Author: naturehydro

A garden may be understood as a place where the ‘geography of the mind meets that of the earth’,making it entirely apropos that the vertical garden finds its origins in the monumentally horizontal prairies and farmlands of the American Middle West, where the topography of Professor Stanley Hart White’s creative intellect meets a seemingly endless geography of flatness. White patented the first known green wall in 1938, prototyping the technology in the backyard of his Urbana residence, yet the concept emerges in his writings and drawings as far back as 1931 as a response to the problem of modern garden design. The significance of this invention has ‘still unrealized provocations’ on the history of gardens and designed landscapes, having been conceived during a trajectory towards modernism in the same geographic region as the Prairie School and American Skyscraper. Although the provenance of this new technological garden is topographically uncanny, the invention itself is pure genius, synthesizing ideas from modern landscape and architectural theory, building sciences, horticulture, and industrial arts alike. White’s vertical garden finds its legal origins in 1937–38, albeit the technological and material precursors to the invention extend back to early horticultural experiments and industrialization of modern building materials. Stanley Hart White, Professor of Landscape Architecture at the University of Illinois Urbana-Champaign from 1922 to 1959, was granted US Patent 2,113,523 on 5th April 1938 for the Vegetation-Bearing Architectonic Structure and System in which he describes the method for creating an ‘architectonic structure of any buildable size, shape or height, whose visible or exposed surfaces may present a permanently growing covering of vegetation’.In six beautifully illustrated pages, Professor White reveals the new art of growing plants within/on a vertical,commercial greenhouse supplies architectonic, substrate-holding frame, and in the process describes a new vertical garden type not fully realized till after his death in 1979.

All that remains of White’s invention are his careful diaries, a series of patents, and his brother E. B. White’s correspondences about Stan’s new invention. Stanley Hart White is best known as an educator who modernized landscape pedagogy at the University of Illinois, influencing the work of Hideo Sasaki, Peter Walker, Richard Haag, and others, through his innovative teaching style and creativity.With the discovery of his patent for the first known green wall, or Botanical Bricks, he may also be credited as an inventor and technological innovator, conceptualizing the vertical garden and pioneering green modernism . White’s thoughts on vegetation-bearing architecture crystallize in his patent of 1938, yet notions of a green wall emerge as early as 1931 in his lectures and writings on the modern garden. Although the intended audiences for White’s early writings on vertical greenery are not yet apparent, the idea of a vegetation bearing garden enclosure preoccupies him for several years as documented in his personal journals, or Commonplace Books, in the University of Illinois Library Archives. Technical aspects of White’s green wall find their clearest articulation in US Patent 2,113,523, filed on 18th August 1937, yet the theoretical dimensions developed as a treatise on modernism and garden design, in which the vertical surfaces of the garden create a backdrop for modern living. In an essay titled ‘What is Modern’, White discusses the green wall as a design solution for the modern garden, allowing for the preservation of a free plan and composition of a garden in the vertical dimension. His references to Walter Gropius, Le Corbusier, Frank Lloyd Wright, Louis Sullivan, Walt Whitman, Charlie Chaplin, Norman Bel Geddes, Adolph Appia, Sheldon Cheney, Walt Disney, and others, situates the work among a group of ‘moderns’ concerned with changing lives through art and architecture.The Vegetation-Bearing Architectonic Structure and System evolved as a response to the problem of modernism in garden design, and is a unique contribution of landscape architecture to this effort, representing a clear translation of garden theory into garden form and legalese.

The prescience of this work is astounding, predicting not only the emergence of the vertical garden in the contemporary built environment, but a method of scholarship in patent development not widely accepted by US universities until the 1970s.The first drawings of a green wall appear in White’s Commonplace Books in 1932 as two humble sketches of a wall section and elevation. White’s elevation sketch shows a wall composed of vertical greenery, decorative insets, lights, fishes, and birds occupying the inset voids. His section sketch reveals a soil filled wall structured with interior irrigation and voids for compositional elements. Annotations in the Commonplace Books Index identify the sketch as an idea for the Century of Progress Exhibition in Chicago, and as his first sketches of Botanical Bricks.White’s inchoate drawings and description of a green wall in 1931–32 mature until his application for the Vegetation-Bearing Architectonic Structure and System on 18th August 1937, where he artfully translates garden theory into United States Patent and Trademark Office legalese with the help of his attorney, Elmer Hovenden Gates of Arlington, Virginia.The new art of vegetation bearing architecture was entirely novel at the time of application, and no citations of prior art are associated with White’s invention. Currently, thirty four international patents cite US Patent 2,113,523 as prior art, encoding an array of inventions from grass cube chairs, to vegetation-bearing gabion walls.Interestingly, White’s lawyer, Elmer Hovenden Gates, and proposed business partner, William M. McPherson, patented related vegetation-bearing technologies within weeks of his submission. More than 50 patents cite the Vegetation Bearing Cellular Structure and System, Vegetation-Bearing Display Surface, and the Vegetation-Bearing Architectonic Structure and System, collectively encoding a diverse ensemble of environmental technologies. The legalese defining this new field offers valuable insights into the founding principles of vegetation-bearing architecture as a chimera of architectonic structure and vegetated system. According to White, architectonics relates to ‘the art of landscaping structure as well as to buildings, but distinguished from the art of plant culture’.Within this architectonic structure, plant growth is supported through a layering of horticultural substrates and reticular materials. In this configuration, the ‘vegetation in its final positions has its roots within the compost while the tops of the vegetation would extend through the reticular surfaces of the units or compounds into the open air where their normal development occurs’.

The patents legalese describes not only the technical specifications of White’s new invention, but also the proposed scope of vegetation-bearing architecture as a new art. This scope is of particular interest with the emergence of the vertical garden and green wall in the contemporary built environment, as the language that defines the new art also encodes innovations in related technologies today.White’s vision of rapidly assembled and scalable plant-bearing architectonic structures adopts principles from modular building and construction techniques, situating his invention among the array of building products developed during the early decades of the twentieth century. The common name Botanical Bricks further reveals White’s notion that his modular building units be integrated into the building arts as commonly as bricks in gardens and architecture. Potential application listed in the patent range from camouflage, concealment, decoration, backgrounds, and screens for use in architectural project . Contrasts between White’s proposed applications, and contemporary notion of vegetated architecture as a sustainable technology is especially salient,vertical grow as language to describe sustainability or environmental performance had not yet been established. The field of vegetation-bearing architecture was rapidly expanded by two contingent patents filed on 4th April and 28th May 1938 by William M. MacPherson and Elmer Hovendon Gates, respectively. These patents reinterpret the structural system proposed by White in which the vegetation bearing units assemble like masonry or bricks, and are essentially self supporting through stacking and repetition . Consecutive to this, patents by Gates and MacPherson envision vegetation-bearing units supported within a structural and load-bearing framework, and vegetation bearing units secured to a self-supporting wall as a veneer or surface system. Although the structural iterations presented by each patent alter the load bearing relationship of the vegetation to the structural element, the new art, as described by White, remains remarkably resilient to these adaptations and contemporary permutations.Although the structural modifications proposed by Gates and MacPherson reorient the relation of the vegetation-bearing unit to the underlying structural system, the basic technology and principles remains intact. The resilience of White’s new art is found in its aptitude for reinterpretation, as he defines a relationship between plants and structure that elucidates their interdependence and interstices. Triangulations between the building sciences, horticultural arts, and landscape theory are seminal to White’s conception of the Vegetation Bearing Architectonic Structure and System. White received a Bachelor of Science in agriculture from Cornell in 1912, his Master of Landscape Architecture from Harvard in 1915.His background in science and design undoubtedly contributed to a synthetic approach to plants, structure, and garden form, yet it is a convergence of material innovations and new scientific discoveries that make the vertical garden possible, and timely, in the 1930s. Innovation in building materials occurred rapidly in early years of the twentieth century, as industrialization and wartime research fueled experiments in architecture and the sciences alike. Material Sciences flourished during World War I and material product lines expanded through the roaring twenties, radically impacting architecture and the production of buildings.New building materials were readily tested, and exhaustive volumes of literature were published to disseminate information in an ever-changing marketplace for architects and designers. Building material bibliographies from the era document rapid integration and research on materials such as armorphy, clay tile, plywood, glass block, masonite, rostone, vinylite, celotex, porcelain, zonolite, to name just a few.Concurrent to the expansion of new materials in architecture, was an expansion of the role of popular science and a belief that technology would improve modern living. In agriculture, new technologies such as hydroponics, popularized in the 1930s, promised to create new productive systems that would increase yields and feed a growing population with increased efficiency.

White’s invention exists at the intersection of these spheres of innovation, where the differentiation between horticultural system and building system begins to blur. As human impact increases in marine and coastal ecosystems , rules, regulations, and policies have struggled to keep up with the need for adequate environmental management . There has long been an emphasis on biodiversity conservation in environmental decision-making , but biodiversity for biodiversity’s sake is not always enough in the face of industry and economic development. However, biodiversity is often considered the source of most ecosystem services , which are the direct and indirect benefits derived from the environment. The concept of ecosystem services was made popular by the Millennium Ecosystem Assessment , and has since gone through many iterations . I adopt the following framework: ecosystems have physical, chemical, and biological structures that support ecological functions , which can be combined with a human perspective to produce ecosystem services. They are divided into four categories: provisioning , regulating , cultural , and supporting . Ecosystem services provide a direct link between natural systems and human well-being, contributing to a tangible rationale for sustainable development, management, and protection of the environment. As a result, they can be a useful tool in environmental decision-making. This dissertation aimed to explore approaches for characterizing ecosystem services in two types of systems subject to human impact: deep-sea habitats and natural storm water treatment systems . The broad scope of systems highlights the context dependence of ecosystem services as well as overarching themes among them. I employed an interdisciplinary approach that was heavily influenced by the ecology and economic disciplines, which facilitated application of results to real questions in environmental management. In the deep sea, there is increasing demand for natural resources , but environmental rules, regulations, and policies are only now being developed for some parts of it . The emerging industry of deep-seabed mining presents a novel opportunity to incorporate ecosystem services into environmental management prior to commercial exploitation. Chapter 2 reviews ecosystem services associated with deep-sea habitats targeted for mining , and the structures and functions that support them. Known examples include fisheries landings , genetic resources , carbon sequestration and storage , and element cycling , but there are likely to be ecosystem services yet discovered. Chapter 2 also discusses how and where ecosystem services can be incorporated into existing deep-seabed mining regulations. Overall, the second chapter of this dissertation creates a framework for studying ecosystem services and for applying them to environmental decision-making.

It considers historical evidence that correlates a decline in Native American health and fertility with ruptures to indigenous food systems following European colonization. It suggests novel and interdisciplinary ways in which advanced undergraduate or graduate level students might examine the correlation between breached indigenous nutritional practices and a decline in Native American health. These learning objectives bring together students of history and natural science in one classroom and entail new ways of synthesizing hitherto separate scholarly enterprises. In light of the most cutting-edge scientific literature on nutrition, metabolic syndrome, and immunology, they require a new consideration of the historical association between Native American health and indigenous food systems. In the centuries after European contact, many Native American communities were forced to move away from diets that had been comparatively high in animal proteins, animal fats, and fat soluble vitamins, and which also often incorporated important starch and plant sources such as wild rice, tubers, chenopods, beans, seeds, maize, squash, berries, and leafy vegetables. Notwithstanding regional variations, the pre-contact Native American diet was thus relatively nutrient dense; incorporating varied macro-nutrients and micro-nutrients through hunting and gathering practices and indigenous forms of horticulture that were subsequently disrupted. Thanks to the deleterious and often deliberate effects of colonization, which can only be understood through careful historical study and analysis, deeply-rooted food systems were ruptured. From as early as the sixteenth century, new post-contact circumstances forced many Native Americans to adopt diets that favored imported European grain cultivars, to maintain greater calorific reliance on New World maize species,square plastic pot and to reduce their consumption of traditionally hunted animals and fish and cultivated plant sources.

It is important to avoid any crude interpretative framework that might “exoticize” pre-contact Native American communities as having avoided any form of managed agriculture, crop monoculture, or organized land husbandry. Recent historical research, after all, has often employed the metaphor of “gardening” to question the notion that pre-contact Native Americans relied solely on hunting and gathering methods for sustenance. It is also imperative to avoid eschewing the distinct variations between indigenous food cultures both during and after the period of European contact: veering between the cultivation of maize, tubers, and starchy seeds alongside hunted animals in the Southwest to a relatively homogenous reliance on fats gathered from hunted meats and fish in the sub-Artic; as well as many gradations in between, such as the cultivation of wild rice alongside more traditional hunting and gathering patterns in the Great Lakes region. Yet this article – and the proposed educational course it defines – attempts at least some degree of generalization in discussing the differences between indigenous food systems and those that were introduced after European contact; and in discussing how students and researchers might view those distinctions in light of the modern scientific literature on metabolic and nutritional health. In questioning crude definitions of pre-contact Native Americans as noble hunter-gatherers – including those that are sometimes used by advocates of ancestral health and Paleolithic nutritional principles – it is important to avoid going to the other extreme by deemphasizing the relative environmental and dietary importance of hunting and gathering systems in many different parts of North America immediately prior to, and even after, European contact. While indigenous agricultural activities were present throughout the American continent, hunter gathering practices were also continued to a far greater extent than in post-Paleolithic Europe and the Middle East – potentially heralding important ecological and nutritional differences between the two regional populations over the following centuries. Those differences may inform our understanding of the role of nutrition in evolutionary health, particularly by comparing the pre and post-contact history of Native Americans. Recent scientific research has suggested that we may be able to locate specific loci in the DNA of some Native Americans that affect their insulin sensitivity. Individuals with certain genetic variants at these loci would be more likely to develop diseases such as diabetes following a move towards a higher carbohydrate diet, as has often taken place from the period of European colonization to the present day.

Examining the Pima Indian community of Arizona as a case study, researchers have found several loci with genetic variants that confer susceptibility to diabetes. For example a genome wide association study by Hanson et al identified polymorphisms in the DNER locus that are associated with increased risk of diabetes in Pima Indians. The Decolonizing the Diet project, and other similar endeavors, thus start with the hypothesis that a return to pre-European contact diets will improve the health of Native American communities, reducing hitherto disproportionately high instances of diabetes, as well as heart disease and other conditions associated with metabolic syndrome. Native American populations, indeed, have often featured as case studies among those scholars who attempt to define a “thrifty gene hypothesis” to explain why some people are prone to diabetes and/or obesity. A “thrifty” genotype, it is suggested, may have been evolutionarily successful for individuals descended from hunter-gatherer populations. Its occurrence would have allowed those populations, particularly child-bearing women, to gain fat more easily during times of abundance . Those with more fat may have better survived times of food scarcity, and thus passed on their genes. But during times of nutritional abundance, they would be more likely to develop metabolic syndromes such as obesity and diabetes, according to the hypothesis. In post-hunter-gathering populations, a similar paradigm has been hypothesized by Sellayah and others, who have suggested that the thrifty genotype may have appeared among those who had “undergone positive selection for genes that favored energy storage as a consequence of the cyclical episodes of famine and surplus after the advent of farming 10 000 years ago.” In any class, project, or research agenda, however, it is important to avoid necessarily deterministic conclusions when assessing the correlation between recent genetic studies and epidemiological data from Native American communities . Firstly, more research is still needed in order to assess whether particular genetic variants for insulin sensitivity are present exclusively or at a higher frequency in Native American populations compared to other populations, or whether they are equally prevalent in other ethnic communities who were not included in present studies. Despite retaining the same genetic variants, those other communities might not suffer from diabetes to the extent of Native Americans. Secondly, moreover, greater genetic susceptibility to insulin insensitivity or any other medical condition need not pre-determine the actual onset of diabetes or other disorders, as is evidenced by the relatively positive health markers among Pima and other Native American communities prior to increasing their consumption of processed and high sugar foods.

The notion that Native Americans have suffered from particular genetic predispositions might also prove problematic in encouraging students, scholars, and researchers to adopt an overly deterministic account of health outcomes,25 liter pot eschewing the disrupting role of human interventions against ancestral food-ways – either in exacerbating Native American susceptibility to metabolic syndromes and/or infectious disease or even as a primary factor in their increasing mortality and declining fertility after European contact. Examining – and problematizing – the link between modern disease susceptibility and genetic predispositions should prepare students for a related scholarly endeavor: assessing the potential tensions and pitfalls associated with the concept of a “biological exchange” of infectious diseases at the period of contact between Europeans and Native Americans . Here too a focus on abstract biological forces risks overlooking the role of human interventions in determining the inevitability of demographic decline in the face of disease. Historians, notably and most famously Albert Crosby, once defined the decimation in number and health of post-contact Native American communities according to a metaphor of biological exchange. Here, we have been told, Native Americans in a “virgin land” were unable to cope with the pathogens inadvertently introduced by Europeans after the arrival of Columbus.These great killer diseases, introduced by germs, spores, and parasites from European and African sources, included smallpox, measles, influenza, bubonic plague, diphtheria, typhus, cholera, scarlet fever, trachoma, whopping cough, chicken pox, and tropical malaria. Yet as the most advanced historical scholarship now suggests, human interventions were necessary to bring about the marked decline in Native American health and fertility, and the increase in mortality, in the centuries after the arrival of Columbus in the western hemisphere – as distinct from the notion of an amorphous biological exchange involving a mismatch between European and Native-American immunity.There is no doubt that Native American communities and Europeans retained different immunities during the period of contact. But suggesting that Native Americans were predisposed to near–total demographic collapse solely due to their relative lack of immunity may lead students and scholars to eschew any further assessment of their nutritional disruption as a co-factor in such a phenomenon; just as modern studies of genetic loci for diabetes might lead researchers to eschew the role of post-colonial interventions in late-nineteenth century and twentieth-century Native American food patterns, which affected their insulin sensitivity above and beyond any genetic predisposition. Scholarship of global infectious disease has shown that societies have most often been able to recover demographically from near collapse following massive outbreaks, usually in around 150 years. Disturbances such as epidemics have tended to result in only short-term demographic decline, with populations returning to pre-disease levels of growth, decline, or stability.

Describing the response to the European “black death”, for example, McNeill points out that “the period required for medieval European populations to absorb the shock of renewed exposure to plague seems to have been between 100 and 133 years.”As Gottfied has demonstrated, fourteenth and fifteenth century Europeans suffered multiple epidemics including the Black Death, Typhus, influenza and measles, yet their populations were able to recover demographically after around a century.Herring has even shown that early twentieth century Native American populations outside reservations were able to recover numbers following influenza, smallpox and measles epidemics. Taking these general studies as a starting point, students and researchers in biology and public health policy would gain a broader understanding of immunology and epidemiology through a joint course with historians and anthropologists. Rather than assuming that certain communities are more prone to metabolic syndromes and diseases, whether from genetic loci or a comparative lack of exposure to certain pathogens, they would be able to consider the ways in which human interventions – particularly in food ways – exacerbated demographic decline in the face of disease; both in terms of reduced immunity prior to infection and reduced ability to fight pathogenic invasion. Let us now consider how students might use case studies in Native American history to illustrate such a phenomenon, before turning to the ways in which contemporary scientific studies might then inform their analysis of the role of nutrition in enhancing or reducing the potential for recovery after mass epidemics. Historians of Native-American health and fertility have drawn methodologically and conceptually from general epidemic studies in order to question the Biological Exchange thesis for demographic decline between the 1500s and 1800s. Near total demographic collapse, according to a developing historiographical consensus, was made possible by the rupturing of ancestral social mechanisms by European colonization – rather than simply as a result of differing immune capabilities among the two populations. The failure of Native American populations to return to pre-epidemic demographic numbers, many scholars now assert, derived from human interventions that accompanied the spread of disease, rather than simply the diseases as singular factors. One such human intervention lay in the domesticated agricultural practices that were prescribed by European colonization after first contact with Native Americans. The disastrous decline in Native American demography was partly affected by the growing mismatch between their long-evolved ecological frameworks and European cattle-pens and agricultural methods. The latter exacerbated the spread of diseases that Native American communities were already struggling to fight off due to their impaired immunity.According to the pioneering paleo-archeological work of Armelagos, such a phenomenon has commonly affected societies as they have transitioned to concentrated agricultural settlement and animal husbandry. Examining paleo-archeological evidence in European and Middle Eastern populations, Armelagos has noted the problems that followed relatively sudden proximity to domesticated animals and to human and animal waste in newly agricultural societies. Such proximity increased the spread of parasitic disease. In previous hunter-gatherer populations, frequent migrations limited the contact of individuals with human waste. In more sedentary populations, concentrated around grain production and domesticated animals, human and animal waste became more likely to contaminate drinking water.

TOC1 and CO regulate circadian rhythm and day-length responses, while RGA represses floral growth in the absence of gibberellic acid. WUSCHEL genes have a well-characterized function in meristem organization and have been shown to interact with AGAMOUS in floral meristem development. AGL11 is important in ovule and seed development, and has been shown to interact with cytokinin to control fruit size. These floral development genes may have specific roles as “entry points” for the SDR into the floral development pathway to regulate the development of a particular sex . Cronk and Müller proposed that ARR17 may act as a feminizing master regulator in Populus through the suppression of PISTILLATA or APETALLA3 MADS-box genes. Importantly, there was no association of either PI or AP3 expression with genomic variation in the SDR, despite the fact that PI shows very high levels of expression in males , further suggesting that the mechanism of sex determination in Salix may be different from Populus. eQTL analysis revealed several loci with an exceptionally high number of trans-eQTL . Intriguingly, the hotspot with the greatest number of trans-eQTL is located in the region on chr07 homologous to the S. nigra SDR, which could implicate its role as an ancestral SDR in Salix, or explain a fitness advantage of a chr07 SDR in S. nigra when linked with sex-dimorphism genes in this region. Approximately 250 kb from this locus on chr07 is Sapur.007G068100, a homolog of AGL32,fodder growing system a MADS-box gene involved in ovule development. The expression of this gene is associated in trans with the chromosome-15 SDR region, further supporting its role as a top-level regulatory gene under direct regulation by the SDR.

Compounds produced from the phenylpropanoid and terpenoid pathways are well-characterized in Salicaceae, and there is evidence that floral volatile, terpenoid, and phenolic glycoside profiles differ substantially between males and females, which affects both pollinator attraction and herbivory, traits that are likely to be evolutionary drivers of diecy and affect cultivar yield. In support of this, we identified five terpenoid pathway genes and 15 phenylpropanoid pathway genes with eQTL in the SDR. These include both genes involved in the core phenylpropanoid pathway, and biosynthesis of specific compounds, including naringenin, flavenol glucosides, and sesquiter penoids . This provides evidence supporting a direct link between the SDR and synthesis of these compounds, by an as-yet unknown mechanism.Of the 45 identified miRNA loci, 18 were male differentially expressed and seven were female differentially expressed. Among the putative targets of these miRNAs were dicer-like genes, squamosa promoter-like genes, andauxin-response factors, and transcription factors providing evidence that miRNAs are likely to be a component of floral sex-dimorphism regulation. Notably, five miRNAs were identified that had no match with any small RNAs in the PmiREN database and could represent genus- or species-specific micro-RNAs. Furthermore, 13 miRNA loci with matches in the pmiREN database were not matched to a known P. trichocarpa miRNA, which is the closest species for which extensive small RNA data are available . This suggests that S. purpurea may utilize different sets of small RNAs in floral development relative to Poplar, which likely has implications on sex dimorphism and determination. Expression results showed that four miR156 and two miR172 homologs have greater expression in male floral tissue. In Arabidopsis, miR156 and miR172 interact to form a gradient that regulates vegetative-to-floral meristem transition through the targeting of squamosa promoter-like genes.

While all copies of both miR172 and miR156 show malebiased expression in catkins, the overall expression of miR156 is greater than miR172 in males. The majority of SPL genes that are targeted are female-up regulated in S. purpurea, including an SPL4 homolog on chr07 that also shows increased methylation in males in its promoter region. These data support that the miR156/172 pathway is upregulated in male catkins and may be responsible for sex dimorphisms . This pathway may play a role in male floral tissue development or differentiation. Importantly, one copy of miR156 is located in the SDR region unique to chr15Z. Alignment of the chr15Z miR156 precursor sequence to chr15W reveals that a single indel is responsible for this chr15Z-specific mapping , which may prevent transcription or processing of this small RNA from chr15W. This could indicate a dosage dependent response in males, which have four copies of this mature miR156 homolog, compared with only three in females. Female-upregulated miRNAs included miR403, which targets AGO2, and miR162, which targets a dicer-like gene. All of these are involved in small RNA signaling and DNA methylation, which is consistent with the enrichment of transcription regulation terms in the female-upregulated genes. This may point toward a role of genome-wide DNA methylation or RNA silencing in regulating sex dimorphism, possibly mediated by AGO4 or DRB1.Pest- and pathogen-free planting stock is essential for successful establishment and future productivity of new orchards and vineyards. Clean stock is also a requirement for intrastate, interstate and international commerce of tree, vine and garden rose planting stock. To ensure the quality of commercially produced nursery stock in the state, the California Department of Food and Agriculture enforces laws and regulations related to the production of certified nursery stock as outlined in the Nursery Inspection Procedures Manual .

Because of the potentially large and long-term impacts on the nursery crop as well as the subsequently planted orchards, vineyards and ornamental landscapes, control of plant-parasitic nematodes in nursery fields is a major focus of the nursery stock certification program. Producers of perennial crop nursery stock in California can meet nematode certification requirements by fumigating the field at the beginning of the nursery cycle using an approved treatment or by conducting a detailed inspection of soil and planting stock at the end of the production cycle. If growers elect to use inspection procedures instead of approved treatments and soil or plant samples are found to contain prohibited nematodes, further sampling is conducted to delineate the extent of the problem, and nursery stock from the affected area usually is destroyed. Preplant soil fumigation thus reduces the economic risk of a nonsalable nursery crop and is used in most tree and garden rose nurseries in California. Grapevine nursery stock also must meet phytosanitary requirements to be certified in California, but in contrast to tree and rose growers, many grape nursery producers elect to use the inspection procedures rather than fumigation. In practice, the risk of nematode occurrence in production of grapevine nursery stock without fumigants is reduced by spring planting, a relatively shorter nursery production cycle and market preference for smaller nursery stock. However, grape nursery operations with sandy soils or sites where grapes have been grown previously often use preplant fumigation practices comparable to tree and rose nurseries to reduce the economic and market risks of not meeting phytosanitary regulations. Most field-grown perennial nursery operations have used methyl bromide for preplant pest control because it effectively diffuses through the soil profile, penetrates roots and dependably provides effective pest control across a range of soil type and moisture conditions. Under the provisions of the U.S. Clean Air Act and the Montreal Protocol,chicken fodder system the import and manufacture of methyl bromide is being phased out because of its deleterious effects on stratospheric ozone. Perennial nursery producers have largely continued using methyl bromide under the critical use exemptions and quarantine/preshipment criteria . However, increasing production costs and international political pressure on CUE and QPS regulations have spurred efforts to identify economically viable alternatives to methyl bromide for the perennial nursery industry. Several factors limit the adoption of alternative fumigants in California nursery systems. First, there are very few fumigant or non-fumigant nematicides available . In the United States only a handful of fumigants are registered, including methyl bromide, 1,3-dichloropropene , chloropicrin, dimethyl disulfide , and methyl isothiocyanate generating compounds. Of these, DMDS is not currently registered in California and has had only limited testing in nurseries. Methyl iodide was registered in California in late 2010, but the federal registration was withdrawn by the manufacturer in early 2012.Of the fumigants registered in the state, only 1,3-D is an approved treatment in nurseries with medium- to coarsetextured soils . However, it is not approved for nurseries with fine-textured soils because the registered rates are not sufficient to provide acceptable pest control.

Most of the alternative fumigants are heavily regulated due to concerns about human safety and environmental quality related to emission of fumigants and associated volatile organic compounds . These concerns have led to a constantly changing regulatory environment, encompassing buffer zones, field preparation requirements, available compounds and rate limitations on a field and air basin level . Uncertainty within the nursery industry about current and pending fumigant regulations presents a continuing challenge to the adoption of methyl bromide alternatives in California. Although fumigation in the perennial crop nursery industry is driven by nematode certification, there are serious concerns that the level of secondary pest control provided by methyl bromide will not be matched by the alternatives. Weed control with many of the available alternatives is generally not as reliable as with methyl bromide . Although weeds can be addressed to a large extent with tillage, hand-weeding, and herbicides, there are likely to be environmental and economic impacts of greater reliance on these techniques. More importantly, many nursery producers are very concerned about the consequences of soil borne diseases that are currently controlled with methyl bromide or methyl bromide and chloropicrin combinations. Reliance on alternatives with narrower pest control spectrums may result in problems with new diseases or the resurrection of old ones. Research has been conducted in recent years to address issues limiting adoption of methyl bromide alternatives in California’s perennial crop nursery industry . As part of the USDA-ARS Pacific Area-wide Pest Management Program for Integrated Methyl Bromide Alternatives, two additional research and demonstration projects were implemented from 2007 to 2010. First, because current and pending regulations greatly affect how and when fumigants can be used, a research station field trial was conducted to simultaneously determine the effects of emission reduction techniques on pest control and fumigant emissions. Second, two trials were conducted in commercial nurseries to test and demonstrate pest control and nursery stock productivity with 1,3-D treatments in an effort to increase grower experience and comfort with available alternatives.A shank fumigation trial was conducted in 2007 at the UC Kearney Agricultural Center , near Parlier, to determine the effect of two fumigation shank types and five soil surface treatments on 1,3-D emissions and control of representative soil borne pests following removal of a plum orchard. Soil texture at the site was a Hanford fine, sandy loam with pH 7.2, 0.7% organic matter, and a composition of 70% sand, 24% silt and 6% clay. The experiment included 10 treatments with 1,3-D in a split plot design with surface treatments as the main plots and two application shank types as the subplots, as well as an unfumigated control and a methyl bromide plus chloropicrin standard for comparison . Individual plots were 12 feet by 100 feet, and each treatment was replicated three times.Fumigants were applied using commercial equipment on Oct. 2, 2007. Methyl bromide with chloropicrin was applied at 350 pounds per acre with a Noble plow rig set up to inject fumigants 10 inches deep through emitters spaced 12 inches apart while simultaneously installing 1-mil high-density polyethylene film. The 1,3-D treatments, at 332 pounds per acre, were applied using either a standard Telone rig with shanks spaced 20 inches apart and an injection depth of 18 inches or a Buessing shank rig with shanks spaced 24 inches apart and the fumigant injection split at 16- and 26-inch injection depths. The Buessing shank also had wings above each injection nozzle to scrape soil into the shank trace and minimize rapid upward movement of the fumigant . Following 1,3-D application, a disk and ring roller was used to level and compact the surface soil before surface seals were applied over the fumigated plots. Average soil temperature at 20 inches during fumigation was 70°F, and soil moisture was 8.2% to 10.5% weight per weight in the top 3 feet. Surface treatments included HDPE film; virtually impermeable film, VIF ; and a series of intermittent water applications . HDPE and VIF film was installed after the disk and rolling operation using a Noble plow rig.

Fumigant concentration and weed density data were subjected to nonlinear regression analysis using Sigma Plot v. 11 .Because application rates tested in 2007 were normal, the rates were sufficiently high to suppress most pathogens and weeds regardless of the film permeability. For this reason, in 2008 we chose to compare fumigant retention under the two films at a range of rates from low to high, to determine if TIF would improve retention and efficacy across that range. At Salinas in 2008, 1,3-D plus chloropicrin concentrations in the 200-poundper-acre treatment were higher 24 hours post-application under TIF than under standard film . The 1,3-D plus chloropicrin concentrations in the 300-pound-per-acre treatment were higher under TIF than under standard film at 8, 24, 48 and 96 hours after application.Generally, there were no tarp effects on plant diameters except at the 1,3-D plus chloropicrin rate of 100 pounds per acre; TIF plants were 9.4 inches compared with 8.3 inches for standard-film plants .The differences were significant in the 1,3-D plus chloropicrin treatments at 100 and 200 pounds per acre . There was a positive correlation between the 8-hour 1,3-D plus chloropicrin concentration and full season fruit yields for each film . The 8-hour fumigant concentration accounted for 49% to 55% of yield variability in the standard and TIF treatments, respectively. Weed densities were higher under standard film than under TIF. At 100 pounds per acre, 1,3-D plus chloropicrin applied under TIF had significantly fewer weeds than the same rate under standard film . The interaction between fumigant rate and film was significant for common chickweed and common purslane ,dutch bucket hydroponic meaning that the survival of each of these two weeds was different under the two films.

The interaction of yellow nuts edge rate by film was not significant , indicating that nuts edge survival was similar under both films. However, we sought to describe the performance of TIF, therefore we evaluated nuts edge separately under both films. Yellow nuts edge tuber survival was less under TIF than standard film at 100 pounds per acre 1,3-D plus chloropicrin, but not at the other rates. Common purslane and common chickweed seed survival were lower under TIF than standard film at 50 pounds per acre 1,3-D plus chloropicrin . Little mallow and knot weed viability were similar under both films . Differences in weed control due to film type were only observed at the lower fumigant doses of 50 and 100 pounds per acre. This is likely due to the fact that TIF retained more fumigant than standard film, which resulted in a higher dose and lower weed seed survival under TIF than standard film . At application rates above 100 pounds per acre, the fumigant concentrations under both TIF and standard films were sufficiently high to kill weeds, so no differences were found between the films.Results of two trials conducted over 2 years indicate that TIF consistently held methyl bromide plus chloropicrin and 1,3-D plus chloropicrin at higher concentrations than standard film . At fumigant rates of 100 and 200 pounds per acre, strawberry fruit yields were higher and weed control was more complete where TIF was used, compared to standard film . This is likely due to the higher fumigant concentrations being held for a longer time under the TIF than under the morepermeable standard film, so that weeds and possibly soil pathogens were more thoroughly controlled. Drip-applied 1,3-D plus chloropicrin under standard film required at least 300 pounds per acre to provide fruit yields comparable to methyl bromide plus chloropicrin . In contrast, 1,3-D pluschloropicrin drip-applied under TIF at 200 pounds per acre had fruit yields and weed control similar to methyl bromide plus chloropicrin, a 33% reduction in 1,3-D plus chloropicrin rate compared to standard film. Similarly, Ajwa et al. found that the rates of drip-applied chloropicrin required to produce strawberry yields similar to methyl bromide plus chloropicrin were 294 and 198 pounds per acre under standard and VIF, respectively, a 48% reduction in chloropicrin. The recent registration of methyl iodide as a soil fumigant by the California Department of Pesticide Registration requires the use of impermeable films . Methyl iodide must be used with impermeable films as approved by CDPR, and TIF is on the list of approved films .

The results presented here further validate that TIF is effective at increasing fumigant retention and may ease some of the burdens of fumigant regulations on end-users, as well as ease concerns of the general public about exposure to fumigants.Monilinia laxa is the main causal agent of brown rot in Europe, leading to important losses of stone fruit in the field and post harvest. The worldwide yearly losses are estimated to be 1.7 M euros for peach and nectarine and 170 M USD for peach, cherry, and plum production. The disease is controlled using several cultural practices , chemical fungicides in the orchard, treatments onto mummified fruit, and post harvest storage at low temperatures. However, the gradual withdrawal of some fungicides driven by concerns about their negative impact on the environment and human health, the constant threat of the emergence of fungicide resistance, and the appearance of novel virulence alleles demonstrate the need for alternative methods for managing brown rot. Prior to infection, M. laxa can remain latent or quiescent on flowers and fruit surfaces until favorable host factors , and environmental factors and other characteristics intrinsic to the stone fruit variety, trigger the disease cycle. During fruit infection, M. laxa can overcome the need for wounds to infect and penetrate the plant cell. As a necrotrophic pathogen, M. laxa relies on the secretion of cell wall-degrading enzymes , such as pectin methyl esterases, and possibly phytotoxins, although these compounds have not been fully identified yet. After penetration, M. laxa colonizes the epidermis of the fruit with hyphae causing the collapse and disruption of cells, lysogenic cavities, and total degradation of the cuticle and epidermis, similar to the lesions caused by M. fructicola. Overall, fruit can be infected at any growth stage, but their susceptibility to brown rot increases with maturation, which results in a short post harvest life. Hence, the activation of immune responses alongside the physicochemical properties of the fruit may determine the pathogen’s ability to infect and spread.

Although these underlying mechanisms have not been fully elucidated, possible explanations could depend on changes in cell wall composition, volatiles, organic acids, and phenolic compounds. We hypothesize that M. laxa is able to adapt its infection strategies according to the nectarine developmental stage, resulting in either quiescent or disease progression, while the plant host can only establish effective defenses to restrict pathogen growth in fruit tissues that have not yet reached full maturity. Here, the fruit responses and pathogenicity mechanisms in the nectarine–M. laxa interaction were investigated as a function of the host developmental stage and time. Nectarine fruit was harvested at two different developmental stages and inoculated with M. laxa. Disease development and ethylene production were assessed for 3 days. Thanks to the recent availability of the M. laxa 8L genome, a comparative transcriptomics study was conducted on the nectarine–M. laxa pathosystem across four time points. This approach allowed us to identify not only host defense responses that were uniquely or highly induced in immature fruit during early infections,dutch buckets system which may partially explain why these tissues are resistant to brown rot, but also key strategies employed by the fungus to either become established in tissues or colonize them, which may be targeted to control brown rot.We visually assessed the development of brown rot over time at two maturity stages of nectarine . Quality parameters were measured and summarized in Supplementary Table S1. Overall, the disease progressed in mature tissues, while only surface discoloration was observed in immature tissues. At the mature stage, tissue maceration was observed on the surface of the fruit at 14 hpi followed by the pathogen penetration of the pericarp tissues between 14 and 24 hpi, and increasing lesion spread at 48 and 72 hpi. Fungal biomass was also estimated in both inoculated and control fruit to complement the visual assessments . Although no symptoms of brown rot disease were visible on the immature fruit surface at any time point, the M. laxa biomass increased from 6 to 14 hpi, when the highest quantity was detected, and then significantly decreased until 72 hpi. Although at early stages of infection , the fungal biomass was not significantly different between immature and mature tissues, it increased exponentially in the mature fruit at later time points, reaching levels approximately twenty times more than the maximum observed in immature fruit. In control tissues, a negligent quantity of the fungal biomass was detected across all time points in both stages. A dual RNA-Seq study revealed the dynamics of the fruit–pathogen interaction at early and late infection time points. The expression of 21,334 nectarine genes and 8364 M. laxa genes was detected across all developmental stages and time points .

The proportion of the total mapped reads for each sample that corresponded to M. laxa strongly correlated with the measurements of fungal biomass. Remarkably, more than 6000 genes were found to be expressed in inoculated immature fruit at 14 hpi and 24 hpi, indicating that the pathogen was active in these tissues but yet it could not cause disease. More genes were detected in mature fruit, increasing across time, from 6565 at 6 hpi up to 8287 at 48 hpi, reflecting the progression of pathogen growth and host tissue colonization.The principal component analyses revealed that in nectarine, PC1 and PC2 clearly separated the samples based on their developmental stage and infection status . Notably, at both development stages, 14 hpi was the time point when the inoculated samples appeared to experience a significant change in their expression profiles compared to the controls. These results demonstrate that early time points are critical for dictating the outcome of the interaction. For M. laxa, PC1 distinguished the samples based on the fruit developmental stage, while PC2 mainly divided the samples between early- and lateinoculation time points . In immature fruit, there was an evident switch in the pathogen’s transcriptional profile after 14 hpi, coinciding with the decrease in fungal biomass, and then continued to change up to 48 hpi. In mature fruit, M. laxa showed a change in gene expression between 6 and 14 hpi, when disease symptoms were first noticed on the fruit surface. Then, between 14 and 24 hpi, the pathogen altered its gene expression in mature fruit once again and retained most of these changes up to 48 hpi. Remarkably, the expression patterns of M. laxa at late time points of infection were highly divergent when infecting immature and mature tissues, suggesting that the pathogen utilizes different survival or infection mechanisms depending on the host developmental stage. A differential gene expression analysis was performed to determine the responses of immature and mature fruit to M. laxa, and to identify specific strategies used by the pathogen at specific times of infection. Nectarine DE genes  were identified in comparisons between inoculated and control fruit for each maturity stage and time point . A total of 4005 DEGs were detected in immature fruit across all time points, and of these the majority were upregulated in inoculated tissues. In immature fruit, the number of DEGs progressively increased over time and peaked at 24 hpi; then, the changes in gene expression appeared to reach a slightly lower plateau at 48 hpi. Mature fruit displayed a stronger transcriptional response to M. laxa infection since a total of 13,855 DEGs were detected at early and late time points. The number of DEGs in mature fruit continuously increased from 6 hpi to 48 hpi, indicating that the host tissues were undergoing a large transcriptional reprograming as the disease progressed. Monilinia laxa DEGs were detected by comparing the expression profiles of the fungus at each time point against 6 hpi for immature and mature fruit, respectively . These comparisons allowed us to depict how the pathogen modified its transcriptional response based on the initial time point of the interaction when gene expression profiles of M. laxa were similar between immature and mature fruit .To study host metabolic pathways altered during M. laxa progression, we performed a functional enrichment analysis for KEGG terms in the upregulated nectarine DEGs at each time point for immature and mature fruit . Figure 3a depicts KEGG terms that were significantly enriched in at least four out of the eight comparisons .

While many articles have attempted to describe the composition of complex bio-stimulants, these descriptions are frequently incomplete since the vast majority of biological molecules that would be present in crude extracts of complex origin, have not yet been characterized and the mere presence of a specific compound does not a priori demonstrate that compound is functional. The composition of most biologically derived bio-stimulant feed stock will also vary with the season of production, species, physiological state of the source organism and growth conditions. Indeed, there is an implication in the marketing of many bio-stimulants that stress conditions experienced by the plant or microbe utilized to produce the bio-stimulant, results in the production of stress metabolites and amino acids with consequent beneficial effects on plant response. In the absence of knowledge of the functional component of a bio-stimulant, changes in composition of a bio-stimulant over time and between batches and commercial sources cannot be interpreted. In the most rigorously prepared bio-stimulants from leading companies, high-throughput analytical methods have been employed to ensure consistent product quality . Methods such as chromatography, mass spectrometry, NMR spectroscopy, elemental analysis, ELISA, spectrophotometry, etc. are typically used for this purpose . The complexity of this challenge is illustrated in the analysis of a four-year algae composition sequence using a profile or finger print technique employing NMR .bio-stimulants have been used at all stages of agricultural production including as seed treatments,strawberry gutter system as foliar sprays during growth and on harvested products. The mode/mechanisms action of “bio-stimulants” is equally diverse and may include the activation of nitrogen metabolism or phosphorus release from soils, generic stimulation of soil microbial activity or stimulation of root growth and enhanced plant establishment.

Various bio-stimulants have been reported to stimulate plant growth by increasing plant metabolism, stimulating germination, enhancing photosynthesis, and increasing the absorption of nutrients from the soil thereby increasing plant productivity . bio-stimulants may also mitigate the negative effects of abiotic stress factors on plants and marked effects of bio-stimulants on the control of drought, heat, salinity, chilling, frost, oxidative, mechanical, and chemical stress, have been observed . Alleviation of abiotic stress is perhaps the most frequently cited benefit of bio-stimulant formulations. The following text describes the primary modes/mechanisms of action that have been demonstrated or claimed for bio-stimulants in the primary scientific literature.An example of a “mechanism of action” would be a stimulation of photosynthesis or the down regulation of a plant stress signaling pathway without an understanding of the explicit biochemical or molecular “mode of action.” For many bio-stimulant products, however, neither a specified mode of action, nor a known mechanism of action, has been identified. The presence of some spurious products in the marketplace compromises the market for all players resulting in the assumption by many, that bio-stimulants as a whole, are “snake oils” , a pejorative term implying the product is of no value. Multi-component bio-stimulants are particularly difficult to reconcile since they may have constituents for which the mode of action is known and components of no known functional benefit. Furthermore, multi-component bio-stimulants will frequently contain measureable but biologically irrelevant concentrations of known essential elements, amino acids, and plant hormones etc., for which the mode of action is known but the concentrations are irrelevant when used at recommended rates. Thus, for many of the multi-component bio-stimulant in the marketplace today, we propose that a demonstration of a clear “mechanism of action” is a more rationale and attainable regulatory goal than requiring an unequivocal demonstration of the “mode of action.”

Insight into the use of the terms “mode and mechanism” of action can be drawn from the pesticide science and pesticide development. In pesticide science, the “mechanism of action” describes the integral of all the biochemical events following application while the “mode of action” characterizes the main features of a bio-active molecule and its specific biochemical action leading to its effect in treated plants . In reference to plant bio-regulators, Halmann suggests that ideally an understanding of the mode of action of plant bio-regulators on the molecular level requires the identification of the receptor site for each regulator, as well as the elucidation of the subsequent reactions. In reality this standard is often not met in bio-pesticide or bio-stimulant products where the identification of the molecular targets of all bio-available compounds within a given extract cannot be easily achieved. The identification of the target binding sites of the natural bio-molecules has, however, proven to be helpful in the design of new insecticidal molecules with novel modes of action . At the present time, given the difficulty in determining a “mode of action” for a complex multi-component product such as a bio-stimulant, and recognizing the need for the market in bio-stimulants to attain legitimacy, we suggest that the focus of bio-stimulant research and validation should be upon determining the mechanism of action, without a requirement for the determination of a mode of action. This is the standard of practice for many pharmacological products. With the development of advanced analytical equipment, bio-informatics, systems biology and other fundamentally new methodologies a more complete understanding of the mechanisms and even possible modes of action of these materials may be achieved in the future. While this proposal suggests that the development and marketing of a bio-stimulant may not require a demonstration of the mode of action, it is still in the interest of the manufacturers of these products to pursue an understanding of the mode of action so that the product can be improved and the use can be optimized for various environments and cropping systems.This is primarily due to the heterogeneous nature of raw materials used for production and the complex mixtures of components contained in bio-stimulant products which makes it almost impossible to identify exactly the component responsible for biological activity and to determine the involved mode of action .

Therefore, focus should be upon the identification of the “mechanisms of action” of bio-stimulants as indicated by general positive impacts on plant productivity through enhancement in processes such as photosynthesis, senescence, modulation of phytohormones, uptake of nutrients and water, and activation of genes responsible for resistance to abiotic stresses and altered plant architecture and phenology . An example of this process is the advances in use of protein-based bio-stimulants for which recent studies have identified the target metabolic pathways and some of the mechanisms through which they exert their effects on plants . To further our understanding of modes/mechanisms of bio-stimulant action we have systematized the stages of bio-stimulants action on plants after their application: penetration into tissues, translocation and transformation in plants, gene expression, plant signaling and the regulation of hormonal status, metabolic processes and integrated whole plant effects. The penetration of amino acids and peptide based bio-stimulants into plant tissues has been investigated using radiolabeled amino acids and mathematical modeling . The components of a bio-stimulant preparation of animal origin,grow strawberry in containers labeled with 14C proline and glycine, were shown to penetrate rapidly into treated leaves and where subsequently distributed to other leaves . The mathematical model based on the “mechanism of diffusion” allows the estimation of the time required for the absorption of a minimal amount of the active component of a bio-stimulant. Furthermore, it describes the process of its transport from the moment of penetration into the leaf until the arrival at more distant tissues . The penetration of protein hydrolysates into a plant tissues occurs via diffusion of protein molecules through membrane pores and is energy-dependent . bio-stimulants must have a good solubility in water or other suitable solvents. This is a precondition for most types of application and for sufficient penetration of active ingredients into internal structures of treated plants. Surfactants and other additives may be required to overcome solubility and uptake limitations including lipophilicity and molecular size of active components . Ultimately a full understanding of the biological activity of complex bio-stimulant preparations will require a detailed understanding of the mechanism of action and effects on plant productivity and the identification of the biologically active molecules and their molecular mode of action . A wide array of molecular methods has been used to attempt to discern the active compounds found in bio-stimulants including microarrays, metabolomics, proteomic, and transcriptomics methods. These technologies have been applied to bio-stimulants to probe changes in gene expression following the application of bio-stimulants . Further research on the effects of complex bio-stimulants and their components on the complete genome/transcriptome of plants will be required to understand the mechanisms of action involved in growth responses and stress mitigation . The search for the mode of action of bio-stimulants is complicated by the observation that many bio-stimulants have been shown to induce genes and benefit productivity only when plants are challenged by abiotic and biotic stress. Experimental methods must therefore be developed to produce relevant and reproducible stress conditions so that the application of any molecular tool to probe gene function produces results that are relevant to the purported effects on plant productivity. The role of signaling molecules in plant response to environmental cues has been an area of active research in plant biology. The process of signal transmission involves the synthesis of signaling molecules , their translocation, their binding to receptors, the resulting cellular responses, and, finally, the degradation of the signaling molecules . When the signaling molecule binds to its receptor, the initial cellular response is the activation of secondary messengers, or intracellular signaling mediators, which cause a further series of cellular responses. Among the substances that may act as secondary messengers are: lipids, sugars, ions, nucleotides, gases, Ca2+, cAMP, cGMP, cyclic ADP-ribose, small GTPase, 1,2-diacylglycerol, inositol-1,4,5- triphosphate, nitric oxide, phosphoinosides, and others . Generally, a membranemediated action is typical for water-soluble compounds, while cytosol-mediated activity is primarily triggered by lipophilic compounds.

Whereas, enzymes interact with their substrates in a geometrical way , signaling molecules are thought to have a topochemical affinity to their receptors. It is assumed that the interaction of such components at the receptor site is cooperative and quantized . The bioactive compounds in some bio-stimulants are also proposed to display signaling activity in plants or induce signaling pathways. Various amino acids , and peptides function as signaling molecules in the regulation of plant growth and development . Peptide signaling is important in various aspects of plant development and growth regulation including meristem organization, leaf morphogenesis, and defense responses to biotic and abiotic stress . specific signaling peptides contained in a plant-derived protein hydrolysate have been shown to affect plant growth and development, defense responses, callus growth, meristem organization, root growth, leaf-shape regulation, and nodule development . Protein hydrolysates from soybean and casein have been shown to act as elicitors to enhance grapevine immunity against Plasmopara viticola . Proteins may also contain hidden peptide sites, “cryptides” or “crypteins” in their amino acid sequence, which may have their own biological activities, distinct from its precursor . Evidence that cryptides can trigger plants defense reactions have recently been demonstrated and there are reports of the isolation of cryptides by hydrolysis of proteins from marine organisms, including seaweeds, and cryptides may be present naturally in a variety of biological derived products .Exogenous amino acids may affect biological processes by acting directly as signal molecules or by influencing hormone action via amino acid conjugation . It has been suggested that amino acid based bio-stimulants are readily absorbed and translocated by plant tissues and once absorbed, they have the capacity to function as compatible osmolytes, transport regulators, signaling molecules, modulators of stomatal opening, and may detoxify heavy metals among other benefits . Sugars and fatty acids and plant lipids are also known to act as signaling molecules and mitigators of stress response in plants . Animal based lipid soluble fractions, have also been observed to produce an auxin-like response , while sugars, sucrose, and its cleavage products , are also known to act as signaling molecules through regulation of gene expression and by interaction with other hormone signals including auxins. In a sunflower meal hydrolyzate, amino acids, humic substances, microelements, and sugars present in the bio-stimulant appeared to coordinate, with auxin-like compounds in complex signaling cross-talk promoting plant growth, enhancing plant transplanting success and increasing final crop yield .

Accordingly, 22–222 tons of mAb would be needed per year, just in the United States. The population of the United States represents ~4.25% of the world’s population, suggesting that 500–5,200 tons of mAb wouldbe needed to meet global demand. The combined capacity of mammalian cell bioreactors is ~6 million liters27, and even assuming mAb titers of 2.2 g L−1, which is the mean titer for well-optimized large scale commercial bioreactors , a 13-day fed-batch culture cycle , and a 30% loss in downstream recovery, the entirety of global mammalian cell bioreactor capacity could only provide ~259 tons of mAb per year. In other words, if the mammalian cell bioreactors all over the world were repurposed for COVID-19 mAb production, it would be enough to provide treatments for 50% of the global population if low doses were effective but only 5% if high doses were required. This illustrates the importance of identifying mAbs that are effective at the lowest dose possible, production systems that can achieve high titers and efficient downstream recovery, and the need for additional production platforms that can be mobilized quickly and that do not rely on bioreactor capacity. Furthermore, it is not clear how much of the existing bioreactor capacity can be repurposed quickly to satisfy pandemic needs, considering that ~78% of that capacity is dedicated to in-house products, many to treat cancer and other life-threatening diseases . The demand-on-capacity for vaccines will fare better, given the amount of protein per dose is 1 × 104 to 1 × 106 times lower than a therapeutic mAb. Even so, most of the global population may need to be vaccinated against SARS-CoV-2 over the next 2–3 years to eradicate the disease, and it is unclear whether sufficient quantities of vaccine can be made available,flood tray even if using adjuvants to reduce immunogen dose levels and/or the number of administrations required to induce protection.

Even if an effective vaccine or therapeutic is identified, it may be challenging to manufacture and distribute this product at the scale required to immunize or treat most of the world’s population . In addition, booster immunizations, viral antigen drift necessitating immunogen revision/optimization, adjuvant availability, and standard losses during storage, transport, and deployment may still make it difficult to close the supply gap. Regardless of the product, the supply of recombinant proteins is challenging during emergency situations due to the simultaneous requirements for rapid manufacturing and extremely high numbers of doses. The realities we must address include: the projected demand exceeds the entire manufacturing capacity of today’s pharmaceutical industry ; there is a shortage of delivery devices and the means to fill them; there is insufficient lyophilization capacity to produce dry powder for distribution; and distribution, including transportation and vaccination itself, will be problematic on such a large scale without radical changes in the public health systems of most countries. Vaccines developed by a given country will almost certainly be distributed within that country and to its allies/neighbors first and, thereafter, to countries willing to pay for priority. One solution to the product access challenge is to decentralize the production of countermeasures, and in fact one of the advantages of plant-based manufacturing is that it decouples developing countries from their reliance on the pharmaceutical infrastructure. Hence, local production facilities could be set up based on greenhouses linked to portable clean rooms housing disposable DSP equipment. In this scenario, the availability of multiple technology platforms, including plant-based production, can only be beneficial.Several approaches can be used to manage potential IP conflicts in public health emergencies that require the rapid production of urgently needed products. Licensing of key IP to ensure freedom to operate is preferred because such agreements are cooperative rather than competitive.

Likewise, cooperative agreements to jointly develop products with mutually beneficial exit points offer another avenue for productive exploitation. These arrangements allow collaborating institutions to work toward a greater good. Licensing has been practiced in past emergencies when PMP products were developed and produced using technologies owned by multiple parties. In the authors’ experience, the ZMapp cocktail was subject to IP ownership by multiple parties covering the compositions, the gene expression system, manufacturing process technology/know how, and product end-use. Stakeholders included the Public Health Agency of Canada’s National Microbiology Laboratory, the United States Army Medical Research Institute of Infectious Diseases , Mapp Biopharmaceutical, Icon Genetics, and Kentucky Bio-processing, among others. Kentucky Bio-processing is also involved in a more recent collaboration to develop a SARS-CoV-2 vaccine candidate, aiming to produce 1–3 million doses of the antigen, with other stakeholders invited to take on the tasks of large scale antigen conjugation to the viral delivery vector, product fill, and clinical development.25 Collaboration and pooling of resources and kno whow among big pharma/biopharma companies raises concerns over antitrust violations, which could lead to price fixing and other unfair business practices. With assistance from the United States Department of Justice , this hurdle has been temporarily overcome by permitting several biopharma companies to share knowhow around manufacturing facilities and other information that could accelerate the manufacturing of COVID-19 mAb products.26 Genentech , Amgen, AstraZeneca, Eli Lilly, GlaxoSmithKline, and AbCellera Biologics will share information about manufacturing facilities, capacity, raw materials, and supplies in order to accelerate the production of mAbs even before the products gain regulatory approval. This is driven by the realization that none of these companies can saThisfy more than a small fraction of projected demands by acting alone. Under the terms imposed by the DOJ, the companies are not allowed to exchange information about 25Press release April 1, 2020.

Yet another approach is a government-led strategy in which government bodies define a time-critical national security need that can only be addressed by sequestering critical technology controlled by the private sector. In the United States, for example, the Defense Production Act was first implemented in 1950 but has been reauthorized more than 50 times since then . Similar national security directives exist in Canada and the EU. In the United States, the Defense Production Act gives the executive branch substantial powers, allowing the president, largely through executive order, to direct private companies to prioritize orders from the federal government. The president is also empowered to “allocate materials, services, and facilities” for national defense purposes. The Defense Production Act has been implemented during the COVID-19 crisis to accelerate manufacturing and the provision of medical devices and personal protective equipment, as well as drug intermediates. Therefore, a two-tiered mechanism exists to create FTO and secure critical supplies: the first and more preferable involving cooperative licensing/cross-licensing agreements and manufacturing alliances, and alternatively , a second mechanism involving legislative directives.Many companies have modified their production processes to manufacture urgently-required products in response to COVID- 19, including distillers and perfume makers switching to sanitizing gels, textiles companies making medical gowns and face masks, and electronics companies making respirators.Although this involves some challenges,ebb and flow tray such as production safety and quality requirements, it is far easier than the production of APIs, where the strict regulations discussed earlier in this article must be followed. The development of a mammalian cell line achieving titers in the 5 g L−1 range often takes 10–12 months or at least 5–6 months during a pandemic . These titers can often be achieved for mAbs due to the similar properties of different mAb products and the standardized DSP unit operations , but the titers of other biologics are often lower due to product toxicity or the need for bespoke purification strategies. Even if developmental obstacles are overcome, pharmaceutical companies may not be able to switch rapidly to new products because existing capacity is devoted to the manufacture of other important biopharmaceuticals. The capacity of mammalian cell culture facilities currently exceeds market demand by ~30% . Furthermore, contract manufacturing organizations , which can respond most quickly to a demand for new products due to their flexible business model, control only ~19% of that capacity. From our experience, this CMO capacity is often booked in advance for several months if not years, and little is available for short-term campaigns. Furthermore, even if capacity is available, the staff and consumables must be available too. Finally, there is a substantial imbalance in the global distribution of mammalian cell culture capacity, favoring North America and Europe. This concentration is risky from a global response perspective because these regions were the most severely affected during the early and middle stages of the COVID-19 pandemic, and it is, therefore, possible that this capacity would become unusable following the outbreak of a more destructive virus. Patents covering several technologies related to transient expression in plants will end during or shortly after 2020, facilitating the broader commercial adoption of the technology.However, PMP production capacity is currently limited. There are less than five large scale PMP facilities in operation, and we estimate that these facilities could manufacture ~2,200 kg of product per year, assuming a combined annual biomass output of ~1,100 tons as well as similar recombinant protein production and DSP losses as for mammalian cells. Therefore, plant-based production certainly does currently not meet the anticipated demand for pandemic countermeasures. We have estimated a global demand of 500–5,200 tons per year for mAbs, depending on the dose, but only ~259 tons per year can be produced by using the current global capacity provided by mammalian cell bioreactors and plant-based systems currently represent less than 1% of the global production capacity of mammalian cell bioreactors.

Furthermore, the number of plant molecular farming companies decreased from 37 to 23 between 2005 and 2020, including many large industry players that would be most able to fund further technology development . Nevertheless, the current plant molecular farming landscape has three advantages in terms of a global first-line response compared to mammalian cells. First, almost two thirds of global production capacity is held by CMOs or hybrid companies , which can make their facilities available for production campaigns on short notice, as shown by their rapid response to COVID-19 allowing most to produce initial product batches by March 2020. In contrast, only ~20% of fermentation facilities are operated by CMOs . Second, despite the small number of plant molecular farming facilities, they are distributed around the globe with sites in the United States, Canada, United Kingdom, Germany, Japan, Korea, and South Africa, with more planned or under construction in Brazil and China . Finally, transient expression in plants is much faster than any other eukaryotic system with a comparable production scale, moving from gene to product within 20 days and allowing the production of up to 7,000 kg biomass per batch with product accumulation of up to 2 g kg−1 . Even if the time required for protein production in mammalian cells can be reduced to 6 months as recently proposed , Medicago has shown that transient expression in plants can achieve the same goals in less than 3 months . Therefore, the production of vaccines, therapeutics, and diagnostics in plants has the potential to function as a first line of defense against pandemics. Given the limited number and size of plant molecular farming facilities, we believe that the substantial investments currently being allocated to the building of bio-pharmaceutical production capacity should be shared with PMP production sites, allowing this technology to be developed as another strategy to improve our response to future pandemics.Nutrients, especially nitrogen and phosphorus , affect terrestrial ecosystem carbon cycling through their regulation of plant and soil microbial activity . Natural terrestrial ecosystems are often nitrogen and phosphorus limited , with a general consensus that temperate and boreal ecosystems are commonly N limited while tropical forests are phosphorus limited . In both cases, strong competition occurs between plants and microbes so that actual nutrient uptake by individual consumers is often less than their demand due to limited supply and uptake of a nutrient by one consumer suppresses the functioning of other consumers . Furthermore, as CO2 concentrations increase, nutrient competition between plants and microbes is expected to intensify. Because elevated CO2 concentrations fertilize plant carbon productivity, plants will require more soil nutrients to facilitate enhanced photosynthesis and for tissues construction . On the other hand, enhanced carbon assimilation dilutes tissues nutrient concentrations and lowers litter quality .

These results suggest that the ihpCP lines, with no evidence of infection, allow virus movement to the susceptible tissues .Therefore, we are currently performing a new assay grafting the susceptible nontransgenic and CP-line scions before CPsV infection, giving kinetic advantage to PTGS establishment.Leprosis is a complex pathosystem that involves an atypical virus, Citrus leprosis virus C , an unusual mite vector, Brevipalpus sp., and host plants belonging to at least 18 botanical families. CiLV-C causes only localized lesions and does not spread systemically within its hosts, originally thought to be restricted to Citrus sp. Most of what was accepted about leprosis until the early 2000’s was shown to be inaccurate. Exactly ten years ago the first sequences of Citrus leprosis virus Cwere obtained and used for the development of an RT-PCR-based method for the diagnosis of leprosis. Since then, much has been done by our and other research groups and more light has been shed onto the pathosystem. Data obtained during the last decade will be discussed in the conference; mainly on CiLV-C taxonomy , variability , serological detection , and biological and molecular relationships with other viruses, with its mite vector and with its natural and experimental hosts, now expanded to dozens of plant species. Ultrastructural studies made on viruliferous Brevipalpus phoenicisdetected presumed Citrus leprosis vírus Cparticles between membranes at the basal part of the caeca, dorsal podocephalic gland and adjacent cells. Their viral nature was confirmed by in situ immunogold labeling using an antibody against the p29 protein of CiLV-C,mobile grow rack the putative capsid gene. No evidence of viral replication was obtained and it was concluded that CiLV-C circulates but does not replicate in the mite.

A meticulous anatomical study was made parallel to this study, to map the sites where CiLV-C was present within the mite. However, how the vírus enters, circulates and exits the mite body is still an open question. For feeding, it is believed that B. phoenicis uses the stylet to perforate the epidermis to reach parenchymal cells below,and injects saliva for a predigestion of the cell content, when CiLV-C must be injected into the host cell. Then the stylet is withdrawn and the cell sap is sucked with the help of cell turgor and the pharyngeal pump, and is delivered through the esophagus to the ventriculus and midgut. This process is being monitored by an adaptation of the EPG . The graphs obtained are being interpreted and tentatively associated with different phases of the feeding process. Using common bean as indicator plant, a series of parameters .Citrus leprosis, transmitted by the tenuipalpid mite Brevipalpus phoenicis, is caused by Citrus leprosis virus C , the type member of the genus Cilevirus. The disease is responsible for losses of ~US$ 80 million every year to the Brazilian citrus industry due to the localized lesions it induces on leaves, stems and fruits, and to severe die back and fruit drop it can cause. The disease occurs in several countries in South and Central America and is spreading towards the north of the Continent, having reached Mexico. Preliminary, unpublished data on the variability of the putative movement protein and replicase-associated protein genes suggest very low genetic variability among isolates. Additionally, the only three complete genome sequences of CiLV-C available in the GenBank, two from Brazilian isolates and one from a Panamanian isolate of the virus, share sequence identities of 99%. Altogether, these data suggest low divergence amongst isolates. However, since Brazil is likely to be the center or origin of this virus, we decided to determine the variability of the p29 ORF of 22 CiLV-C isolates from nine Brazilian states and one isolate from Argentina. RT-PCR products were cloned, sequenced and compared among them and with the GenBank sequences of the virus. Overall, p29 sequence identity ranged from 98% to 100%, with the exception of one isolate from São José do Rio Preto, São Paulo State. The sequence identity of this isolate ranged between 85% and 86% when compared to the other 25 sequences available. This shows that, even though CiLV-C variability may be considered low, there are isolates with significantly higher variability infecting citrus in Brazil.

It is even possible that other species of cilevirus causing leprosis-like symptoms are present in the country, as recently reported in Colombia. The ecology of plant pathogens of perennial crops is impacted by the long-lived nature of their immobile hosts. Over time, host plants are subject to changes to the genotype structure of pathogen populations that may impact disease spread and management practices; examples include the local adaptation of more fit genotypes, or the introduction of novel genotypes from geographically distant areas via human movement of infected plant material or insect vectors. We studied the genotype mixture of Xylella fastidiosa populations causing disease in sweet orange and coffee crops in Brazil at multiple scales, using fast-evolving molecular markers . Results show that populations of this bacterial plant pathogen were regionally isolated, as were the hosts. Independently of host and geographic origin the data suggest that the populations evolved locally and were not the result of migration. At a smaller spatial scale , results suggest that X. fastidiosa isolates within plants originated from a shared common ancestor, indicating that despite the long-term exposure of trees to infection, infection occurred only once, even though the vector visited the plant multiple times. It is possible that systemically infected trees are less susceptible to new invasions by competing X. fastidiosa genotypes. In summary, new insights to the ecology of this economically important plant pathogen were obtained by sampling populations at different spatial scales and from two different hosts. Xylella fastidiosa is a plant pathogenic bacterium that causes diseases in many different crops. The mechanism of pathogenicity of this bacterium is associated with its capacity to colonize and form a biofilm in the xylem vessels of host plants. There is no method to control this pathogen in field. In this study, we investigated the inhibitory effect of N-Acetylcysteine , a cysteine analogue used mainly to treat human diseases, on X. fastidiosa under different experimental conditions. Concentrations of NAC over 1 mg/mL reduce bacterial adhesion to glass surfaces, biofilm formation and the amount of EPS. The minimal inhibitory concentration of NAC was 6 mg/mL. NAC was supplied to infected plants in hydroponics, by fertigation, and adsorbed to organic fertilizer . HPLC analysis indicated that plants absorbed NAC at concentrations of 0.48 and 2.4 mg/mL, but not at 6 mg/mL. Sweet orange plants with typical CVC symptoms and treated with NAC in hydroponic solutions showed clear symptom remission and reduced the bacterial population to less than 5% compared with untreated plants, as analyzed by quantitative PCR and bacterial isolation.

Fertigation and NAC-fertilizer experiments were done to simulate a condition closer to that normally used in the field. For both, significant symptom remission and reduced bacterial replication rate were observed. Using NAC-Fertilizer the lag for resurgence of symptoms on leaves after interruption of the treatment was increased to around eight months. This is the first report on the effect of NAC as an anti-bacterial agent against a phytopathogenic bacterium. The use of NAC in agriculture might be a new and sustainable strategy for controlling plant pathogen bacteria. A prophage is bacteriophage DNA integrated into a bacterial chromosome or existing as a plasmid inside the bacterial cell. It provides important biological traits of bacteria that may involve virulence,ebb and flow tables environmental adaptation, strain specification and genome evolution. Genome sequence analyses indicate that both Candidatus Liberibacter asiaticus and Spiroplasma citri harbor prophages. Ca. L. asiaticus is associated with citrus Huanglongbing and S. citri causes citrus stubborn disease . Ca. L. asiaticus is not culturable in vitro. S. citri is culturable but the process is highly challenging. For these reasons, knowledge of the biology of the two bacteria is very limited. To study the prophage diversity of Ca. L. asiaticus in China where HLB has been endemic for over 100 years, 12 consecutive open reading frames in a prophage of a Florida Ca. L. asiaticus isolate were selected and primers were synthesized. Using these primers with PCR, 150 Ca. L. asiaticus samples from southern China were examined. At least three prophage types were detected, indicating a possible rich pool of Ca. L. asiaticus prophage in southern China. Since S. citri was culturable, pure culture of nine S. citri strains were obtained and whole genome sequences were generated. Two prophage/phage genes were selected and their abundance in each of the 9 whole genome sequences was estimated. Copy number of one gene varied from 13 to 154. Copy number of the other gene varied from 11 to 56. These results suggest that in addition to the chromosomal form, prophages of S. citri may also exist in extrachromosomal forms. Overall, these studies demonstrate that prophages are important constituents of both Ca. L. asiaticus and S. citri. More information on prophages are needed for better understanding of the two fastidious prokaryotes important to world citrus industry.The impact of the COVID-19 pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 was foreshadowed by earlier epidemics of new or re-emerging diseases such as SARS , influenza , Middle East Respiratory Syndrome , Ebola , and Zika affecting localized regions . These events showed that novel and well-known viral diseases alike can pose a threat to global health. In 2014, an article published in Nature Medicine stated that the Ebola outbreak should have been “a wake-up call to the research and pharmaceutical communities, and to federal governments, of the continuing need to invest resources in the study and cure of emerging infectious diseases” . Recommendations and even new regulations have been implemented to reduce the risk of zoonotic viral infections , but the extent to which these recommendations are applied and enforced on a regional and, more importantly, local level remains unclear. Furthermore, most vaccine programs for SARS, MERS, and Zika are still awaiting the fulfillment of clinical trials, sometimes more than 5 years after their initiation, due to the lack of patients .

In light of this situation, and despite the call to action, the SARS-CoV-2 pandemic has resulted in nearly 20 million infections and more than 700,000 deaths at the time of writing based on the Johns Hopkins University Hospital global database.1 The economic impact of the pandemic is difficult to assess, but support programs are likely to cost more than €4 trillion in the United States and EU alone. Given the immense impact at both the personal and economic levels, this review considers how the plant-based production of recombinant proteins can contribute to a global response in such an emergency scenario. Several recent publications describe in broad terms how plant-made countermeasures against SARSCoV-2 can contribute to the global COVID-19 response . This review will focus primarily on process development, manufacturing considerations, and evolving regulations to identify gaps and research needs, as well as regulatory processes and/or infrastructure investments that can help to build a more resilient pandemic response system. We first highlight the technical capabilities of plants, such as the speed of transient expression, making them attractive as a first-line response to counter pandemics, and then we discuss the regulatory pathway for plant-made pharmaceuticals in more detail. Next, we briefly present the types of plant-derived proteins that are relevant for the prevention, treatment, or diagnosis of disease. This sets the stage for our assessment of the requirements in terms of production costs and capacity to mount a coherent response to a pandemic, given currently available infrastructure and the intellectual property landscape. We conclude by comparing plant-based expression with conventional cell culture and highlight where investments are needed to adequately respond to pandemic diseases in the future. Due to the quickly evolving information about the pandemic, our statements are supported in some instances by data obtained from web sites . Accordingly, the scientific reliability has to be treated with caution in these cases.The development of a protein-based vaccine, therapeutic, or diagnostic reagent for a novel disease requires the screening of numerous expression cassettes, for example, to identify suitable regulatory elements that achieve high levels of product accumulation, a sub-cellular compartment that ensures product integrity, as well as different product candidates to identify the most active and most amenable to manufacturing in plants .

Differences in personal success rate may be because of the amount of regular practice, critical evaluation to determine the cause of success or failure, sharp tools, and attention to the most minute of details.The practice of shoot-tip grafting to eliminate viroids and deleterious pathogens has been very beneficial to the citrus industry world-wide over recent decades. This not only allowed for the safe movement of new varieties around the world but also the use of quality-inducing root stocks like citranges. It has also extended the productive life of citrus orchards and in some cases improved the horticultural performance of cultivars and thereby economic value to farmers. However, the single minded goal of removing all possible pathogens from the plant material seems to have occasional deleterious effects on the horticultural performance of some cultivars. In practice, new citrus varieties are usually discovered via chance mutation in commercial orchards. Such varieties result from old clone material, usually containing one or more viruses/viroids, necessitating clean-up, as specified by relevant Plant Improvement Schemes prior to commercial release. However, the ever-improving science of virus detection and elimination makes the achievement of totally sterile plant material a reality. It is suggested that this influences the horticultural expression of such pure varieties. The authors will present information aimed at demonstrating that complete virus/viroid elimination may satisfy the regulatory hurdles for commercial release but also in some cases result in the loss of economic value. Financial return to the orchard is dependent on a few very basic aspects: early production of good yields of edible fruit with high pack-outs. Altering one or more of these characteristics may mean the difference between a winner and a loser. Beneficial and/or relatively non-harmful aspects of specific viroids/pathogens to horticultural value in fruit trees/horticulture will be outlined.

An argument will be put forward for the characterization of certain citrus viroids relative to their role in horticultural expression and the potential reintroduction of purified strains after the clean-up process to ensure maintenance of such economic value. This presentation aims at stimulating debate and is not intended to criticize or lay blame.There is a large diversity of viruses and viroids that infect citrus trees,stacking pots some of them well characterized and known, belonging to the genera Closterovirus, Ilarvirus, Capillovirus, Sadwavirus, Mandarivirus, Marafivirus, Spirovirus , Badnavirus and viroids belonging to the genera Apscaviroid, Cocadviroid, Hostuviroid and Pospiviroid. In addition, there are a large number of well-known graft transmissible diseases of unknown etiology, which suggests that there exist unknown viral agents infecting citrus. Although the diagnosis of known agents is straightforward by serological or molecular methods, the detection of viruses without prior knowledge remains a challenge. Currently, only biological indexing is able to address the detection of the majority of graft-transmissible diseases. However, the development of next generation sequencing technologies allows exceptional sequence data generation at a fraction of the cost of biological indexing, dramatically modifying the diagnostic landscape. Virus-derived small interfering RNAs from citrus trees showing different symptomatologies were analyzed by Illumina sequencing. De novo contigs generated by different bio-informatic software were analyzed against the Genbank database, using Blastn, Blastx and Tblastx. Those nucleotide sequences, which had some homology to viral sequences, were used along with all siRNAs to reconstruct the genomes of various viral agents through iterative mapping against contigs. Along with known viruses and viroids, new citrus Bunyavirus, Badnavirus and Luteoviridae species were successfully identified. NGS is a powerful technology that could greatly simplify the screening, routine diagnosis, detection and characterization of citrus pathogens, providing knowledge to generate new diagnostic tools and having the potential to rapidly replace biological indexing.Citrus stubborn disease , caused by the bacterial pathogen Spiroplasma citri, is of major concern for citrus production around the world. The development of a reliable virulence assay for S. citri is urgently needed, but has been proven to be extremely challenging. With the current project we investigated the mechanical inoculation of plants using an S. citri axenic culture. A secondary culture of S. citri isolate C189 was injected with a vaccination gun or slashed into the stems of citrus and periwinkle seedlings.

The progression of the infection was monitored using a newly developed TaqMan quantitative polymerase chain reaction assay targeting the S. citri spiralin gene, as well as an antibody ScCCPP1 detecting a secreted protein of S. citri . Our results indicated that S. citri enThered the phloem tissues and established infection in both citrus and periwinkle. Bacterial cells could be detected by qPCR in plant tissues from newly emerged tissues away from the original inoculation site 1-7 weeks post inoculation. The ScCCPP1 antibody verified the qPCR results by producing positive reactions for the same time frame. S. citri detection past 5-7 weeks post mechanical inoculation became erratic by qPCR while the ScCCPP1 antibody continued to produce positive results. These data indicate that S. citri may have failed to maintain a long term infection in these plants while S. citri secreted proteins were still present in the inoculated plants. This is the first record of successful or at least partially successful mechanical transmission of S. citri from axenic culture directly into host plants. Further improvement on the inoculation procedures to achieve long term infection and overcome the possible loss of S. citri pathogenicity due to in vitro subcultures is underway. Viroids are small, non-coding, non-encapsidated, single-stranded, covalently closed RNAs that replicate autonomously when inoculated in their plant hosts in which they may elicit diseases. Presently, more than thirty viroids have been described and classified into two families, Pospiviroidae and Avsunviroidae. In the case of citrus, the viroid journey began in 1934 with the report on the xyloporosis disorder of Palestine sweet lime followed by the reports on the cachexia disease of Orlando tangelo and the exocorThis disorder of trifoliate orange root stock in 1948. The etiological agents of these diseases, that were known to be graft-transmissible, were considered to be viruses until viroids were described as a new class of plant pathogens with the discovery of the Potato spindle tuber viroidand the Citrus exocorThis viroidin the early 1970s. CEVd was characterized after its transmission to gynura . However, since nucleic acid technologies were limited in the 1970s, ongoing studies relied on the use of experimental herbaceous hosts displaying viroid symptoms and yielding high viroid titres. This approach prevented the identification of other citrus viroids with narrower host ranges until the following decade. Following the adoption of ‘Etrog’ citron as an indicator for biological indexing of exocor.This, in the late 1970s, the range of mild to moderate and severe symptoms observed after graftinoculation with field isolates were erroneously considered as evidences for the existence of CEVd strains.

In the mid-1980s, and after the use of a double gel electrophoresis system and silver staining,grow lights four additional viroid-like RNAs with distinct electrophoretic mobilities were identified and assigned the Latin numerals I through IV. Citrus viroidI, -II, -III, and -IV were further characterized and shown to have distinct biological and molecular properties, thus considered individual viroid species separate from CEVd. During the following two decades there was much discussion regarding the names adopted for citrus viroids. Presently, after lengthy field characterization experiments and according to the criteria of the International Committee of Virus Taxonomy, the following has been accepted for the five citrus viroids characterized in the mid-1980s. All identified citrus viroids belong to genera of the Pospiviroidae family and share some structural and replication properties. CEVd is the causal agent of the exocorThis disease and its name refers to the exocorThis disease as originally described for the trifoliate root stock. CEVd belongs to the Pospiviroid genus . The available data shows that differences in virulence are host dependent and associated with certain nucleotide changes located in a specific region of the viroid RNA.CEVd is the largest among citrus viroids and has the unique property of spontaneously increasing in length its RNA genome by terminal repeats after prolonged infections on specific solanaceous hosts. Hop stunt viroidis the causal agent of the cachexia and xyloporosis disease and its name refers to the stunting effect induced in hops. HSVd belongs to the Hostuviroid genus and it has a wide host range. The HSVd variants identified in citrus have a size ranging from 296 to 301 nucleotides and only variants containing specific RNA sequences, also known as cachexia expression motif, cause cachexia disease on sensitive citrus such as mandarins and some of their hybrids. Citrus bark cracking viroidbelongs to the Cocadviroid genus and its name refers to the symptoms induced on trifoliate rootstock. CBCVd is the smallest of the known citrus viroids and is closely related to CEVd. Citrus bent leaf viroidand Citrus dwarfing viroidbelong to the Apscaviroid genus . The CBLVd name refers to the symptom induced in ‘Etrog’ citron whereas CDVd refers to the size reduction of citrus trees propagated on trifoliate rootstock. Two additional citrus viroids, also belonging to the Apscaviroid genus, have been identified since the 1980s. Citrus viroid Vinduces mild reactions in ‘Etrog citron’ however, synergism with other Apscaviroids results in enhanced citron symptoms. CVd-VI also induces mild reactions in ‘Etrog citron’ and has chimeric features related to CDVd, CEVd, and CBCVd. The effects of CVd-V and -VI on citrus under field conditions are still unknown. In the late 1990s, the term ‘transmissible small nuclear ribonucleic acid’ was introduced to describe well-characterised citrus viroid RNA species that do not induce distinct disease syndromes in most citrus hosts but rather act as regulatory genetic elements modifying tree performance to the benefit of the grower. Since then, the TsnRNA-Ia, -IIa, and -IIIb have been studied in lengthy replicated field trials providing inTheresting results for reduced tree height and canopy volume, enhanced fruit size and increased yield per canopy volume as well as achievement of high density plantings in the absence of any adverse effects in fruit quality or tree longevity. It is important to note however, that such results have been achieved only with specific scion/rootstock/TsnRNA combinations /trifoliate/TsnRNA-IIIb and clementine /Carrizo citrange /TsnRNA-Ia+IIa+IIIb.

TsnRNAs had no effect on various other scion root stock combinations /C. macrophylla and Oroblanco /Citremon or even on root stocks such as Carrizo citrange when used as seedlings. In the following decades, the advent of molecular biology provided a variety of new tools for viroid research and detection. Methods such as imprint and blot hybridization, reverse transcription and polymerase chain reaction followed by cloning and sequencing or single-strand conformation polymorphism and transient or transgenic expression of viroid RNA in planta, in combination with in vitro transcription and plant inoculation or protoplasts transfection and more recently deep sequencing and a real time quantitative PCR protocol for the universal detection of citrus viroids, have transformed our diagnostic capacity. Even though, ‘Etrog’ citron and sPAGE remain the golden standard for citrus viroid detection, since it can detect all viroid-like molecules regardless of available RNA sequence information, the need for the development of robust, quick, reliable and economical viroid detection methods is always current. Nowadays, the open trade agreements, the global movement of citrus germplasm and competition of citrus producers, in combination with the ever-changing quarantine regulations and the constant need for pathogen-tested citrus propagative materials, make the use of modern molecular technologies for citrus viroid detection a necessity. With continuing research much information has been generated regarding viroid replication , host processing , evolution and population structure , cell to cell and long distance movement , biologically active RNA secondary structures , and mechanisms involved in pathogenesis and symptom expression . However, a series of interesting and challenging practical and basic science questions remain open. Are viroids associated with the gummy bark disease of sweet orange? Are viroids associated with “Wood pitting Gum pocket- Gummy pitting” observed on trifoliate rootstock? Are viroids associated with the Kassala disease of grapefruit? Do modern molecular viroid detection methods need to replace bio-indexing? In the absence of true dwarfing citrus species and rootstocks and in the face of serious citrus production cost and disease challenges, is the use of TsnRNAs for dwarfing and high-density plantings feasible in commercial scale and ethical? In the absence of any viroid encoded proteins, are viroids using a novel process for the suppression of the gene silencing plant antiviral mechanism?

Overall, the distinctive domain features of the SBE3 predicted protein, and the implifications for functionality may complicate current views of SBE function, but these features may also provide an opportunity to deepen our mechanistic understanding of starch biosynthesis and regulation.Starch metabolism is tightly regulated by plants’ internal clock and the external day-night shifts, especially in photosynthetic organs where transitory starch turnover occurs on a daily basis. The transcriptional response of the SBE genes follows the circadian rhythm in photosynthetic, and, in some cases, storage tissues. Cis-elements related to circadian control and light responsiveness were universally present in all the horticultural SBEs examined . Hormones, such as abscisic acid , ethylene, salicylic acid , jasmonic acid , and sugar signals have been reported to regulate SBE activity in cereal and horticulture crops. In addition, transcription factors that belong to the WRKY, MYB, bZIP, AP2/EREBP families, may bind to their cognate cis-elements in the 5′ upstream regions of SBEs to activate or suppress transcription.However, information on the transcriptional regulations of SBE is fragmented, and putative hub genes or master regulators have not been identified . Systemwide surveys of cis-elements and TFs in combination with in vitro and in vivo experiments could shed light on, and unearth such regulatory networks.Te amylose-to-amylopectin ratio influences the textural, cooking, and nutritional properties of starchy foods, and the functionality of starch-derived biomaterials. Most of this structure-function analysis has been performed on starches isolated from cereals and tubers. However, the relative proportions, and molecular structure of amylose and amylopectin in unripe fruit may have unique properties that could have specialized applications distinct from these well-characterized starches. There may be additional markets for fruit starches if premature harvest occurs, or is desirable, due to climactic events. Starch,vertical rack system or the proportion of the amylose fraction of starch, is used as a common ripening biomarker for apple, banana , and pear. This marker relies on the ability of amylose to physically interact with iodide to form a triiodide blue-black complex.

Starch can also influence the quality of fruit juice. Although starch is degraded to sugars when fruit ripens, this conversion is not complete. Ripe fruit processed for juice therefore contains starch, which is treated with amylases for clarification. Further, the amylose content of the remanant starch in some fruit processed for juice, may alter juice viscosity.Prepackaged leafy greens are convenient and healthy, and are popular options for salads in western countries. Metabolism in this horticultural product can be considered over distinct phases in its lifecycle: pre- and postharvest. In developing spinach, the photosynthetic organ, i.e., the leaf, fixes carbon, and partitions a large portion ~20% to starch biosynthesis during the light period under lab conditions. Starch accumulates linearly across the daytime at an almost constant rate . During the night, the leaf starch is degraded into sugar, to maintain plant metabolism, resulting in an empty polysaccharide reserve before the next light period. In Arabidopsis, the expression of SBEs and the changes of amylopectin and amylose show a similar trend, but there is variation in when SBE transcripts peak. Although there is no information on SBE transcriptional levels in spinach during the diel, there may be some similarities with Arabidopsis because the pattern of leaf starch accumulation is comparable in spinach and Arabidopsis.Harvested green produce are stored in optimized packaging under limited light exposure conditions which restricts new energy and carbon input from photosynthesis. However, respiratory activity, which is the carbon skeleton generation process for cellular metabolites, although reduced, does not stop. In detached leaves, the starch can be broken down to glucose, and sugars become the main source of fuel for cellular metabolism and ATP generation in the early stage of respiration. In the late stage of the respiratory process, the depleted sugars will be replaced by proteins, lipids, and membranes, triggering leaf senescence and cell death. This results in undesirable produce quality and ultimately, in produce loss. Preharvest and post harvest starch content may determine post harvest energy reserves and influence the time span that buffers the onset of senescence, thus influencing shelf-life of harvested green leaves.

Correlations between leaf starch content and post harvest longevity have been found. For example, lettuce and red chard harvested at the end of the day, when leaf starch content was highest, had a longer extended shelf-life than organs harvested at other times of day. This may not be true of all varieties e.g., salad roquette. Starch also correlated with improved shelf-life quality after light exposure to detached leaves in vegetables such as Chinese kale and lettuce. The accessibility of sugars from the degraded starch may relate to leafy-green quality, and the upregulation of SBEs would convert amylose to the more catabolically available amylopectin, providing a more readily available source of sugar.Te amylose-to-amylopectin ratio in Arabidopsis influences flowering time and reproductive growth, key markers of development, and fitness. Whether starch molecular structure and composition influences the preharvest growth of leafy greens in a similar way, remains unknown, but it seems likely.Potato, sweet potato, and cassava are generally considered as high glycemic index foods because the starch in their storage organs is easily digested to sugars when consumed, leading to a rapid increase in blood sugar level. It is established that high GI food exacerbate metabolic disorders such as diabetes and obesity. In contrast, multidisciplinary experimental research shows that digestion-resistant starch could increase the healthful microbial communities of the gastrointestinal tract, reducing the occurrence of constipation, and lowering the risk of colon cancer. Altering potato starch composition is a viable way to increase ‘dietary fber’ content and to enhance colonic health. This can be achieved by either physical, chemical, or enzymatic modifications of purified starch, e.g., etherification, esterification, or by fine-tuning the activity of starch biosynthetic enzymes. Reduction or knockout of SBEs in a range of species have reliably led to an increase in the resistant starch content in various species including horticultural crops e.g., potato, sweet potato, and cassava. Interestingly, SBE2 is not the dominant isoform expressed in storage tubers and roots, but it exerts a major function in amylopectin synthesis. Very high levels of RS can be achieved by the combined suppression of SBE1 and SBE2, but with a yield penalty. The transcriptional profiles and functions of SBE3 are unclear in the developing tubers . In addition, potato tubers suffer from a post harvest disorder: cold-induced sweetening . Potato tubers are stored at low temperatures to extend shelf life and to meet year-round demand. However,nft growing system sugars accumulate from starch breakdown, a process referred to as CIS.

Although a problem for the potato industry, CIS could be a mechanism to allow tubers to cope with chilling stress. CIS negatively affects the quality of fried or baked potato products: reducing sugars react with free amino acids at high temperature cooking through the Maillard reaction, to form carcinogenic acrylamide. Changes in the enzymes involved in starch biosynthesis and degradation are involved in CIS. SBEs are actively expressed in CIS susceptible tubers, and in StVInvsilenced, CIS-resistant tubers, SBEs transcriptional level were suppressed. Naturally occurring high RS potato varieties, also, have less susceptibility to CIS.Therefore, evidence points to a positive association of SBE activity with CIS severity in some potato genotypes.Starch is a major component of the dry mass of fruits at commercial harvesting time. Starch is transiently synthesized and stored in unripe fruits with a peak just before ripening. Starch appears to be a critical feature of climacteric fruit metabolism, known for their bursts of respiratory activity and ethylene production upon ripening. Climacteric fruits contain more starch, and, more active starch biosynthesis than non-climacteric fruit after anthesis. In tomato, the functional genomics model for feshy climacteric fruit, starch fulfilled 40% of the carbon needed for respiratory processes based on a constraint-based flux model. Experimental evidence from post harvest metabolism also supports the model: tomato fruits stored post harvest under low or chilling temperatures undergo bursts of stress-related carbon dioxide and ethylene production when allowed to recover at room temperature, with an accompanying and corresponding decrease in starch reserves. A similar inverse relationship between starch content and respiratory activity was observed in ripening banana, ginger rhizomes sunberry, apple and durian. The relationship between The issue starch content and respiration may not be perfectly linear in all species, e.g., in stored ginger, starch showed a biphasic accumulation pattern as respiration progressed, a trend not seen in other The issues examined . Furthermore, the relationship between these variables may also differ among genotypes within a species. Apart from climacteric characteristics, after the onset of ripening, starch content plummets sharply accompanied by starch decomposition into soluble sugars, and the total soluble sugar content continues to rise proportionally . This dynamic metabolic process had been reported for both climacteric and non-climacteric species including tomato, apple, banana, plantain, mango, kiwifruit, pear, and strawberry. Adequate storage of the starch-derived soluble sugars, is essential to produce an acceptably favored horticultural produce of appropriate sweetness. Accompanying the starch-sugar dynamics, amylopectin-to-amylose ratio , also changes interactively . Te difference in the AP/AM ratio in fruit development is expected to influence the structure of starch and its degradability. In the ripening tomato, the rate of decrease of amylose was greater than that for amylopectin Tus, the AP/AM ratio increased dramatically during ripening, in concert with the increase in soluble sugar content and fruit color change from green to red. This phenomenon where the proportion of amylopectin increases relative to amylose, was also evident in ripening apple and banana. It is possible to speculate that of the available starch left during fruit ripening, the amylose, or longchained amylopectin was converted into amylopectin whose branch-like structure has a much higher susceptibility to enzyme attack, allowing the rapid process of starch degradation into soluble sugars and supply for respiration. However, this mechanism may not be universal for all fruit.

For example, the changes in AP/ AM ratio in kiwifruit are similar to those in developing potato tubers, where the ratio of AP/AM almost remains constant during tuber development. In ripening tomato fruit with sharp increases in AP/AM, up-regulation of SBEs transcriptional expression is expected. Among SBEs, the class 2 SBE has the major effect on altering starch compositions. Elevated expression of SBE2 transcripts does parallel the changes in the AP/AM in ripening tomato, apples, and banana. We propose that ultimately, this change in glucan structure indirectly contributes to favor, quality, and commodity value.Starch, in general, plays an essential role in balancing the plant’s carbon budget as a reserve of glucose that is tightly related to sucrose metabolism and sugar signaling pathways. Starch is considered as an integrative mediator throughout the plant life cycle, regulating plant vegetative growth, reproductive growth, maturation and senescence, and response to abiotic stresses. This comprehensive regulation is achieved by changes in the synthesis and degradation of starch to balance glucose levels, after developmental and environmental triggers in different organs. Transitory starch and its biosynthesis have been well studied in the model plant Arabidopsis, but little research has been conducted on post harvest leafy greens. Quality metrics such as shelf-life, favor, color, firmness, and texture are of consumers’ choice, and they are related to the limited pools of storage compounds in detached leaves, which cells rely on to maintain basic cellular activities. A hypothesized function for the starch in packaged leaves could be presented as such: starch may act as a buffer against sugar starvation, and protect against cellular autophagy, by serving as an alternative energy source. If the biosynthesis and degradation of starch could be adjusted in a controlled way, then the modulated release of sugars may influence the post harvest shelf-life in detached leafy greens . A continuous, paced supply of sugars may preserve vacuolar nutrients and water content, leaf cellular structure and integrity, and, thus extend the ‘best by’ post harvest date of the produce. Although the eco-physiological role of amylose is poorly understood in Arabidopsis, the AP/AM ratio may set a threshold for the optimum usage of starch. SBE action in leafy crops may differ from those in Arabidopsis given the dissimilar numbers of their isoforms and domain features . Modifying the quantity and quality of the starch in leafy greens such as spinach, lettuce, and watercress, by targeting starch biosynthetic enzymes, may provide evidence to its post harvest function in terms of produce longevity.

Automated control offers the ability for continuous imaging and manipulation of media conditions with high temporal resolution. One notable example of a microfluidic device for rhizosphere studies is the RootChip, which uses the micro-valves in a PDMS device to control the fluidics . The first study using the RootChip grew 8 Arabidopsis plants on a single device with micro-valves but by the second iteration, the throughput has been doubled indicating rapid technological advances in the field. In addition, all these studies demonstrated spatiotemporal imaging at single-cell resolution and dynamic control of the abiotic environments in the rhizosphere. Another microfluidics-specific application to rhizosphere study is to use the laminar flow to generate the spatially precise and distinct microenvironment to a section of the root as demonstrated by Meier et al. . A young Arabidopsis’ seedling was sandwiched and clamped between two layers of PDMS slabs with microchannel features to tightly control synthetic plant hormone flow with 10 to 800 µm resolution to the root tip area, enabling observations of root tissues’ response to the hormones. As many root bacteria produce auxin to stimulate the interactions with the root, this study showed the possible mechanism of microbiome inducing the interaction by stimulating root hair growth. Another application of laminar flow utilized the RootChip architecture by adding the two flanking input channels to generate two co-laminar flows in the root chamber,hydroponic farming subjecting a root to two different environmental conditions along the axial direction to study root cells adaptation to the microenvironment at a local level . These studies revealed locally asymmetrical growth and gene pattern regulations in Arabidopsis root in response to different environmental stimuli.

Microfluidic platforms have also been successfully employed to study the interactions between the root, microbiome and nematodes in real time . In the systems, additional vertical side channels are connected perpendicularly to the main microchannel to enable introduction of microorganisms and solutes to the roots in a spatially and temporally defined manner . A recent microfluidic design incorporated a nano-porous interface which confines the root in place while enabling metabolite sampling from different parts of the root . These studies demonstrated the potential of microfluidics in achieving spatiotemporal insights into the complex interaction networks in the rhizosphere. Despite several advantages of microfluidics in rhizosphere research as described above, some challenges remain. All the microfluidic applications grow plants in hydroponic systems where clear media is necessary for the imaging applications and packing solid substrates in the micro-channels is not trivial. The microscale of the channels limits the applications of these devices to young seedlings. Thus, interrogating the microscale interactions in bigger, more developed plants is not possible with current microfluidic channel configurations. In addition, technical challenges such as operating the micro-valves and microfabrication present a barrier to device design and construction for non-specialists. Fabricated ecosystems aim to capture critical aspects of ecosystem dynamics within highly controlled laboratory environments . They hold promise in accelerating the translation of lab-based studies to field applications and advance science from correlative and observational insights to mechanistic understanding. Pilot scale enclosed ecosystem chambers such as EcoPODs, EcoTrons and EcoCELLs have been developed for such a purpose . These state-of-the-art systems offer the ability to manipulate many parameters such as temperature, humidity, gas composition, etc., to mimic field conditions and are equipped with multiple analytical instruments to link below ground rhizosphere processes to above ground observations and vice versa .

Currently, however, accessibility to such systems is low as there are only several places in the world which can host such multifaceted facilities due to the requirement of significant financial investments. Switching back to lab-scale systems, a recent perspective paper calls for the need to standardize devices, microbiomes and laboratory techniques to create model ecosystems to enable elucidation of molecular mechanisms mediating observed plant-microbe interactions e.g., exudate driven bacterial recruitment . Toward this goal, open source 3D printable chambers, termed Ecosystem Fabrication devices, have been released with detailed protocols to provide controlled laboratory habitats aimed at promoting mechanistic studies of plant-microbe interactions . Similar to a rhizotron setup, these flow through systems are designed to provide clear visual access to the rhizosphere with flexibility of use with either soil or liquid substrates . Certainly, there are many limitations to these devices in that they are limited to relatively small plants and limit the 3D architecture of the root system. Still, an advantage with the EcoFAB is that its 3D printable nature allows for adaptations and modifications to be made and shared on public data platforms such as Github for ease of standardization across different labs and experiments . In fact, a recent multilab effort showed high reproducibility of root physiological and morphological traits in EcoFAB-grown Brachypodium distachyon plants . The development of comparable datasets through the use of standardized systems is crucial to advancing our understanding of complex rhizosphere interactions. Open science programs such as the EcoFAB foster a transparent and collaborative network in an increasingly multidisciplinary scientific community. Specialized plant chamber systems are necessary for nondestructive visualization of rhizosphere processes and interactions as all destructive sampling approaches tend to overestimate the rhizosphere extent by 3–5 times compared to those based on visualization techniques . Nonetheless, plants in such chambers are still grown in defined boundaries and suffer from inherent container impacts.

For instance, studies have pointed out that container design significantly influences root growth during early developmental stages and leaves lasting impacts on plant health and phenotype . The majority of the lab-based chambers are also centimeter scale and are unlikely to replicate exact field conditions in terms of soil structure, water distribution, redox potential or root zone temperatures . While comparisons between chamber-grown and pot-grown plants show similar outputs , studies comparing plants grown in confined spaces to those directly grown in the field are missing. A recent review mapped the gradient boundaries for different rhizosphere aspects and found that despite the dynamic nature of each trait, the rhizosphere size and shape exist in a quasi-stationary state due to the opposing directions of their formation processes . The generalized rhizosphere boundaries were deducted to be within 0.5–4 mm for most rhizosphere processes except for gases which exceeds > 4 mm and interestingly, they are independent of plant type, root type, age or soil . Bearing this in mind, our assessment of the different growth chambers revealed possible overestimation of rhizosphere ranges in some chamber set ups. For instance, the use of root-free soil pouches representing rhizosphere soil despite being cm-distance away from the rhizoplane. This prompts the need for careful evaluation of new growth chamber designs to ensure accurate simulation of natural rhizosphere conditions. To date,ebb flow tray many rhizosphere microbiome studies and growth chambers systems focus on the impact of plant developmental stage, genotype and soil type on microbial composition and function . On the other hand, predation as a driver in the rhizosphere microbiome remains understudied. For instance, protists are abundant in the soil and are active consumers of bacteria and fungi and play a role in nutrient cycling yet remain an overlooked part of the rhizosphere . Viruses are also pivotal in modulating host communities thereby affecting biogeochemical cycles but their influence in the rhizosphere is poorly studied . These predatorprey interactions in the rhizosphere deserve in-depth studies which can be facilitated by these specialized growth chambers. Another area worth investigating in the rhizosphere is in anaerobic microbial ecology. At microbially relevant scales, soils primarily exist as aggregates . Aggregation creates conditions different from bulk soil, particularly in terms of oxygen diffusion and water flow resulting in anoxic spaces within aggregates and influences the microbial community . The rhizosphere is also rich in a wide range of compounds which can serve as alternative electron acceptors such as nitrate, iron, sulfate and humic substances in the absence of oxygen . However, most anaerobic studies in the rhizosphere focus only on aqueous environments such as water-logged paddy soils despite biochemical and metatranscriptomic evidence pointing to the possibility of anaerobic respiration in the rhizosphere . To fully understand biogeochemical cycles in the rhizosphere, it is imperative to investigate rhizosphere processes in the microscale and to include localized redox conditions as one of the influencing parameters. Microfluidic platforms with its fast prototyping capabilities can be helpful in creating growth chambers designed to stimulate these redox changes. In the study of the rhizosphere microbiome, genetic manipulation strategies are foundational in deep characterization of microbial mechanisms and current manipulation techniques require axenic isolates. However, the uncultivability of a significant portion of soil microorganisms continues to hamper efforts in gaining mechanistic knowledge. Even for culturable isolates, the process of isolation introduces selective pressure and disturbance to the community with inevitable loss of information on spatial interactions. A recent innovation in gene editing technologies using CRISPR-cas systems demonstrated in situ editing of genetically tractable bacteria within a complex community .

Coupled with the use of transparent soil-like substrates , the application of such a technique for the editing of in situ rhizosphere microbiome while preserving spatial and temporal associations would indeed bring invaluable insights. Specialized growth chambers using 3D fabrication and microfluidic technologies are primed to facilitate such innovations. Finally, this review revealed that while similarities exist among the different growth chamber systems, many of these systems are bespoke. This makes it difficult to replicate experiments and determine reproducibility which are important cornerstones of scientific advancement. The complexity of rhizosphere interactions also warrant that computational models are essential to gain a better understanding of system level processes . However, predictive modeling requires data from standardized approaches to be comparable between experiments. Thus, future growth chamber systems and designs are encouraged to follow the open science framework to enable standardization to an extent, such as in the case of EcoFABs . New root phenotyping technological developments are needed to overcome the limitations of traditional destructive root investigation methods, such as soil coring or “shovelomics” . Mancuso and Atkinson et al. provide extensive reviews on the methodological advances on non-destructive root phenotyping, including Bioelectrical Impedance Analysis , planar optodes, geophysical methods, and vibrating probe techniques. These techniques aim to mitigate key limitations of traditional root phenotyping, especially addressing the need for a better and more convenient characterization of the finer roots and root functioning. Advances in non-invasive and in-situ approaches for monitoring of root growth and function over time are needed to gain insight into the mechanisms underlying root development and response to environmental stressors. Geophysical methods have been tested to non-destructively image roots in the field. Ground Penetrating Radar approaches have been used to detect coarse roots . Electrical Resistivity Tomography and ElectroMagnetic Induction approaches have been used to image and monitor soil resistivity changes associated with the Root Water Uptake . Recent studies explored the use of multi-frequency Electrical Impedance Tomography to take advantage of the root polarizable nature . Despite these advantages, geophysical methods to date share common limitations regarding root characterization. Geophysical methods developed to investigate geological media: in the case of roots they measure the root response as part of the soil response, see Fig. 1a for the ERT acquisition. Because of the natural soil heterogeneity and variability the resolution and signal characteristics of geophysical methods strongly depend on soil type and conditions. As such, interpretation of the root soil system response is non-unique, hindering the differentiation between roots of close plants and the extraction of specific information about root physiology from the electrical signals. Unlike geophysical methods, the BIA for root investigation developed to specifically target the impedance of plant tissues, limiting the influence of the growing medium. A practical consequence is that BIA involves the application of sensors into the plant to enhance the method sensitivity. BIA measures the electrical impedance response of roots at a single frequency or over a range of frequencies . The measured BIA responses have been used to estimate root characteristics, such as root absorbing area and root mass . A key assumption is that current travels and distributes throughout the root system before exiting to the soil , with no leakage of current into the soil in the proximal root position . It is only in the former case, that the BIA signal would be sensitive to root physiology.