Month: April 2023

Therefore, leaf and other vegetative litter should be considered as potential sources of Phytophthora, including pathogenic species, whether they are found in natural streams or other surface waters. Alternatively, the suitability of local vegetation may be a determinant of what Phytophthora species become established or prominent in streams.Current farming and food systems confront and are implicated in multiple challenges and unsustainable changes, including biophysical dimensions such as climate change , environmental pollution, escalating losses of biodiversity, and deteriorating ecosystem services . Social forces and structures as well as unsustainable socioeconomic processes also strain present capacities to manage growing population pressure, unplanned urbanization, food and nutrition insecurity, dietary shifts, and health disparities associated with poverty, and growing inequality among multiple stakeholders, including women, youth, migratory workers, and indigenous peoples . Both urban and rural actors are impacted in relation to land ownership and land use change issues and drivers underpinning global industrial agriculture and connected food systems. Human activity has approached critical limits over an increasing number of the so-called Planetary Boundaries , beyond which the functioning of ecosystem services may be substantially altered, increasing the risk of destabilizing life on our planet . Agriculture and food systems are both a villain and a victim in approaching or breaching PBs, and this is already impacting the ability to farm and produce food. How can humanity sustainably grow nutritious food and return to a safe operating space within the PBs? As an alternative to this scenario,hydroponic growing supplies a growing number of studies and reports indicate significant potential gains from transitioning toward agroecological agriculture as a way of nourishing current populations sustainably while allowing for future generations to support their livelihoods .

One core quality of transitioning to agroecological farming systems is the regenerative trend of increased “outputs” per unit “input” for a more efficient agriculture for using and conserving diversity on a long-term basis, through the use and combination of different agricultural techniques in ways which restore and nourish the soil and enhance the local environment, instead of continuously degrading it. In addition, the diversification strategy makes food producing systems resilient to external shocks and influences, such as floods or droughts, using, for example, approaches built on the principles and science of agroecology . There is growing evidence that such production systems allow for lower cost and more diverse fruit and vegetable supply . Furthermore, conventional thinking about food is increasingly being challenged, shifting from being regardedonly as a commodity toward becoming acknowledged for its nourishment, social and cultural values, the links it creates between people, and its deep connectedness with ecosystems, ecosystem services, and natural resources . The current globalized industrial food system exhibits the same drivers which impact and shape farming industries and food production, and underscores the importance of focusing on how food flows into food systems, and which structures and related policies are shaped to support and reinforce current farming as well as food systems . It is not only conventional and industrial production of animal feed, genetic material, or major commodities such as wheat, rice, coffee, sugar, maize, and chicken which are controlled and shipped across continents by large trans-national corporations. Our globalized industrial food systems sometimes also include food which originates from farming systems based on organic farming regulations and principles like the IFOAM principles, calling for more coherent, equitable and holistic food systems, and applying agroecological farming methods. In other words, the intentions behind such farming systems and their contributions to agricultural and environmental sustainability are not always extended to food systems, which generally contribute to out-competing local produce, distorting prices and producing huge amounts of food waste and other waste.

This can be seen as a contradiction and emphasizes the importance of thinking of not only organic and agroecological production, but also has consequences for thinking the principles into the entire food systems. At the same time, there are many examples of organic farming and food as well as agroecology presenting alternatives to the industrial farming and food systems , and by increasing and emphasizing this, we can move toward a food system that falls within the PBs. This calls for profound analyses of how agroecological food systems function, and how they can contribute to coherent, resilient and equitable production and exchange of food, while human and social capitals are built up throughout the food systems, and resources are cycled rather than transported through, from or to disconnected parts of the systems. How can such food systems meet challenges such as losses of complex and system-oriented, context-relevant knowledge about farming and food, and how can they contribute to re-connect consumers and the food that they eat across urban-rural settings in city-region food systems? An increasing number of papers and reports link agroecology and food systems , referring to the fact that agriculture and food systems are intricately linked, and to a large extent driven by the same global structures. Given the intricate and mutually-reinforcing relations between agriculture, food, and socioeconomic systems, the present article aims to characterize and explore how the concept of agroecology stimulates the conceptualization of agroecological food systems, or perhaps even a more inclusive term like “socio-agroecological food systems.” Food systems following the principles of agroecology calling for resilience, multi-functionality , equity, and recycling of resources face particular challenges and have significant options for impacting sustainable development in city regions . This needs to be seen in a light where an increasing amount of the global population lives in urban areas, from smaller towns with a few thousand inhabitants, to mega-cities of millions of people. Urbanization has changed diets and nutrition, while food consumption has become detached from food production worldwide . Taking a systems approach to reconnecting these gaps requires major changes in consumption patterns, resource management and social responsibility, if everybody is to be nourished in agroecological food systems.

We aim to explore the connections and linkages between the concepts of agroecology and food systems, and focus particularly on how the food system framework can locate and ground the concept of agroecology within a rural– urban landscape setting. This exercise requires us to critically examine the reciprocal flows and the multiple environmental, social, and governance related connections needed for an agroecological food system transformation.A food system is a system that involves activities, social and institutional structures, and processes related to the production, distribution, exchange, and consumption of food . Agricultural systems are part of food systems, integrated in ecosystems, and constituted socioecological systems . Over the past few decades, the understanding of food systems has clearly developed as result of the development of a more and more globalized food system . Ericksen compared some features of “traditional” versus “modern” food systems, and addressed the governance of different food systems, with or without support for local production, and Foran and co-authors point to the existence of different concepts of how food systems are constructed, with examples from so-called developing countries. The structure and governance of the food system clearly influences consumption patterns by providing both producers and non-food-producing consumers with options of availability. The range of social and environmental welfare outcomes stemming from food system activities were also discussed and visualized in Ericksen ,flood table and Jennings and co-authors analyzed how planned and well governed city-region food systems could contribute to different aspects of food security for different groups of citizens, stable incomes, circular economies, and resilience at various levels. Characterizing a food system can follow through its different social aspects and arrangements, like the type and degree of contact between those who grow and produce food and those who receive and eat the food without participating in the production of it, or who and how many people are involved in the cycle between the soil and the plate. Where local food systems with short supply chains have potential for involving resource feedback loops, raising collective awareness among different actors within the food system, and give possibilities for mutual learning , a larger and decoupled food system lacks the direct interaction and feedback, reduces exchange of experiences and knowledge, or the embeddedness inherent in a localized food system. A decade of research on New York’s Chinatown produce economy gives an example of the importance of this connectedness: the studies revealed that 80-plus produce markets offered an incredibly diverse assortment of lower-cost produce because they are connected to a web of nearby, independently-run small farms and wholesalers . In a food chain , a product flows through different steps, where various forms of transformation may occur, and connection and feedback loops between these different steps may not necessarily exist. In such systems, farmers or industrial food producers can risk becoming producers of “food from nowhere,” as expressed by Bové and Dufour , and later unfolded by Campbell , and “consumers” can become reduced to a non-informed and non-responsible person, only “consuming food no matter of origin,” as a contrast to so-called “food citizens” defined as a consumer who makes decisions that support a democratic, economically just and environmentally sustainable food system, with a possibility of being actively involved in the food system at different levels . The call and practice of re-localizing of food systems is similarly seen as a harbinger of rural– urban reciprocity as consumers and producers are re-embedded physically and socially in the food system while raising awareness of their respective impacts on one another .Agroecology is widely acknowledged equally as a science, a practice and a movement . Its academic roots go back nearly 100 years, drawing on the fields of agronomy, horticulture, and ecology.

Through the view of agricultural systems as ecosystems, agroecology combines these disciplines and has subsequently incorporated further disciplines of cultural, human, and social sciences in a wider systems approach. It has existed as an explicit concept since the 1930s, evolving through the 1970s by increasing awareness of practices, focusing on indigenous knowledge and emerging social movements. These tenets position agroecological paradigms as both an alternative to chemical, mono-cultural or industrial farming, and as a catalyst for conventional agriculture to adopt more sustainable approaches. Agroecological systems are considered to be built on the principles of natural ecosystems and are seen as multifunctional and functionally integrated systems of complementary and dynamic relations between living organisms and their environments. In Table 1, some well-explored key characteristics related to agroecology are listed. The functions of natural ecosystems, in terms of energy and nutrient flow, as well as the dynamics of adjusting and being resilient to constantly changing surroundings and regulating populations, clearly are different from an agroecosystem. The latter are altered by and reacting to human dominance, or at a more extreme end, are disconnected or isolated from pre-existing energy and nutrient flows . Over the past decades, many academic agroecologists have increasingly stressed the importance of considering the human and social systems as integrated parts of the agroecological system. Building complex systems involves extensive human knowledge, experience, and community collaboration. Blay-Palmer and co-authors point to how the benefits of sharing place-based knowledge and good practices can help in joining forces for transforming food systems at a wider scale. The scale of an agroecological system can be large or small, but the scope of agroecological farming activities is wide; the majority of the population of smaller-scale family farmers are often considered to be applying agroecological farming approaches, and are currently estimated to produce food nourishing 50–70% of the global population, and supply up to 80% of the food in Sub-saharan Africa and Asia . With regard to human livelihood and scale related to agroecological systems, Walter Goldschmidt found that rural communities with more, smaller farms saw higher human well being than those with fewer, larger farms in settings of North-American farming in the middle of last century. This has been questioned by modernist scholars, but has also seen numerous studies supporting its conclusions over time, and it certainly has never been strongly refuted . As the example above on research in New York’s Chinatown produce economy showed, the diversity of production was found directly related to the proximity of supply and lower cost of healthy food. Another argument for how the resilience of an agroecosystem includes environmental elements as well as social and institutional elements is raised by Gonzales De Molina who refers to Holling, Berkes, and Folke and Holt-Giménez : “The resilience of an agroecosystem does not depend solely on its productive arrangements.

Collections also have been useful in determining whether the establishment and early stages of invasion are linked to single or multiple introductions. For example, Russello et al. used genetic evidence obtained from natural history specimens to infer the origin of monk parakeet populations in the USA and to link the invasion success of this species to propagule pressure exerted by the pet trade industry. Voucher specimens are useful for testing evolutionary hypotheses through data gathered from examination of trait and molecular variation. Molecular methods can be used to examine genetic variation of introduced populations and to reconstruct patterns of genetic change over time. For example, Hartley et al. used DNA extracted from vouchers to determine that blow flies were preadapted to rapid evolution in response to organophosphate insecticides. Also, phenotypic changes that occur during the different stages of invasions can be examined using natural history collections. Zangerl and Berenbaum used herbarium specimens to examine changes in phytochemistry of an invasive plant over a 152-year time period after introduction. In accordance with the enemy release hypothesis , they found that insect damage was nonexistent during the establishment phase of this species, and in accordance with the evolution of increased competitive ability hypothesis , they found that defense compounds of plants from the introduced range were significantly lower than those of plants from the native range. Further, defense compounds increased after the accidental introduction of a specialist insect herbivore from the native range. Another approach to examine factors that contribute to invasion success is to study a group of introduced species, both invasive and noninvasive. For example, Suarez et al. examined unintentionally introduced ant species from port-of-entry samples stored at the National Museum of Natural History. They found that 12% of 232 introduced species have become established in the USA, and that the probability of establishment was influenced by propagule pressure and nesting habit of ant species. Similar investigations of intentional introductions,hydroponics growing system such as bio-control agents and horticultural plants , also may provide important information on species-level ecological traits as well as phylogenetic patterns and evolutionary processes related to invasion success.

Natural history collection data are not quantitative and include species occurrences only . In addition, especially when dealing with few samples, there is a concern about how representative the samples are of the introduced populations. In some cases, these concerns can be alleviated and relative abundances of invaders can be determined from passive sampling techniques that indiscriminately collect specimens . Also, relative abundances may be inferred using specimens as a random sample of the associated community. For example, changes in the composition of pollen loads collected from bumble bee specimens reflected changes in abundance of an invasive weed in northwestern Europe . Similarly, insect and other animal specimens could be used to examine invasive parasitoids, parasites, and pathogens, and plant specimens could be used to examine invasive herbivores and pathogens. Despite the limitations of natural history collections, numerous studies have demonstrated the utility of these collections in the study of invasion biology.Natural history collections from museums and herbaria contain a wealth of data that may be used in the study of biological invasions. For example, Suarez and Tsutsui estimated that more than 100 million insect specimens are contained in just 11 entomological collections in the United States. Worldwide, natural history collections contain billions of specimens that have been collected over hundreds of years and these collections are continuing to grow . Natural history collections provide a valuable source of preserved biological materials ranging from whole organisms to DNA libraries and cell lines. Collection specimens are associated with, at minimum, information on the date and locality of collection, and often have additional information, including associated observational data and physical samples derived from specimens, such as frozen tissues and DNA extracts. Furthermore, much of the data housed in natural history collections recently has been digitized and is available through a number of searchable databases and online resources. Biodiversity informatics is an emerging field of science, and great strides have been made to link available genetic, species, and ecosystem level data, and make these data available electronically to users worldwide . The Invaders Database System , National Biological Information Infrastructure , Global Biodiversity Information Facility are just a few examples of online data portals and resources that provide access to a global network of biodiversity information, including data on voucher specimens located in natural history collections found throughout the world .

The Invaders Database System is focused on the Pacific Northwest region of the USA and combines manually entered herbarium records dating back to 1877 with records from regional literature, extension agents, and state agriculture departments, providing presence data that allow researchers to examine historical spread.For example, GBIF provides access to 285 data providers, 7445 datasets, and nearly 175 million searchable records. Some online data sources, such as Lifemapper and the Ocean Bio-geographic Information System provide links to data from a number of collections as well as tools for mapping and predicting species distributions using linked data. Such online resources will only continue to enhance the accessibility of data; however, many natural history collections are still making efforts to digitize available data. Thus, invasive species researchers should be aware that there may be a number of local, regional, and taxon- specific collections containing voucher specimens with potentially important data that are not yet summarized electronically.Improvements to natural history collection data accessibility are well underway, as many curated collections are being digitized and made available on the internet. Digitization of collection data is important for invasive species researchers who may want to use these collections, and the linking of many collections through a data portal or centralized database increases the power of available data. To facilitate the study of early-stage invasions, we recommend that researchers and field collectors, who often are very familiar with the flora or fauna within the regions they study, collect and deposit voucher specimens in the appropriate natural history collection when new or rare species are detected, in particular those species of foreign origin. Further, if an introduced species is observed in a new habitat, it would be especially useful to collect multiple individuals and to record the number of individuals observed in the population. Also voucher specimens for biological control introductions and new horticultural introductions should be deposited in the appropriate natural history collection with pertinent data, including geographic source of origin. In particular, we recommend that natural resource managers and researchers introducing bio-control agents deposit voucher specimens with data including the number of individuals introduced, the original source population of agents, the laboratory where they were reared, and the location of introduction. Because substantial efforts are being made to digitize and link data from natural history collections through centralized data portals and databases, these vouchers may be especially useful for future investigations.

Considering the potential benefits of UA for improved ecosystem functioning, in this review we discuss the ability of UA to support local and landscape level biodiversity, the role of UA in providing ecosystem services,mobile vertical grow tables and the agenda for future UA research.Urban agriculture activities are diverse and can include the cultivation of vegetables, medicinal plants, spices, mushrooms, fruit trees, ornamental plants, and other productive plants, as well as the keeping of livestock for eggs, milk, meat, wool, and other products. This definition points to the fact that UA is not solely for food production, but for a wide range of needs of the local community, including medicinal and ornamental plants. The different types of UA allow for a diverse set of vegetation structures to contribute to the edible landscape in a range of community types, and this wide range of products means that UA systems are highly heterogeneous in size, form, and function. A description of different types of UA can be found in Table 1, with associated images in Fig. 1. Community or allotment gardens often represent small-scale, highly-patchy, and qualitatively rich semi-natural ecosystems and are usually located in urban or semi-urban areas for food production . Private gardens are primarily located in suburban areas and may be the most prevalent form of urban agriculture in cities. Privately owned gardens cover an estimated 22–27% of the total urban area in the UK , 36% of urban area in New Zealand , and 19.5% of the urban area in Dayton, Ohio, USA . Easement gardens are located within private/community properties, but are often regulated by the local government. Urban easements are established with the purpose of improving water quality and erosion control , but they can include a wide array of biodiversity, depending on management type . Roof-top gardens are any garden established on the roof of a building and can be both decorative and used for agriculture. Urban orchards are tree-based food production systems that can be owned and run privately or by the community. Increasingly, schools and hospitals are establishing fruit trees that provide crops, erosion control,shade, and wild life habitat, while producing food for the local community . Many UA systems may fit into more than one category. For example, both private gardens and community gardens may exist as rooftop gardens, and community orchards may exist within community gardens.Urban landscapes are typically highly simplified, intensively developed ecosystems with low levels of native biodiversity . However, urban green spaces such as UA can bring diverse green infrastructure back into the urban system, providing vegetative structure and biodiversity for ecosystem function and services across fragmented habitats and spatial scales . UA may be especially important for biodiversity conservation in cities because vegetative structural complexity in simplified landscapes contributes disproportionately more to conservation than in more natural landscapes . Just as in agricultural systems where more complex agri-environment management can have a larger effect on biodiversity when implemented in simple agricultural landscapes than more complex landscapes , UA provides many opportunities for re-vegetating the landscape at the local scale within a vegetatively depauperate urbanized landscape. Urban agriculture has the potential to support biodiversity not only within UA sites, but also nearby due to a landscape-mediated ‘spill over’ of energy, resources, and organisms across habitats. Such spill over may be an important process for the persistence of wildlife populations in human-dominated landscapes because it allows for resource acquisition and re-colonization events . At the same time, chemical, water, and animal movement is bi-directional, and intensified management implemented in backyards, such as pesticide application, extensive pruning, frequent mowing and other disturbances, can limit the capacity of gardens to maintain rare or sensitive insect species . Thus, it is important to understand that not all biodiversity is necessarily “good” biodiversity, and there may be a number of disservices that come from UA as well . Here, we examine the ability of UA systems to support vegetative, insect, and vertebrate biodiversity .The wide variety of UA types in practice allow for considerable variation in vegetative complexity and diversity. Domestic gardens vary widely in features that may promote plant biodiversity,such as ponds,moss, ground cover, and varied vascular vegetative structures . For example, tropical home gardens have stratified vegetation similar to those seen in multi-stratified agroforestry systems and can thus provide a large amount of planned and associated biodiversity . The diversity of vegetation types within home gardens has been documented in Santarem, Brazil, where 98 plant species were identified in 21 urban gardens and included a large diversity of fruit trees and shrubs , ornamental plants , vegetable/herb plants , and medicinal plants . In Leon, Nicaragua, 293 plant species belonging to 88 families were recorded across 96 surveyed home gardens, ranging in habit and taxonomic origin . In Hobart, Australia, 12 distinctly different garden types with different species, habits, and canopy heights were documented in front and backyard gardens , and a similar survey conducted in Toronto found 25 woody plant species and 17 different herbaceous plant species per backyard garden . In an example from five UK cities, more than 1000 species were recorded in 267 gardens, exceeding that recorded in all other local urban and semi-natural habitats .In Stockholm, allotment gardens are older than many backyard gardens, often representing lush, well-managed flower-filled areas ranging in size . Such areas are often extremely rich in plant diversity, with more than 440 different plant species recorded in a single 400 m2 allotment garden .

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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