In order to compare between experiments, central and public data storage is fundamental. The use of standardized metadata combined with open controlled vocabularies or ontologies is crucial to being able to interoperate between different data types. The “findable, accessible, interoperable, reusable” data principles are aimed at improving the data ecosystem to allow researchers to better locate and integrate data. In the phytobiome sphere, the National Microbiome Data Collaborative is a new initiative to make microbiome data FAIR and aims to use standards such as the Environment Ontology to describe environmental characteristics of samples and the microbial ecosystems embedded within them. One of those limitations surrounding reference databases is the paucity of experimentally validated data that links microbial and plant metabolism, protein function, and DNA sequence. As an example, whereas microbial genes are assigned putative functions based on sequence homology, their actual activity may deviate from these annotations leading, to incorrect interpretations and predictions of ecosystem function . Furthermore, standardized analysis is critical and can be achieved by using centrally updated, state-of-the-art software tools. KBase, the U.S. Department of Energy Systems Biology Knowledge base, has offered a public data storage and analysis dashboard that allows the generation of so-called narratives in which a dataset undergoes a string of analyses . In addition,vertical farming tower for sale large-scale field datasets are increasingly taking advantage of supercomputer resources and ML algorithms that are required to filter noise and generate sensible interpretations from billions of data points.The development of the above mentioned technologies and experimental platforms will improve our understanding of the plant–microbe–atmosphere–soil ecosystem at high spatial and temporal resolution. The combination and integrated use of the discussed tools will further provide opportunities for novel approaches to plant root microbiome research.

An example of an integrated approach is the combination of UAVs equipped with advanced imaging capabilities to study QTL or GWAS populations growing in the field. This would streamline and scale current experimental procedures, so that new genetic markers for various above- and below ground phenotypic characteristics could be identified. These, in turn, could be correlated to microbiome community profiles in roots and leaves. Another example is the combined usage of SynComs and single plants in EcoFabs for advanced root and microbe imaging resolved over space and time complemented with metabolite analysis,enabling systematic examination of the role of specific microbes and metabolites in modifying root architecture. This approach can help identify novel, specific microbial products that can be used to influence important plant traits known to affect field performance . Microbial model systems can then be engineered to produce promising compounds for tests on plants in soil. Finally, we foresee EcoPODs and EcoTrons being used for time-series experiments that span several weeks and months, possibly years, in which high-throughput omics together with continuous environmental sensor measurements can provide in depth yet broad-scale datasets that can be used for training artificial intelligence algorithms related to biogeochemical cycling in relationship to climate. Due to the many direct and indirect ties between local plant– microbe–soil–ecosystem well-being and systems-wide ecological health, technological improvements in phytobiome research are directly translatable to improvements in climate change research. The above mentioned advances in instrumentation and methodology push precision agriculture and precision phytobiome research forward and allow for improved and more sustainable crop productivity under rapidly changing and increasingly extreme climatic conditions. These advances will have impacts in food and energy security and bio-safety as well as environmental conservation and bio-remediation.Seasonally dry tropical forests are dominated by deciduous species coexisting with a small number of evergreen species . Trees withstand the dry season through two mechanisms of drought resistance: desiccation delay and desiccation tolerance .

Two important traits related to desiccation delay are leaf shedding which reduces water loss, and depth of rooting , which determines the sources of water and nutrients used by vegetation . Although previous reports suggested that evergreen species access relatively deeper water sources than deciduous species ,more recent reports suggest that access to water is more related to tree size than phenology . However, there is relatively little information regarding differences among deciduous species having different timing or leaf shedding behavior, even though it is well known that leaf senescence behavior varies greatly among tropical dry forest tree species. Flushing and leaf abscission result from complex interactions between plants and their environment; in many species, the main abiotic factors driving these processes are solar radiation, air relative humidity, vapor pressure deficit, precipitation and soil water content . Four main categories of leaf shedding phenology have been proposed by Williams et al. : evergreen species, which retain a full canopy throughout the year; partially deciduous species, which lose up to 50 % of their canopy during the dry season; semi-deciduous species, which lose more than 50 % of their canopy during the dry season; and deciduous species, in which all leaves are lost during the dry season as they remain leafless for at least 1 month. Most tropical dry forest species are thought to deploy the majority of their root systems relatively deep in the soil profile where moisture tends to be greater and of longer duration . However, in northern Yucatan the hard upper limestone layer, beginning immediately below the shallow soil, impedes root growth, limiting downward growth to crevices and rhizoliths, and the occasional cavities filled with soil material . Rock crevices allow roots to grow far deeper than they would in unfractured bedrock . Thus, in the seasonally dry tropical forests of northern Yucatan, the ability of tree species to grow deep roots and access additional sources of water beyond topsoil could be a crucial characteristic related to variation in phenology and the relative abundance of contrasting tree species. Sources of water used by trees can often be identified by comparing the isotopic composition of water from stems with potential water sources, because there is usually no isotopic fractionation of either hydrogen or oxygen isotopes during water uptake . When trees take water from more than one source, the proportion of water absorbed from each source can be calculated using isotope mixing models .

Such models were developed to cope with multiple sources and allow the input of ancillary data that are known about the system to constrain model outputs, thereby providing results that are restricted to real possibilities. Sources of water used by native trees in northern Yucatan have been studied using these approaches, and large variation in the depth of water uptake among deciduous and evergreen species has been observed . Furthermore,hydroponic vertical farm using these same isotopic approaches along a forest age chronosequence in northeastern Yucatan, evergreen trees were found to access deeper water sources than deciduous species in early succession . Thus, integrating rooting depth as a component of tropical dry forest tree strategies appears especially promising in complex karstic Yucatecan soils. Water-use efficiency , the ratio of carbon gained in photosynthesis relative to water loss during transpiration , is another key factor when considering the costs and benefits of a deep rooting system. Leaf carbon isotopic composition can be used to assess WUE in certain circumstances, and is often positively related to WUE because a high photosynthetic rate per unit stomatal conductance is usually associated with relatively low internal CO2 concentration and reduces discrimination against 13CO2 by rubisco . Although d13C has been used alone to infer WUE, its combination with analysis of isotopic composition leaf organic oxygen improves interpretation of leaf d13C values by allowing analysis of whether variation in d13C is due to changes on the photosynthetic activity or stomatal activity . When humidity increases, the isotopic enrichment of leaf water decreases, causing a reduction in d18O . Theory and empirical data also demonstrate that d18O correlated negatively with stomatal conductance . In shallow soils of northern Yucatan, Querejeta et al. showed that individuals of the same tree species differing in age had different WUE, with younger trees having greater WUE than older ones, indicating that these techniques hold promise for integrating potential differences in water sources with leaf physiological activity. This study focuses on phenological variation between two dominant tropical dry forest species in relation to the depth of water uptake. We hypothesize that the late deciduous habit in P. piscipula and the early deciduous habit in G. floribundum may be determined by their ability to take water from different sources. P. piscipula may have access to deeper sources than G. floribundum. However, due to the restrictions for root growth imposed by the hard bedrock, both species will likely extract most of their water from shallow sources. We also hypothesize that differential use of water sources is linked to key ecophysiological measures of plant performance, including the timing of leaf fall, leaf size, leaf water potential and the balance of carbon gain and water loss as interpreted by leaf stable isotopic composition.Topsoil, calcium carbonate rock layers, soil pockets and plant tissue samples were collected in three sampling campaigns: October 2007 , January 2008 and May 2008 . Topsoil, bedrock and soil pocket samples were obtained from recently exposed walls. Sampling of the lower portion of the walls was restricted by the rock materials produced during blasting events, moreover, roots were not often observed growing in this layer; thus, soil pockets and rock samples were taken only from 0 to 5 m depth.

Because topsoil was removed before rock blasting, soil samples were taken from areas up to 40 m away from the wall being sampled. In addition, gravimetric water content was evaluated by taking 20 samples from topsoil and each rock layer, and a variable number of samples from soil pockets depending on presence of these features in the wall being sampled and drying at 105 C. The eight water sources considered go down from the upper soil layer down to the ground water . Non-transpiring woody shoots were collected from five individuals of each species from vegetation within the quarry. Four stem samples of 5–10 mm width and 50–80 mm long were obtained from each tree. The same trees were sampled at each sampling campaign. No leaves or green tissue were included in the sample to avoid contamination of xylem water by isotopically enriched water that had undergone evaporation from the plant . Samples were preserved in hermetic capped vials wrapped with parafilm and stored in the freezer until processed. Groundwater samples were taken from an open well 2 km from the quarry. Water was extracted from topsoil, soil from pockets, rock and plant stem samples using a cryogenic vacuum distillation line for at least 60 min for stems and 40 min for soil and rocks . Water content of soil, bedrock and tree stems was calculated from sub-samples taken right before the water extraction and oven dried for 24 h at 80 C and 105 C . Stable isotopic composition of oxygen and hydrogen analyses were conducted at the Centre for Stable Isotope Bio-geochemistry at the University of California, Berkeley using a chromiumreactor interfaced with a continuous flow isotope ratio mass spectrometer . d18O and d2 H values were reported in delta notation relative to the Vienna Standard Mean Ocean Water . Values of substrate and stem water d18O and d2 H were plotted in a bi-variate relationship with the Meteoric Water Line determined for this region by Socki et al. , to evaluate the role of water sources across seasons. The contribution to tree water uptake from the different sources during the three sampling campaigns was calculated using Iso-Source software . This software calculates ranges of source-proportional contributions to a mixture based on the isotopic signatures of the mixture and each of the sources. Both d18O and d2 H data were used for model calculations. The data set for modeling was grouped as follows: Laja 0–200 cm was disregarded because is not a significant source of water at any time , February data were not analyzed to avoid misinterpretations due to unsampled water sources , ground water was taken out of the analyses because root systems of P. piscipula and G. floribundum were not observed growing beyond 5-m depths and, bedrock from 200–400 cm was grouped as a single source because their isotopic values were very similar. Because sampled trees were growing \1 km from where rock samples were taken, we assumed that the isotopic compositions of rock were similar in both places.

Please refer to other reviews that discussed aspects of this process . Rhizobacteria was shown to induce the accumulation of sesquiterperne synthase transcripts . Sesquiterperne, used in cosmetics and perfumery, is one component of vetiver root essential oil synthesis. The authors showed that the root associated bacteria metabolized the vetiver oil and produced additional compounds, which suggested that each distinct rhizobacteria community contributed to a signature composition of commercial vetiver oil. For two decades, it was thought that the plant pathogenic fungus Rhizopus microsporus produced the antimitotic polyketide macrolide rhizoxin. Partida-Martinez and Hertweck uncovered that it was actually an endosymbiont, Burkholderia sp., of the fungus that produced rhizoxin. The authors demonstrated that in the absence of Burkholderia, no rhizoxin was produced in the fungal culture. Transmission electron microscopy demonstrated that Burkholderia sp. was localized in the fungal cytosol . Rhizoxin inhibits mitosis and leads to cell cycle arrest, and has potential as an antitumor drug. Further investigation elucidated rhizoxin derivative structures and obtained stable analogues by inhibition of a putative P-450 monooxygenase . Ryan et al. summarized applications for bacterial endophytes, including production of bio-materials such as poly-3-hydroxybutyrate and poly-3-hydroxyalkanoate . Catalán et al. showed that diazotrophic endophyte Herbaspirillum seropedicae could accumulate 36% of its biomass as PHB and could constitute a cost-effective mean for producing bio-material.

Organic contaminants naturally biodegrade,plastic pots 30 liters except for chlorinated compounds such as polychorinated biphenyl and 4-chloronitrobenzene , which tend to be persistent and recalcitrant in the environment. Recent reports on chlorinated compound degrading rhizosphere bacteria are presented. Natural PCB-degrader bacterial populations were cultured from several plant species growing in a contaminated site. High numbers of culturable PCB-degrader colonies were isolated from roots of Austrian pine and goat willow . Most of the PCB metabolizing bacteria are Rhodococcus sp. However, the isolates were first selected based on cultivability and morphology thus possibly leading to a biased representation of PCB-degrading bacterial communities. In addition, since the PCB degradation capability of these strains was tested in liquid medium, the benefits of plant association was not addressed. Narasimhan et al. investigated the role of plant secondary compounds in stimulating rhizobacteria growth and PCB removal efficiency. The authors identified that the Arabidopsis root exudates consisted mostly of phenylpropanoids, such as flavonoids, lignins and indole compounds. Wild type and mutant Arabidopsis lines over producing flavonoids sustained higher counts of flavonoid-utilizing P. putida strain than bacteria grown on Arabidopsis mutant not producing flavonoids. More interestingly, close to 90% of PCB was degraded in soil adhering to the roots indicating that direct contact with roots and the exudates resulted in bacteria growth and bio-degradation enhancement. Genetically engineered microbes were also applied towards remediation. The PCB degradation efficiency of a recombinant strain of P. fluorescens expressing a bph operon under the control of nodulation genes from Sinorhizobium meliloti, was examined . Resting cell PCB-degradation experiments indicated that the recombinant strain metabolized different cogeners of PCB more efficiently than the Burkholderia sp. strain LB400. However, no plant-bacteria potted experiments were performed to measure PCB degradation. Böltner et al. enriched rhizosphere microbes and demonstrated that four Sphingomonas strains were capable of rhizoremediating hexachlorocyclohexane .

Potted experiments showed that 30% of lindane was removed with corn seedlings inoculated with the Sphingomonas strains. Whereas, in unplanted soil, sterile planted soil, and uninoculated-unplanted soil, less than 3% of lindane was removed. Liu et al. successfully inoculated a gfp-tagged strain of Comamonas sp. onto the roots of alfalfa as demonstrated by quantitative competitive -PCR and confocal laser scanning microscopy . The rhizosphere community shifted due to addition of 4CNB and the Comamonas strain was characterized using denaturing gradient gel electrophoresis . In outdoor potted experiments, the symbiosis enhanced phytotoxicity resistance, and removed 4CNB faster than the plant control without the strain in a 24-hour period. However, the control without the strain removed 60% of the 4CNB compared to 99% in experiment with strain inoculation. It seemed likely that if the experiment was extended beyond the 24-hour measurements, the 4CNB removal might be similar between the control and the treatment.However, certain volatile organic compounds such as trichloroethylene and BTEX are released into the atmosphere through the plant’s vascular system. The endophyte-plant interaction was used to degrade these volatile organic pollutants and minimize evapotranspiration . Barac et al. demonstrated that the genetically modified endophytic strain of Burkholderia cepacia together with yellow lupine reduced evapotranspiration of toluene by 50-70%, and reduced phytotoxicity. The same group inoculated poplar cuttings with two strains of B. cepacia, an endophyte and a soil isolate, expressing the toluene monooxygenase , and showed significantly less toluene being transpired in the poplar inoculated with the endophytic B. cepacia . In addition, the authors indicated evidence for horizontal gene transfer of the recombinant plasmid encoding the toluene degradation pathway from the inoculant B. cepacia to the endogenous microbial community, and suggested that it would be possible to eliminate selection of an appropriate endophyte since the bio-degradation gene will be transferred to the endogenous microorganisms.

However, the authors touched on the issue of low horizontal transfer efficiency but did not elaborate on solutions for the lack of control over the time scale of the transfer and recipients of the genes. The authors also did not comment on the risks associated with the spread of exogenous genes into a new environment. However, the risk caused by a gene originally isolated from the environment, such as the tom gene should be low compared to risks from synthetic genes. Moore et al. isolated endophytic bacteria from two poplar varieties that were grown at a field trial site phytoremediating BTEX in the groundwater. There were differences in spatial compartmentalization of strain localization in the root, shoot and leaves suggesting there were species-specific and non-specific associations between bacteria and plants. However, the study did not examine the reasons for the strain localization and association with the plants and whether remediation efficiency was affected by bacterial community. Heavy metal contamination is a persistent problem since the metals, unlike organic compounds, do not biodegrade. Several recent reviews address role of plant growth promoting rhizobacteria in metals remediation , and various plant bacteria systems that have been applied towards remediation of metals contamination . Removal of metals from soil or groundwater using rhizosphere bacteria and plants has been demonstrated recently. Willowseedlings inoculated with different strains of rhizosphere Streptomyces and Agromyces and the strains ability to produce indole acetic acid and siderophore was measured. No correlation was observed between IAA and siderophore production and metal accumulation. Only plants inoculated with Agromyces terreus exhibited reduced phytotoxicity due to cadmium and zinc. Metal accumulation in leaf biomass was measured . However, the data did not demonstrate a marked difference between the inoculated plants and the non-inoculated control. Perhaps,round plastic pots most of the phyto accumulated metals were immobilized in the roots where most of the rhizosphere strains would be located. No root metal concentrations were measured. Wu et al. utilized an engineered symbiosis between recombinant P. putida and sunflower plants for adsorption of cadmium. The recombinant P. putida expressed a synthetic phytochelatin, EC20, and exhibited inherent cadmium resistance. The inoculation of the P. putida strain enhanced plant growth and resulted in 40 % more cadmium accumulation from hydroponic solutions than the non-inoculated control. No field study was conducted. Ryan et al. demonstrated that a recombinant strain of P. fluorescens F113rifPCB with arsenic resistance genes and PCB degradation capabilities protected M. sativa when grown in soil supplemented with sodium arsenate. No arsenic removal nor PCB degradation efficiency were assessed with this plant-bacteria system. Since most of the metals tend to accumulate in the top 20 cm layer of the soil , grass species with high fine-root biomass providing large surface area for bacteria colonization and metal accumulation, in the top soil layer, would be ideal for remediation of metal contaminated sites. The plants serve as concentrators of metals, and further treatment of the plant biomass accumulated with metals would be required. Overall, the use of the plant-bacteria system would be more cost-effective than excavation of the soil contaminated with low levels of heavy metals. The successful demonstrations of laboratory-scale phyto- or rhizo- remediation do not always translate into adequate removal of contaminants in field-scale. The reasons include heterogeneity of the field site, unpredictability of environmental conditions, inability to sustain bacterial population and the remediating microbes being out competed by endogenous organisms. There are several reasons that plant-bacteria interactions are advantageous when applied to remediation. The plant-associated bacteria population would be more competitive than the native soil microorganisms since plant exudates provide nutrients.

Plants would act as natural pumps that draw contaminated soil pore water towards the plant-associated remediating bacteria. Rhizoremediation is an ideal strategy for cleanup of mixed-contaminants. Recombinant rhizosphere bacteria with specific genes targeting pollutants present at the site can be inoculated into plant roots. For example, Lee et al. engineered two strains of rhizobacteria with TCE degradation capability, and surface expression of synthetic phytochelatins for improved heavy metal resistance, thus enhancing the rate of TCE degradation. An excellent review by Gerhardt et al. addressed strategies to overcoming challenges of field application of phyto- or rhizo-remediation such as stressors to the microbes, complexity of the field conditions, regulatory acceptability and the use of genetically modified organisms . Research that look at the impact of GMOs on native bacteria and bacterial community changes post introduction of willow trees for rhizoremediation of PCB contamination are starting to emerge and will elucidate questions regarding the impact on the native microfauna. Studies that compare remediation efficiencies of natural attenuation, bio-augmentation, phytoremediation and rhizoremedation, under field conditions , assess bacterial colonization and activity on plant roots, and measure allometric relationships of tree trunk size and various root parameters provide invaluable information for determining field application options and evaluating contaminant removal. More extensive, long-term field comparisons of control plots with treatment plots for remediation efficiency are necessary to validate laboratory observations and to gain public and regulatory agency confidence for this promising application. Another application of plant-microbe interaction drawing increasing attention is carbon sequestration, where atmospheric C is deposited as plant root material, incorporated into the soil microorganisms and soil organic matter. It is hypothesized that increase in CO2 leads to an increase in rhizodeposition, and wider C/N ratio thus retarding decomposition . Several reviews are dedicated to presenting methods for measuring CO2 fluxes in different soil compartments , mechanism of root carbon stabilization , effects of elevated CO2 on below ground carbon storage , and carbon sequestration by roots . This nascent field is still at the exploratory stage where most of the research is focusing on understanding the effects of elevated CO2 levels on microbial community, below-ground plant material production, and biomass decomposition, as well as land management practices and plant species on the long term potential of C rhizodeposition. Several researchers explored the possibility of different land management practices to enhance C sequestration. Bailey et al. compared ex situ incubations of soil samples from five different ecosystems . The restored prairie samples had the highest total soil carbon and also the highest fungal-to-bacterial activities . The authors asserted that increased F:B ratios correlated with higher amount of carbon stored in the soil. Also, that invasive land management decreased fungal biomass and thus the carbon stored in the soil. Soil samples were collected and CO2 respiration experiments were conducted in a laboratory setting over a 6-hour period, thus the measurements might not be representative of field conditions. Heinemeyer et al. described contradicting results in their in situ study of the ability of Lodgepole pine associated mycorrhiza to store soil C over a period of 1- year, where the fungus was thought to return plant surplus C directly back to the atmosphere. Verburg et al. compared the net ecosystem carbon exchange between the atmosphere and two experimental non-native cheat grass varieties in a 2-year study in the Desert Research Institute’s Ecologically Controlled Enclosed Lysimeter Laboratories. They showed that fertilization increased C uptake initially, however, C loss through soil respiration was also enhanced. Bazot et al. found similar results, at a grassland ecosystem of Free Air CO2 Enrichment is Eschikon, Switzerland, where increased N supply to the plants enhanced allocation of fixed C to the shoots and reduced below ground carbon allocation and rhizodeposition.

All of the farms in our survey follow agroecological production practices which include a focus on building soil health through, most commonly, cover cropping, compost application, and no-till practices. These practices produce synergistic effects of adding fertility to the soil through organic matter amendments and boosting water holding capacity. Soil building practices are a response to the impetus to remediate toxins present in urban soils , a prerequisite to intensive cultivation and unique consideration of the urban farm environment. Overall, production practices on our urban farms seek to conserve, protect and enhance natural resources. Our survey respondents described numerous strategies for enabling diversified, intensive production of fruits, vegetables, and other agricultural products. These strategies span both short and long-term, from planting in raised beds with imported soil, to building soil health in situ via heavy applications of compost, manure, and cover crops for several years leading up to vegetable crop production. There is a growing interest in using no-till practices, which are among the suite of practices associated with “carbon farming” for enhancing soil carbon sequestration . This illustrates a synergistic opportunity for urban food policy and urban climate policy, showing where urban food production and city Climate Action Plans 4 can converge and generate mutual support . Farmers are also engaged in innovative resource recycling and resource use efficiency and other strategies to enhance resilience such as installing rainwater catchment systems in concert with swales and soil health practices to optimize use of this scarce resource.

Farms are planting native flowers and shrubs to attract beneficial insects,fodder sprouting system rather than purchasing chemical inputs for pest management. From a city planning perspective, the impetus to remediate storm water overflows and maintain corridors for essential pollinators are two priorities that can be met through incentivizing and planning spaces for UAE.East Bay urban farms reflect multiple scales and forms of diversity including agrobiodiversity, organizational and participant diversity, diversified sources of capital, labor and land arrangements, as well as diversified modes of exchange. Diversity among operations technically doing the same thing- growing food in cities- signals the fluid, flexible, peripheral, and at times revolutionary nature of urban food production spaces, which may conflict with or resist the institutional, political-economic status quo . Urban farms rely on diverse revenue streams from their diversity of activities beyond sale of produce. These activities, including educational services and community events, are important to elevate in policy conversations. Valuing and therefore protecting urban food production spaces requires thinking differently about them in a context like the San Francisco Bay Area. One stakeholder suggested considering urban farms as museums, providing essential cultural and educational offerings to city residents . The quality of the food and the value of the education, health, and community building, are strong arguments for including urban farms in an urban-agroecological framework for city planning and efforts to improve CFS. The diversity of land access agreements and labor sources used by urban farmers in the East Bay underscores equity considerations in urban agroecological transitions. Farms rely heavily on donated land and volunteer and citizen labor. Even 50% of the for-profit enterprises reported relying on volunteer labor, speaking to both the precarious economics of running an economically viable for-profit food production business in the city, and the interest among young people and aspiring farmers in gaining agroecological cultivation skills through arrangements where they donate their labor free of charge. Volunteer labor substitutes for revenue to a certain degree, allowing farms to exist and distribute food informally without needing to generate much revenue or provide many jobs.

In the UA literature, reliance on volunteer labor comes under criticism for being a product of the “neoliberal city,” where responsibility for action falls to the individual rather than the state, and the equity concerns around who is able to volunteer their time are problematized . By reporting the common use of volunteers on East Bay urban farms, we do not seek to promote or valorize this practice, but rather recognize it as a necessary interim step occurring in our study context in the absence of dramatic local government intervention or radical reforms to address community food insecurity: those who are willing and able are participating through civic engagement in urban farms to produce, harvest and distribute healthy food to those in need. Many volunteers are retired or recent graduates, seeking opportunities to contribute meaningfully to their communities. The volunteers we have communicated with generally report positive experiences and enjoyment from their time digging in the soil. Despite this, it is vital to acknowledge that the goals of food sovereignty underlying agroecology, especially the Nyéléni declaration, imply that food producers need to be able to earn a living to secure other basic needs, farm revenue is needed to sustain operations, and community members need to be able to pay. However, in cities where wages are stagnating relative to the cost of living and the right to remain is under threat to rising property values and rents , affordability of food impacts growers and consumers alike. The critique in the literature against charity in the food system is that the dependence on charitable donations in the food space are a patch for the destructive neoliberal state, which has shifted the burden of social well-being onto the nonprofit sector. Heynen, critiquing the depoliticization of hunger and poverty through charity, asserts that “[c]harity, however well intentioned, has become the means by which the welfare state was successfully rolled back” . At the same time, in exploring the radical democratic politics of groups like Food-Not-Bombs, Heynen describes the kind of anarchist philosophy of mutual aid and cooperativism through food sharing that we see in the East Bay agroecosystem.

Farms are not just distributing food to the hungry in hidden basements or exploiting free labor, but engaging in highly visible work, inviting those who visit or consume farm outputs to work, cook, learn, teach, share and get political. The reality is that growing food in cities has particular challenges, increasing the costs of farming on top of issues already outlined regarding the cost of land and labor . We find that the importance of donated money and time to further the anti-hunger and advocacy efforts of farms is not counter to the transformational goals of AE broadly or UAE in particular. In this way, we seek to nuance the premise that volunteer labor is universally problematic and counter-productive to radical food system reform efforts, aligning instead with some urban agroecological scholarship that argues for improved work-life balance through living wage jobs that afford more people opportunities to pursue hobbies and interests and volunteer their time supporting community efforts that align with their values . Pimbert outlines three dimensions of urban agroecological transformation that are needed, including economic, with new forms of organization and relocalized wealth production as well as “creation of free time for citizens to shape and re-govern urban spaces” . Volunteerism has a place in a transformed, equitable, environmentally sustainable local food system,microgreen fodder system although reliance on it as the primary source of labor is undesirable. Our findings around labor in particular stand in contrast to the often-referenced benefit of urban agriculture as a job creation tool . At least in the current political economic landscape of the East Bay, urban farms do not generate enough economic revenue or city investment in order to hire many full time positions; this remains a goal of many operations and opportunity for policy intervention, especially with respect to enhancing the resilience of urban agroecosystems to economic disturbance.Farms in our case study display a strong focus on reducing hunger and promoting food equity, namely through culturally appropriate diets, and the emphasis on human and social values. Due to the plethora of produce going home with volunteers, circulating at neighborhood crop swaps, and gleaned or harvested by community members that is not weighed and tracked before it is consumed, it is understandably difficult to quantify the “food security” impacts of urban agriculture . While food security may be difficult to quantify, it is nevertheless being addressed by urban farms in unique ways . In school gardens, for example, produce that is not used for classroom cooking demonstrations sometimes goes home with students or families excited to find culturally relevant crops growing in their neighborhood. Supporting healthy, diversified and culturally appropriate diets are an important element of agroecology. The diversity and quality of produce grown, especially when it is an item that might not otherwise be available to a family in a “food desert,” contribute greatly to the value produced on urban farms. One farmer interviewed described how one school garden site serves students from Hispanic, African American, Middle Eastern, Asian, and Eastern European families. The garden teacher spoke about the diversity of crops relevant to various cultural food traditions; for example, the chayote plants were of particular interest to Latinx students excited to bring them home to their mothers, while African American students eagerly collected bunches of collards, and Middle Eastern mothers came to the garden in person to collect fava beans and figs. In this way, urban agroecology contributes to food security and nutrition as well as biodiversity. It also serves to reaffirm cultural identity and a sense of place for immigrant and refugee families.

Agroecology places a strong emphasis on human and social values, such as dignity, equity, inclusion and justice contributing to improved livelihoods of [urban] communities . Our study demonstrated that the majority of farm respondents placed food security, education, and environmental sustainability above profit, sales and yield. Forty percent of respondents self-identified as “Educational” farms, and most others offer educational workshops and demonstrations as part of their focus on horizontal knowledge-sharing.The majority of our study respondents were also women. As a grassroots movement, urban agroecology can empower women to become their own agents of change.Our results suggest the opportunity to reconceptualize and refocus the urban food policy discussion in U.S. cities around urban agriculture in a way that includes and values their social, educational, and cultural services. Urban farms are recreational and cultural heritage sites bearing comparison to public parks and museums, while also producing invaluable healthy food in areas that most need it. They provide important respite, social connection, and stress reduction to urban residents, often particularly in need of peaceful spaces. In the words of one farmer, “Urban farms can be havens of peace, health, and community, but it requires heavy involvement and advocacy from those communities for the long term in order to be successful” . Agroecology calls for responsible and effective governance to support the transition to just, equitable and sustainable food and farming systems. In an urban environment, this requires the creation of enabling policies that ensure equitable land access and producer control over access to land, especially among the more vulnerable and historically marginalized populations. Land access is expressed most frequently as an obstacle to scaling urban food production by survey respondents, and it is certainly more of a challenge for lower-income and minority groups interested in cultivating their own “commons” . There are examples among our East Bay survey respondents of collective governance at the farm and community level, such as one farm site which is owned cooperatively by three non-profit organizations that collectively serve minority and formerly incarcerated populations, aspiring beginning farmers, and the local community through a cooperative goat dairy, fruit tree nursery, and annual vegetable production plots. City and county governance bodies have an opportunity to strengthen the resilience of urban agriculture operations and opportunities for farmer collaboration by providing subsidies and incentives for social and ecosystem services. City-level efforts to compensate or recognize farmers for ecosystem services such as soil remediation and carbon sequestration, for example, are not yet realized. Further examples of responsible governance from our data include recommendations for public procurement programs to source food from aggregated urban produce . Our respondents are engaged in circular and solidarity economies, key features of agroecology, including bartering, sharing, and exchanging resources and produce with those in their social networks. They are also interested in collaborating in a localized effort to strengthen the link between producers and consumers by aggregating produce and sharing distribution .

In the dark, the STM cell densities were similar to that of WT M. loti . However, high intensity blue light prevented the growth of WT M. loti, but after 72 h, the growth of the STM strains was partially restored. The A610 values of the WT and STM strains were significantly different at 144 h . We also measured growth parameters in plants inoculated with the STM strains and exposed to blue light. For the shaded roots, no significant difference between the WT M. loti and STM strains was observed in shoot length , root length , or nodule number . For the unshaded plants, the nodule number of plants inoculated with WT M. loti was 40% of that of the shaded roots. For plants inoculated with the STM strains, the nodule count in unshaded roots was >65% of the number for the shaded roots . Taken together, these results suggest that both sets of rhizobial blue light receptors are required to inhibit nodulation.Because blue light perception by both the root and rhizobia leads to an inhibition of nodulation, we inoculated RNAi plants with the STM mutants under the same growth conditions used in Table 1 and assessed nodulation status 21 dai. As shown in Table 2, root nodules were formed in cry-RNAi plants with M. loti or in EV plants inoculated with STM strains, supporting the results shown in Table 1 and Figure 5H. The nodulation of RNAi-targeted Ljcry1A and Ljcry2B plants when inoculated by mllpp1 or mlphl was increased compared with EV plants inoculated with mllpp1 or mlphl. In RNAi-targeted Ljcry1A and Ljcry2B plants inoculated with the mllpp1 strain or the mlphl strain,dutch buckets nodulation was enhanced compared with EV plants inoculated with a WT M. loti strain .

The increase in nodule number was additive, indicating that the inhibition of nodulation by light is caused by blue light perception by both the host plant roots and rhizobia.Previous studies showed that nodulation in Trifolium subterraneum L. Woogenellup is either inhibited or not inhibited in the light, depending on the rhizobial strain . On the other hand, in Pisum sativum and P. vulgaris , root nodule numbers were decreased when roots were exposed to light. Our data support both of these seemingly disparate conclusions because, in L. japonicus, inhibition of nodulation by light is caused by blue light perception by both the host plant roots and rhizobia. Bonomi et al. reported that a short time exposure of rhizobia to white light prior to inoculation enhanced nodulation in Pisum sativum. In our study, a light treatment was given after inoculating with rhizobia, which may explain the differences in results. Using a split-root system, we found that the number of nodules formed on unshaded root systems was significantly lower compared with shaded root systems and even on a single root, which showed that nodule inhibition was not systemic . Recently, Chen et al. reported that the shoot lengths of Oryza sativa seedlings were inhibited by blue light and, in Lactuca sativa, shoot biomass was also decreased under blue light . Furthermore, shoot and hypocotyl lengths of lettuce, radish, and pepper decreased in response to increasing the quantity of both blue light and red light . Although the number of nodules did not differ between the red and blue treatments, the shoot lengths of blue light–treated plants in shaded L. japonicus roots were significantly decreased compared with red light–treated plants . In lettuce, root growth was decreased by blue light irradiation . In contrast, in rice, root growth was not different between roots either exposed to blue light or not . Our observation of root growth was that no large difference occurred whether the roots were shaded or unshaded in L. japonicus .

Taken together, these results show that overall shoot growth is inhibited by blue light irradiation, whereas the effect of blue light irradiation on root growth depends on the plant species. Under both white and blue light, a significant difference was seen in the shoot lengths of inoculated plants that had their roots shaded compared with uninoculated plants grown under the same conditions. These data lead us to conclude that the shoot growth of shaded roots was positively affected by the presence of root nodules. We also found that the inhibition of nodulation in white light was caused by its blue component and that root nodule number reduction under blue light was not related to the lack of a carbon source . Similarly, for isolated P. vulgaris roots, blue light inhibited nodulation more than red light or white light did . Our data are thus consistent with the absence of a significant effect of red light on root nodule number in Pisum sativum and, also, show that number of root nodules following blue light treatment was significantly reduced . Further implicating the key role of blue light, we found that nodulation was significantly increased in roots depleted of cry1A and cry2B . Because the expression of all the Ljcry1 and Ljcry2 genes was down-regulated in both cry1A and cry2B RNAi plants, we conclude that blue light inhibits nodulation via one or both cryptochromes 1 and 2. On the other hand, reduced expression of the three phototropin genes in L. japonicus MG20 had no effect on nodulation, indicating that blue light perception affects nodule development in the roots of this legume through cryptochromes and not phototropins. We also demonstrated that M. loti growth is inhibited specifically by blue light and that the rhizobial blue light receptors LPP1 and photolyase are involved in its suppression . Moreover, the growth of the STM strains under blue light did not lead to a full recovery of the levels of the dark-grown strains , which means that both rhizobial photoreceptors are involved. Finally, when the Ljcry1A and Ljcry2B-targeted RNAi plants were inoculated with the STM strains, an additive increase in nodule number was observed .

These results thus demonstrate that the inhibition of nodulation by light is caused by blue light perception by both host plant roots and rhizobia. With regard to nodulation, the number of infection threads per plant in L. japonicus is severely reduced in plants grown under blue light . Nodule size classes also differed between the shaded and unshaded plants. In the latter, a large population of smaller-sized nodules and only a few large ones developed on the illuminated roots compared with the shaded controls , suggesting that the inhibition of L. japonicus MG20 root nodulation under blue light results from one or both reduced or abnormal infection. Nodule fresh weights are also lower in the illuminated plants. At least two hypotheses can be invoked to explain these results. The first is based on the fact that light perception reduces rhizobial growth, resulting in insufficient size of the rhizobial population needed for inducing the earliest stages of nodule formation. This scenario is consistent with the studies on Rhizobium leguminosarum bv. viciae 3841 in which white light was reported to interfere with the synthesis of bacterial surface determinants that are needed for competent infection and nodulation of pea . Inhibition of attachment to root surfaces might also result in fewer infection events with a concomitant reduction in nodule size and weight,grow bucket which we observed. An alternative or additional mechanism is based on the observations of Grobbelaar et al. that light perception affects nodule development independently of the early stages of attachment, root hair deformation, and infection thread formation, which would mean that darkness is not required for nodule initiation but for nodule organogenesis. Our data are also consistent with this mechanism because we observed a larger size class of nodules in the 0.5- to 1-mm range and a reduction in the group of 1-mm-sized nodules in the shaded vs. unshaded roots. In addition, the fresh weight per nodule under shaded conditions was greater than when the roots were illuminated. However, the fact that fewer mature nodules were observed on unshaded roots could solely be due to the abortion or lack of the initial nodulation attempts under blue light. We are currently examining which of these two mechanisms is more likely to explain the downstream effects of blue light in the M. loti–L. japonicus symbiosis. Higher plants developed avoidance mechanisms to survive under conditions of biotic or abiotic stress. It is well-recognized that plants utilize light as the trigger for these responses. For example, in the case of anthogenesis and light-induced germination, the plant determines the timing by light perception through phytochromes and cryptochromes to increase the possibility of survival of descendants . In root negative phototropism, plant roots bend opposite to the light direction via phototropins . In the shade avoidance syndrome, plants grow taller or bend toward the light to avoid shade through the action of phytochromes . In shade avoidance in the case of root nodulation and mycorrhization, host plants suppress these interactions under suboptimal light conditions through the phytochrome system to avoid expending energy . Therefore, we can consider inhibition of nodulation by light as one of several avoidance responses plants use to conserve energy in response to environmental stress. Sun et al. reported that the light from the surrounding environment of herbaceous plants enters the interior of the stem. Then, via an internal light-conducting system, light of wavelengths between 710 and 940 nm, which includes far-red light, is conducted axially and efficiently from leaves and stems toward underground roots, whereas lower wavelengths of light , such as blue, are not.

These authors also stated that this internal light environment might be of crucial importance for the phytochrome-regulated metabolic activities of plant stems and roots. Because the phytochrome system is involved in the response to FR light conducted to the root, another system, such as blue light perception, may be utilized for avoidance mechanisms in response to stress. Clearly, more studies are needed.As nanotechnology rapidly evolves,engineered nano materials are entering air, waters, soils,and sediments where they could adversely affect organisms to ecosystems.Actual environmental impacts of ENMs have not been documented, and there are uncertainties about the potential for, and how to evaluate, impacts.ENM ecotoxicology elucidates hazards and their mechanisms.The scope overlaps with conventional ecotoxicology, although ENMs are particulate and diverse,with varying cores, native or acquired surface chemistries,conditional agglomeration or dissolution,and size- plus composition-dependent electronic properties,affecting their reactivity and biological interactions.Focusing ENM ecotoxicology invokes exposure scenarios relevant to ENM production,use,disposal,and product release .Scenarios consider environmental fate and transport,bio-availability,and ENM uptake into ecological receptors.In conventional chemical toxicology, observed and perceived exposures often diverge. In ENM ecotoxicology, water is emphasized, while soil and sediment impacts have received less attention.Where do exposures occur? What ENM forms and quantities are involved? Which ecological receptors are affected?Which local exposure conditions prevail? As with conventional chemical risk assessment, such questions unite hazard and exposure assessments.Standardized test regimens do not derive from scenarios, since ENM test conditions are predefined for standardized end points.Ideally, ENM hazards are studied at realistic exposures for ecologically relevant receptors. An example would be studying real soils under controllable yet realistic conditions, that is, in greenhouses or lysimeters.However, requiring absolute realism in all ENM ecotoxicology would pose scientific challenges associated with measuring ENMs analytically in environmental media; measuring toxicity across a representative range of environmental conditions; characterizing environmental ENM forms and their transformations so that toxicity is measured for representative materials; ENMs altering physical or chemical exposure media conditions; and few efficient approaches for estimating hazards and exposures necessary to evaluate risks before ENM products develop. Another challenge is internal to the scientific community: multiple dissimilar working definitions of environmental relevance intruding on scholarship, including peer review.Environmental relevance remains undefined, leading to categorization of research around a few selected concepts.Previously, over 600 published studies were examined to compare modeled or measured environmental concentrations of ENMs versus concentrations administered in ENM ecotoxicity assessments.The study found nominal concentration disparities, but also infrequent testing at low ENM concentrations. The study noted uncertainties in ENM exposure modeling, and that other toxicity testing conditions beyond ENM concentration including aqueous chemistry, biological receptor, system complexity, and ENM form relate to real-world conditions. However, the study did not establish what constitutes environmental relevance in the ecotoxicology of ENMs.

NA has been shown to be an important divalent metal chelator and is involved in metal transport and homeostasis in plants.Previous evidence also showed over expression of genes involved in NA synthesis increased nickel tolerance in tobacco and Arabidopsis thaliana.Liao et al. revealed that NA and histidine have the highest binding constants for Cu2+ in chicory and tomato.Thus, the observed up-regulation of NA and histidine in leaves is a possible Cu2+ detoxification mechanism for lettuce plants. Phytochelatins and metallothioneins have been shown to play an important role detoxifying excess Cu2+.PCs are synthesized from reduced glutathione in a transpeptidation reaction.In addition, GSH is involved in a plethora of cellular processes, including defense against ROS,and sequestration of heavy metals.Glycine and glutamate are the main constituents of GSH. The observed elevated levels of glycine may indicate GSH and PCs are upregulated to detoxify excess Cu2+. Pidatala et al. also observed that glycine and glutamate increased in response to Pb. Increasing Tolerance. In addition to chelating copper, lettuce plants must employ other strategies to increase their tolerance to higher copper levels. The level of spermidine and putrescine, which are important polyamines, were elevated in all treated plants . Previous studies showed that putrescine and spermidine play an important role in plant stress response to diverse environmental stresses by acting as antioxidants to scavenging free radicals.Therefore, elevated polyamines may contribute to enhanced tolerance of lettuce to copper. In this study,hydroponic nft channel ethanolamine levels were increased in all NP treated plants. Rajaeian et al. suggested that EA increased salt tolerance of tobacco plants by stimulation of antioxidative responses.

Kogan et al. showed pretreatment with ethanolamine enhanced the tolerance of Helianthus annuus L. to salt stress.Elevated K+ is another possible important tolerance mechanism. Increased K+ in plants can lower ROS production by reducing activity of NAD oxidases and maintaining photosynthetic electron transport.K+ has been implicated in regulating plant stress responses. Guard cells take up mainly K+ .It has been demonstrated that K+ triggers the stomata to open. In guard cells of open stomata, K+ was 2−4 times higher, malic acid 6 times higher, and citric acid 3 times higher, compared to closed stomata.It has been repeatedly hypothesized that organic acid synthesis would accompany stomatal opening.The accumulated K+ in guard cells may promote stomata opening. Increased stomatal opening/ transpiration is expected to promote photosynthesis and thereby increase plant growth.Borowski and Michalek showed that foliar application of potassium salts to spinach leaves resulted in more intensive gas exchange in leaves and, as a consequence of that, increased leaf yield. Antioxidant Defense. As reported before, Cu generates ROS in cells through the Fenton reaction. Our study also showed ROS was triggered by Cu2 NPs . ROS scavenging enzymes and antioxidant molecules are a common plant response to ROS stress.Phenolic acids and ascorbic acid are important low molecular antioxidants.Previous studies indicate ROS stress increases accumulation of antioxidant molecules.Up-regulated low molecular weight antioxidants can serve as scavengers of free radicals to protect plants from oxidative damage.Interestingly, our results showed the levels of three phenolic compounds and dehydroascorbic acid, which are important antioxidant molecules, were significantly decreased in all nanopesticidetreated lettuce leaves . GABA levels also decreased. It is possibly that biosynthesis of these metabolites was activated in response to ROS stress induced by Cu at an early stage of defense. However, since the stress was sustained for one month, this induced the imbalance between ROS and the antioxidant defense system.

Therefore, the antioxidant system was impaired due to the continuously generated ROS and limited ability to regulate them.Exposure to copper-based nanopesticides is likely to increase. For lettuce, exposure via foliar application, as intended, did not result in visible leaf damage. In fact, in several cases leaf biomass increased significantly. Cu2 nanopesticides can clearly enter stomata, even when aggregated. We demonstrated that Cu was translocated to the roots, although almost all the Cu mass was accumulated in leaves. Despite no visible damage, metabolomics revealed some significant changes in levels of amino acids, organic acids, carbohydrates and other important metabolites, particularly in leaves. The effect in roots was much smaller. The plants may be up-regulating some of these metabolites to increase the tolerance of plant to Cu2 nanopesticide. Metabolomics can be used as a sensitive and powerful tool to understand the response of plants to nanoparticles at a molecular level. However, it is not clear if the observed metabolic changes were entirely induced by Cu ions or if NPs also contributed. Future work should address how best to use Cu ions as a control at the same level of bio-availability, to better distinguish the contribution of nano-Cu from that of the Cu ions to the observed metabolomics changes.Micro-irrigation has become the optimal standard for irrigation and fertigation of horticultural crops in Australia, due to increased water scarcity and higher costs of fertilizers over the last decade. Intensive fertigation schedules have been developed to increase yield and quality of many permanent horticultural crops, including mandarin. This combines drip irrigation and fertigation to deliver water and nutrients directly to the roots of the crop, with the aim of synchronizing the applications with crop demand and maintaining the desired concentration and distribution of ions and water in the soil . The overall aim of these interventions is to develop an irrigation and nutrient management program that increases yield and fruit quality, while reducing leaching. The fundamental principle of drip fertigation is to apply water and nutrients regularly to a small volume of soil at a low application rate and at a high frequency to closely meet crop demand .

However, the potential for movement of water and mineral nutrients, especially nitrogen , below the root zone and into the ground- and then surface-waters using these approaches is still high. This is due to a number of factors: amount and intensity of precipitation, the large amounts of water and nutrients being applied, the limited capacity of roots to take up these nutrients, and to the ability of irrigators to manage drainage and hence leaching. Citrus is one of the important horticultural crops being grown under intensive fertigation systems in Australia. The vast majority of citrus plantings are oranges , with the rest split between mandarins , lemons and limes , and grapefruit . About 75% of the Australian citrus industry is located in the Murray-Darling Basin, utilising the lighter-textured free-draining soils adjacent to the Murray, Darling and Murrumbidgee rivers,nft growing system and thus potential off-site effects of poorly managed fertigation may have wider implications. Irrigated horticulture has, in general, been identified as the major source of nitrogen in drainage waters in the Murray Darling Basin . A significantly high nitrate level has been reported in drainage water and soil solution under grapevines in the Murray Darling Basin. These values are significantly higher than the Australian environmental trigger value for nitrate . Leaching of nitrates from soils under perennial horticulture may pose a potential threat to groundwater. The main sources of nitrate in mandarin production are mineral fertilizers. Nitrate is removed from the soil by plant uptake or through decomposition by micro-organisms in the process of denitrification. In well-aerated soils typical of this region, denitrification is often negligible because of a lack of favourable conditions . Nitrate, being an anion, moves freely in these mineral soils, and hence has the potential to leach into groundwater and waterways if fertigation is not well scheduled . Several researchers have reported substantial leaching of applied N in citrus cultivation under field conditions . Syvertsen and Jifon found that N leaching was higher under weekly fertigated orange trees than under daily or monthly fertigated trees. Syvertsen and Sax reported that increasing the number of fertigation events could significantly reduce N leaching. However, they observed 38–52% leaching of N from fertilizer, and the nitrogen use efficiency ranging between 25% and 44% in Hamlin orange trees. Other researchers have reported that nitrate accumulates toward the boundary of the wetted volume for most combinations of drip emitter discharge, input concentrations, and volumes applied. These studies suggest that there is a need for efficient tools, capable of describing and quantifying nitrate leaching, as well as nitrate uptake by crops, which in turn would help in designing and managing drip irrigation systems and achieving a high N fertilizer use efficiency, thereby limiting the export of this nutrient as a pollutant to downstream water systems. In addition to nitrate leaching, salinity is also an important factor influencing the sustainability of the citrus production worldwide, as citrus species are relatively salt sensitive. The reported value of the average threshold electrical conductivity of saturation extract and slope for oranges are 1.7 dS m 1 and 16%, respectively . Salt damage is usually manifested as leaf burn and defoliation, and is associated with accumulation of toxic levels of Na+ and/or Cl in leaf cells. Under drip irrigation there are many factors influencing the distribution of soil water and salts, and hence the water use efficiency , such as water quality, dripper discharge rate , irrigation water depth , and irrigation frequency . Simulation models have been valuable research tools in studies involving complex and interactive processes of water flow and solute transport through the soil profile, as well as the effects of management practices on crop yields and the environment .

HYDRUS-2D has been used extensively in evaluating the effects of soil hydraulic properties, soil layering, dripper discharge rates, irrigation frequencies, water quality, and timing of nutrient applications on wetting patterns and solute distribution . Although these studies demonstrate well the importance of numerical modelling in the design and management of irrigation and fertigation systems for various crops, most studies involving salinity and nitrate leaching are based on either an analysis of hypothetical scenarios, or are carried out for annual crops. Hence, there is a need to carry out modelling studies for perennial horticultural crops such as mandarin, using experimental results from field studies involving modern irrigation systems such as drip. The objectives of the present investigation were to evaluate water, salt , and nitrate movement in soil below young mandarin tree using HYDRUS-2D, and to evaluate various irrigation and fertigation strategies for controlling deep drainage and nitrate leaching, whilst maintaining soil salinity below the threshold for mandarin. This approach will help us understand the best irrigation and fertigation management practices to be adopted in future practical applications, with the goal to increase root water and nutrient uptake.The field experiment was conducted at the Dareton Agricultural and Advisory Station , located in the Coomealla Irrigation Area, 3 km from Dareton and 10 km from Went worth in New South Wales . The research station forms part of the Sunraysia fruit growing district of NSW and Victoria located in the Murray Darling Basin. An experimental site with an intensive fertigation system, consisting of various mandarin varieties budded onto a number of root stock varieties , was established in October 2005. The trees were planted at a spacing of 5 m-2 m. The actual monitoring and measurements were initiated in August 2006. The trees were managed and fertilized following current commercial practices, although the amounts of applied fertilizer varied. The soils of the site are alkaline , with red sandy loam from the surface to 90-cm depth, and loam below . The total organic carbon content is very low in the first 30 cm, and below 0.25% in the remainder of the root zone. The climate is characterized as dry, with warm to hot summers and mild winters. The total rainfall during the experimental period from 21 August 2006 to 20 August 2007 was 187 mm , which was slightly below average for the area.Mild frost conditions occur during the winter months. Weather data were collected from an automated weather station located within the research station.The HYDRUS-2D software package was used to simulate the transient two-dimensional movement of water and solutes in the soil. This program numerically solves the Richards’ equation for variably-saturated water flow, and advection–dispersion equations for both heat and solute transport. The model additionally allows specification of root water uptake, which affects the spatial distribution of water, salts and nitrate between irrigation cycles. The solute transport equation considers the advective–dispersive transport in the liquid phase, as well as diffusion in the gaseous phase.

Of special interest have been further investigations of alterations in this kinetic pattern that occur as the membrane complex is gradually disassembled. The disappearance of the period 4 behavior is a sensitive indicator of the loss of ability to. store oxidizing equivalents; however, under many circumstances rapid rereduction of the primary electron donor continues to occur by components that have yet to be identified. Photo system I reaction centers behave qualitatively differently from those of PS II. Instead . of the large increase in fluorescence yield seen upon closing PS II reaction centers through illumination or chemical reduction of the electron acceptors, it has been difficult to demonstrate any contribution to this variable fluorescence from PS I. Now, using isolated and enriched PS I reaction center preparations we have seen a small decrease in fluorescence yield associated with closing the reaction centers. By contrast with the behavior of PS II, this variable fluorescence’ occurs’ upon oxidation of the primary electron’ donor chlorophyll, P700. These results for both· PS I and PS II are’ consistent with, but· do not prove, a model in which the increased fluorescence yield comes from the reversal of the primary process leading to the repopulation of the chlorophyll excited singlet state following charge recombination. We are carrying out time-resolved fluorescence decay measurements to try to define the mechanism more precisely. !.r Model studies were carried out on electron donor/acceptor complexes synthesized by covalent linkage of a photo-excitable ruthenium complex to a benzoquinone. The linker consisted of oligo peptides containing zero to four prolines.

By monitoring the effect of increasing Si the physical separation of the electron donor from the electron acceptor using fluorescence yield and time-resolved fluorescence relaxation,ebb and flow tray we could follow the competing electron transfer in the range from 10-9 to 10-6 sec. It is clear from these studies, carried out by Miller Research Institute Fellow Dr. Kirk Schanze, that electron transfer can occur rapidly and efficiently across longer non-aromatic bridging groups than had been suspected. In ongoing studies using kinetic EPR spectroscopy we will investigate the lifetime of the charge-separated species that simulates the initial stages of photosynthetic charge separation. Much evidence has now accumulated that the storage of the four oxidizing equivalents needed to produce 02 from water photosynthetically occurs in a complex containing four manganese atoms. The nature of this complex, which is presumed to involve interaction with a protein environment, is still unknown. Even the kinetics is in dispute. Optical spectroscopic changes have been interpreted to result from three successive one-electron oxidations from Mn+3 to Mn+4 prior to O2 release, whereas EPR, X-ray spectroscopy and other approaches favor a mechanism with different oxidation state changes for the three preliminary steps. We have used X-ray absorption and EXAFS, in conjunction with low temperature EPR measurements to investigate these questions. Absorption edge energy studies on Mn are suggestive that, So, the most reduced state of the complex~ has the .same oxidation state for Mn as Sl, where one electron has been removed by the PS II reaction center. Presumably this electron has come from an associated ligand that does not directly involve Mn. The second step, from Sl to S2, definitely involves Mn oxidation, perhaps by two equivalents transferredone to the reaction center and one to rereduce the ligand. The third step, from S2 to S3, again . involves no apparent change in the Mn oxidation state, but the final step S3 to S4 and back to So returns the complex back to the fully reduced state. The state S4, which should be the most oxidized but is unstable, has not yet been trapped for investigation.

EXAFS measurements give information about the local coordination environment of the Mn in the water splitting complex. Two of the three shells previously identified for Sl are attributed to 0 or N atoms, and the third shell to Mn atoms. Further enhancement of signal quality has revealed a fourth shell, apparently also resulting from Mn. A possible model involving a distorted cubic arrangement with Mn and either 0 or N atoms at alternate comers has been proposed by Brudvig and Crabtree [Proc. Natl. Acad. Sci. USA 83, 4586-4588 ] to account for these and other spectroscopic data. We find that the coordination in Sl does not differ significantly from that in S2, by the EXAFS criteria; however, analogous investigations of S3 are still under way. Previous proposals that sulfur or chlorine atoms are involved as Mn ligands are not consistent with the EXAFS data. We have also shown that resolved super hyperfine structure of a low temperature EPR signal associated with S2 does not result from interactions with CI nuclei in the immediate coordination environment. Until the successful isolation of the water-splitting complex has been accomplished, further spectroscopic studies such as these will be our principal source of information about the enzyme that accomplishes this important aspect of photosynthesis. The information gained is expected to be valuable for designing bio-mimetic systems for using light to split water into hydrogen and oxygen as alternative energy sources. It is of interest that the genes for Crt biosyntheis are regulated differently from the other photosynthetic’ genes in response to light intensity and O2 • We have found that under steady-state high O2 the level of mRNA from the Bam Ill-H fragment was relatively high and it increased when the cells were shifted from anaerobic to aerobic conditions. We have evidence suggesting that crt A in the Bam HI-H is activated by O2 • This gene is responsible for the oxidation of spheroidene to spheroidenone. Such regulation in response to O2 may be related to the function of Crt. It has been long known that Crt has two functions: harvesting light and protecting cells including the photosynthetic apparatus from photooxidative damage which only occurs in presence of both high light and O2 • Activation of crt A and other Crt genes by O2 may be part of the protective mechanism by which Crt scavenges O2 radicals in the cell.

Here we have also found another type of protective mechanism in response to high light. That is, in contrast to the decrease in the levels of mRNA for LH, RC and Bchl biosynthesis, an increase in light intensity raised the levels of mRNA from a number of crt genes located in the Bam HI-M, Bam Ill-G and Bam Ill-H fragments. Although the increase in transcription of crt genes in response to high light seems to be a plausible protective mechanism, we do not rule out the possibility that regulation may also occur post-transcriptionally, including the activation of Crt biosynthetic enzymes by light and O2 • The 0.4 kb small transcript from the Bam Ill-I also showed a response to high light in the opposite fasion to the mRNAs for LH and RC. It did not hybridize to either regions genetically mapped as crt F or crt E . The response of this transcript to O2 is very similar to that of mRNAs for LH and RC, but is very different from that of mRNAs for Crt biosynthesis. On the other hand, transcript level increases in response to high light as do the mRNAs for Crt biosynthesis except to a greater extent, but it is very different from that ‘of mRNAs for LH and RC in this respect.Other interesting results in this study are the finding of multiple transcripts for the RC-H as shown by Northern hybridization. The 1.2 and 1.4 kb transcripts of the RC-H gene, 764 bp in size, are probably initiated from the middle of the ORF F1696. We do not know whether they have different initiation sites,4×8 flood tray or if the 1.2 kb transcript is the product of processing of the 1.4 kb transcript. The relatively long 5′ non-coding region may contain important regulatory sequences. Assay for transcripts from all of the ORF putative genes near the LH and RC gene clusters in the Bam Ill-C -EcoRI-B, and Bam Ill-F fragments resulted in the detection of mRNA from only C2397. We suggest that the putative genes in both fragments are either not expressed under the define growth conditions, or their mRNAs, if present, are below the limits of detection by our methods. Our research has been directed towards the understanding of the mechanism controlling the coordinate expression of genes encoding photosynthetic components. While the photosynthetic processes are primarily carried out in the chloroplasts of eucaryotic photosynthetic cells, the genes encoding the components are distributed into both the nuclear and the chloroplast genome. The coordination of expression of these physically separated genomes is the subject of our investigations. We are determining if the chloroplast or its encoded components play a role in the light response of nuclear genes whose produc.ts· are involved in photosynthesis. The photosynthetic apparatus is a highly organized and specialized structure made up of many components. The activity of the system is dependent on the activity and arrangement of the individual components. The cell must balance the production of the individual components to optimize the photosynthetic activity of the system.

This balancing act is not simply a maintainence of equal numbers of all components since some components in the apparatus are needed in great excess over others. Also, control must be exerted on the production of these components as a function of time. For example, the production of Light Harvesting Chlorophyll Binding Protein must not begin before chlorophyll biosynthesis since the protein is rapidly destroyed in the absence of chlorophyll. Even though the chloroplasts of photosynthetic organisms contain their own genome, many of the functions carried out in the organelle are encoded in the nucleus. Some of the proteins which function in chloroplast are encoded partially in the chloroplast and partially in the nucleus. The expression of both these genomes is regulated by light. Light stimulates mRNA synthesis as well as DNA synthesis in Euglena gracilis. Presumably” this light response is mediated by photo pigments in the cells which receive the light and convert it into a signal which ‘control nucleic acid activity in both genome compartments. We are studying a series of bleached mutants of Euglena which lack various photo pigments as well as have different levels of chloroplast DNA. Using these variants, we hope to determine the involvement of the photo pigments and chloroplast DNA in the stimulation of nuclear DNA and RNA synthesis. First, we are characterizing the cell cycle response to light of these various mutants by flow cytometry. Wild type Euglena conditioned in the resting medium in the dark can be stimulated to reenter the cell cycle by light. We are comparing the kinetics of reentry into the cell cycle of the bleached mutants with that of the wild type to discern the role of the photo pigments and chloroplast supplied functions in regulating the DNA replication response in the nucleus. We are also comparing the light stimulated expression of nuclear encoded chloroplast functions in wild type vs bleached mutants to determine if the chloroplast and/or the missing photo pigments play a role in the regulation of the nuclear genes. Can nuclear genes coding for photosynthetic components be regulated by light in the absence of functional chloroplasts? At present we are using heterologous probing to clone the nuclear genes for the light harvesting chlorophyll alb proteins in Euglena. We have been testing conditions that will allow plant protein-DNA complex formation. Tobacco root and leaf protein extracts have been tested for their DNA-binding capacities after • _ separation of the proteins by electrophoresis and transfer to a nitrocellulose membrane. We have seen a number of proteins which show DNA-binding capacities, and have noted large differences in the DNA-binding pattern of extracts from the two tissues. We are presently characterizing the nature of these proteins and assessing the contribution of chloroplast DNA-binding proteins to the binding pattern observed in the case of the leaf extracts. Even if a major contribution in this difference comes from the leaf chloroplastic proteins, the two smaller MW proteins present only in the root extract represent a true difference in the DNA binding patterns of the proteins from root and leaf tissue.

The outcome of this R&D effort will be the identification of hardware design approaches and efficient software algorithms that can be implemented as part of an ITS Upgrade and similar detector systems in other collider detectors. We have established a number of benchmark observables of heavy-flavor production at high and low-energy scales that became a strategic part for the future heavy-ion program at the LHC. Moreover, these observables connecting the precision of the single particle tracking and full jet reconstruction put stringent requirements on the performance of the new Si-detector developed for experiments at high collision rates. A major achievement was to establish a working environment for massive computing simulations using the High-Performance Computing clusters . The lightweight framework combines the conventional computing strategy with the possibility to leverage high capacity and parallelism within the HPC for the future simulation tasks. These developments, utilizing the multi-node and multi-core architecture will provide us with the necessary framework for the studies of the time-stamp based digitalization software for the purpose of future detectors. We have started the first tests of the novel setup. On the hardware side of the project, the main performance benchmarks of the principal elements of the readout system have been identified. The processing chip architecture will be defined within the first quarter of 2015 we will be able to devise the necessary hardware design approaches during the second year of the LDRD funding. Enabling more efficient flow of electrons across the boundary between living and human-made systems is critical for bio-energy technologies, including harvesting energy from wastewater and efficient synthesis of fuels from sunlight and CO2. Over the last 20 years, proteins that mediate electron transfer across this abiotic-biotic interface have been identified, purified,mobile vertical grow tables and structurally characterized in isolation. Yet, there remains a dearth of information about the in operando structure of the protein-material interface.

Our overall goal is to address these critical structure-function knowledge gaps so that we can redesign proteins for more efficient electron transfer to materials. The Mtr pathway of Shewanella oneidensis MR-1 is currently the best understood extracellular electron transfer pathway. It consists of a periplasmic decaheme cytochrome c , an outer membrane non-cyt c porin-like protein and an outer membrane decaheme cyt c . The outer membrane decaheme cyts c are the most unique and important components of this pathway since these are the proteins that transfer electrons to abiotic surfaces such as minerals or electrodes. However, there is little information about the protein-material interaction and there is no information on which amino acid residues of cyt c are recognized or associated with the material. This project seeks to uncover mechanisms of interaction between these cyt c and materials . Obtaining adequate amounts of outer membrane decaheme cyts c for structural studies is challenging because of intrinsic difficulty of expressing decaheme cyts c and their low solubility. This year we overcame these challenges to express and purify two variants of the decaheme cyt c, MtrF. First, we expressed a lipidated MtrF with a histidine-tag at its C-terminal. This protein was then purified in the presence of mild detergent using both affinity chromatography and ion exchange chromatography to greater than 90% purity with a net yield of ~1 mg protein/L culture. To eliminate the need for detergent, which will in turn facilitate structural characterization, we also expressed an MtrF variant lacking any lipid modification. To do so, we created a DNA construct containing the MtrB signal sequence, followed by the MtrF coding sequence and a Cterminal histidine-tag. This protein was secreted directly into the culture media, and we successfully purified it to greater than 90% purity at a yield of ~0.5 mg/L culture using a single affinity chromatography step.

We also characterized both the lipidated and non-lipidated MtrF to assess whether they are fully matured decaheme cyts c. UV-Vis spectroscopy of these proteins shows the characteristic peaks of c-type cytochromes at 408, 535, and 552 nm. Additionally, Electrospray Ionization Mass Spectrometry shows that the mass of these proteins is equal to the expected mass of decaheme MtrF. Thus, we conclude that these proteins are indeed fully matured MtrF. Lastly, we have established assays to measure the binding affinity of MtrF to inorganic materials. Specifically, we have shown that the fluorescence of MtrF is quenched upon exposure to certain materials. This measurement will allow us to determine the partition coefficient of MtrF for different materials, and thus greatly add to our understanding of the thermodynamics of proteinmaterial binding. Our most significant accomplishment has been the development of a flexible and inexpensive method for assaying the phenotypes of thousands of genes in parallel using transposon mutagenesis and DNA barcode sequencing. The key to the approach is the introduction of random DNA barcodes into the transposon. A mutant library for a given microbe is characterized a single time using the time-consuming and expensive TnSeq protocol. All subsequent assays to measure mutant fitness for thousands of genes in parallel only require the quantification of the DNA barcodes, a simple and inexpensive assay termed BarSeq. To date, we have applied RB-TnSeq to 21 diverse bacteria and generated over 3,000 whole genome mutant fitness profiles, representing ~9 million gene fitness measurements. We have identified phenotypes for over 20,000 bacterial genes including thousands of genes with no previous known function. We are using these data to predict gene function in diverse species using correlations in mutant fitness across hundreds of diverse growth conditions. In the current year of LDRD funding, we are working to extend the RB-TnSeq to additional, diverse microorganisms relevant to bio-fuel production, bioremediation, and nutrient cycling including archaea. In addition, we are working to increase the efficiency of transposon mutagenesis in diverse microorganisms by engineering new vector variants.

Lastly, we are developing computational tools to enable the microbiology community at large to mine our functional genomic datasets and to globally infer gene function across all sequenced microbial genomes using homology-based methods. There is a strategic imperative for investment in bio-manufacturing infrastructure at Berkeley Lab. In the 2013 State of the Union address, Barack Obama said: “I’m announcing the launch of three more of these manufacturing hubs, where businesses will partner with the Departments of Defense and Energy …. And I ask this Congress to help create a network of fifteen of these hubs and guarantee that the next revolution in manufacturing is Made in America.” While the complete specifications for these fifteen national manufacturing hubs have yet to be announced, given the Administration’s 2012 National Bio-economy Blueprint, it is very likely that there will be at least one biological manufacturing hub. This FY14 lab-wide strategic LDRD aims to place Berkeley Lab in a dominant position from which to lead a competitive effort that brings a bio-manufacturing hub to Northern California. While Berkeley Lab is uniquely positioned to leverage DOE investments in the JGI, NERSC, KBase, JBEI, and the ABPDU for bio-manufacturing competitive advantage,mobile vertical farm the capabilities and expertise at these facilities have yet to be integrated and successfully demonstrated as a ‘one stop-shop’ from target molecule identification to industry-ready microbial production strains. Operationally, this requires bio-manufacturing pipeline component standardization and interoperability; the ability to generate, QC, and track large numbers of DNA constructs; seamless integration of microbial strain construction with high-throughput functional assessment; and machine intelligence to learn from previous successes and failures to drive forward the next design iteration. This strategic LDRD aims integrate existing and develop new Berkeley Lab capabilities and expertise to create revolutionary bio-manufacturing infrastructure. This infrastructure will enable the rapid design, implementation, and assessment of target molecule production by iteratively uncovering and resolving critical biosynthesis bottlenecks. This LDRD aims to tackle a stress-test and a challenging biosynthesis demonstration project to drive a compelling success story narrative, while in parallel addressing key infrastructure gaps. Demonstrating the ability to go from target molecule to functional construct within a year for important and valuable targets will cement Berkeley Lab’s leadership in bio-manufacturing. Our major accomplishment in FY14 was to design and very nearly complete the DNA construction of a refactored actinorhodin antibiotic pathway . This is an impressive feat, as it constitutes a record-breaking refactored secondary metabolite pathway at 22 genes and 30 kbp in length. [In early FY15, we completed and validated the construction of the refactored actinorhodin pathway, transformed the construct into a modified Streptomyces coelicolor host lacking its native actinorhodin pathway, and detected the production of actinorhodin.] Sample preparation and analytical mass spectrometry methods were developed for actinorhodin and other secondary metabolites including violacein, our selected biosynthesis stress-test project target. The completion of the DNA construction of a 60,000-variant refactored violacein pathway combinatorial library is anticipated in the first half of FY15. Significant progress was also made in the development of machine learning algorithms for processing the violacein combinatorial library production data, and in the further development of DNA construction process tracking and assistance software.We succeeded in collecting “pseudo-SINBAD” data from our synchrotron beamline after careful re-alignment of the spindle. Collecting back-scattered reflections and extracting anomalous differences form them proved tractable, and indeed easier than expected.

The problems of non-isomorphism and reflecting crystal design proved to be significant, and we have new projects now investigating these two fronts. Non-isomorphism is a problem that must be overcome for any future multi-crystal macromolecular crystallography endeavor to move forward, and this fact is growing in appreciation in the XFEL and synchrotron communities alike. We discovered that ambient humidity can have a dramatic impact on isomorphism, and we are preparing a manuscript on this phenomenon, which suggested apparatus for controlling it. The design of a high-angle soft-X-ray monochromator also requires further development. This technology is critical for self-seeding in the soft and “tender” X-ray FELs currently under development, so we anticipate significant interest in it. What we learned is that the art of polishing and etching these crystals is absolutely key to preventing severe degradation of the beam emittance at each bounce, but existing manufacturing methods are optimized only for silicon and new etching protocols must be worked out for alternative materials. We also found that the current status of dynamical theory of reflection is severely lacking in the soft X-ray region, and we re-formulated this theory from first principles. We believe a highZ and large-cell material is optimal if crystals of sufficient quality can be prepared. Bismuth Telluride is a promising candidate. Nitrogen is an essential component of proteins and consequently a key element for life and cell development. Mineral N is often limited for plants, which consequently reduces plant growth and biomass yield. While this practice has been partly responsible for the ‘green revolution,’ it has come at high environmental and economic costs. In natural ecosystems, plants have developed strong relationships with microbes to cope with the low availability of essential nutrients such as N. For example, plant rhizospheres contain N2-fixing bacteria that are able to fix atmospheric N2 without the requirement of forming symbiotic association with a host-plant, however several of them depend on plant root exudates for carbon supply. Furthermore, endophytic bacteria colonizing root, stem and leaves of plants with N2-fixing function have been identified in several plants. The aim of this project is to optimize benefits from interactions between plants and free N2-fixing bacteria communities to provide adequate amounts of assimilable N to host-crops. This would reduce fertilizer consumption and carbon footprint of feed stock production, and greatly improve the sustainability of biomass production. Several additional N-fixing strains were isolated from plant isolates were phylo-typed based on 16S rDNA analyses. The genome sequences of several diazotrophic strains isolated were obtained through collaboration with JGI. Genomic comparison and extensive phenotypic characterizations were performed with a few of these strains. Most of the endophytes were capable of utilizing a broad range of carbon substrates. We analyzed the ability of the endophytic diazotrophs to fix atmospheric N2 under varying temperature in-vitro.Results indicated that N-fixation occurred optimally under optimal growth conditions. In addition, biochemical and genomic studies revealed that a bacterial strain could produce from a plant exuded compound the plant growth promoting plant hormone. This discovery led us to the engineering a plant to boost bacterial production of the plant hormone . Coupled with the ability to provide fixed N2 to the plants, this is an extremely desirable trait that can be utilized for sustainable agriculture of biofuel crops such as Switchgrass.

Our second significant accomplishment was that we developed methods of addressing and removing image artifacts on a very large number of images, allowing extraction of the porespace for analysis. All FIB-milled samples of heterogeneous samples have artifacts resulting from imperfect beam shape and the nature of sample heterogeneities, and these have previously hindered extraction of porespace. Third, we successfully simulated single-phase flow through the identified and discretized porespace from one of our largest image sets and are processing our largest image set for flow simulation. The overarching aim of the proposed research is to improve understanding of the degree to which ecological and land management processes influence the physical Earth system and feedback to climate change. The specific goals of the current proposal are aimed at evaluating and improving models of land-atmosphere interactions and climate change field experiments intended to reveal ecosystem responses to climate change. Land surface models are used to represent the properties of vegetation and soils that influence weather and climate through their effects on energy, water and greenhouse gas fluxes. Until recently, land management and cropping systems have not been represented well by these models even though crop growth and management practices have potentially large impacts on many aspects of weather and climate. We are using ground based and satellite observations, including from the ARM site and Ameriflux network towers in agricultural regions to validate and improve a coupled atmosphere-land surface model, WRF-CLM. Such model improvements will improve predictions of weather and climate in agricultural regions,hydroponic nft and in regions undergoing changing land management.

Initial efforts focused on biogeophysics have laid the groundwork for evaluation and improvement of modeled biogeochemistry. Climate change experiments are used to quantify vegetation responses to altered climate states, and mechanisms underlying the responses. Infrared heating is increasingly used to manipulate temperatures. This method has rarely been evaluated in terms of its effects on winter and early spring conditions, nor have comparisons been drawn across multiple sites with the same treatment implementation. We are using data from existing experiments in the Rocky Mountains to examine snowpack and soil responses to the climate manipulations. A key accomplishment in FY14 has been submission of papers describing a regional climate-land surface model that includes dynamic crop growth and irrigation, and its application to assess effects of irrigation decline on heat waves in agricultural regions. The first paper is still in review at Climate Dynamics, while the second was rejected by Geophysical Research Letters and is in revision for Environmental Research Letters. Another analysis was completed to evaluate how irrigation influences land-atmosphere coupling strength. This analysis was presented at the AGU Fall Meeting in December 2014 and is in preparation for a journal submission in FY15. Further progress was made in spinning up an offline version of CLM with crops in the Southern Great Plains – a complex agricultural region with intensive observations. With Noah Molotch we revised and submitted a manuscript for publication describing the effects of experimental heating on snow accumulation and melt, and comparing this to a similar site further south and to a snow pack model. The paper was recently accepted for publication pending minor revision at Forest and Agricultural Meteorology. We also conducted initial analysis of gap-filled microclimate data from multiple warming experiments for two separate papers; one focused on the relationship between spatial variance in soil temperature and the mean over the growing season, and another describing heating effects on soil microclimate. The first analysis was presented in a poster by PhD candidate Danielle Christiansen at the MtnClim meeting in Utah in October 2014.

The second was included in invited talks at Utah State and Penn State Universities in 2014. The purpose of this project is to observe—in situ, and over a range of length- and timescales—how changes in chemical conditions affect the conformation and reactivity of natural organic matter relevant to both soil and shale systems. We believe an ambitious but attainable goal will be to explore such chemistry at the smallest relevant scales, from NOM aggregates and ultimately to single macromolecules. Natural organic matter is a complex mixture of organic molecules and associated metals and is ubiquitous in the near earth surface environment. In the form of soil organic matter and dissolved organic matter, NOM chemistry impacts practically every meaningful ecological process in soils, sediments, ground waters, surface waters, and marine systems. This reduced carbon is important on a mass basis for global carbon cycling, and its’ long-term fate is a major outstanding question for predicting the feed backs associated with global climate change. As kerogens in sedimentary rocks, NOM chemistry plays a key role in energy production capacity, efficiency and associated environmental impacts. A core approach in this project is the application of recent technical developments from the Schuck group in surface-enhanced and tip-enhanced Raman scattering for nanoscale analysis.Raman scattering was chosen as the central analytical technique because it has good sensitivity to organic matter composition and compatibility with aqueous environments. We will also plan to take advantage of the technique of nano-FTIR spectroscopy and X-ray ptychography to explore the nano-scale structure of organic matter in natural shales. The results are highly complementary to information obtained by electron and X-ray imaging and spectroscopy.

Our most significant accomplishment to date has been to show that the naturally occurring iron oxide, magnetite, can be used as a SERS substrate to probe the interaction of organic molecules with natural mineral surfaces. We believe this development is the first demonstration of SERS behavior in a natural metal oxide and will be broadly useful to the study of NOM dynamics. A manuscript on these results has been prepared by Namhey Lee and is currently under review at Journal of Physical Chemistry Letters. We have also collected preliminary ptychography data on a natural shale sample. While data processing is still underway, we believe that this too is a world unique measurement and will have important implications for our understanding of the nano-scale structure of shale materials. Urban farm systems are a dynamic assembly of interdependent systems , but there is a fundamental lack of an understanding of how these systems work independently and as a group. Consequently there is only a vague understanding of how commercially and environmentally sustainable urban farming systems can be designed and operated. While there are many innovations around the country in each of the areas described above, for urban farming efforts to be replicable, scalable and cost effective, rigorous scientific knowledge needs to inform their design and implementation. To have impact at scale, technology needs to be developed and tested in partnership with those who would be the on-theground urban farmers. We propose a partnership with three cities: Oakland, CA; Toledo, OH; Chicago, IL. The goal would be to develop a rich relationship with existing urban agricultural efforts already going on in these cities and use these relationships to inform ongoing development decisions; test and fine tune new technologies as they are developed, and understand the implementation framework that will support adoption of proposed technologies. Specific tasks that would be conducted in partnership with these places would include: Initial scan of existing urban agriculture efforts and assets: Each of the proposed cities already has existing urban agricultural efforts. Understanding the scope and scale of these efforts, the innovations that are already being put into play,hydroponic channel and the challenges and barriers being experienced will be an important grounding for this work. Creation and regular engagement of a partner’s group in each city. We propose in each city to create a partner’s group which would include between 8 to 10 groups involved in urban agricultural efforts. Quarterly meetings with these partner groups would inform larger project decision-making. Testing of new urban agriculture methods, strategies or tools: As the work evolved these partner groups would serve as the on-the-ground testers of new technologies. Although CO2 has been used as an injection fluid for enhanced oil recovery for decades, CO2-EOR remains inefficient. The primary limitation of CO2-EOR effectiveness has been the low viscosity and high mobility of CO2 that results in injected CO2 bypassing oil resulting limited displacement efficiency. After decades of extensive research and practice on ways to reduce CO2 mobility, the CO2-bearing foam approach seems more promising. A variety of surfactants have been designed for generating foams, however, their commercial applications are still lacking. The major barriers limiting industrial applications of CO2 foam-EOR include the high costs of surfactants, compare to the price of oil. Concerns over environment impacts also exist for some of these surfactants. If a CO2-compatible surfactant that overcomes these limitations can be identified, such a discovery will benefit oil recovery, while at the same time increase the incentive to implement carbon capture and increase the capacity of geologic carbon sequestration in depleted oil reservoirs. The overall objective of this project is to develop a new and unconventional material, which is less expensive, non-toxic and highly effective as a substitute of conventional synthetic surfactants, for increasing CO2 viscosity and control its mobility in EOR. The Earth’s near surface sediments contain deposits of natural organic matter produced primarily from degradation of plants.

We hypothesize that these organic deposits contain a large fraction of surfactant-like material. This material can be extracted, and dispersed in the form of nanoparticulates when re-dissolved in water. These nano-particulates can stabilize supercritical CO2- brine foams, and are more cost-effective and environmentally friendly than synthetic surfactants currently used in CO2 foam-EOR. Our most significant accomplishment has been to identify the sources of NSS , and all has been successfully tested as source materials for NSS extraction . We have developed a method to produce the NBS. The NBS is capable of generating and stabilizing dense scCO2-in-water foams containing CO2 at up to 80 volume% with controllable effective viscosities up to 40 cP, three orders of magnitude higher than that of pure CO2 at the same pressure and temperature. We measured interfacial tension of NSS liquid solutions paired with N2, and paired with scCO2 as function of NSS solution concentrations. The NBS performs as an effective surfactant, significantly reducing interfacial tensions between the N2-water and the supercritical CO2- water interfaces. We have also just built a modified high pressure and high temerature foam generator and successfully tested foam-stability vs. temperature. The NSS is a N2- and CO2- compatible and high quality surfactant. We are in the process of optimizing NSS extraction method, estimating extraction costs, and optimizing laboratory CO2-EOR tests in cores. The purpose of this project is to develop a small suite of highly energy-efficient and low-cost medical devices, in order to enable affordable healthcare in rural Sub-Saharan Africa and South Asia. The specific devices developed as a part of this—identified in LIGTT’s “50 Breakthroughs” study—include an infant warmer , and continuing development of a portable solar-powered vaccine refrigerator . For the infant warmer, the key technical questions that needed to be overcome were the choice of a safe, low-cost phase change material which could maintain the appropriate temperature for more about 6 hours; a low-cost, comfortable , safe and sturdy external material; and an overall system design. For the vaccine refrigerator, the key technical question was the appropriateness of thermoelectric materials to effectively and efficiently transfer heat away from the vaccine chambers. Early-stage, conceptual architectural design has suffered radical changes in recent years, due to development on powerful modeling tools that allow fast, parametric modeling of complex building geometries, rapid-prototyping of 3D models, and links to diverse performance simulation tools. As parametric design progressively becomes the preferred method for early-stage conceptual design, the use of Generative Design Methods represents the next step in this process, by introducing performance-based optimization into architectural design, namely in terms of low-energy design. As proofs of concept, this project explored the implementation of Generative Design Methods for optimization at multiple design scales, namely: 1) Design optimization and prototyping of a light redirecting system; main research questions addressed were: a) Currently available methods/tools to use in generative design optimization; b) Reliability and error margins introduced by these methods; c) obstacles for more accurate methods; 2) Whole-building early stage design, for multi-criteria lighting, heating and cooling energy optimization. Light Redirecting System: The system simulated was built in light-weight aluminum, with a 3M high reflectance film applied on top of louvers , and a lower face coated in 70% light reflectance white mate paint, to reduce glare to occupants.

Experimental data from an aeroponic system were selected to evaluate the described approach to model the influence of temperature on the vertical penetration of the root system. The experimental setup in an aeroponic system with no soil and no water movement has the advantage of evaluating the effects of temperature on root growth while limiting the effects of other factors. The experiment was conducted in a greenhouse with bell pepper at the Jacob Blaustein Institutes for Desert Research, in Midreshet Ben-Gurion, Israel . The objective was to evaluate the effect of three different root zone temperatures on root and plant growth. Six bell pepper plants were cultivated from 7 Jan. to 20 Feb. 2015 in aeroponic pots mounted on top of the aeroponic systems. The aeroponic apparatus comprised circular pots made from plastic material with a diameter of 50 cm and a depth of 14 cm. Within each thermally isolated aeroponic pot, misters were fixed to produce the desired fine mist sprayed directly onto the plant roots. The computer-controlled spraying varied from 8-s sprayings at 1-min intervals, depending on the growth stage of the plants and the temperature of the greenhouse. Three different water temperatures were applied to each treatment, namely 7, 17, and 27°C. The air and root zone temperatures were measured daily. The air temperatures in the greenhouse were 25°C during the day and 18°C at night. The treatments were replicated twice, leading to six tanks in total. The maximum rooting depth of each plant was observed four times during the 44 d of plant growth. In the aeroponic systems,rolling bench all other factors that affect the growth and development of roots were rendered insignificant during the treatment.

The implemented modeling approach to simulate the temperature dependent vertical root penetration was evaluated by comparing modeling results with the measured maximum rooting depths in the experimental aeroponic system with bell pepper. Because two approaches were implemented in HYDRUS to describe both time-dependent potential root growth and the temperature stress factor, four combinations are thus available to describe the temperature-dependent root growth. All four models were tested against measured maximum rooting depths to validate their ability to describe the temperature- and time-dependent vertical root penetration. The four combinations are summarized in Table 3. Were the these root growth models able to properly simulate the influence of temperature on vertical root penetration, a single combination of model parameters for each model that could reproduce the measured maximum rooting depths for all temperature treatments would have to exist. An overview of the model parameters that have to be specified for each model is given in Table 4. Table 4 shows that, depending on the model, four or six parameters have to be specified to model temperature-dependent root growth. In a complex soil water flow model such as HYDRUS, four to six additional parameters can significantly increase the calibration effort and parameter uncertainties. The temperature-dependent modeling approach was tested outside of the HYDRUS implementation and within a MATLAB environment. The goal of the evaluation was to determine whether the combination of the time-dependent root growth functions and the temperature stress functions were able to reproduce the measured rooting depths under the given boundary conditions.

A global sensitivity analysis was conducted using the Sobol¢ method to reveal the key parameters of each model and to determine the contribution of the uncertainty of each parameter to the uncertainty of the model output. The Sobol¢ method is based on variance decomposition and provides the impact of each parameter and its interactions with other parameters on the model output . This type of global sensitivity analysis can be applied to nonlinear and non-monotonic models and is a widely used tool for sensitivity analysis studies. Its ability to account for interactions between model parameters is an important advantage of the Sobol¢ method .An overview of studies using the Sobol¢ method for sensitivity analysis in hydrological modeling was provided by Song et al. . The method has already been applied with the HYDRUS software package by Li et al. , Brunetti et al. , and Wang et al. . Sobol¢ proposed that the total variance of the model output can be decomposed into component variances of individual parameters and their interactions. The first-order sensitivity index quantifies the main effect of the ith parameter, Xi . This sensitivity index denotes the part of the total variance due to Xi without considering the interactions with other parameters. The total-order sensitivity index additionally includes the proportion of the variance due to the interactions of Xi with the other parameters. The values of the indices vary from 0 to 1, where 0 stands for no influence and 1 for a high influence on the variance.The number of parameter sets in the sensitivity analysis of the four models was set to 10,000. This number was initially set higher than in studies of Brunetti et al. and Zhang et al. to avoid a time-consuming convergence analysis of the sensitivity analysis and to achieve a higher accuracy of the sensitivity analysis, which increases with an increasing number of model runs.

The p parameter depends on the considered model and is either four or six . To calculate the sensitivity indices for all i = 1, …, p parameters, Matrix AB i has to be evaluated p times. The total number of model runs required to calculate the sensitivity indices for all parameters of each model were M = N. Archer et al. suggested using bootstrap confidence intervals to evaluate a suitable accuracy of the sensitivity estimates. For this reason, each estimation of the sensitivity indices was repeated 500 times to evaluate the 25th and 75th percentiles of the sensitivity indices. The small number of repetitions is due the fact that the sensitivity analysis of four models, each including four or six parameters, requires a high level of computational effort. Evaluated percentiles were used only as an additional parameter to assess the sensitivity indices .Rather than using experimental data, which may be subject to various errors and effects of various factors, the sensitivity analysis was performed using a hypothetical data set in which observed rooting depths LObs were generated by running all four models with a predefined parameterization. The cardinal temperatures Tmin, Topt, and Tc were set to 8, 23, and 27°C, respectively. A complete list of parameter values for the model runs is provided in Table 5. The boundary conditions were similar to those of the aeroponic experiments. The temperature was set to 22°C during the first 14 d and to either 7, 17, or 27°C during the remaining time period of 150 d. Additionally,grow table hydroponic a fourth scenario was considered with a temperature of 37°C after the first 2 wk to make sure that the specified cardinal temperatures would lie within the applied boundary conditions to determine their influence on the model output. Each model was thus executed four times with four different temperature boundary conditions to generate data for Lsim in Eq..The Differential Evolution Adaptive Metropolis algorithm was used for optimizing the model parameters and for model calibration. The DREAM algorithm is based on Bayesian statistics; it runs multiple different Markov chains to generate a random walk through the search space. Based on a proposal distribution, the sampler evolves to the posterior distribution by iteratively finding solutions with stable frequencies stemming from the fixed probability distribution . The Gaussian likelihood function was used to summarize the distance between model simulations and corresponding observations. The residuals were assumed to be independent and normally distributed while the measurement error was neglected.Eight Markov chains were run with a set of 5000 generations. The initial state of each chain was sampled from a Latin hypercube. The parameter space of each parameter was defined by using the same boundaries as were used for the sensitivity analysis . These parameter limits also define the search domain for the predominantly physically based parameters . The calculation time for the DREAM optimization of a single model was approximately 10 min, with no parallelization needed on a machine with the following specifications: Intel Core i7–4710HQ CPU with 2.50 GHz of RAM and 12 GB of storage.We evaluating the effects of different factors on root growth with two examples using the new root growth module in HYDRUS-1D and HYDRUS-2D.

Then we analyzed the collected experimental data and the evaluation of the temperature-dependent root growth modeling approach. Therefore, we first collected data on the experimental outcomes. Second, we carried out the sensitivity analysis to evaluate the sensitivity of the modeling results to various input parameters and identified which parameters need to be fitted and which can be set to values from the literature. Third, we used the DREAM optimization approach to analyze the collected experimental data while considering the results of the sensitivity analysis.We used two hypothetical examples that demonstrate the implemented root growth model and the impact of various environmental factors on root growth. In the first example, we used HYDRUS-1D and simulated optimal root growth as well as root growth restricted due to low water availability, temperature, texture, and bulk density. In the second example, we used HYDRUS-2D to again simulate optimal root growth and then root growth affected by a nonuniform distribution of water contents due to asymmetrical irrigation.Figure 2 shows examples of the development of simulated root systems under the influence of various environmental factors compared with the potential development of the root system, independent of environmental factors . The soil profile was considered to be homogenous, consisting of 10% sand, 50% silt, and 40% clay and having the bulk density of 1530 kg m−3. The parameters for the soil hydraulic functions of van Genuchten and Mualem were estimated from the textural information using the Rosetta module of HYDRUS-1D. Time-dependent root growth was simulated using the function of Borg and Grimes . Model 2 from Table 2 was selected to describe the potential root length density distribution. The maximum potential rooting depth was set to 120 cm, and root growth was considered for 90 d . The upper and lower boundary conditions were set to atmospheric boundary conditions with surface runoff and free drainage, respectively. In Scenario 1, the development of the root length densities was simulated for drought conditions, which may cause aeration stress and root senescence . Potential evaporation and transpiration rates were set to increase stepwise at the beginning and remain constant at the end of the simulation. Irrigation took place at irregular intervals and covered only 25% of the potential evapotranspiration. Due to low water availability, the root system was underdeveloped compared with the reference simulation when no restrictions on root growth were considered . The root length density in this scenario is reduced compared with Scenario 0, mainly close to the soil surface because the drought stress is highest in soil horizons with the highest root water uptake. Scenario 2 considered the effects of texture and bulk density on the development of the root length densities and vertical root penetration. The results show that due to the increased soil strength , the potential maximum rooting depth of 120 cm was not reached. The additional stress due to the soil strength negatively influenced the vertical penetration of roots, in addition to the effects of root senescence due to the drought stress in Scenario 1. Scenario 3 considered the influence of different root zone temperatures on the root length density development. In this case, only the option to consider the influence of temperature on root growth was enabled. The development of the root system was simulated using the root growth function of Borg and Grimes in combination with the temperature stress function of Jones et al. . The parameters Tmin, Topt, and t m were set to 5°C, 35°C, and 90 d, respectively. The temperature throughout a soil depth of 150 cm was set to a constant value of 10°C. The temperature at the soil surface was set to 25°C during the first 14 d and to 30 and 7°C in Scenarios 3a and 3b, respectively, until the end of the simulation. The results show that the root system in Scenario 3b, which was exposed to temperatures close to Tmin, was underdeveloped compared with the root system in Scenario 3a, which was exposed to temperatures close to Topt.

Additionally, we wanted to test the contribution by each Phytophthora species to leaf decomposition and to determine if there was any difference depending on leaf senescence based on their differing trophic adaptations. While the inclusion of natural stream water in these experiments is an imperfect approximation of natural conditions, namely in excluding both shredder organisms and other microorganisms eliminated in the holding period, it had the potential to reflect the interaction of the inoculated Phytophthora species with bacteria, protozoa, fungal communities, and possibly micro-invertebrates that persisted in stream water. Though the effect of stream water treatment was not statistically significant in the models, a noticeably higher occurrence of P. ramorum on brown leaves in sterile stream water treatments compared with non-sterilized stream water additions and also higher detection of P. ramorum spores by baiting in sterile compared to non-sterile stream water treatments both indicated that there was some difference between the two treatments. Though a much greater diversity of organisms likely influences this system under natural conditions, our previous research exposing leaves in natural streams demonstrated both P. ramorum and clade 6 Phytophthora species effectively colonize California bay leaves under natural conditions. As expected, based on previous work, both Phytophthora species rapidly colonized more than 60% of the leaf area of green leaves in both experiments. That P. ramorum also persisted on green leaves at high levels for the entire 16 weeks despite the loss of approximately 40% of leaf biomass stands in contrast to our previous findings where its colonization of leaves peaked within a few weeks after exposure in natural streams,ebb and flow table but then rapidly dropped to very low levels as colonization by clade 6 Phytophthora species rose and persisted at high levels.

This is evidence that the reduced recovery of P. ramorum from green leaves in natural streams as decomposition progressed was due to displacement from saprotrophic organisms like clade 6 Phytophthora species. Unfortunately, P. ramorum was completely suppressed from colonizing leaves in combined inoculations with P. gonapodyides and it could not be determined if the pattern observed in field experiments would occur under these simulations when both species were present. The suppression of P. ramorum colonization of green leaves in combined Phytophthora inoculations—consistent across all three experiments—was surprising because both species were effective at colonizing leaves when inoculated alone. One explanation could be that sporulation of P. gonapodyides from mycelial mats occurred earlier than that of P. ramorum and that the latter was therefore precluded from leaves because in all experiments, full colonization of green leaves by P. gonapodyides occurred very rapidly. Indeed, in the first experiment, colonization of P. gonapodyides occurred more rapidly on green leaves than that of P. ramorum . However, baiting two days after inoculation in the second experiment showed that P. ramorum spores wereactive in the microcosms where it was inoculated alone, but almost absent in the combined inoculation microcosms. This suggests that the presence of P. gonapodyides itself may have suppressed sporulation by P. ramorum. The rapid leaf colonization by P. gonapodyides in these microcosms also contrasts with the slower colonization that was observed in natural streams and may be an artifact of high inoculum loads and the relative abundance of substrate. The aim of these experiments was to characterize the capacity of each organism for growing and reproducing from each type of leaf rather than estimating typical colonization and decomposition in streams. Though logistically more difficult to prepare and standardize for an experiment of this magnitude, using sporangia or zoospore inoculum rather than mycelial mats may overcome the problem of uneven inoculum activation, the success of which we have experienced in smaller scale experiments.

Alternatively, the use of colonized plant tissue instead of mycelial mats as a source of inoculum may also produce a different outcome from the suppression of P. ramorum that we found with this approach in mixed inoculations. Interestingly, the kind of succession observed in field experiments did occur in a few control microcosms into which both Phytophthora species were accidentally contaminated . However, the limited occurrence and unknown relative quantity of original inoculum precluded more substantial evaluation. In any case, the suppression of P. ramorum sporulation in treatments where P. gonapodyides was present raises the question of what mechanism was responsible for the effect. It also furthers the impression that P. gonapodyides and other clade 6 Phytophthora species may have a moderating effect on the presence of P. ramorum in streams. The green leaves that we used were of mature cuticle and collected in midwinter and late summer for the first and second experiments, respectively. While some seasonal variation in susceptibility to P. ramorum infection has been reported in California bay leaves, the physical and chemical properties of mature leaves have also been reported to be relatively consistent throughout the year. Our results were similar for both experiments, and therefore, any variation in the leaves was overcome by experiment factors. The extensive colonization of brown leaves by P. gonapodyides and their limited colonization by P. ramorum is consistent with previous work where we showed that the former is a competent saprotroph while the latter is relatively ineffective at colonizing dead tissue. A significant discovery in this work was that P. ramorum colonized yellow, senescent leaves that were still fresh and had an intact cuticle to nearly the same degree as it did green leaves. At this stage, though chloroplasts and most of the protein content are gone from leaves, the cells are expected to be still alive, while in brown leaves that have dried the cells are no longer biologically active.

In fact, colonization of the yellow leaves by P. ramorum was not quite as extensive as its colonization of green leaves in the second experiment, which ran more or less concurrently and in which green and brown leaves were maintained in separate microcosms , though the difference between the separate experiments was not analyzed statistically. Though green leaves are shed into streams as a relatively low proportion of total litter, yellow leaves, often shed directly into streams from trees, constitute a much greater proportion of leaf litter in streams . This indicates that a great proportion of leaf litter in the streams is suitable for colonization by P. ramorum, and conforms to the regular recovery of this pathogen from natural leaf litter. Furthermore, the degree of colonization of yellow leaves by both Phytophthora species remained persistent throughout the 16 weeks, as with green leaves in the other experiments,growing lettuce hydroponically suggesting that the same kind of succession may be expected in these leaves as seen with green leaves in natural streams. Also consistent with previous findings with leaves colonized in naturally infested streams, leaves colonized by both Phytophthora species were generally conducive to sporulation as detected by baiting from the microcosms. Phytophthora gonapodyides was consistently recovered from P. gonapodyides-only and combined Phytophthora species inoculation treatments where it had colonized all green and brown leaves at all sampling points. The results from baiting of P. ramorum spores from microcosms were less regular, but nonetheless, mostly successful from microcosms containing colonized green or yellow leaves and occurred minimally from microcosms containing brown leaves which were colonized at only very low levels. The relatively less frequent recovery of P. ramorum by baiting from microcosms with non-sterilized stream water, not observed for P. gonapodyides, may be the consequence of P. ramorum not being well adapted to sporulation in biologically active aquatic environments or relying on different environmental signals. Nevertheless, these results confirm that both of these Phytophthora species can sporulate from colonized, decomposing leaves, whether green, yellow or brown leaves. Furthermore, at least under these conditions, their spores persisted for weeks and even months after any visible substrate was available, though the effect occurred more definitively and for longer with P. gonapodyides. As P. gonapodyides is not known to produce long-term survival structures, the question arises of how P. gonapodyides persisted so long in the microcosms in the absence of leaves. This observation also stands in contrast to our successful elimination of Phytophthora spores from original stream water collections simply by holding the water at cool temperatures for approximately three weeks. The observed persistence of spores of both Phytophthora species may be the result of an abundance of zoospore cysts due to the compact nature of the microcosms, or perhaps because the spores originated from propagules that would not have been suspended in the water column of the flowing streams. While oomycetes have been acknowledged as decomposers in aquatic environments until recently they have primarily been regarded as acting on non-cellulosic detritus such as insect and animal tissue. As most Phytophthora species are known as plant pathogens, the recent evidence that they may also degrade plant tissue in detritus is not surprising. Parasitism is considered an early characteristic in the evolution of oomycetes, but the possible evolution of a saprotrophic lifestyle from parasitic precursors has been considered for fungi and oomycetes.

Clade 6 Phytophthora are known to be opportunistic pathogens. Stradling saprotrophic and parasitic lifestyles, stream-resident Phytophthora may play an important role in the early breakdown of leaves and vegetative matter that still contain living cells. As facultative pathogens,clade 6 Phytophthora species can enter living cells and open intact tissues to further colonization by other saprotrophic organisms with less ability to penetrate living tissue. This is analogous to the paradigm of ‘conditioning’ of vegetative litter by pioneer microbial species, though in this case with respect to secondary saprotrophic microorganisms that could not on their own overcome physical and chemical protections still present in senescent but still alive leaf tissue. Our results were consistent with this hypothesis, as green leaves decayed more slowly in the absence of Phytophthora. It is uncertain why in the first experiment green leaves in the treatments with no Phytophthora inoculation decomposed very little over the entire 16 weeks of the experiment. In this experiment, both green and brown leaves were maintained together in microcosms, and it is possible that leachate from the leaves, particularly the brown leaves, may have had an inhibitory effect on some microorganisms. In the second experiment, leaves were leached prior to being deployed in the experiment, and also green and brown leaves were kept in separate microcosms. Green leaves in non-inoculated controls in the second experiment lost biomass to a degree ultimately similar to that of inoculated treatments, albeit at a slower rate. This indicates that other organisms were present that could initiate the decomposition of green leaves through the presence of Phytophthora accelerated it. We attempted additional isolations from some samples of leaves on acidified potato dextrose agar medium and found that the leaves in both controls and inoculated treatments were generally well colonized by a multitude of fungi . The fact that similar fungi occurred on leaves from microcosms prepared with both sterile and non-sterilized stream water suggests that many of these fungi were present on the leaves before entering streams as leaf litter .Additionally, overall there were no differences in decomposition rates between treatments with sterile or non-sterilized stream water added. Decomposition was also similar for leaves colonized by either Phytophthora species, indicating that, though P. gonapodyides is a better adapted saprotroph, both species had a similar effect on the decomposition of live, green and yellow leaves. This would be consistent with Phytophthora having the effect of opening integral tissue to colonization by other saprotrophs that then push decomposition forward. Finally, it is interesting that the presence of fungi in these leaves did not affect the persistence of P. ramorum throughout the experiments, suggesting that they are using different resources and that the successive displacement of P. ramorum in previous work may be specific to competition with other Phytophthora species or similar organisms. Under natural conditions, leaves would be exposed to a greater diversity of organisms, including other oomycetes such as Phytopythium species. As P. gonapodyides can colonize dead leaf tissue, it could be expected that it would contribute to leaf decay in brown leaves as well. This was not observed, as loss of biomass in brown leaves was the same in all treatments unaffected by Phytophthora colonization. The fact that P. gonapodyides substantially colonized brown, senesced leaves, but did not increase the rate of biomass loss raises the question as to what resources the organism uses in this substrate.