Month: May 2023

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.

Rhizoboxes offer the advantage of localized sampling in soil using sorption media such as paper and membrane filters, compound specific ion exchange binding resin or micro-suction cups placed closed to root zones of inTherest to collect exudates . Moreover, in a rhizobox fitted at the bottom with a porous rootimpenetrable membrane, a root mat is allowed to be formed which is then further transferred onto a collection compartment . The collection compartment containing soil could then be cut into thin slices parallel to the membrane to represent differing distances from the rhizosphere . While this approach can be used to investigate exudate release and sorption under soil conditions, the root mat growth generalizes exudate production in terms of the whole root system and occludes spatial exudation patterns. In a hybrid set up by Oburger et al. , the rhizobox is transplanted to a second specialized rhizobox for continued vertical root growth. This specialized rhizobox consists of a nylon membrane close to the transparent side to restrict root growth into the soil except for root hairs . This creates a vertical flat root mat onto which localized exudate samples can be collected. A comparison of this novel set up to conventional collection methods showed that amino acid exudation rates were most varied among the different methods , further highlighting the need for specialized chambers. Nonetheless, successful implementation of these chambers is still limited to fast-growing plants which can form active root mats. The high density of root mats could also lead to unnatural root exudate levels and an overestimation of rhizosphere effects. In addition, care has to be given to the choice of membrane as selective sorption of certain root exudates onto the membrane may also occur . Free-living nematodes are ubiquitous in the soil. They are beneficial to the plants by playing a role in nutrient cycling and in defense against insects and microbial infections through signaling interactions with the roots .

Conversely, infections by parasitic nematodes in the roots increase the plant’s susceptibility to stress and other pathogenic bacteria, fungi,growing hydroponically and viruses creating major losses in crop productivity . With an impending rise in nematode infections due to climate change, understanding nematode behavior and interactions in the rhizosphere becomes important to develop appropriate biocontrol methods to ensure long term food security . Traditional nematode studies are performed in petri dishes with agar or culture media . However, these substrates do not accurately emulate the physical textures and heterogeneity of soil and create homogenous solute and temperature gradients which could impact nematode behavior and interactions with the roots . Indeed, nematode motility speed and dispersal decreased in substrates more closely mimicking sand . On the other hand, studying nematode behavior in the soil is a difficult endeavor as its near-transparent body and small size makes it almost indistinguishable from soil particles. Cross-sectioning and staining infected roots make it possible for nematode visualization but they are destructive and provide only static snapshots of cellular changes or nematode behavior during infections . On the other hand, microscopy rhizosphere chambers provide non-invasive detection and observation of nematode activity in the rhizosphere . The roots in these chambers grow between a glass slide and a nylon membrane . The membrane restricts movement of roots except root hairs into the soil while the transparent glass enables microscopy of the roots at high resolution . Coupled with fluorescently stained nematodes, microscopy rhizosphere chambers allowed for non-destructive in situ observations of nematode infection in its host species over the entire life of the parasite . Nonetheless, staining nematodes is an additional challenge as nematode cuticles are impermeable to stains . This can, however, be alleviated by using advanced imaging technologies which eliminates the need for staining.

A recent study demonstrated live screening of nematode-root interactions in a transparent soil-like substrate through the use of label-free light sheet imaging termed Biospeckle Selective Plane Illumination Microscopy coupled with Confocal Laser Scanning Microscopy . Using this set up, researchers were able to monitor roots for nematode activity at high resolution and suggest its possible use in rapid testing of chemical control agents against parasitic nematodes in soil-like conditions . Fungal communities in the rhizosphere are involved in the degradation of organic matter in the soil and subsequent nutrient turnover affecting plant health as well as the microbial community . Fungal biomass often reaches a third of total microbial biomass carbon and almost all terrestrial plants are able to form symbiotic associations with mycorrhizal fungi . The majority of these associations are with arbuscular mycorrhiza fungi  which penetrate into root cortex cells to form highly branched structures . The investment of photosynthetic carbon by plants to AMF is rewarded with increased nutrient availability made possible by the extended hyphal network in the soil. For instance, up to 90% of phosphorus uptake in plants can be contributed by symbiosis with AMF . AMF networks in the soil also influence water retention and soil aggregation further impacting plant growth . Moreover, next-generation sequencing technologies and advances in imaging techniques have greatly improved our knowledge on the taxonomical and functional properties of fungal communities in the rhizosphere . However, these methods are optimized for fine scale analysis and are not capable of assessing the foraging capabilities of hyphal networks which can span across centimeter to meter scales. Toward this end, several researchers have used compartment setups with physical barriers created by 20–37 µm nylon membranes which restrict movement of roots but not mycorrhizal fungi. This separation creates root-free and plant free soil compartments connected only by mycorrhizal fungi to examine the transport of various compounds across these compartments. Using this set up, the importance of mycorrhizal fungi in the flow of different elements such as carbon , nitrogen and phosphorus between plants, soil and microbes over centimeter distances have been validated.

Repeated disruption of the hyphal connections also led to a decreased resistance in plants to drought stress . The membranes can also be placed horizontally to create different depth gradients to investigate hyphal contributions to water uptake . In some studies, an additional 1.5–3 mm air gap is created between two membranes with a wire net to restrict solute movement between two chambers . A common feature of these set ups is the size-exclusion membranes which proved to be critical in distinguishing fungal hyphae processes in the rhizosphere soil. In addition to AMF interactions, a split root set up, which separates the roots of one plant into halves, can be introduced to investigate the systemic response of plants . In essence, the split-root system directs the growth of the roots to generally two different growth conditions and enables the investigation of whether a local stimuli have a local or global response which can be observed at the root or shoot level . Split-root systems are widely studied and have been adapted to rhizoboxes as well as to pots and tubes . In the rhizosphere, plants host a wide diversity of bacteria on the surface of the root as well as within roots in the vascular tissues . Due to its abundance and importance,ebb flow tray the bacterial community in the rhizosphere is perhaps the most widely studied among other microbial members in the rhizosphere ecosystem. While the study of endophytic bacteria requires inevitable destructive sampling due to its localization, several non-destructive approaches have been developed to study microbes inhabiting the rhizoplane. One of the most widely studied plant-microbe interactions in the rhizosphere is that of the symbiotic relationship between legumes and rhizobia . Once a potential nodule forming bacteria is isolated, it is often required to authenticate its nodule forming phenotype by inoculating on host plants. However, conventional methods such as the use of soil pouches do not allow long term incubation, while “Leonard jars,” consisting of two stacked glass jars forming the top soil layer and the bottom nutrient solution layer, can be expensive and time consuming . A recent study challenges this by describing the use of clear plastic CD cases as minirhizotrons with potential for use in phenotyping root traits such as legume formation, and demonstrated innovation that democratizes research opportunities in rhizosphere research . Other microbial interactions in the rhizosphere, however, may not result in visible changes to the root system and often rely on next-generation omics technologies. As such, physical separation of the rhizosphere from the bulk soil becomes paramount in elucidating changes to microbial community and interactions. One approach to this end is the use of nylon bags with differing pore sizes . The nylon bag restricts the movement of roots and the soil inside the bag is then regarded as the rhizosphere soil to compare against the surrounding root free bulk soil . Developing further on this concept, Wei et al. designed a specialized rhizobox that allowed repeated non-destructive sampling by adding individual nylon bags of root-free soil surrounding the root compartment which are then used as a proxy for the rhizosphere . These methods allowed easy distinction of the rhizosphere and the bulk soil but, we now know that the rhizosphere community is not only distinct from the bulk soil but also varies with type, part and age of the root, largely as a consequence of varying root exudation patterns . Studying this phenomenon in situ in the soil requires separation of desired roots from others without disturbance to plant growth or soil. To address this, researchers have used a modified rhizobox design with a side compartment to regulate root growth and quarantine specific roots from the main plant chamber . This additionally creates easy distinction between old and new roots and allows testing on specific quarantined roots despite plant age. A study using this set up showed specific microbial chemotaxis toward different exudates on an individual root whereas another showed spatial and temporal regulation of niche differentiation in microbial rhizosphere guilds .

Similar physical perturbations to regulate root growth in response to microbial stimuli have also been applied in the microscale and are explored in the next section. Our assessment of the major growth chambers showed that most of the systems applied share similarities in basic structural components such as in the use of two parallel sheets in rhizobox based devices. While these growth chambers brought many of the rhizosphere processes to light, limitations do exist. One limitation is with the scale of applicability. Most of these growth systems are mesoscale and can easily reproduce potscale studies but may not be easily translatable to interactions occurring at the microscale nor recapitulate processes occurring at field-relevant scale. The next section describes advances in technology resulting in a new wave of unique devices making use of microfluidic processes and fabricated ecosystems which are specifically made to investigate specific rhizosphere processes.A complex web of biochemical processes and interactions occur in microscale dimensions in the rhizosphere. Having the ability to interrogate and manipulate these microscale processes and environmental conditions with high spatiotemporal resolution will elucidate mechanistic understanding of the processes. Microfluidics has proven to be a powerful approach to minimize reagent usage and to automate the often-repetitive steps. The microscale of the channels also allows precise control of reproducible conditions utilizing the laminar flow and automated fluidic operations . In addition, the microfluidic devices are well integrated with conventional imaging techniques by using a glass slide or coverslip as a substrate bonded with polydimethylsiloxane . These characteristics, as well as the ability to rapidly prototype and reproducibly manufacture using soft lithography technique, have enabled new ways of interrogating and studying the rhizosphere environment in a reproducible manner.They have an opening port, sometimes with pipette tips inserted into the PDMS body where the seed of the seedling rests and a microchannel where the primary root grows into. The dimension of the channel depends on the type and age of the plant. For example, an Arabidopsis thaliana’s seedling is typically grown in a microfluidic device up to 10 days, with chamber dimension around 150 to 200 µm in height, whereas the Brachypodium distachyon seedling chamber is 1 mm in height due to its thicker roots . Media and/or inoculation of the microbiome is achieved through additional channels to the main chamber. The PDMS body with the channels is typically bonded on a 50 mm by 75 mm microscope slide, and is made to accommodate multiple plants to increase throughput.

The International Seed Testing Association has as one of its important objectives to develop and test methods used to quantify seed germination. Existing methods to test seed viability include X-ray analysis, tetrazolium staining, a cut test, in some cases the careful extraction and sterile culture of zygotic embryos under aseptic in vitro conditions, but germination tests are probably the most widely used. Each method has advantages and disadvantages and consequently no single method provides a definitive means of accurately quantifying seed viability. For example, while relatively new and non destructive the X-ray assessment of seeds only indicates whether seeds are structurally intact , not whether the seeds are actually alive so while the seeds can be placed back into storage following assessment the use of this test on its own does not provide any certainty as to whether any structurally sound seeds are specifically alive. Although the germination test is probably one of the most accurate ways to assess seed lots, it is labor intensive, time consuming , and it may dependent upon knowledge concerning the optimal germination conditions and dormancy breaking requirements of the species in question. In addition, a significant concern from a seed bank management perspective is the fact that germination tests are ‘‘irreversible’’, so if seeds are viable and therefore germinate, the genetic information that they represent will be lost if not grown to maturity which may not possible in large seed banks. This is a particular concern with critically endangered species, as seed stocks may be exceptionally rare and in some cases impossible to source again. With currently available seed viability tests being largely irreversible, generally destructive, and sensitive to subjective data interpretation,hydroponic grow systems there is a pressing need for development of non-destructive and quantitative methods to assess viability and germination of precious seed stocks.

Several studies have demonstrated the potential of reflectance based spectroscopy methods in studies of plant seeds, including detection of internal infestations by weevils in dry field peas, classification of near isogenic maize lines, ageing of cabbage seeds, classification of near isogenic maize lines, differentiation between black walnut shell and pulp, sorting of lettuce seeds , and viability of horticultural seeds. These spectroscopy studies are based on the fundamental assumption that reflectance data acquired from the seed coat provides indicative information about the quality/ germination of the given seed. The research objective is therefore to identify portions of the wavelength spectrum, in which seeds show a significant and measurable change in certain parts of the examined reflectance spectrum and associated that change in reflectance with certain traits, such as, germination . A wide range of classification methods have been used as part of using reflectance data to characterize seeds and food products; these classification methods include support vector machine, variogram analysis, partial least square analysis, and linear discriminant analysis. LDA is based on discriminant functions, which are linear combinations of features and with one function for each target class. For each observation, a discriminant score is calculated and the observation is assigned to the class for which the discriminant function generates the highest discriminant score. In this study, we used hyperspectral imaging data to determine the germination of seeds from three native Australian tree species [Acacia cowleana Tate , Banksia prionotes L.F. , and Corymbia calophylla K.D. Hill & L.A.S. Johnson ]. These species represent the Australian flora with many different species held in seed banks across Australia as well as internationally including a number of critically endangered taxa. Seeds were exposed to standardized rapid ageing conditions, and at each assessment point were subjected to germination testing and hyperspectral imaging. We hypothesized that there would be detectable difference in seed coat reflectance between germinating and nongerminating seeds, and that changes in reflectance profiles would be most pronounced in spectral bands near known pigment peaks involved in photosynthesis and/or near spectral bands used in published indices to predict chlorophyll or nitrogen content in leaves.

The potential benefits of developing accurate machine vision systems to automate non-destructive monitoring of seed germination are discussed in the context of management of seed banks, botanic gardens, and implementation of vegetation restoration programs.A push-broom hyperspectral camera was mounted 40 cm above the seeds, and hyperspectral images were acquired with the spatial resolution of 50 pixels per mm2 under artificial lighting . The main specifications of the hyperspectral camera were: Firewire interface , 12 bit digital output, 240 spectral bands from 392 to 889 nm by 640 pixels . The objective lens had a 35 mm focal length with a 7 field of view, and it was optimized for the near-infrared and visible near-infrared spectra. During hyperspectral image acquisition in the lab, RH was between 30% and 40% and temperature 19–22C. A piece of white Teflon was used for white calibration. reflectance value was referred to relative reflectance and compared to that obtained from white Teflon. Colored plastic cards were imaged at all hyperspectral imaging events, and average reflectance profiles from these cards were used to confirm high consistency of hyperspectral image acquisition conditions .Immediately after hyperspectral imaging at each sampling time, the sub-samples of seeds were transferred to Petri dishes with semi solidified water agar for germination testing. Each seed was sequentially placed onto the dish and individually labelled with a permanent marker . Petri dishes were sealed with plastic film, wrapped in aluminium foil and incubated in a 15C growth chamber and checked weekly for germination. For determination of the predicted time required for germination to decline to 50% probit analysis was performed in Genstat version 10.0 . Seeds were aged at 60  C rather than 45C as is more common in seed ageing experiments. P50 values were calculated using the original data then adjusted to 45C by multiplying 60C P50 values by 8.44 as described in previous studies.Pixels with R660 reflectance values outside the stipulated ranges were excluded before average reflectance profiles were generated for each seed. We obtained 200 average reflectance profiles from each species , and relationships between results from the germination tests and hyperspectral imaging data were tested using LDA classification.

To select an optimized subset of the 10 ‘‘best’’ spectral bands, we conducted a forward stepwise LDA based on all average reflectance profiles for each of the three seeds species, and only these 10 spectral bands were used. Although not presented in this study, we conducted additional classification analyses with both more or fewer spectral bands,rolling benches and negligible classification accuracy was gained by including more than 10 spectral bands, and significant classification accuracy was lost by reducing the number of spectral bands. Thus, using the ‘‘best’’ 10 spectral bands was considered an optimum for this particular application. classification accuracies of each LDA was based on independent validation, as the original 200 average reflectance profiles and germination test results from each seed species were randomly divided into 80% training data and 20% independent validation data, This random division of data into training and validation data was repeated five times, and we calculated the average classification accuracy from the five randomized divisions of each data set.The effect of experimental ageing showed that within 0– 10 days, germination of Acacia and Corymbia seeds was above 90%, but both species of seeds showed a considerable decrease in germination after 10–20 or 20–30 days of experimental ageing, respectively . From the onset of ageing, there was an exponential decline in germination of Banksia seeds, and none of the seeds germinated after 30 days of experimental ageing. Germination for all three species was below 10% after 30 days of ageing, with B. prionotes seeds showing the steepest decline in germination with a P50 of only 7.0 days . For comparison, the P50 values for A. cowleana and C. calophylla seeds were 19.3 and 22.9 days respectively. Adjusted P50 values for all three species were 163.3, 59.1 and 193.3 days for C. calophylla, A. cowleana and B. prionotes respectively. With 25 seeds and eight sampling events , we obtained the following numbers of germinating and non-germinating seeds: Acacia , Banksia , and Corymbia .Average reflectance profiles acquired after 5 , 20 , and 20–30 days ofstandardized rapid ageing were used to illustrate the difference in reflectance between germinating and non-germinating seeds on days with similar numbers of seeds in both categories. Average reflectance profiles from the seed coat of the three species showed similar pattern with relative reflectance values commencing around 0.10 in spectral bands near 400 nm and gradually increasing reflectance in spectral bands near 900 nm. Loss of germination caused a decrease in reflectance in Banksia and Corymbia seeds, while it caused an increase in reflectance in Acacia seeds. We wish to highlight that it was virtually impossible to distinguish germinating and non-germinating seeds on the basis of visual inspection.

This statement is confirmed by the fact that reflectance values across the visual part of the spectrum were very similar. Based on independent validation of LDA classifications, we found that germination of Acacia and Corymbia seeds could be classified with over 85% accuracy, while it was about 80% for Banksia seeds . Regarding Acacia and Banksia seeds, we obtained similar classification accuracies of germinating and non-germinating seeds, but for Corymbia the classification accuracy associated with non-germinating seeds was considerably higher than for germinating seeds. We examined the relationships between days of ageing and classification accuracies, and it was revealed that the classification accuracy of : Corymbia seeds was above 80% at all time points, the classification accuracy of Acacia seeds was above 90% in the beginning and end of the study period but was below 65% around the time point with 50% germination, and Banksia seeds was below 80% during the time period with a marked decline in germination but around 90% in the beginning and end of the study period. In other words, seeds sampled during the gradual decrease in germination, in the transition from germination to non-germination, were generally classified with lower accuracy.Despite growing inTherest in use of reflectance based methods to determine the germination of horticulture and agriculture seeds, we are unaware of any published studies involving the assessment of native seeds for conservation or vegetation restoration purposes. We demonstrated that the germination of seeds can be accurately classified on the basis of commonly used classification methods, such as, LDA. Thus, it may be possible to replace time consuming and destructive germination tests with non-destructive reflectance based technologies as part of improved management of seed banks. The germination of Acacia and Banksia seeds decreased from above 90% to below 20% in about 10 days of experimental ageing. The decline in germination of Corymbia seeds was less pronounced from over 90% to about 10% in 20 days. Numerous studies have demonstrated successful use of reflectance-based spectroscopy as part of studies into seed germination. Shetty et al. used near-infrared spectroscopy to classify viable/ non-viable cabbage and radish seeds of different sizes with classification accuracies exceeding 90%. Similarly, Ahn et al.used a combination of hyperspectral imaging and fluorescence lighting to discriminate between viable and non-viable Brassica seeds with over 90%accuracy. Ahn et al. used the Fourier transform near-infrared reflectance technology technique to classify viable/non-viable water melon [Citrullus lanatus ] seeds and obtained classification accuracies also exceeding 90%. Finally, Esteve et al. used near-infrared reflectance technology to detect heat and frost damage to corn and to differentiate viable and non-viable corn and soybean seeds. The authors concluded that only heat damage could be accurately predicted. The exact associations between seed coat reflectance and primary seed metabolism are not known, so it is not possible to provide much more than speculations about the importance of certain changes in seed coat reflectance. However, some important insight may be gained by using knowledge gathered from reflectance studies of plant leaves. For instance, it is known that important plant pigments have maximum peaks at particular wavelengths: chlorophyll a , and carotenoids . In addition, there is a wealth of simple two spectral band indices used to estimate chlorophyll content in leaves, including : R430/ R680, R672/R550, R710/R760, R750/R550. Finally, there is a large body of research into use of reflectance based methods to quantify nitrogen content in leaves, and these have recently been reviewed. An important study analyzed the correlation between reflectance in spectral bands between R447–R1752 nm and leaf N accumulation in rice and wheat. The authors found that leaf N accumulation was strongly correlated with reflectance at R660, R810, and R870 nm.

Eight of the nine markers showed highly significant association with die back resistance, consistent with the Tvr1 gene being located in this region. Although the threshold for declaring association significant was set at p < 0.001, most of the associations were significant at p ≤ 0.00001. The only exception was marker Cntg4252, where the most significant association reached only p = 0.0042. The low association between SNPs at this marker and die back resistance was somewhat unexpected, since Cntg4252 co-segregated with the resistance allele in the × Salinas mapping population. While unexpected, it is not uncommon that markers closely linked with a trait in a mapping population do not show association when tested on a set of diverse accessions. This problem is well documented in potato, where markers linked to the Gro1 and H1 resistance genes in the mapping population were tested on 136 unrelated cultivars. The Gro1-specific marker was not correlated with the resistance phenotype, while H1-specific marker was indicative of resistance in only four cultivars. A similar example can be shown for lettuce, where markers most tightly linked to the cor resistance gene were the least useful for diagnostic when tested in a large collection of cultivars. There are several other examples of markers tightly linked to resistance genes, but whose use present problems in material different from the original in which they were identified.Therefore, an important requirement for any molecular marker used in MAS is not just its applicability in a specific cross, but its association in a wide gene pool. From SNPs that were significantly associated with die back resistance, the best fit was observed for those located in marker Cntg10192. This is the second of two markers, the other being Cntg4252, that co-segregated with the resistance allele in the mapping population. It is intriguing that one of the two markers co-segregating with the Tvr1 allele in the mapping population showed no significant association in a set of diverse accessions,30 litre plant pots bulk while the other showed a perfect match. Although these two markers were not separated in the intraspecific population, the linkage map developed from the Salinas × UC96US23 cross indicates that they are 1 cM apart.

Therefore it is possible that testing more RILs from the intraspecific population would separate the two markers and Tvr1. Association of SNPs from marker Cntg10192 with the resistance allele was validated in a larger set of 132 diverse accessions from several horticultural types. The marker-trait association was observed not only in L. sativa, but also in two L. serriola accessions included in the study. However, while the susceptible haplotype is identical in both species , the resistant haplotypes are different . To investigate the relationship between Tvr1 and the resistance observed in L. serriola, we screened 119 F8 RILs from the Salinas × UC96US23 population for resistance to die back. If Tvr1 and the resistance locus from UC96US23 were distinct and unlinked, approximately 25% susceptible offspring would be observed. However, since all RILs were resistant to the disease , we concluded that the resistance locus in UC96US23 is either allelic or linked to Tvr1. The same conclusion was reached for the resistance locus in the primitive romaine-type accession PI491224. The three resistance loci are associated with three distinct haplotypes; resistance in cv. Salinas with R1, in PI491224 with R2, and in UC96US23 with R3. Even though all 200 L. sativa accessions from the two testing sets showed the same haplotype-resistance association, it is unlikely that the EST from which this marker was derived is directly involved in die back resistance. A search for protein similarity in the NCBI database indicates that Cntg10192 is similar to the copper ion binding protein from castorbean and the plastocyanin-like domain-containing protein from Arabidopsis . The annotated functions of these two proteins do not imply an obvious role in plant-pathogen interactions.Moreover, the two substitutions at marker Cntg10192 that are the most significantly associated with die back resistance are synonymous, coding the same amino acid. Assuming that marker Cntg10192 is not directly involved in the resistance, it is probable that a recombinant genotype will eventually be identified. On the other hand, marker-trait associations can be very strong between some tightly linked alleles. For example, Rick and Forbes [51] documented linkage between allozyme Aps1 and tomato resistance gene Mi that did not break in as many as 30 backcross generations. Chromosomal linkage group 2 contains a large cluster of resistance genes that confer resistance to downy mildew and lettuce root aphid.

However, the Cntg10192 marker is well separated from thiscluster on the Salinas × UC96US12 map. Moreover, Tvr1 is one of the few resistance genes that was not at a genetic position coincident with any type of candidate resistance gene so far mapped in lettuce. Thus, it is possible that Tvr1 is different from the common types of pathogen recognition genes.We used HRM to directly detect sequence variations in PCR amplicons. High-resolution melting curves were recorded by the slow and steady heating of PCR products in a Light Scanner instrument.The method worked well for most of the analyzed markers, however, in a few cases, alleles could not be distinguished. When this occurred, we applied two alternative approaches to increase sensitivity through heteroduplex formation. In one approach, the heteroduplex formation was facilitated through mixing of samples prior to PCR. For example, if one sample contained DNA from cv. Salinas only, the other one would contain a mix of DNA from both cv. Salinas and Valmaine. The second alternative used an unlabeled probe 20 bp to 35 bp long that was designed for the region carrying the SNP. The probe was included in the PCR mix prior to cycling but was not consumed during amplification due to 3′ block. Genotyping was accomplished by monitoring the melting of probe-target duplexes post-PCR as described in Light Scanner manual. Both of the above alternatives improved allele detection; however, the probe-target duplex approach appeared to be more sensitive.Emerging plant diseases represent a continuous threat to economically important crop plants. Pandemics that affect supplies of grains and other annual subsistence crops are of particular concern . Diseases of perennial fruit trees are often very important for producers, due to the high costs of establishing intensively cultivated orchards and the extended amount of time needed to recover from outbreaks of diseases for which no effective chemical controls are available. For annual crops, a sensitive genotype can be replaced with disease-resistant varieties within a relatively short period of time. Diseases affecting fruit trees and vines may cause considerable losses through the gradual accumulation of newly affected trees over a number of years. This review describes how an important disease emerged, probably not from ingress of a new pathogen, but more likely from changes in cultural practices. It brings together information from a range of literary sources on the historical background of the emergence of xyloporosis and the linkage of this epidemic event with the history of citrus cultivation and with the natural history of Hop stunt viroid and other citrus viroids endemic to the Near East and the Mediterranean region.

Reichert and Perlberger reported on a “new” disease, xyloporosis, which appeared in 1928 among many of the newly planted citrus groves of Shamouti sweet orange trees that had been grafted onto Palestinian sweet lime , the most commonly used root stock in the British mandate-ruled area of Palestine. These authors described 3 essential phases of xyloporosis. First,wholesale plant containers small depressions appear on the stem bark of the root stock with small conoid pits with interfacing brownish pegs in the inner part of the bark. These symptoms often appear within 1 year of grafting and are most noticeable close to the bud union. In the second stage these symptoms intensify: The wood becomes discolored and, typically, the young tree becomes bent over and its leaves show symptoms typical of trees with root rot. In the third stage, there is blackish discoloration on the bark, the bark splits and leaves are small and yellow. Eventually, the branches wilt and die. Following intensive observations of a large number of infected trees of different ages in different planting areas, Reichert and Perlberger concluded that xyloporosis was present in all parts of the country and was not associated with any specific horticultural practice. Furthermore, although some differences were noticed among trees subject to different edaphic conditions, the disease could not be associated with any specific soil problem. The disease symptoms intensified as the trees aged. Seed source did not affect disease incidence. Pathological tests indicated that the disease was not associated with culturable fungal or bacterial pathogens. These observations led the authors to suggest 2 possible etiological causes of the disease: an unprecedented physiological disorder or an unknown viral pathogen. A major contribution to xyloporosis research was the observation of transmission by grafting of the cachexia disease affecting mandarins to a number of hosts, including a few that showed xyloporosis-like symptoms . These results suggested that the names of the 2 diseases, xyloporosis and cachexia, are synonymous and, based on the convention of prioritizing scientific names, the name cachexia was proposed for both diseases. Cachexia was found throughout most or all citrus-growing areas, particularly among Mediterranean varieties in Florida. Attempts by Norman and Childs to spread the cachexia disease via 5 different insect species failed and Olson showed that cachexia is not transmitted through seed. Later studies, reviewed by Bar-Joseph , showed that not only xyloporosis, but also exocortis and 3 other citrus viroids, were not seed transmitted to citron seedlings. The finding by Calavan and Christiansen that ‘Parsons Special’ mandarin showed more distinct symptoms than PSL supported the notion that the disease agents that cause xyloporosis and cachexia on different hosts are closely similar or identical. Recently, Langgut et al. reported the finding of typical citron pollen grains among the extracts from one layer of plaster, deposited to prevent leakage from an ancient irrigation pool belonging to a royal palace garden at present-day Kibbutz Ramat Rachel, near Jerusalem. Archeological evidence dated the construction of the facility to the Persian period and provides the first physical evidence for the earliest cultivation of citrus in the province of Judea. Initially, local growers refrained from grafting citron trees and were not familiar with the grafting technique .

The second edition of this book reported that some growers had started practicing grafting of citron on the PSL root stock, instead of propagation as seedling plants. Grafting was necessary because of the emergence of the destructive phytophthora gummosis disease, which rapidly decimated non-grafted trees and entire citrus industries throughout the Mediterranean basin . The dependence on grafting to save trees from diseases coincided with the selection of the Shamouti orange, which was obtained from a bud mutation of the local orange . Unlike the fruit of its seedy parent, the Shamouti fruit was almost seedless and these trees were propagated by grafting onto root stocks of an easily rooting citrus species, the PSL.Citrus cultivation expanded beyond the coastal plain , where the Shamouti orange on PSL root stocks was performing excellently, to new production areas just 10 km east of Jaffa, where all of the trees rapidly succumbed to the phytophthora gummosis disease. Phytophthora damage on PSL was initially blamed on the heavier soils of the new planting areas. However, when local citriculture expanded to sandy soils the root rot problems continued despite the improved edaphic conditions. It took almost 50 years for researchers and growers to realize that PSL root stocks made from cuttings were different from juvenile PSL seedlings, which were more sensitive to phytophthora root rot, and that the source of this difference was the absence in PSL seedlings of the viroid load commonly present in the root stocks that were made from cuttings. The positive effect of citrus viroid infection, the acquired resistance of phytophthora-sensitive root stocks induced by viroid infection, was first noticed by Rossetti et al. in Brazil. They noted that trees grafted onto Rangpur lime seedlings were succumbing to gummosis while those infected by the citrus exocortis viroid remained unaffected. Later studies using viroid-free and viroid infested buds of Shamouti grafted on PSL and also of other citrus stionic combinations confirmed this observation. Solel et al. showed that viroid infection also provides citron and Rangpur lime with tolerance to another serious fungal disease, mal secco . In retrospect, the change in susceptibility to phytophthora root rot could now be associated with the shift from the traditional practice of raising PSL root stocks from cuttings to producing root stocks from seed.

Only the smaller GFP variants moved beyond this zone . To add further complexity to protein trafficking and regulation, phosphorylation, and glycosylation are required for pumpkin CmPP16 to interact and form a stable complex with the mobility-endowing protein, Nt-NCAPP1, prior its phloem trafficking . Discrepancies in observed mobility from one study to another could be attributed to phosphorylation and glycosylation since earlier studies did not take these post-translational, covalent modifications into consideration. Two groups have demonstrated that non-endogenous proteins are retained in the root stock. The Gastrodia antifungal protein expressed by transgenic plum root stocks under the control of the constitutive CaMV35S promoter was identified in roots by immunoblot, but not in the soft shoot or leaf tissues of grafted, WT scions. This suggested that GAFP-1 was not moving into the WT-scion tissues of transgrafted plum trees . In the other example, transgenic watermelon root stocks over-expressing a cucumber mottle mosaic virus coat protein gene were transgrafted with WT watermelon. Protein expression and mRNA levels were detected in the transgenic root stock but not in the non-transgenic scion . Detection limits of the techniques utilized were not reported in either of these studies. A poke weed antiviral protein was expressed in transgenic Nicotiana tabacum root stocks and provided resistance to potato virus X in NN and nn grafted non-transgenic scions. However, the antiviral protein was detected only in the root stocks and not in the grafted scion tissues . The basis for resistance expression in this situation is not clear. Protein translocation from a transgenic root stock to a WTscion will likely depend on the species and/or type of protein in the transgene construct. Should proteins encoded by transgenes manage to migrate to the scion,plants in pots ideas their longevity is a consideration. For example, NPTII and GUS proteins have estimated half-lives of 6–7 min and 36 h, respectively, in planta .

If NPTII were translocated to scions it would be lost rapidly, but the GUS protein would not be reduced to 1% of the initial level accumulated in scions for 10 days. Research on the production of proteins encoded by transgenes in root stocks for delivery to scions arguably is more advanced than analogous work with the use of nucleic acids. For example, researchers at the University of Florida have engineered grape root stocks that deliver hybrid lytic peptides to control bacterial and fungal diseases .Work in our lab has shown that delivery of a protein that inhibits microbial maceration of plant cell walls is possible . While advances to date have focused on delivery of single gene products with specific functions to scions, future advances may target transport of transcription factors that influence expression of multiple genes,which could coordinate concerted scion responses to complex challenges such as pathogens, pests, or abiotic stresses.Proteins that are delivered to and function in the apoplast can provide protection against pathogens, particularly those pathogens that target the cell wall. In many plant–microbe or plant– pathogen interactions, the plant cell walls are a major obstacle to colonization or expansion within plant tissues. To overcome this barrier, most fungal pathogens produce a variety of enzymes, which degrade the host cell wall. Polygalacturonases  are often the first enzymes secreted during the infections . PGs cleave α- linkages between d-galacturosyl residues in pectic homogalacturonan, causing cell separation and tissue maceration. Botrytis cinerea expresses six PGs during infection and growth on plant hosts and the PG-inhibiting protein produced in pear fruit , inhibits some but not all of these PGs . Given the importance of PGs in pest and pathogen interactions with plants, it is not surprising that PGIPs are components of the defenses against invasion by pathogens and pests . Tomato foliar and ripe fruit resistance to the fungal pathogen, B. cinerea, is improved about 40% by the constitutive over-expression of pPGIP in tomatoes . The Miridae insect, Lygus hesperus, produces PGs that cause damage to alfalfa and cotton flflorets and PGIPs can inhibit these PGs and may, therefore, reduce the damage to plant tissues . The nematode, Meloidogyne incognitacausing root knot disease expresses PGs , but it is not known if they can be inhibited by PGIPs. PGIPs expressed in root stocks, therefore, are potential anti-pathogen proteins thatcould be delivered from the root stock to the scion in transgrafted plants. Our work has shown that pPGIP expression reduces the effects of Pierce’s Disease in grapevines, caused by the bacterium, Xylella fastidiosabecause it inhibits the X. fastidiosa virulence factor, PG .

As with other vascular pathogens, the X. fastidiosa PG contributes to disease development by digesting the polysaccharides in the pit membranes of the xylem network. When intact, these so-called “membranes” help to prevent the pathogen’s vessel-to-vessel spread from the initial sites of infection of grapevines . Because pPGIP inhibits the X. fastidiosa PG and because pPGIP can enter the xylem, PGIPs in the xylem of both the root stock and the scion could provide protection against other PG-utilizing pathogens in the water transport system. We have observed that when pPGIP-expressing transgenic plants are used as root stocks onto which non-expressing scions are grafted, the pPGIP protein, but not the pPGIP-encoding nucleic acids, are exported to the scion, crossing the graft union via the xylem system . In grafted tomato plants expressing pPGIP in the root stock, pPGIP protein has been detected in scion leaves . Similarly, in grafted grapevines, we have observed the pPGIP protein in the wild-type scion tissue grafted onto pPGIP-expressing root stocks . Furthermore, we have observed that expression of pPGIP in root stocks reduces pathogen damage in scion tissues . Thus, defense factors in roots can be made available to scions via grafting, improving the vigor, quality, and pathogen/pest resistance of the food-producing scion and its crop.DNA barcoding is an effective tool to identify many plant species rapidly and accurately. However, there is no single universal barcode that can be successfully used to identify all plants to the species level. Consequently, two alternative strategies have been proposed to distinguish among plant species: the first one is the use of complete chloroplast genomes, named ‘super-barcoding’, and the second one is an approach that involves searching for mutational hotspots, or using comparative plastid analyses to find loci with suitable species-level divergence. Analyses of entire chloroplast genome sequences provide an effective way to develop both of these strategies. In most angiosperms, the chloroplast genomes are inherited maternally and have a consistent structure, including two inverted repeats , one large and one small single copy region. Te chloroplast genome always contains 110–130 genes that exhibit a range of levels of polymorphism. Thus, chloroplast genome sequence data are extremely valuable for studies of plant population genetics, phylogeny reconstruction, species identification, and genome evolution.

The Ranunculaceae is a large family, which includes approximately 59 genera and 2500 species. Many plants of Ranunculaceae are pharmaceutically important. The genus Pulsatilla Adans. consists of about 40 species which are distributed in temperate subarctic and mountainous areas of the Northern Hemisphere. There are always long, soft hairs covering plants of Pulsatilla species. Most of the fowers of Pulsatilla are large and showy, and therefore the genus has horticultural importance. The fowers are solitary and bisexual. In one flower, there are always six tepals, numerous stamens and carpels, with the outermost stamens resembling degenerated petals, excluding P. kostyczewii. In China, there are eleven species of Pulsatilla. Some species of Pulsatilla have been used in traditional Chinese medicine for many years, such as for “detoxifcation” or “blood-cooling”, because Pulsatilla species contain numerous secondary metabolites, including phytosterols, triterpenoid saponins and anthocyanins. At the same time, all members of Pulsatilla produced the lactone protoanemonin. In Europe, some species of Pulsatilla are rare, endangered and endemic. Those taxa are protected due to their small populations and disappearing localities,container size for blueberries and those species have been placed on the Red Lists of Endangered Species. Taxonomically, Pulsatilla is an especially complex and challenging group. In all treatments published before, three subgenera have been recognized: subgenus Kostyczewianae , subgenus Preonanthus, and the largest subgenus Pulsatilla. However, the intragenic morphological variability of Pulsatilla was especially complicated. Te recognition and identifcation of wild Pulsatilla species is particularly difcult based on traditional approaches. Molecular markers are significant to explore the phylogenetic relationships of the genus Pulsatilla. Phylogenetic relationships between Pulsatilla and closely related genera have been dedicated during the past years. Previous studies have attempted to identify these species among Pulsatilla with universal molecular markers, but the species resolution was relatively low. In this study, we present seven complete cp genomes from two subgenera of Pulsatilla obtained through next-generation sequencing and genomic comparative analyses with four previously published cp genome sequences of Pulsatilla from NCBI, with Anemoclema glaucifolium as the out group. We identify microsatellites , larger repeat sequences, and highly variable regions, with the aim of developing DNA barcodes and testing the feasibility of phylogenetic analyses of Pulsatilla using the chloroplast genome.In most angiosperms, the IR regions of cp genomes of angiosperms are highly conserved, but the expansion and contraction of IR region boundaries are ever present. At the same time, several lineages of land plant chloroplast genomes show great structural rearrangement, even loss of IR regions or some gene families. The expansion and contraction in IRs are significant evolutionary events, because they can change gene content and chloroplast genome size. Expansion of the IRs has been reported in Araceae. Sometimes, the size of LSC increases and that of SSC decreases, becoming only 7000 bp in Pothos. At the same time, a linear chloroplast genome was also reported in some groups, e.g. maize. Expansion and contraction of the IR regions can also lead to duplication of certain genes or conversion of duplicate genes to single copy, respectively. Changes in the size of the IRs can also cause rearrangement of the genes in the SSC as recently observed in Zantedeschia. The Pulsatilla chloroplast genomes were compared to previously published data and showed typical Anemoneae genome structure. As reported for Anemoclema, Anemone, Clematis and Hepatica, the IR regions of genus Pulsatilla are roughly 4.4 kb longer than those of other genera of the family Ranunculaceae, such as Aconitum, Coptis, Talictrum, Megaleranthis, Ranunculus, and Trollius. The gene orders located within the IR-SSC and IR-LSC boundaries are similar among tribe Anemoneae but diferent from those of other genera of Ranunculaceae . We compared the IR/SC boundary regions of Pulsatilla, and the junction positions are very similar and conserved within genus Pulsatilla. In the four boundary regions of seven Pulsatilla cp genomes, the LSC/IRa and IRb/LSC border was in the intergenic region, and the adjacent genes is rps36, rps8 and rps4, respectively. The genes ycf1 andψycf1 have crossed the SSC/IRb and IRa/SSC boundary, respectively, which was also found in Monsteroideae. The pseudogene ycf1 has been found in other groups. The IR regions were highly conserved, with nucleotide diversity values in those regions less than 2%.Chloroplast genome markers, especially several universal chloroplast regions, have been widely used in plant systematics and identifcation at multiple taxonomic levels. Highly suitable polymorphic chloroplast loci have been identifed and designed as unique markers in diferent groups. However, relationships within the genus Pulsatilla have not been well resolved because of the lowpolymorphism of these universal markers. In order to facilitate identification of closely related species of Pulsatilla, we sought to identify highly variable regions of the chloroplast genome, as previously described. As a result, we identified nine divergent hot spot regions, including six intergenic spacer regions and four protein-coding regions. Most commonly employed loci, e.g. trnL-trnF, trnH-psbA were not selected in our finding. The nine highly variable regions included 684 variable sites, including 181 indels. However, these indels are not suitable for the phylogenetic inference because Maximum likelihood model used only substitutions not indels. Their nucleotide diversity values ranged from 0.00802 to 0.02212. The region of ccsA-ndhF showed the highest variability, the next most variable regions were rps4-rps16, ndhC-trnV, and psbE-petL. Te diversity level of two protein-coding regions was the lowest. Among the nine divergent hotspot regions, the ndhI is difcult to align. There are large numbers of indels in ndhI and the intergenic spacer between ndhI and ndhG, these regions were not considered suitable for the phylogenetic inference of the Pulsatilla. Thus, we selected eight regions, four in the LSC and four in the SSC, with relatively high variability as potential molecular markers for the study of species identification and phylogeny in Pulsatilla.