia, land use/land cover of Andisols is primarily native rainforest, tea plantation, horticultural crops, terraced paddy fields and other food crops. Land-use conversion from tropical rainforest to agriculture has taken place over long periods of time ; however, no rigorous studies have examined changes to Andisol soil properties over these time periods. In addition, several studies have examined microbial biomass carbon and CO2 measurements in topsoil horizons, however, MBC and CO2 measurements in subsoil horizons have been ignored although these measurements are crucial for explaining the exceptionally high carbon stocks in Andisols. Given the several unique properties of Andisols, it may be expected that these soils are more resilient to land-use change and agricultural management practices. Therefore, we hypothesize that the unique soil properties of Andisols lessen the negative impacts of land-use change from tropical forest to agriculture on soil physical, chemical and biological properties. The objective of this study was to take advantage of long term, land-use/land management changes to examine changes in several physical, chemical and biological properties of Andisols in tropical Indonesia following conversion of rainforest to tea plantation and horticultural crops.Samples were pretreated with H2O2 to remove organic matter and dispersed with dilute Na-hexametaphosphate. Silt- and clay-sized fractions were measured after sedimentation according to Stokes law. The sand fraction was separated from the clay and silt fractions by wet sieving through a 0.05 mm sieve. Water retention at various tensions was determined using a pressure plate. Plant-available water holding capacity was estimated as the volume fraction of water retained between 33 and 1500 kPa. A sample of < 2-mm , air-dry soil was placed on a porous ceramic plate and wetted by capillary action; gravimetric water content was measured following attainment of equilibrium at 33 and 1500 kPa. Soil pH was measured 1:2 in H2O and 1.0 M KCl. Phosphate retention was determined using the method of Blakemore et al. and the Bray-1 extraction was used as an estimate of available P . Exchangeable cations were displaced by 1 M NH4OAc at pH 7.0, then the cations were measured in the supernatant using an atomic absorption spectrometer . The cation exchange capacity was determined in 1 M NH4OAc after extraction of NH4 + by 10% NaCl as a measure of CEC. Base saturation was calculated as the sum of base cations by 1 M NH4OAc divided by CEC. Sulfate-sulfur was extracted using monocalcium phosphate as outlined by Schulte and Eik and available micronutrients were determined by DTPA extraction . All weight percent data were reported on an oven-dry basis .