Month: May 2023

Hormones are of central importance for the regulation of metabolic processes and plant development in a complex system of interacting hormones and cofactors, the functions of which are closely intertwined and mutually dependent . bio-stimulants developed from humic substances, complex organic materials, seaweeds, antitranspirants, free amino acids , and crude extracts of lower and higher plants have been frequently demonstrated to have an effect on plant hormonal status . While hormone-like compounds may be present in bio-stimulants, it is also possible that de novo synthesis of hormones may be induced by such preparations in treated plants and amino acids, glycosides, polysaccharides and organic acids are contained in many bio-stimulants and may act as precursors or activators of endogenous plant hormones . Hormones or hormone-like effects could therefore be responsible for the action of natural bio-stimulants derived from microorganisms, algae, higher plants, animal, and humate based raw material . Information on currently available bio-stimulants gives some insight into the possible biochemical and molecular genetic effects of bio-stimulants derived from different natural raw materials . Many published reports are available suggesting various bio-stimulants improve plant productivity through increased assimilation of N, C, and S , improved photosynthesis,hydroponic nft channel improved stress responses, Thered senescence, and enhanced ion transport . bio-stimulants are also reported to increase free amino acids, protein, carbohydrates, phenolic compounds, pigment levels, and various enzymes .

The protective effect of many bio-stimulants against biotic and abiotic stresses has been associated with a reduction of stress-induced reactive oxygen species, activation of the antioxidant defense system of plants, or increased levels of phenolic compounds . While it is clear that many biologically derived bio-stimulants contain small molecular weight compounds that are involved in signaling events and may directly influence plant metabolic processes, it remains unclear how an exogenous soil or foliar application of an uncharacterized product can have predictable and beneficial responses in plants. It is well-known, for example, that application of exogenous plant hormones or compounds that disrupt hormone function can have markedly negative effects on plants and that optimization of PGR materials and their applicaitons requires precise information on dosage and timing. Application of bio-stimulants for which the dosage and efficacy of the functional compounds is unknown, cannot, therefore, be expected to result in predictable plant responses and identification of molecules with effects on plant metabolic processes is not, in of itself, a sufficient explanation for the function of a bio-stimulant. It is also uncertain why the application of a bio-stimulant with purported function as a PGR, signaling molecule or other discrete compound would be superior to, or more easily controlled, than a direct application of the purifified product itself. Modern crop production requires a balance of high and consistent productivity with maximum safety for consumers, agricultural workers, and the environment . While some bio-stimulants have been analyzed with regard to unwanted side effects including negative impact on the natural environment most bio-stimulants have not been fully characterized but have been regarded as generally recognized as safe on the basis of the biological origin of their constituents . Generally, bio-stimulants are assumed to be biodegradable, non-toxic, nonpolluting and non-hazardous to various organisms.

While this may be a rational conclusion for many formulations derived from biological materials such as seaweed extracts and their components , higher plants , chitin and chitosan it is not clear that this is a valid assumption for microbial products or products that would not normally be present in agricultural fields. bio-stimulants have been utilized as bio-remediants and have been shown to improve ATP levels and phosphatase and urease activity , and hence increase the rate of degradation of xenobiotics in the soil and to enhance beneficial soil microbial communities under semi-arid climates . bio-stimulants may also help reduce the amount of potentially risky agrochemicals including reducing the use of fertilizers and pesticides . Most compounds contained in bio-stimulants are natural constituents of terrestrial and aquatic ecosystems and metabolites of plant and microbial origin and as such most are generally regarded as safe, particularly at the low rates at which they are typically applied. Thus, it has been proposed that bio-stimulants can be positioned as eco-friendly products for sustainable agriculture . In many countries, however, bio-stimulants are not subject to rigorous toxicological screening and there remains the potential for the persistence of human pathogens in materials of animal origin and for the synthesis of novel compounds of unknown function or toxicology during the manufacturing process. Even though there have been relatively few rigorous demonstrations of the benefit of bio-stimulants, and to a large extent the mode of action of these products remains uncertain, the industry for bio-stimulants is substantial and rapidly growing. Though many recent “market” studies show that the market for these products is growing at a remarkable rate, the validity of these analyses must be considered with care as they frequently do not provide an explicit definition of term “bio-stimulants.” The value of the European bio-stimulants market ranged from e200 to e400 million in 2011, e500 million in 2013 and may grow to more than e800 million in 2018 with annual growth potential in 10% and more . France, Italy, Spain are the leading EU countries in the production of bio-stimulants .

In North America, the bio-stimulant market was valued at $0.27 billion in 20131 , and is expected to grow at a growth rate of 12.4% annually, to reach $0.69 billion by 2018, the USA is the largest producer and consumer of bio-stimulants in the region . In 2014, the USA market was assessed at $313.0 million and is projected to reach $605.1 million by 20192 , at a CAGR of 14.1% . The bio-stimulants market in the Asia-Pacific was valued at $0.25 billion in 2013, and is expected to grow at a CAGR of 12.9% annually, to reach $0.47 billion by 2018. China and India are key countries playing a significant role. The Southeast Asian & Australasian bio-stimulants market was valued at $233.8 million in 2015, and is projected to reach $451.8 million by 2021. The market in Latin America was valued at $0.16 billion in 2013, and is expected to grow at a CAGR of 14.4% annually, to reach $0.32 billion by 2018 . This market is mostly concentrated in Brazil and Argentina. The regional market shares of the global bio-stimulants market6 are: EU—41.7%, North America—21.5%, the Asia-Pacific region—20%, Latin America—12.9%. Globally, it bio-stimulants were valued at $1402.15 million in 2014 and are projected to have aCAGR of 12.5% reaching $2524.02 million by 2019, largely as a consequence of growing interest in organic products. Wu summised that “the global bio-stimulants market is projected to reach $2.91 billion by 2021, with a CAGR of 10.4% from 2016 to 2021. In terms of area of application, the bio-stimulants market is projected to reach 24.9 million hectares by 2021 and is projected to grow at a CAGR of 11.7% from 2016 to 2021” .” The bio-stimulant industry faces many problems and challenges. Until recently bio-stimulant products based on natural raw materials and particularly waste stream has mainly been developed based on observational and less commonly,nft growing system empirical data. While many contemporary bio-stimulants have been shown to be effective in practice, very few bio-stimulants can claim to understand the mechanisms or modes of action . Furthermore, while bio-stimulants can be categorized by source of origin, this is frequently inadequate as very substantial differences can exist between products even within a common feed stock origin. The challenge to bio-stimulant science is further exacerbated since composition and content of active substances in the original plant raw material can be affected by many factors including the location and growing conditions, season, species, variety, organ, and the phase of growth . Similarly, the response of the target crop can be expected to vary across crops and environments. One solution to this problem is to derive the raw materials for the bio-stimulant under highly regulated conditions. This approach has been successfully implemented by leading seaweed producers and fermentation based products that have developed harvesting and manufacturing processes that ensure uniformity of product performance through time. The development of a product with uniformity of response is not, however, a guarantee that the product is optimized for biological efficacy. To address these issues, developments in -omics approaches will be critical in accelerating the discovery of mode of action of bioactive compounds and optimizing their use. Metabolomics, phenomics and agronomics represent the integration of gene expression, protein interactions, and other regulatory processes as they impact on plant productivity and thus are more appropriate tools for discovery in this field than mRNA, transcripts, or proteins analyzed in isolation . Integrative, multidisciplinary approaches using tools from transcriptomics in conjunction with metabolomics and biochemical analysis are necessary to establish the mechanism of action and to identify the active components in the extracts . The difficulty in identifying modes of action and subsequent standardization of composition of multi-component bio-stimulants based on natural raw materials will continue to hamper the use, certification and registration of bio-stimulants. The solution to this problem will require the collaborative efforts of specialists from different fields: chemists, biologists, plant physiologists, industrial manufacture, sales and distribution and those with expertise in practical agricultural production .

Products with a single active substance represent a simpler construct in which the physiological effects and mechanism of action can be more readily determined and hence certification and registration is simpler. The multi-component composition of many preparations, however, are much more difficult to characterize , though they may offer novel insight into biological synergy , multi-functionality and emergence which may be crucial to product efficacy . In the absence of a functional rationale for every constituent in a multi-component bio-stimulant, it is likely that there will be molecules present that may positively or negatively influence plant productivity. Currently, it is almost impossible using available chemical-synthetic, and genetic engineering approaches to reproduce the full suite of molecules and complexes of biologically active substances that are present in most bio-stimulants. Many have noted the state confusion in the field of bio-stimulants and this has resulted in the opinion that much of the bio-stimulant market is not based on science or efficacy and that many products are little more than recycled waste products sold on the basis of pseudoscience and marketing. Indeed, research on several bio-stimulant products has shown them to be ineffective or to contain inactive, unstable or inconsistent properties with several showing negative effects compared when contrasted with well-designed controls . For example, foliar and root application of a product containing amino acids from animal origin have been reported to cause severe plant-growth depression and negative effects on Fe nutrition while a second product containing amino acids from plant origin stimulated plant growth . In another report that tested several bio-stimulant products it was concluded that “none of the bio-stimulant products tested achieved a sufficient degree of pathogen control to warrant replacement of or supplementation with conventional synthetic fungicides” , and there have been demonstrated positive and negative impacts and overall questions of the economic feasibility of the use of humic substances for increasing crop yields . Since biological systems are inherently complex, and given that most bio-stimulant products have not been characterized and have received relatively little replicated and rigorous independent validation, it is perhaps not surprising that many products are ineffective or highly variable in response. Nevertheless, there are a significant number of rigorous independent reports of benefits from some bio-stimulant formulations and market growth data demonstrates that there is a good deal of support for these products within agricultural producer communities. That such market growth has occured, even in the absence of a known “mechanism of function” suggests that there are aspects of plantmetabolism and productivity constraints that are not understood but are potentially important if we are to achieve the goal of increased global food production. The market euphoria that is taking place in the bio-stimulant industry recognizes these unknowns and bio-stimulants are viewed by many innovators and investors as a mechanism to conduct broadscale, if unfocussed, discovery of novel biologically derived molecules. Much as the exploration of marine organisms, and plants and microbes from diverse ecosystems has led to the discovery of novel pharmaceuticals, so too the development of bio-stimulants from the broad range of source materials, holds significant promise of discovery.

Decomposing lower quality litter implies that soil microbes may need to immobilize nutrients to maintain their stoichiometric balance . In addition, under elevated CO2 conditions, available nutrients will progressively move from fast cycling tissues to slow cycling tissues , which induces progressive nutrient limitation that further exacerbates nutrient limitations. Although increased external nutrient inputs and accelerated nutrient mineralization rates under warming soil conditions may enhance soil nutrient availability and partly ease plant– microbe nutrient competition, these additional nutrients may be insufficient to satisfy the enhanced plant nutrient demands . To investigate nutrient competition and its effects on the terrestrial carbon cycle, different theories of plant-soil nutrient competition have been developed and implemented in Earth System Models . However, theoretical justification and observational support for these theories are rarely discussed, which may have resulted in large biases in modeled nutrient and carbon cycling . To reconcile this inconsistency between theory, observations, and models, we focus on one overarching question in this study: Is there an observationally consistent, theoretically supported,ebb and flow trays and mathematically robust theory that is simple enough to implement in ESMs while accurately representing plant–microbe competition for nutrients? To answer this question, we first survey four existing nutrient competition theories and their implementation in ESMs .

In Results, we discuss in detail these four competition theories: CT1, no direct competition; CT2, microbial decomposers outcompete plants; CT3, competition depends on pore-scale soil fertility heterogeneity; and CT4, plant–microbe relative demand controls competition. Then we describe a new theory of nutrient competition based on Equilibrium Chemistry Approximation kinetics . We test our new theory together with other existing competition theories against a unique observational data set of N competition in a grassland ecosystem.To inform the development of ESM land models, observations have to satisfy two criteria. First, observations should capture plant and microbe competition at the whole-soil level, because the significance of microsite heterogeneity diminishes at this spatial scale. Second, measurements should target short-term nutrient uptake, thus enabling relatively clear separation of the instantaneous competitive interactions from other ecosystem dynamics that occur over longer time scales . To our knowledge, the only experiment that meets these two criteria was conducted in an alpine meadow ecosystem with homogeneous vegetation cover using a 15N tracer . In this experiment, the investigators randomly selected 90 10 cm diameter microplots within a 25 × 25 m area and added 0.32 g N/ m2 in the form of 15N−NH4 + or 15N−NO3 − with three soil depths treatments. 15N was injected at 2.5 cm, 7.5 cm, and 12.5 cm depth for 0–5, 5–10, and 10–15 cm treatments, respectively. The isotopic tracer was sampled 24 and 48 hours later, which informed partitioning of added nitrogen between microbes and plants. Furthermore, the grassland system has a very high rooting density, which allows us to isolate competitive interactions in the root zone from transport limitations so that observed competition patterns are directly comparable with theoretical models .

For our model evaluation, we extracted the data points from Fig. 4 of Xu et al. using the matlab script GRABIT .2Among the four existing theories surveyed, the traditional Nutrient Competition Theory assumes that plants and microbes do not compete for nutrients. This theory presumes that plants can assimilate carbon directly from the atmosphere but rely on nutrients released from soil microbial activity, so plants are carbon rich but nutrient limited . Conversely, because soil microbes decompose soil organic matter to obtain carbon and nutrients , they are relatively nutrient abundant but carbon limited . A second reason ecologists hypothesize that plants and microbes do not compete is that microbes can directly use organic N during decomposition , while plants primarily use inorganic N . However, depending on their carbon use efficiency and biomass stoichiometric imbalances against substrates , microbes do immobilize inorganic nutrients and thus directly compete with plants, creating the first contradiction against the CT1 theory. Further, plants may also utilize some low molecular weight amino acids through mycorrhizal fungi associations or direct root uptake , which creates a second contradiction to the theory. However, no existing ESMs apply CT1 to represent nutrient competition . The second theory posits that microbial decomposers out-compete plants in nutrient acquisition. This theory assumes that microbial nutrient uptake is extremely efficient , and microbes assimilate as much nutrients as they can during decomposition, provided they are not carbon limited. When carbon is limited, mineral nutrients are released as a “waste product” . This concept leads to the classic idea that plants can only use “leftover” nutrients after microbial demands are satisfied , which is why measured net mineralization rates are commonly used as a proxy for plant-available nutrients . However, no evidence exists to support its validity at the whole-soil or ecosystem level.

In contrast, 15N labeling studies have demonstrated that plants can continuously acquire inorganic nutrients, even when both plants and microbes are nutrient limited .CT2 has been applied in several ESMs. HadGEM2 and GFDL assume that soil microbial decomposers always outcompete plants and have priority for available nutrients . IPSL and BNU-ESM also assume that microbial immobilization has priority, but apply this priority to the estimated gross mineralization flux in the current model time step, as opposed to the nutrient pool. The third competition theory applies the emerging perspective that plant–microbe nutrient competition depends on the spatial heterogeneity of soil nutrient fertility, and therefore plants do not completely lose the competition at the whole-soil or ecosystem level. In a heterogeneous soil medium, inorganic nutrients move from nutrient-rich microsites toward nutrient-limited microsites , with roots potentially intercepting the nutrients . CT3 has been integrated into very fine-spatial scale models that explicitly consider the role of microsite soil nutrient heterogeneity, nutrient diffusion, root–microbe interactions , and microbe–microbe competition . In these models, plants do not completely lose the competition with microbes because they can take advantage of fine-scale spatial gradients between immobilizing and mineralizing microbes. The emergent responses from these models indicate that nutrient diffusion rates, sink strength , and competitor spatial distributions are the most important factors affecting plant competitiveness. However, these models’ fine spatial resolution is not directly applicable to ESMs. In ESMs, each soil column is assumed to be a well-mixed environment of nutrients and competitors. Such an assumption is currently necessitated, at least, by limited computational power and observations. Although ESM spatial resolutions likely will become finer, simulating microsite-level soil heterogeneity will remain impractical in the near future. In addition, a model based on CT3 may have high explanatory value but low predictive value, because it requires fine resolution observations of soil heterogeneity . The fourth nutrient competition theory has been applied in several ESMs. In these ESMs, plant nutrient demand is simulated based on potential Net Primary Production in the absence of nutrient constraints and the plant C to N ratio ; an analogous approach is taken for microbial nutrient demand. When soil nutrient supply is insufficient to saThisfy these demands,4×8 flood tray both plant and microbial demands are reduced in proportion to their respective demands . The actual NPP is then calculated by rescaling NPP demand with the reduction factor. This “relative demand” theory implicitly assumes that the consumer with higher demand will be relatively more competitive. While being simple, the CT4 predicted plant nutrient uptake is mechanistically inconsistent with measurements , although Goll et al. argued that the “demand-driven” approach requires fewer model parameters. The ESMs that apply CT4 include CLM-CN and NorESM , CLM-CNP , and JSBACH-CNP . The ECA-based nutrient competition theory.—The final competition theory is based on the concept of substrate–enzyme interactions applied to multiple nutrients and consumers . Substrate-enzyme kinetics theory has been extensively applied to model enzyme mediated processes ; however, its potential to represent plant and microbe nutrient competition is under-appreciated. Previous work that used the Michaelis-Menten equation to calculate nitrogen uptake only considered one carrier enzyme and one substrate at a time. To our knowledge, this study is the first that successfully extends classic enzyme kinetics theory to reconcile inconsistencies between observed plant and microbe nutrient competition and predictions from existing ESM competition theories. The substrate–enzyme view of plant nutrient uptake posits that first, extra-cellular enzymes, primarily produced by microbes, degrade polymers into monomers and inorganic nutrients, and second, plants produce nutrient transporter enzymes specialized for each mineral nutrient .

These plant-membrane-bound transporter enzymes react with substrates and form complexes , which are then transported into the root cell, after which the transporter enzymes are freed for the next substrate acquisition. Similar uptake mechanisms have been identified for microbes . This “nutrient carrier enzymes mediating uptake” conceptual framework allows us to extend the classic enzyme kinetics theory to represent plant– microbe competition based on the Equilibrium Chemistry Approximation theory and functional traits of the organisms. The same idea has provided the theoretical basis for the classic Michaelis-Menten type representation of nutrient uptake . However, classic enzyme theory and the resulting MM kinetics only consider one carrier enzyme and one substrate at a time, making them theoretically unable to represent competitive interactions among multiple consumers for multiple substrates. The novelty of the ECA approach is that it systematically considers multiple nutrient carrier enzymes and substrates together, and provides a rigorously derived and theoretically supported mathematical solution . It is also more accurate than the MM kinetics for cases involving only one carrier enzyme and one substrate . No current ESMs apply the ECA theory, although we are integrating it into the ACME Land Model . This work represents our first attempt to demonstrate its utility for plant–microbe nitrogen competition.The ECA representation of nutrient competition provides a theoretical and modeling construct that resulted in very good comparison with the nitrogen uptake partitioning. These predictions demonstrate that integrated across the soil profile, plants were less competitive than microbial decomposers; plant competitiveness against microbes is a spatially distinct property and there is no simple coefficient that can scale their “competitiveness”; the ECA framework offers a theoretically consistent approach to continuously update individual competitiveness; plant competitiveness is controlled by functional and structural traits ; and in the topsoil, plants might out-compete microbes and consequently suppress microbial nutrient uptake. Of course, applying the ECA competition to ESMs comes at the cost of introducing new parameters and additional uncertainty associated with those parameters. However, the ECA approach does not necessarily increase overall model uncertainty . In fact, ECA competition largely reduced the uncertainty in global-scale predictions by considering essential processes that govern system dynamics . We argue that an analogous result occurred in this analysis, i.e., that the uncertainty reduction in model structure overwhelmed uncertainty associated with new model parameters. In addition, most of the ECA parameters are kinetic parameters, which can be directly measured or optimized , implying that targeted experiments and model calibration could further reduce parameter uncertainty.Nutrient competition constantly occurs between plants and microbes in natural terrestrial ecosystems and it will likely intensify under climate change . Therefore, two fundamental questions arise: what controls the partitioning of limited nutrient resources between plants and microbes and how should short-term competition be modeled? Regarding the first question, we highlight the very few observations available to quantitatively partition nutrient acquisition by plants and microbes, and contend that such observations are critical to improve carbon-climate feedback predictions. As we showed here, the detailed 15N tracer experiment used in this study allowed us to evaluate the existing and newly developed plant–microbe N competition hypotheses, because the experiment was conducted at the plot scale and 15N was directly injected in the rooting zone . Thus, most of the observed plant N uptake pattern reflected the direct competition between roots and microbes, via nutrient carrier enzymes quantity and quality. Regarding the second question, we show here that plant and microbial nutrient uptake can be mechanistically explained as different nutrient transporter enzymes reacting with soil nutrients in a competitive manner. By linking plant root and microbial biomass density to nutrient transporter enzyme abundances, our new competition theory produces qualitatively correct competition patterns with literature-derived parameters from other ecosystems, and is easy to calibrate for specific ecosystems. Further, the linkage of nutrient competition with plant and microbial traits will allow a model to represent the competitors’ dynamic allocation of resources to acquire necessary nutrients.

A major advantage of plants in this respect is the ability to test multiple product candidates and expression cassettes in parallel by the simple injection or infiltration of leaves or leaf sections with a panel of Agrobacterium tumefaciens clones carrying each variant cassette as part of the transferred DNA in a binary transformation vector . This procedure does not require sterile conditions, transfection reagents, or skilled staff, and can, therefore, be conducted in standard bio-safety level 1 laboratories all over the world. The method can produce samples of even complex proteins such as glycosylated monoclonal antibodies for analysis ~14 days after the protein sequence is available. With product accumulation in the range of 0.1–4.0 g kg−1 biomass , larger-scale quantities can be supplied after 4–8 weeks , making this approach ideal for emergency responses to sudden disease outbreaks. Potential bottlenecks include the preparation of sufficiently large candidate libraries, ideally in an automated manner as described for conventional expression systems, and the infiltration of plants with a large number of candidates. Also, leaf-based expression can result in a coefficient of variation >20% in terms of recombinant protein accumulation, which reduces the reliability of expression data . The variability issue has been addressed to some extent by a parallelized leaf-disc assay at the cost of a further reduction in sample throughput . The reproducibility of screening was improved in 2018 by the development of plant cell pack technology,flood table in which plant cell suspension cultures deprived of medium are used to form a plant tissues surrogate that can be infiltrated with A. tumefaciens in a 96-well microtiter plate format to produce milligram quantities of protein in an automated, high-throughput manner.

The costs can be as low as €0.50 per 60-mg sample with a product accumulation of ~100 mg kg−1 and can typically result in a CV of <5% . These costs include the fermenter-based upstream production of plant cells as well as all materials and labor. The system can be integrated with the cloning of large candidate libraries, allowing a throughput of >1,000 samples per week, and protein is produced 3 days after infiltration. The translatability of cell pack data to intact plants was successfully demonstrated for three mAbs and several other proteins, including a toxin . Therefore, cell packs allow the rapid and automated screening of product candidates such as vaccines and diagnostic reagents. In addition to recombinant proteins, the technology can, in principle, also be used to produce virus-like particles based on plant viruses, which further broadens its applicability for screening and product evaluation but, to our knowledge, according results had not been published as of September 2020. In the future, plant cell packs could be combined with a recently developed method for rapid gene transfer to plant cells using carbon nanotubes . Such a combination would not be dependent on bacteria for cloning or gene transfer to plant cells , thereby reducing the overall duration of the process by an additional 2–3 days . For the rapid screening of even larger numbers of candidates, cost-efficient cell-free lysates based on plant cells have been developed and are commercially available in a ready-to-use kit format. Proteins can be synthesized in ~24 h, potentially in 384-well plates, and the yields expressed as recombinant protein mass per volume of cell lysate can reach 3 mg ml−1 . Given costs of ~€1,160  ml−1 according to the manufacturer LenioBio , this translates to ~€400 mg−1 protein, an order of magnitude less expensive than the SP6 system , which achieves 0.1 mg ml−1 at a cost of ~€360  ml−1 based on the company’s claims. Protocol duration and necessary labor are comparable between the two systems and so are the proteins used to demonstrate high expression, e.g., luciferase.

However, the scalability of the plantcell lysates is currently limited to several hundred milliliters, and transferability to intact plants has yet to be demonstrated, i.e., information about how well product accumulation in lysates correlates with that in plant tissues. Such correlations can then form the basis to scale-up lysate-based production to good manufacturing practice -compliant manufacturing in plants using existing facilities. Therefore, the cell packs are currently the most appealing screening system due to their favorable balance of speed, throughput, and translatability to whole plants for large-scale production. In any pandemic, the pathogen genome has to be sequenced, made publically available, and freely disseminated in the global scientific community to accelerate therapeutic and vaccine development. Once sequence information is available, a high priority is the rapid development, synthesis, and distribution of DNA sequences coding for individual viral open reading frames. These reagents are not only important for screening subunit vaccine targets but also as enabling tools for research into the structure, function, stability, and detection of the virus . Because many viral pathogens mutate over time, the sequencing of clinical virus samples is equally important to enable the development of countermeasures to keep pace with virus evolution . To ensure the broadest impact, the gene constructs must be codon optimized for expression in a variety of hosts ; cloned into plasmids with appropriate promoters, purification tags, and watermark sequences to identify them as synthetic and so that their origin can be verified ; and made widely available at minimal cost to researchers around the world. Not-for-profit plasmid repositories, such as Addgene and DNASU, in cooperation with global academic and industry contributors, play an important role in providing and sharing these reagents.

However, the availability of codon-optimized genes for plants and the corresponding expression systems is often limited . For example, there were 41,247 mammalian, 16,560 bacterial, and 4,721 yeast expression vectors in the Addgene collection as of August 2020, but only 1,821 for plants, none of which contained SARS-CoV-2 proteins. Sharing plant-optimized SARS-CoV-2 synthetic biology resources among the academic and industry research community working on PMPs would further accelerate the response to this pandemic disease. Screening and process development can also be expedited by using modeling tools to identify relevant parameter combinations for experimental testing. For example, initial attempts have been made to establish correlations between genetic elements or protein structures and product accumulation in plants . Similarly, heuristic and model-based predictions can be used to optimize downstream processing unit operations including chromatography . Because protein accumulation often depends on multiple parameters, it is typically more challenging to model than chromatography and probably needs to rely on data-driven rather than mechanistic models. Based on results obtained for antibody production, a combination of descriptive and mechanistic models can reduce the number of experiments and thus the development time by 75% , which is a substantial gain when trying to counteract a global pandemic such as COVID-19. These models are particularly useful if combined with the high-throughput experiments described above. Techno-economic assessment computeraided design tools, based on engineering process models,rolling benches can be used to design and size process equipment, solve material and energy balances, generate process flow sheets, establish scheduling, and identify process bottlenecks. TEA models have been developed and are publicly available for a variety of plant-based bio-manufacturing facilities, including whole plant and plant cell bioreactor processes for production of mAbs , antiviral lectins , therapeutics , and antimicrobial peptides . These tools are particularly useful for the development of new processes because they can indicate which areas would benefit most from focused research and development efforts to increase throughput, reduce process mass intensity, and minimize overall production costs.The rapid production of protein-based countermeasures for SARS-CoV-2 will most likely, at least initially, require bio-manufacturing processes based on transient expression rather than stable transgenic lines. Options include the transient transfection of mammalian cells , baculovirus-infected insect cell expression systems , cell-free expression systems for in vitro transcription and translation , and transient expression in plants . The longer term production of these countermeasures may rely on mammalian or plant cell lines and/or transgenic plants, in which the expression cassette has been stably integrated into the host genome, but these will take months or even years to develop, optimize, and scale-up. Among the available transient expression systems, only plants can be scaled-up to meet the demand for COVID-19 countermeasures without the need for extensive supply chains and/or complex and expensive infrastructure, thus ensuring low production costs . These manufacturing processes typically use Nicotiana benthamiana as the production host and each plant can be regarded as a biodegradable, single-use bioreactor . The plants are grown either in greenhouses or indoors, either hydroponically or in a growth substrate, often in multiple layers to minimize the facility footprint, and under artificial lighting such as LEDs. In North America, large-scale commercial PMP facilities have been built in Bryan, TX , Owensboro, KY , Durham, NC , and Quebec, Canada . The plants are grown from seed until they reach 4–6 weeks of age before transient expression, which is typically achieved by infiltration using recombinant A. tumefaciens carrying the expression cassette or by the introduction of a viral expression vector such as tobacco mosaic virus , for example, the GENEWARE platform . For transient expression by infiltration with A. tumefaciens, the plants are turned upside down and the aerial portions are submerged in the bacterial suspension.

A moderate vacuum is applied for a few minutes, and when it is released, the bacteria are drawn into the interstitial spaces within the leaves. The plants are removed from the suspension and moved to an incubation room/chamber for 5–7 days for recombinant protein production. A recent adaptation of this process replaces vacuum infiltration with the aerial application of the A. tumefaciens suspension mixed with a surfactant. The reduced surface tension of the carrier solution allows the bacteria to enter the stomata, achieving a similar effect to agroinfiltration . This agrospray strategy can be applied anywhere, thus removing the need for vacuum infiltrators and associated equipment . For transient expression using viral vectors, the viral suspension is mixed with an abrasive for application to the leaves using a pressurized spray, and the plants are incubated for 6–12 days as the recombinant protein is produced.Depending on the batch size , the vacuum infiltration throughput, and the target protein production kinetics, the infiltration/ incubation process time is 5–8 days. The inoculation/incubation process is slightly longer at 6–13 days. The overall batch time from seeding to harvest is 33–55 days depending on the optimal plant age, transient expression method, and target protein production kinetics . Importantly, plant growth can be de-coupled from infiltration, so that the plants are kept at the ready for instant use, which reduces the effective first-reaction batch time from gene to product to ~10–15 days if a platform downstream process is available . The time between batches can be reduced even further to match the longest unit operation in the upstream or downstream process. The number of plants available under normal operational scenarios is limited to avoid expenditure, but more plants can be seeded and made available in the event of a pandemic emergency. This would allow various urgent manufacturing scenarios to be realized, for example, the provision of a vaccine candidate or other prophylactic to first-line response staff.The speed of transient expression in plants allows the rapid adaptation of a product even when the process has already reached manufacturing scale. For example, decisions about the nature of the recombinant protein product can be made as little as 2 weeks before harvest because the cultivation of bacteria takes less than 7 days and the post-infiltration incubation of plants takes ~5–7 days. By using large-scale cryo-stocks of ready-to-use A. tumefaciens, the decision can be delayed until the day of infiltration and thus 5–7 days before harvesting the biomass . This flexibility is desirable in an early pandemic scenario because the latest information on improved drug properties can be channeled directly into production, for example, to produce gram quantities of protein that are required for safety assessment, pre-clinical and clinical testing, or even compassionate use if the fatality rate of a disease is high . Although infiltration is typically a discontinuous process requiring stainless-steel equipment due to the vacuum that must be applied to plants submerged in the bacterial suspension, most other steps in the production of PMPs can be designed for continuous operation, incorporating single-use equipment and thus complying with the proposed concept for biofacilities of the future .

Are the viroid-like molecules the evolutionary link between the RNA and DNA world? We hope that the current and the future generations of citrus scientists will carry on the 80 years old journey of viroid research and that they will provide exciting answers, new discoveries, and even more questions for scientific advancement.Citrus exocorThis viroid , is a member of the genus Pospiviroid within the Pospiviroidae family. CEVd is the causal agent of exocortis disease characterised by bark scaling and dwarfing symptoms in sensitive citrus hosts commonly used as rootstocks and limiting crop production of the grafted commercial species. Since symptom expression takes 3-5 years to show on inoculated trifoliate orange Raf the biological characterization of CEVd usually relies on the use of indicator plants. Etrog citron has been widely used for indexing purposes, bio-amplification of the viroid RNA and strain characterization. Following infection with CEVd, Etrog citron displays a characteristic syndrome that includes severe stunting, leaf epinasty and midvein necrosis in 3-6 months. Furthermore, the effect of a severe isolate of CEVd on the gene expression of Etrog citron has been examined, using a citrus cDNA microarray approach that revealed that infection triggered important changes in chloroplast, cell wall, peroxidase and symporter activities. However the changes in gene expression found in CEVd infected Etrog citron may not be necessarily responsible for the bark scaling symptoms that are the most characteristic exocortis syndrome in trifoliate orange. Four trifoliate orange seedlings that were graft inoculated with a severe strain of CEVd and were planted in a field plot in June 1993,led grow lights developed the characteristic bark scaling symptoms, yellowing of the twigs and remained stunted as compared with the non-inoculated controls.

In 2010 plant material was collected in order to accomplish a gene expression analysis using the same genome-wide 20-K cDNA microarray developed under the Citrus Functional Genomic Project . For that, the gene expression profiles of the CEVd infected trifoliate oranges and uninfected control ones were compared. Subsequently, Significance Analysis of Microarrays was performed to identify those genes differentially expressed in both conditions and lastly, they were classified with gene ontology analysis by using the Blast2GO tool . As expected, results showed similarities and differences with those obtained for Etrog citron that could account for some of the differences found in the symptomatology. We developed a semi-automated, high throughput, RNA extraction and purification procedure optimized for citrus tissues. The system utilizes the SPEX SamplePrep’s Cryo-station and Geno Grinder 2010, and the Applied Biosystems’ MagMAXTM Express-96 along with a modified 5x MME-96 Viral RNA Isolation Kit. The detection of viral RNA with reverse transcription quantitative real time polymerase chain reaction requires high quality RNA as defined by concentration, purity, and integrity. RNA concentration and purity were assessed by spectrophotometry at 260 nm, and 260/280 and 260/230 ratios, respectively. The RNA concentration of 304 citrus samples ranged from 14.4 to 886.0 ng/μl with an average of 180.082 . The majority of the samples had concentrations ≥50ng/μl while 89.1% of the samples had concentrations of 50-400 ng/μl. The RNA purity ratios were higher than the desirable 1.8 for all samples tested with a mean value of 2.404 while 98% of the samples had 260/280 ratio ≥2.0. The 260/230 ratios had a broader variation in comparison to the 260/280. The 260/230 ratio mean value was 1.883 . The majority of the samples had the desirable 260/230 ratio of 1.8-2.0 .

The remaining 27% of the samples had 260/230 ratio of 1.0-1.7 and the remaining 11% had 260/230 ratio ˂1.0. Subsequent experimentation with adjusted grinding and washing buffers significantly improved and standardized the 260/230 ratios to the desirable values of 1.8-2.0.The RNA integrity of 23 samples was evaluated by 118 RT-qPCR reactions targeting the mRNA of the NADH dehydrogenase citrus gene. The mean Ct value of the RT-qPCR was 21.948 with maximum and minimum values 28.5 and 16.29, respectively. The cost of supplies for the presented RNA extraction and purification procedure was estimated at US$4.03 per sample. Citrus vein enation disease is characterized by woody galls on the trunk and vein enations on the leaves of susceptible citrus species. This disease is graft transmissible and naturally spread by aphid species in a persistent manner. With the aim of identifying the putative virus associated with vein enation, small interfering RNAs from three different field citrus trees that tested positive to citrus vein enation by indexing and that are located in Valencia, Tenerife and Gran Canaria, Spain, were analyzed by next generation sequencing Illumina technology. Bioinformatic analysis of individual samples allowed the identification of a new Luteoviridae present in the three samples. Bioinformatic analysis using the CLC Genomic Workbench, Velvet, Geneious and Bowtie software allowed the reconstruction of a sequence of 5077 nucleotides, corresponding to a new viral species for which the name Citrus vein enation virus is proposed. Open reading frames were identified coding for an hypothetical protein, the polymerase, the aphid transmission protein and the coat protein. Protein homology analyses for these ORFs showed similarities with Luteoviridae members: 44% , 66% , 62% and 70% , respectively. Specific primers and a TaqMan probe based on the new sequence were designed for real-time RTPCR detection of the agent.

The method allowed the successful detection of this virus in plant material and in various aphid species, even using direct systems of sample preparation. This novel diagnostic could greatly simplify and reduce the cost of routine detection of this highly prevalent disease in certification and sanitary programs.It is widely assumed that fleshy fruits are involved in mediating the attraction of seed dispersal organisms and the avoidance of consumption by seed predators. It is thought that the primary function of secondary metabolites present in immature fruits is to defend them against all types of potential consumers. Changes in size, texture, taste, aroma and color occur during ripening. Frugivores include not only legitimate dispersers such as vertebrates and birds but also less appreciated but more abundant consumers of fleshy fruits, microbes. Plant volatile organic compounds comprise a wide diversity of low-molecular-weight secondary metabolites, including terpenoids. In general, flowers and fruits release the widest variety of VOCs, with emission rates peaking before pollination and at ripening. Sweet orange fruits accumulate mainly terpenoids in mature peel oil glands,vertical grow system and Dlimonene accounts for about 97% of their content. In nature, D-limonene content is usually low in orange fruits during the 2 to 3 months post-anthesis; it then drastically increases when the fruit is still green but contains seeds and remains at a high level until the fruit becomes fully mature. To investigate the role of VOCs in mature fruit interactions with specialized pathogenic microorganisms, we have generated transgenic orange plants carrying a D-limonene synthase gene in antisense configuration. Transgenic expression caused a dramatic decrease in the accumulation of D-limonene in fruit peels, being about 80-100 times lower in AS samples than in empty vector transgenic ones. A global gene expression analysis of these fruits linked the decrease of D-limonene to the upregulation of genes involved in innate immunity. Additionally, this caused the activation of J jasmonic acid signalling and metabolism upon challenge with different economically important fungal and bacterial pathogens, which led to strong general resistance against Xanthomonas citri subsp. citri, Penicillium digitatum and PhyllocThista citricarpa in AS orange peels, indicating that D-limonene and related terpene accumulation not only attract legitimate seed dispersers but also facilitate infection by specialized microorganisms.Citrus tristeza viruscauses one of the most devastating diseases of citrus worldwide inducing the death of sweet orange, mandarin, lime and grapefruit trees budded on sour orange. The availability of a CTV-resistant rootstock with the sour orange attributes of productivity, fruit quality and tolerance to abiotic stresses would be a major benefit to the citrus industry worldwide. The objective of the field trial was to evaluate the response to CTV of 10 sour orange transgenic lines carrying CTV-derived sequences. They were obtained in the laboratories of IVIA, Spain and planted at the INTA Experiment Station in Concordia, Argentina where CTV is endemic and efficiently transmitted by the brown citrus aphid .

Rooted cuttings of transgenic sour orange lines were budded with non-transgenic and virus-free Valencia Late sweet orange . Valencia trees budded on tolerant rootstocks as well as on non-transgenic sour orange were planted as controls. Trees were planted in a complete randomized design with two trees per plot and 5 replications. Every six months imprints were taken to determine the progress of CTV infection in each tree. Based on direct immuno printing-ELISA, differences in disease progress were observed till June 2012 on the different transgenic rootstocks. By December 2012 the percentage of diseased trees was over 80%. The sudden increase in disease progress in the last semester could be due to post-freeze effects. Four years after planting, almost 100 % of the trees are CTV infected, showing stunted growth and yellowing of foliage. Trees from each transgenic line were grouped according to symptom severity in the field. The better looking trees were those of two of the ten transgenic lines carrying CTVderived sequences.Citrus tristeza disease was reported in Northeast Argentina in 1930 and in the Northwest in 1947. Later, millions of citrus trees on sour orange died from quick decline in both citrus regions. The most efficient vector, Toxoptera citricida and other aphids are present and consequently, the disease is endemic. Nowadays, citrus varieties are only grafted on tolerant rootstocks. Independent of rootstock, grapefruit is affected by stem pitting, and disease expression is severe in some selections. Biological characterization of Citrus tristeza virusisolates from Northwest Argentina has been carried out since 2008 in the Centro de Saneamiento de Citrus of the EEAOC although molecular identification of isolates has not been performed thus far. In order to identify isolates, a reverse-transcription polymerase chain reaction was performed. Five sets of genotype-specific CTV primers within the open reading frame -1a of well recognized genotypes were used for characterization. CTV isolates were collected from Citrus limon, C. sinensis, C. paradisi, C. reticulata C. reshni, C. latifolia, C. macrophylla, Poncirus trifoliata and Troyer citrangeaccording to the following criteria: species or cultivars of the source tree, visual symptoms on the source tree, and symptom expression in greenhouse tests with Mexican lime , Pineapple sweet orange , sour orange and Duncan grapefruit indicator plants. Most of the source trees showed no remarkable symptomatology in the field tree. Of the five CTV genotypes analyzed, severe genotypes were widely distributed, whereas mild isolates were detected at a very low incidence. The genotypes T3 and VT were predominant in mixed infections, independent of host species and variety. Data obtained are relevant because they complement existing information for CTV biological diversity in Northwest Argentina. This is the first characterization and classification of northwestern CTV isolates.Twelve Citrus tristeza virusisolates, collected from 5 different orchards in Hunan, China, were simultaneously characterized by RT-PCR of the p23 gene, capillary electrophoresis of single strand conformation polymorphisms of the p23, p25 and p27 genes as well as multiple molecular marker analysis with four standard isolates, namely T3, T30, T36, and VT. Results from RT-PCR of the p23 gene indicated that all the isolates were virulent. CE-SSCP and MMM characterization revealed high levels of genetic diversity among the isolates ranging from single genotype infections to highly mixed infections. Out of 12 isolates, 11 contained the T3 genotype, three being single T3 genotypes, two contained T3+VT and six had T3+VT+T36+T30. The remaining isolate was the VT+T36+T30 genotype. T3 and VT, reported to be virulent genotypes, are widely distributed in Hunan Province. As the isolates were from trees showing stem pitting, the single T3 profile of CE-SSCP was likely to be related to stem pitting. Further confirmation is to be performed with more samples showing obvious stem pitting symptoms. In young orchards, single genotype isolates are usually detected, and mixed ones are found in old orchards.The characterization by the three different molecular methods resulted in consistent results with some inconsistency among different methods. In the latter case, sequencing should be conducted for further characterization.Despite millions of trees being indexed by ELISA, Citrus tristeza disease continues to spread worldwide, confirming that quarantine restrictions, eradication, and tristeza-free propagation material are not enough to combat the virus once it becomes established in an area.

Resistant starch is a popular nutritional additive to produce food with enhanced quality attributes, i.e., higher fiber content, and starchy horticultural commodities are similarly attractive. Te yield penalty of high amylose crops may be alleviated by picking an ideal AP/AM ratio through a coordinate change in the relative balance of starch biosynthetic enzymes. In the case of potato, it is plausible that down regulation of SBEs not only produces healthy fiber-starch, but also lessens the CIS severity and acrylamide problem . However, the sugars derived from starch during CIS may be an adaptive mechanism to enhance plant chilling tolerance. Rapid sugar accumulation upon cold stress have been reported in fruit. Te sugars freed from starch may promote metabolic activity and serve as an osmoprotectant, thus alleviating chilling injury. Te major functional SBEs were found to be upregulated in cold-stressed banana fruit, potato tuber, and Arabidopsis leaf , which may facilitate the ‘sugaring’ process. Modulating SBE activities may alter the rate of sugar released from the highly digestible starch polymers, thus changing the fruit/tuber cold responses. In fruiting species, the importance of ‘transitory-storage starch’ may be underestimated due to the lack of enough direct knowledge of its function, gained from experimental data. Tomato serves as a functional genomics model for feshy fruit, as it is easily transformed and genetically manipulated. Te putative function of ‘transitory-storage starch’ in fruit ripening, respiration, and sweetness enhancement may be revealed by engineering AP/AM ratio through over expression or suppression of SBEs. We hypothesize that high amylose, resistant starch tomato fruit may have reduced available starch, sugars,nft hydroponic system and changes in fruit ripening and other processes that are dependent on starch as a carbon supply and source of energy post harvest.

Tomato SBEs may not reflect the functionality of all fruit SBEs, but it would produce fundamental knowledge and expand our understanding of species-, organ- and developmental-specific regulations of the core starch biosynthetic enzymes.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. Resistant starch is a popular nutritional additive to produce food with enhanced quality attributes, i.e., higher fiber content, and starchy horticultural commodities are similarly attractive. Te yield penalty of high amylose crops may be alleviated by picking an ideal AP/AM ratio through a coordinate change in the relative balance of starch biosynthetic enzymes. In the case of potato, it is plausible that down regulation of SBEs not only produces healthy fiber-starch, but also lessens the CIS severity and acrylamide problem . However, the sugars derived from starch during CIS may be an adaptive mechanism to enhance plant chilling tolerance. Rapid sugar accumulation upon cold stress have been reported in fruit. Te sugars freed from starch may promote metabolic activity and serve as an osmoprotectant, thus alleviating chilling injury. Te major functional SBEs were found to be upregulated in cold-stressed banana fruit, potato tuber, and Arabidopsis leaf , which may facilitate the ‘sugaring’ process. Modulating SBE activities may alter the rate of sugar released from the highly digestible starch polymers, thus changing the fruit/tuber cold responses. In fruiting species, the importance of ‘transitory-storage starch’ may be underestimated due to the lack of enough direct knowledge of its function, gained from experimental data. Tomato serves as a functional genomics model for feshy fruit, as it is easily transformed and genetically manipulated. Te putative function of ‘transitory-storage starch’ in fruit ripening, respiration, and sweetness enhancement may be revealed by engineering AP/AM ratio through overexpression or suppression of SBEs. We hypothesize that high amylose, resistant starch tomato fruit may have reduced available starch, sugars, and changes in fruit ripening and other processes that are dependent on starch as a carbon supply and source of energy post harvest.

Tomato SBEs may not reflect the functionality of all fruit SBEs, but it would produce fundamental knowledge and expand our understanding of species-, organ- and developmental-specific regulations of the core starch biosynthetic enzymes.Cell size correlates strongly with key aspects of cell physiology, including organelle abundance and DNA ploidy. Maintenance of uniform size may also underlie the efficient functioning of tissues and organs. While cells employ diverse strategies to regulate their size in different situations, it is unclear how these mechanisms are integrated to provide robust, systems-level control. In budding yeast, a molecular size sensor restricts passage of small cells through G1, enabling them to gain proportionally more volume than larger cells before progressing to Start . In contrast, size control post-Start is less clear. The duration of S/G2/M in wild type cells has been reported to exhibit only a weak dependence on cell size, so larger cells would be expected to add a greater volume than smaller ones. Yet it is also the case that even large mother cells produce smaller daughter cells, suggesting that additional regulation may play a role during S/G2/M, either by limiting bud growth rate or shortening the duration of budding. There is also conflicting evidence regarding the molecular size control mechanisms that might operate during S/G2/M, such as whether the kinase Swe1,mobile grow rack the budding yeast homolog of fission yeast Wee1, regulates growth by sensing bud size or bud morphogenesis. Furthermore, while G1 size control mechanisms act on cells smaller than their set-point size, no mechanisms have been clearly defined to limit size as cells become larger. Since physiological perturbations can result in abnormally large cells, mechanisms must exist to ensure cells that grow too large are able to return to the set-point volume after successive rounds of growth and division. Underscoring the importance of this aspect of size homeostasis, tumor cells lacking functional size-homeostasis pathways often grow far larger than normal. In sum, how cells regulate size during S/G2/M and whether such mechanisms might enforce an upper limit on cell volume remains an important open question. To gain further insight into post-Start size control, we prepared ‘giant’ yeast using two approaches to reversibly block cell cycle progression but not growth: optogenetic disruption of the cell polarity factor Bem1 or a temperature sensitive cdk1 allele. We reasoned that giant yeast would satisfy pre-Start size control while enabling us to uncover post-Start size-limiting mechanisms though the identification of invariant growth parameters . Upon release from their block, giant mothers reentered the cell cycle and populations of their progeny returned to their unperturbed size within hours. Volume regulation in these cells was inconsistent with two major classes of size control mechanisms: an ‘adder’ specifying a constant volume increment added over the course of a cell cycle and a ‘sizer’ specifying daughter cell volume. Instead, our data support a ‘timer’ mechanism that specifies the duration of S/G2/ M across the full range of daughter sizes. Our data thus provide evidence that cell size homeostasis is maintained by at least two separable mechanisms of size control: a pre-Start size sensor enabling size-dependent passage through Start, and a post-Start timer ensuring that daughters are smaller than their mothers. Together, these mechanisms ensure that yeast populations generated from cells at either size extreme rapidly return to a set-point within only a few cell division cycles.To achieve reversible control over cell size in the budding yeast S. cerevisiae, we first took advantage of the light-responsive PhyB/PIF optogenetic system to control the localization of Bem1, a cell polarity factor. In this “optoBem1” system, red light illumination relocalizes the PIF-Bem1 fusion protein to mitochondria-anchored PhyB . Light induced Bem1 re-localization produces an acute loss-of-function phenotype where cells fail to form a site of polarized Cdc42 activity, fail to initiate budding, and instead undergo continuous isotropic growth.

Strikingly, this effect is quickly reversed upon illumination with infrared light, which releases PIF-Bem1 from the mitochondria within seconds. Upon release, cells form a bud within minutes and proceed to cytokinesis . The PIF-Bem1 fusion protein appears to fully recapitulate normal Bem1 function: when it is not sequestered to the mitochondria, overall cell sizes and cell growth rates are similar to an isogenic wild type strain. We performed additional experiments to more completely characterize optoBem1 giant cells. Our initial experiments quantifying the growth of red light-illuminated optoBem1 cells revealed two sub-populations of cells that grew at different rates . We hypothesized that cell growth rates differed depending on the cell cycle phase at the time of Bem1disruption. Indeed, we found that synchronizing optoBem1 cells before red light stimulation led to unimodally-distributed growth . Furthermore, restricting our analysis to measure growth only following entry into G1 yielded a unimodal distribution . We also observed that a substantial fraction of optoBem1 yeast burst as they become increasingly large , and hypothesized that cell lysis may be a result of large cells’ increased susceptibility to osmotic pressure. Supporting this hypothesis, growing cells in high-osmolarity media containing 1 M sorbitol decreased the frequency of cell lysis without affecting the rate of isotropic growth . We therefore supplemented our media with sorbitol for all subsequent experiments involving optoBem1-arrested cells. Finally, to test whether growth was isotropic during the entire time period, we pulsed cells with fluorescent Concanavalin A to mark the existing cell wall, followed by a washout of free FITC-ConA. We found that cells exhibited uniform dilution of FITC-ConA around their surface, consistent with isotropic growth .Prior studies have established that unperturbed, freely-cycling budding yeast cells appear to exhibit an exponential growth in volume over time. However, most of this growth is localized to the bud, with only a minor contribution from the mother cell’s isotropic growth during G1.Since distinguishing between growth patterns is difficult to achieve during the growth interval of normal sized yeast, we reasoned that the ability to prepare isotropically-growing yeast with volumes spanning an order of magnitude would permit high-quality measurements of this growth law, and potentially reveal processes that limit cell growth as size increases.We imaged optoBem1 cells during red light illumination at multiple z-planes and used a custom code to automatically measure cell diameter every 10 min over a 12 h period. Following entry into G1 after Bem1 arrest, we found that isotropically-growing optoBem1 cells exhibited a linear increase in cell diameter over time, corresponding to a rate of volume growth proportional to 3 . Since these volume increases also show a strong correlation with protein content, as assessed by fluorescence , our data suggest that the growth we observed primarily arises from increases in cell mass rather than cell swelling . This result is inconsistent with two classic models of cell growth: a constant growth law, where volume increases linearly over time; and exponential growth, where the rate of growth is proportional to the cell’s current volume. In contrast, a linear increase in cell diameter is the expected result for volume increasing in proportion to cell surface area .

Despite the physiological relevance of the BIA assumptions and the number of BIA studies, a suitable solution for the characterization of the current pathways in roots is missing. Thus far, only indirect information obtained from invasive and time-consuming experiments have been available to address this issue . Mary et al. , and Mary et al. 2020 tested the combined use of ERT and Mise A La Masse methods for imaging grapevine and citrus roots in the field. An approach hereafter called inversion of Current Source Density was used to invert the acquired data. The objective of this inversion approach is to image the density and position of current passing from the plant to the soil. The current source introduced via the stem distributes into “excited” roots that act as a distributed network of current sources . Consequently, a spatial numerical inversion of these distributed electric sources provides direct information about the root current pathways and the position of the roots involved in the uptake of water and solutes. The numerical approach used to invert for the current source density is a key component required for such an approach. Mary et al. used a nonlinear minimization algorithm for the inversion of the current source density. The algorithm consisted of gradient-based sequential quadratic programming iterative minimization of the objective functions described in Mary et al. . The algorithm was implemented in MATLAB, R2016b, using the fmincon method.

Because no information about the investigated roots was available,flood and drain tray the authors based these inversion assumptions and the interpretation of their results on the available literature data on grapevine root architecture. Consequently, Mary et al. highlighted the need for further iCSD advances and more controlled studies on the actual relationships between current flow and root architecture. In this study, we present the methodological formulation and evaluation of the iCSD method, and discussits applications for in-situ characterization of current pathways in roots. We perform our studies using laboratory rhizotron experiments on crop roots. The main goals of this study were: 1) develop and test an iCSD inversion code that does not rely on prior assumptions on root architecture and function; 2) design and conduct rhizotron experiments that enable an optimal combination of root visualization and iCSD investigation of the current pathways in roots to provide direct insight on the root electrical behavior and validate the iCSD approach; and 3) perform experiments to evaluate the application of the iCSD method on different plant species and growing medium that are common to BIA and other plant studies.The relationship between hydraulic and electrical pathways has been the object of scientific debate because of its physiological relevance and methodological implications for BIA methods .The distribution of the current leakage is controlled by 1) the electrical radial and longitudinal conductivities , and 2) by the resistivity contrast between root and soil.

With regard to σcr and σcl, when σcl is significantly higher than σcr, the current will predominantly travel through the xylems to the distal “active” roots, which are mostly root hairs. Based on the link between hydraulic and electrical pathways, this is consistent with a root water uptake process where root hairs play a dominant role while the more insulated and suberized roots primarily function as conduits for both water and electric current . On the contrary, if the σcr is similar to σcl, the electrical current does not tend to travel through the entire root system but rather starts leaking into the surrounding medium from root proximal portions. The coexistence of proximal and distal current leakage is in line with studies that suggest the presence of a more diffused zone of RWU, and a more complex and partial insulation effect of the suberization, possibly resulting from the contribution of the cell-to-cell pathways . Soil resistivity can affect the distribution of the current leakage by influencing the minimum resistance pathways, i.e., whether roots or soil provide the minimum resistance to the current flow. In addition, soil resistivity strongly relates to the soil water content, which, as discussed, affects the root physiology. Therefore, information on the soil resistivity, such as the ERT resistivity imaging, has the potential for supporting the interpretation of both BIA and iCSD results. Dalton proposed a model for the interpretation of the plant root capacitance results in which the current equally distributes over the root system. Because of the elongated root geometry this model is coherent with the hypothesis of a low resistance xylem pathway . Numerous studies have applied Dalton’s model documenting the predicted correlation between root capacitance and mass . In fact, recent studies with wheat, soy, and maize roots continue to support the capacitance method .

Despite accumulating studies supporting the capacitance method, hydroponic laboratory results of Dietrich et al. and other studies have begun to uncover potential inconsistencies with Dalton’s assumptions. In their work, Dietrich et al. explored the effect of trimming submerged roots on the BIA response and found negligible variation of the root capacitance. Cao et al. reached similar conclusions regarding the measured electrical root resistance . Urban et al. discussed the BIA hypotheses and found that the current left the roots in their proximal portion in several of their experiments. Conclusions from the latter study are consistent with the assumption that distal roots have a negligible contribution on root capacitance and resistance. Because of the complexity of the hydraulic and electrical pathways, their link has long been the object of scientific research and debate. For recent reviews see Aroca and Mancuso ; for previous detailed discussions on pathways in plant cells and tissues see Fensom , Knipfer and Fricke , and Findlay and Hope ; see Johnson and Maherali et al. in regard to xylem pathways. See Jackson et al. and Hacke and Sperry for water pathways in roots. Thus, above discrepancies in the link between electrical and hydraulic root properties can be, at least to some degree, attributedto differences among plant species investigated and growing conditions. Among herbaceous plants,hydroponic equipment maize has been commonly used to investigate root electrical properties . For instance, Ginsburg investigated the longitudinal and radial current conductivities of excited root segments and concluded that the maize roots behave as leaking conductors. Similarly, Anderson and Higinbotham found that σcr of maize cortical sleeves was comparable to the stele σcl. Recently, Rao et al. found that maize root conductivity decreases as the root cross-sectional area increases, and that primary roots were more conductive than brace roots. By contrast, BIA studies on woody plants have supported the hypothesis of a radial isolation effect of bark and/or suberized tissues.Plant growing conditions have been shown to affect both water uptake and solute absorption due to induced differences in root maturation and suberization . Redjala et al. observed that the cadmium uptake of maize roots grown in hydroponic conditions was higher than in those grown aeroponically. Tavakkoli et al. demonstrated that the salt tolerance of barley grown in hydroponic conditions differed from that of soil-grown barley. Zimmermann and Steudle documented how the development of Casparian bands significantly reduced the water flow in maize roots grown in mist conditions compared to those grown hydroponically. During their investigation on the effect of hypoxia on maize, Enstone and Peterson reported differences in oxygen flow between plants grown hydroponically and plants grown in vermiculite. The results reported above and in other investigations are conducive to the hypothesis that root current pathways are affected by the growing conditions, as suggested in Urban et al. .

For example, the observations by Zimmermann and Steudle and Enstone and Peterson may explain the negligible contributions to the BIA signals from distal roots under hydroponic conditions . At the same time, the more extensive suberization in natural soil and weather conditions could explain the good agreement between the rooting depth reported by Mary et al. based on the iCSD and the available literature data for grapevines in the field. To minimize these ambiguities and to develop a more robust approach for non-invasive in-situ root imaging, we aim to develop iCSD inversion code that does not rely on prior assumptions on root architecture and function and use rhizotron experiments to validate the iCSD approach.The phrase “inversion of Current Source Density” was introduced by Łęski et al. to describe the 2D imaging of current sources associated with the brain neural activation. Similar inversion methodologies have been developed for the interpretation of the self potential data, where the distribution of naturally occurring currents is investigated . With regard to active methodologies, Binley et al. developed an analogous approach for detecting pollutant leakage from environmental confinement barriers. Although there are physical and numerical intrinsic differences between application of the iCSD to detect brain neuronal activity and current pathways in roots, we decided to adopt the term iCSD as the general physical imaging of current source density remains valid. With iCSD, we indicate the coupling of ERT and MALM through the proposed numerical inversion procedure for the imaging of the current source density, and its correlation with root architecture. We introduce the necessary aspects regarding the ERT and MALM methods in this section. However, we direct the inTherested readers to more in-depth discussion about the ERT method , and to Schlumberger and Parasnis with regard to the MALM method. In the following discussion we use ρmed to represent the 2D or 3D distribution of the electrical resistivity in the growing medium . CSD represents the 2D, or 3D, distribution of the Current Source Density within the same medium. In the case of roots, the CSD is controlled by the current conduction behavior of the roots, specifically by the leakage pattern of the root system . Both ERT and MALM are active methods. In these methods the current is forced through the medium by applying a potential difference between two current electrodes. In ERT, both current electrodes are positioned in the investigated medium, while for MALM the positive current pole is installed in the plant stem,similar to BIA . The potential field resulting from the current injection depends on CSD, resistivity of the medium , and boundary conditions. The boundary conditions are known a priori and their impact on the potential field can be properly modeled. In ERT, the current sources correspond to the electrodes used to inject current, allowing us to invert for ρmed. Then, the iCSD accounts for the obtained ρmed and explicitly inverts the MALM data to obtain current source distribution.The rhizotrons used in this study were designed to enable the concurrent direct visualization of the roots and electrical measurements. Rhizotron dimensions were 52 cm × 53 cm × 2 cm , see Fig. 2. Figure 2a shows the rhizotron setup with 64 silver/silver chloride electrodes located on the back viewing surface. The viewing surfaces were covered with opaque material to stop the light from affecting the development of the roots. The back viewing surface was removable, allowing homogeneous soil packing for the plant experiments and convenient access to the electrodes. Besides the top opening, the rhizotrons were waterproof to enable hydroponic experiments and controlled evapotranspiration conditions during the soil experiments and plant growth. All the experiments were performed in a growth chamber equipped with automatic growth lights and controlled temperature and humidity. The temperature varied with a day/night temperature regime of 25/20 °C. The humidity ranged from 45 to 60%. For both ERT and MALM methods, the electrical potential field is characterized by a set of potential differences measured between pairs of electrodes. It is important to properly arrange the electrodes on the rhizotron viewing surface and design a suitable acquisition sequence to obtain a good sensitivity coverage of the investigated system . This is particularly true for the iCSD, as both ERT and MALM acquisitions affect its result. The 64 electrodes were arranged in a 8 by 8 grid on the back viewing surface of the rhizotron, leaving the front surface clear for the observation . For the ERT, the designed arrangement of the electrodes offers a good compromise between a high coverage on the central part of the rhizotron, which encompasses the root zone, and a sufficient coverage on the rhizotron sides to avoid an excessive ERT inversion smoothness. For the MALM, the arrangement of the electrodes is highly sensitive to the position of the investigated current sources.

Downstream firms transform the paste in final consumer products. According to the Food Institute, at the end of the process, raw material account for 39%-45% of total production cost. According to the ERS, there was a radical structural change in the processing industry in the late 1980’s and early 1990’s. A period of relatively high prices in the late 1980s triggered new investments. This finally resulted in excess supply and decreasing prices. As a consequence, many processors went bankrupt and the whole industry was restructured. The current structure is the result of such adjustments.A brief industry description highlights two key points prior to the estimations. Price expectations. The majority of production is sold under contract. This has two implications: i) producers know prices when planning production, so we do not need to model expectations; rather we assume perfect information, ii) the actual contract price is unobservable, being industry private information.We use the spot price as a proxy for the real contract price. However, since the measurement error is likely to be correlated with the error terms in the production equations we use an instrumental variable approach. The instrument is the previous year’s spot price, which is correlated with the current spot price, but uncorrelated with random shocks in current production. Structural change. The industry underwent structural changes from the late ‘80s until the early ‘90s. Much of the change is likely due to continued expansion in food-service demand, especially for pizza, taco,4×8 flood tray and other Italian and Mexican foods .

Increased immigration and changes in America’s tastes and preferences have contributed to rising per capita tomato use . Commercial varieties were developed to expedite packing, shipping, and retailing in the processing market. Mechanical harvesting and bulk handling systems replaced hand harvest of processing tomatoes in the California in the 1960’s as the new varieties were introduced. Increases in yields are due to the development of higher yielding hybrid varieties and improved cultural practices such as increases in use of transplanting . The hypothesis of structural change was tested on both the supply and demand side.The acreage model was estimated assuming a partial adjustment process. Price expectations have been modeled using the previous year’s price for the period 1960-1987 and a two-year lagged price before the period 1988-2002. This was done because after the structural change, the prices exhibits an alternate pattern, so that the current price is negatively correlated with the previous year, but positively correlated with two periods before. Finally we tested the influence of the processing industry on the fresh tomato acreage, by using the price of processing tomato as a regressor. What accounts for the structural break in 1987 in fresh tomato acreage? Much of the increase in California acreage can be explained as a response to changes in consumption patterns, according to the USDA. In terms of consumption, tomatoes are the Nation’s fourth most popular fresh-market vegetable behind potatoes, lettuce, and onions. Fresh-market tomato consumption has been on the rise due to the enduring popularity of salads, salad bars, and sandwiches such as the BLT and subs. Perhaps of greater importance has been the introduction of improved tomato varieties, consumer interest in a wider range of tomatoes , a surge of immigrants with vegetable-intensive diets, and expanding national emphasis on health and nutrition.

After remaining flat during the 1960s and 1970s at 12.2 pounds, fresh use increased 19 percent during the 1980s, 13 percent during the 1990s, and has continued to trend higher in the current decade. Although Americans consume three-fourths of their tomatoes in processed form , fresh-market use exceeded 5 billion pounds for the first time in 2002 when per capita use also reached a new high at 18.3 pounds. Because of the expansion of the domestic greenhouse/hydroponic tomato industry since the mid-1990s, it is likely per capita use is at least 1 pound higher than currently reported by USDA . One medium, fresh tomato has 35 calories and provides 40 percent of the U.S. Recommended Daily Amount of vitamin C and 20 percent of the vitamin A. University research shows that tomatoes may protect against some cancers.The own price elasticity of tomatoes is estimated to be -0.32, which is highly statistically significant. Therefore demand for fresh tomatoes is relatively inelastic with respect to changes in retail prices. The own-price elasticity of carrots is -0.53 and for lettuce it is -0.71. The estimate of the own price elasticity of cabbage is positive at 0.12, which is counter intuitive. This finding, however, is not statistically significant. The estimated second-stage expenditure elasticities are all positive and range in values from 0.89 to 1.44. In all cases the expenditure elasticities are statistically significant. All of the cross prices elasticities are negative indicating that the four fresh vegetables are complements. Only the complementarities between tomato quantity with carrot and lettuce prices are statistically significant.Models for both fresh and processed tomatoes were developed and estimated. An almost ideal demand subsystem was estimated for four fresh vegetables that included tomatoes, carrots, lettuce, and cabbage. The second-stage own-price elasticities were all inelastic except for cabbage which was unexpectedly positive. The conditional expenditure or income elasticites varied from 0.89 for fresh tomatoes to 1.44 for carrots. All of the cross-price elasticities were negative indicating that the four fresh vegetables aregross complements. A plausible explanation for this is that the four commodities are used in salads, especially given that no significant complementarities were found with respect to fresh cabbage.

Ordinary least squares and instrumental variable techniques were used to obtain estimated partial adjustment acreage functions of processing tomatoes. The estimated short-run own-price elasticity estimates were between 0.47 and 0.41. Chow tests confirmed a possible structural break in the acreage function for processed tomatoes around 1988. One possible explanation of the break is the increase use of contracts around this time period. Estimated own-price elasticities for processed tomatoes in the production function varied between 0.45 and 0.55. Producers respond to prices increases in a positive manner, in accordance with theory. With respect to demand for processing tomatoes, the own-price elasticity was estimated to be – 0.18 and the cross-price estimated elasticity of tomato paste on processing tomatoes was 0.16. Thus, as the price tomato paste increases the derived demand for processed tomatoes increases, as expected. For the second period the estimated own-price elasticity in the acreage equation was 0.23 indicating that producers respond positively to increases in prices. The short-run elasticity of fresh tomato production with respect to price was 0.22 prior to 1987 and 0.27 after 1987. Thus, through out the sampling period, the own-price elasticity in the fresh tomato production function was found to be inelastic. The exchange of CO2 between forested ecosystems and the atmosphere has received significant attention in recent years in the context of global carbon cycling. In contrast, the role of forests as sources or sinks of less abundant carbon trace gases such as methane , methanol,indoor garden and other volatile organic carbon compounds is relatively poorly understood. It is challenging to measure the atmospheric exchange of such gases because of low fluxes and high spatial variability, yet scaled over large areas the mass flux of these compounds is sufficient to influence atmospheric chemistry and climate. Methane is particularly noteworthy because it is an important greenhouse gas, contributing about 20% of current radiative forcing, and a key compound governing hydroxyl radical concentrations that regulate much atmospheric chemistry. This paper provides a brief review of recent evidence suggesting that our knowledge of CH4 production in upland forests is insufficient to meet the demand for accurate accounting of radiatively active gas sources. It was motivated by the groundswell of interest that followed the first report of CH4 production by aerobic plant tissues . We begin with an overview of CH4 cycling because the topic is unfamiliar to many tree physiologists.Our current understanding is that CH4 is an end product of organic carbon degradation performed by a consortium of microbes in an O2-free environment . After a series of hydrolytic and fermentation reactions that simplify complex organic matter, microorganisms within the domain Archaea—the methanogens—produce CH4 as a respiratory end product of either H2 oxidation coupled to CO2 reduction, or acetate fermentation. Because methanogens are poor competitors for H2 and acetate, their activity is suppressed by other microbes that couple oxidation of the same electron donors to the reduction of nitrate, ferric iron and sulfate . Exposure to O2 inhibits methanogens indirectly by regenerating oxidized forms of N, Fe and S that support competing microorganisms, and directly through O2 toxicity.

Methane can be produced in soils without being emitted to the atmosphere because it is also consumed by aerobic microorganisms that oxidize CH4 to CO2. Methanotrophic bacteria grow by coupling the oxidation of CH4 to the reduction of O2. They are ubiquitous in soils and explain why upland soils are generally net CH4 sinks . Despite much research on methanotrophs in upland soils, there are no isolates of these organisms to date and little is known about their ecology. To our knowledge, no one has investigated the possibility that methanotrophs exist on the surfaces of upland plants. However, they occur symbiotically on Sphagnum tissues where they provide CO2 to support photosynthesis . Methane is produced abiotically from combustion of organic carbon during biomass burning and by thermal alteration of sedimentary organic carbon. It has been proposed that CH4 is produced abiotically in aerobic plant tissues .Despite generally inhospitable conditions, there is abundant evidence of methanogenic activity in upland soils. Andersen et al. used a 14CH4-labeling technique to infer that two forest soils produced CH4 even though the soils as a whole were net CH4 sinks. von Fischer and Hedin used a stable isotope technique to make direct measurements of gross CH4 production in 130 soil cores from 17 sites and found that even dry, oxic soils produced CH4. Aerobic forest and agricultural soils have been reported to switch from net CH4 uptake to CH4 emission in the presence of a compound that blocks CH4 oxidation . Finally, upland soils incubated anaerobically begin producing CH4 within days or weeks . Collectively, these studies suggest that upland soils harbor populations of methanogens and are capable of becoming net sources of CH4 when sufficiently wet. The possibility of CH4 production in upland soil microsites is consistent with the occurrence of denitrification and Fe reduction in upland soils, and observations that acetate, a CH4 precursor, is found in upland soils . Although studies of methanogen isolates suggest they are extremely O2 sensitive, other evidence suggests that they can tolerate a certain amount of O2 . Methanogens have been reported to survive long periods in dry and oxic soils , perhaps protected from O2 by reactive soil minerals . The evidence that upland soils can support low rates of methanogenesis suggests that CH4 oxidizing bacteria consume CH4 from two sources, the atmosphere and the soil itself . The juxtaposition of these sources may explain a puzzling observation about the response of CH4 fluxes to changes in soil water content. Andersen et al. reported that an intact upland forest soil core left uncovered at room temperature changed from a net sink for atmospheric CH4 to a net source. Isotopic data showed that CH4 oxidation fell to almost zero over this period, suggesting that CH4 oxidizing bacteria attached to soil surfaces were more sensitive to soil drying than methanogens buried in the anaerobic center of soil aggregates. The cessation of CH4 oxidation could have been caused by a physiological drought response among methanotrophic bacteria, more rapid CH4 diffusion from the soil to the atmosphere due to low tortuosity , or both. In other circumstances, decreases in soil water content can enhance CH4 oxidation in upland soils by increasing CH4 diffusion from the atmosphere into soil pore spaces . In addition to microsites, anaerobic conditions occur in saturated zones that coincide with the water table surface. Soils with a deep source of CH4 have a soil CH4 concentration profile characterized by two maxima—one at the soil surface and the other near the water table—separated by a minimum. Such profiles have been observed in a variety of upland ecosystems, including desert , temperate hardwood forest and temperate coniferous forest . It is possible that plants transport CH4 from a deep groundwater source through the transpiration stream, effectively bypassing the zone of CH4 oxidation . The most direct evidence of methanogenesis in upland soils is that they occasionally emit CH4 to the atmosphere.

We plotted the highlighted spectral bands into Fig. 3, and it is seen that the selected spectral bands in each LDA-based classification were located near known pigment peaks involved in photosynthesis and/or near spectral bands used in published indices to predict chlorophyll or nitrogen content in leaves. Thus, it appears that seed germination may be successfully classified based on reflectance in narrow spectral bands associated with the primary metabolism function and performance of plants. Although this study has demonstrated proof of concept in the potential utilization of machine-vision systems for managing ex situ seed resources several questions remain. Seeds in this study were aged under artificial conditions so the seed coat changes detected using hyperspectral analysis may not be indicative of changes that occur to seeds when stored under standard seed bank conditions. Across the plant kingdom, seeds vary greatly in colors, color patterns, shapes, and sizes. In addition, they have species-specific responses to environmental conditions. Different classification algorithms were therefore used for each of the three seed species, and successful use of this technology among other native plant species will require development of species specific classification algorithms. Availability of machine-vision systems to automate non-destructive assessments of seed germination may greatly improve the management of seed banks in future once further more detailed assessments of the technology are undertaken. Indeed, it may also be possible that such machine-vision systems can be used in advanced research into seed dormancy and other studies of seeds and their responses to environmental conditions and thus provide fresh insights into the underlying seed biology and physiology.

California organic strawberry producers face the challenge of controlling soil borne diseases,ebb and flow bench notably Verticillium wilt caused by Verticillium dahliae, Fusarium wilt caused by Fusarium oxysporum and charcoal rot caused by Macrophomina phaseolina . The main approach used to control these pathogens is crop rotation, however, many of the vegetable crops that can economically be rotated with strawberry are also hosts to one or more of the pathogens. Brassica species, notably broccoli have been found to suppress V. dahliae , but other key crops like lettuce are hosts. Furthermore, microsclerotia of V. dahliae can persist in the soil for many years. There is a clear need for additional disease control strategies and two that have shown promise are anaerobic soil disinfestation and use of mustard seed meals . Anaerobic soil disinfestation was developed in Japan and The Netherlands and has been shown to control soilborne pathogens and nematodes in strawberries. Previous studies conducted were aimed at optimizing ASD for California strawberry systems, and in conventional systems ASD was shown to be consistently effective at suppressing Verticillium dahliae in coastal California when 20 t ha-1 of rice bran was pre-plant incorporated and 75 to 100 mm of irrigation was applied in sandy-loam to clay-loam soils . California berry growers have started to adopt ASD at a commercial scale with ASD acreage increasing from 2 ha to over 400 ha in the last four years , the majority in organic production.It has also been evaluated in strawberry systems and showed neutral to positive effects on fruit production and disease suppression when used alone or in combination with other non-fumigant approaches such as ASD. Here we report on a recent rotation experiment testing ASD and MSM in a long term organic field site, and some large scale field demonstrations carried out in two different regions of California.In June 2011, a 4 replicate randomized block split-plot experiment with crop rotation as the main plots and ASD, mustard seed meal , ASD+ MSM, and untreated control as sub plots was established at the Center for Agroecology and Sustainable Food Systems organic farm on the University of California, Santa Cruz campus.

After strawberries, a legume/cereal mix winter cover crop was planted and followed with summer lettuce in all treatment plots. For management details see Zavatta et al. . Briefly broccoli ‘Gypsy’ and cauliflower ‘Snow crown’ were grown from June to September 2011 as main plots which were split prior to planting strawberries. For ASD plots, 20 t ha-1 of rice bran was applied to the bed surface and rototilled to 15 cm depth. For MSM plots 3.4 t ha-1 MSM was incorporated. For ASD+MSM plots, 16.9 t ha-1 of rice bran and 3.4 t ha-1 of MS were applied. ASD and ASD+MSM plots were drip irrigated to create and sustain anaerobic conditions for 3 weeks with a total of 108 mm of water applied. Strawberry plants ‘Albion’ were transplanted in November 2011, and fruit yield monitored from 20 plants bi-weekly from April to September 2012. A legume/cereal cover crop , 45% vetch , 10% rye was planted in all plots in the fall 2012 and grown until the following spring at which time they were mowed and incorporated into the soil. Romaine lettuce ‘Salvius’ was grown in the summer 2013. The numbers of viable V. dahliae microsclerotia in 0-15 cm of soil were estimated using a modified Anderson sampler and NP10 selective medium before and after each crop, and pre- and post-ASD treatment. V. dahliae infection on strawberry plants was evaluated at the end of the growing season for four plants per plot. A split plot ANOVA was used for statistical analysis. Two non-replicated large-scale ASD demonstration trials were conducted at a grower’s farm in Watsonville, California during the 2012-2013 growing season. One trial was on conventionally farmed land and the other in a nearby organic field. Main treatments were ASD using rice bran at 20 t ha-1 or RB 10 t ha-1 + Molasses 10 t ha-1, and subtreatments with or without pre-plant fertilizer. For the conventional field, a 673 kg ha-1 of slow release fertilizer was added pre-plant, and for the organic site feather meal at a rate of 1,122 kg ha-1. For ASD, RB 20 or 10 t ha-1 was broadcast onto the assigned plots and rototilled to a depth of 15 cm. Beds were formed, drip tapes and plastic mulch applied, and the first irrigation began two days later. Mol was diluted with water at 1:2 to 1:5 in a water tank prior to application, then 6.5 t ha-1 was applied through the drip tapes. Seven days later,hydroponic growing the remaining Mol was applied in the same manner. All plots were intermittently drip irrigated for three weeks from the first irrigation, with total irrigation amount of 60-75 mm. Soil Eh at 15 cm depth was monitored continuously using ORP sensors connected to a datalogger. Strawberries were harvested from 4 sections of each plot bi-weekly from March 19 to October 28, 2013.

A second strawberry demonstration trial was established in Oxnard, CA, in August 2014 with unreplicated 0.4 ha blocks of treatments of ASD RB 20 t ha-1, ASD RB 15 t ha-1, MSM 5.6 t ha-1 and grower standard practices. A 2.24 t ha-1 of pre-plant organic fertilizer was applied only to the grower standard plot. The same treatments, except for the RB 15 t ha-1 plot, had been applied to the same plots for the previous strawberry crop which was followed by a wheat cover crop incorporated in early August 2014. Instead of RB 15 t ha-1, the previous treatment had been ASD using RB 6.7 t ha-1 plus MSM 4.5 t ha-1 as Csources. RB and MSM were both applied on August 25 by broadcaster then beds shaped, drip lines added and TIF plastic tarp laid down. Approximately 200 mm of water was applied during ASD. Anaerobic conditions and soil temperatures were measured during ASD as above, and strawberries were harvested by the grower from January to May 2015. Plant mortality was measured by counting dead or nearly dead plants in each plot. Soil samples were taken from 0 to 6 inch depth in all plots post-treatment for microbial analysis by USDA-ARS WA using real-time quantitative PCR and terminal restriction fragment length polymorphism analysis. There was no significant effect of previous crop on the growth and yield response of strawberries irrespective of the disease management strategy used . However, when averaged across all previous crop treatments yields were highest in the ASD treatment with RB+MSM as the carbon source and ASD with RB only . This was likely due to a combination of enhanced fertility in the early season and disease suppression in the later season. Indeed disease severity was significantly reduced with the ASD treatments . What is particularly inTheresting, however, is that the numbers of V. dahliae microsclerotia present in the soil were still lower in the ASD treatments almost 2 years later . This is despite a cover crop being grown and incorporated and a lettuce crop produced in the interim, and suggests a long term suppression of disease by the ASD treatment. This agrees with observations by Goud et al. that ASD provided long term suppression of V. dahliae, but in their case the soil was undisturbed during the intervening 3 years. As discussed below, microbial community changes associated with ASD have been found to persist for many months and may be responsible for the longer term suppression. Excellent anaerobic conditions were created in this field demonstration , in both the organic and conventional fields and were associated with excellent strawberry yields . Previous work found that around 50,000 mV h below 200 mV was necessary for good control of V. dahliaand this threshold was greatly exceeded in both fields. In the conventional fields yields were equivalent to the adjacent fumigated areas, and in both cases there was little or no additional yield benefit from adding pre-plant fertilizer. Rice bran and MSM both contain large amounts of nutrients which can substitute for preplant fertilizer, however, current work is focusing on either reduced rates of application or alternative carbon sources to avoid potential excess losses of nutrients into the environment. Treatments connected by a line are not significantly different at p<0.05 . Main plot treatments are previous crop: fallow, broccoli or cauliflower; and sub plots: ASD=ASD 20 t ha-1 rice bran, ASD+MC=ASD 16.9 t ha-1 rice bran + 3.4 t ha-1 mustard seed meal, MC=3.4 t ha-1 mustard seed meal, UTC = untreated control. Macrophomina phaseolina is present at this location and is a serious concern for strawberry production. In both years of the demonstration ASD greatly improved crops yields relative to the grower’s standard practice and reduced disease severity as measured by crop mortality . The second year trial again demonstrated that no pre-plant fertilizer was necessary at ASD RB 20 t ha-1 plot. There was a significant shift in fungal community composition in soils post ASD, relative to the MSM and the grower standard . However, prior to treatment the following year and after an intervening wheat cover crop, the ASD RB9 treatment still clus.

Thered together and were distinct from the other plots again indicating longer term changes in the soil following ASD. Following the second year with ASD treatment soil fungal communities again showed very distinct clustering based on treatment . This pattern has been observed in other field trials and we are in the process of identifying which species become more prevalent following ASD and if this is related to type of carbon source. Initial screening of NextGen sequencing data suggest that enhanced strawberry yields in response to ASD conducted using rice bran was associated with elevated detection of sequences representing various genera within the Flavobacteria known to have anti-fungal properties. In this opinion-based article, we discuss how indirect effects of drought may adversely affect both the performance of systemic insecticides and also lead to increased risk of insect pests developing behavioral insecticide resistance. Furthermore, we argue that the possible adverse effects of drought on the performance of systemic insecticides has to be given increased research attention, as climate change will likely lead to increased severity and frequency of drought in many agricultural regions. The Food and Agriculture Organization of the United Nations defines a pesticide as “Any substance or mixture of substances intended for preventing, destroying, or controlling any pest … The term includes substances intended for use as a plant growth regulator, defoliant, desiccant, or agent for thinning fruit or preventing the premature fall of fruit.” . Systemic insecticides are chemicals absorbed by plants and distributed internally via the vascular system, delivering the insecticide to untreated plant tissues .

California has the largest and most complex agricultural labor market in the United States, reflecting seasonal employment demands, the predominance of immigrant workers and the significant role of labor contractors in matching workers and jobs. Whether measured in sales, production or acres, California agriculture expanded in the 1990s . Farm sales reached $27 billion in 2000, with about 77 million tons of crops produced on 8.8 million acres. More than half of these sales were in fruits and nuts, vegetables and melons, and horticultural specialties , such as flowers and nursery products. Rising yields meant that more tons of vegetables were produced from the same acreage, while acreage of fruits and nuts rose from 2 million acres in 1990 to 2.4 million acres in 2000, a 19% increase over the 1990s. Many FVH commodities are labor intensive, with labor accounting for 15% to 35% of production costs. Most of the workers employed on FVH farms are immigrants from Mexico, and a significant percentage are believed to be unauthorized . In recent years, several proposals have aimed to reduce unauthorized worker employment in agriculture . In September 2001, Mexican President Vincente Fox called for a U.S.-Mexico labor migration agreement so that “there are no Mexicans who have not entered this country [U.S.] legally, and that those Mexicans who come into the country do so with proper documents. Regularization does not mean rewarding those who break the law. Regularization means that we give legal rights to people who are already contributing to this great nation.” President George Bush agreed: “When we find willing employer and willing employee,plastic pot manufacturers we ought to match the two. We ought to make it easier for people who want to employ somebody, who are looking for workers, to be able to hire people who want to work” .

The United States and Mexico appeared close to agreement on a program to legalize farm and other workers before September 11, 2001. However, after the war on terror was declared, the momentum for a newguest-worker program and the legalization of immigrants already in the country slowed. In summer 2003, there were several new proposals for a migration agreement with Mexico to legalize the status of currently unauthorized workers and allow some to earn immigrant status by working and paying taxes in the United States. There is little agreement, however, on what impacts such a program would have on California’s farm labor market. We used a unique database to examine farm employment trends in California agriculture. The data suggests that: about three individuals are employed for each year-round equivalent job, helping to explain low farm worker earnings; there was a shift in the 1990s from crop farmers hiring workers directly to farmers hiring via farm labor contractors ; and there is considerable potential to improve farm labor market efficiency, by using a smaller total workforce with each worker employed more hours and achieving higher earnings.California employers who pay $100 or more in quarterly wages are required to obtain an unemployment insurance reporting number from the California Employment Development Department . The EDD then assigns each employer or reporting unit a four-digit Standard Industrial Classification or, since 2001, a six-digit North American Industry Classification System code that reflects the employer’s major activity . Major activities are grouped in increasing levels of detail; for example, agriculture, forestry and fisheries are classified as a major industrial sector and, within this sector, SIC 01 is assigned to crops, 017 to fruits and nuts and 0172 to grapes. We defined “farm workers” as unique Social Security numbers reported by farm employers to the EDD, and then summed their California jobs and earnings. This enabled us to answer questions such as how many farm and non-farm jobs were associated with a particular SSN or individual in 1 year, and in which commodity or county a person had maximum earnings.

We adjusted the raw data before doing the analysis. Farm employers have reported their employees and earnings each quarter since 1978, when near universal UI coverage was extended to agriculture. Although it is sometimes alleged that farm employers, especially FLCs, do not report all their workers or earnings, there is no evidence that under reporting of employees or earnings is more common in agriculture than in other industries that hire large numbers of seasonal workers, such as construction. We excluded from the analysis SSNs reported by 50 or more employers in 1 year . We also excluded wage records or jobs that had less than $1 in earnings and jobs, or that reported earnings of more than $75,000 in one quarter. These adjustments eliminated from the analysis 2,750 SSNs, 62,571 wage records or jobs and $803 million in earnings. These exclusions were about 0.25%, 2.7% and 6.1% of the totals, respectively, and are documented more fully in Khan et al. . There is no single explanation for the outlier data we excluded. In some cases, several workers may share one SSN, while in others our suspicion that a SSN had “too many” jobs may represent data-entry errors. During the 1990s, the Social Security Administration cleaned up SSNs, including threatening to fine and reject tax payments from employers with too many mismatches between SSNs and the names associated with those SSNs, which should have reduced the number of SSNs reported by employers. We think the rising number of SSNs reflects more individuals employed in agriculture, not more noise in the data.Agricultural employment can be measured in three major ways: at a point in time, as an average over time or by counting the total number of individuals employed over some period of time.If 100 workers are employed during each month and there is no worker turnover from month to month, then point in time, average and total employment is 100. However, agricultural employment during the six summer months may be 150, versus 50 during the six winter months, meaning that point, average and total employment counts differ.

We began with all SSNs reported by agricultural employers , summed the jobs and earnings of these SSNs within each SIC code, and assigned each SSN to the four-digit SIC code in which the worker had the highest earnings. This means that a SSN reported by a grape employer as well as by an FLC would be considered a grape worker if his highest-earning job was in grapes. Farm workers had a total of 1.5 million farm jobs in 1991, 1.7 million in 1996 and 1.8 million in 2001. One-quarter also had at least one non-farm job — about 407,000 workers were both farm and non-farm workers in 1991, 453,000 in 1996 and 697,000 in 2001 . The total California earnings of persons employed in agriculture were $11.1 billion in 1991, $12.0 billion in 1996 and $15.8 billion in 2001 . The share of total earnings for farm workers from agricultural employers was 77% in 1991, 77% in 1996 and 71% in 2001, indicating that in the late 1990s,black plastic plant pots wholesale farm workers tended to increase their supplemental earnings via non-agricultural jobs. Average earnings per job were highest in livestock, $13,800 per job in 2001. There was little difference between average earnings per job in agricultural services and crops . Average earnings per job were higher for the non-farm jobs of agriculture workers than for agriculture jobs .In 2001, California’s farm workers held 2.5 million jobs, including 1.8 million jobs with agricultural employers. These agricultural jobs included 630,000 in crops, 69,000 in livestock and 1.1 million in agricultural services. The agricultural services sector includes both farm and non-farm activities, such as veterinary and lawn and garden services; FLCs accounted for 70% of the employees reported by farm agricultural services. Fruits and nuts accounted for 53% of the crop jobs, dairy for 39% of the livestock jobs and FLCs for 58% of the agricultural services jobs. The major change between 1991 and 2001 was the drop of 54,000 jobs in crop production and increase of 313,000 jobs in agricultural services. We placed SSNs in the detailed commodity or SIC code that reflected the maximum reported earnings for the worker, and considered workers to be primarily employed in the SIC with maximum earnings. In 2001, there were 877,000 primary farm workers, and they included 322,000 reported by crop employers, 50,000 reported by livestock employers and 504,000 reported by agricultural service employers. Fruit and nut employers accounted for 47% of the crop-reported workers, dairy for 40% of the livestock-reported workers and FLCs for 44% of the agricultural services–reported workers. The major change between 1991 and 2001 was the increase in number of SSNs with their primary job in agriculture — from 758,000 to 877,000. There was a slight drop in the number of workers reported by crop employers, a slight increase in livestock workers and a sharp 135,000 increase in agricultural services workers, anchored by a 59,000 increase in workers reported by FLCs in 2001. Most farm workers had only one job. In 2001, 53% of the SSNs were reported by only one employer to the EDD, 26% were reported twice, 12% three times, 5% four times and 4% five or more times.

During the 1990s, about 65% of farm workers were reported by one agricultural employer only, 17% to 21% by two agricultural employers, 5% by at least two agricultural employers and one non-farm employer, and 9% to 12% by one farm and one non-farm employer. In the three-digit SIC codes representing more detailed commodity sectors, 60% to 83% of the employees had only one job. For example, in 2001 79% of the employees reported by dairy farms had one dairy farm job, while 7% also had a second agricultural job — 3% had a dairy job, a second farm job and a non-farm job, and 11% had a non-farm job in addition to the dairy job. About two-thirds of the employees of FLCs and farm management companies had only jobs with one such employer; 22% had another farm job; 6% had an FLC job, another farm job and a non-farm job; and 6% had a non-farm job in addition to the FLC job. Even more detailed four-digit SIC codes showed the same pattern: the commodities or SICs most likely to offer year-round jobs such as dairies and mushrooms had 70% to 80% of employees working only in that commodity, while commodities or SICs offering more seasonal jobs, such as deciduous tree fruits and FLCs, had 53% to 63% of employees working only in that commodity. At the four-digit, SIC-code level, the five largest SICs accounted for about 45% of the agricultural wages reported.Agricultural employers paid a total of $11 billion in wages in 2001, an average of $10,200 per worker . Earnings were highest for the 64,000 workers primarily employed in livestock; they averaged $14,800, followed by those primarily employed by crop employers and those employed by agricultural farm services, custom harvesters and FLCs . There was considerable variation in earnings among workers in agricultural farm services: workers in soil preparation services averaged $21,100 in 2001, versus $12,700 for crop preparation services for market and $4,400 for FLC employees. The average earnings of primarily farm workers varied significantly, even within detailed four-digit SIC codes — in most cases, the standard deviation exceeded the mean wage . Median earnings were generally less than mean earnings, reflecting that higher wage supervisors and farm managers pulled up the mean. If the workers in detailed commodities are ranked from lowest-to-highest paid, the lowest 25% of earners in an SIC category generally earned less than $4,000 a year. For example, among workers primarily employed in vegetables and melons in 2001 , the first quartile or 25th percentile of annual earnings was $3,000. This reflects relatively few hours of work — if these workers earned the state’s minimum wage of $6.25 an hour in 2001, they worked 480 hours. The 25th percentile earnings cutoff was lowest for those employed primarily by FLCs, only $634, suggesting that FLC employees receiving the minimum wage worked 101 hours. The highest 25th percentile mark was in mushrooms , $9,491, which reflects 1,519 hours at minimum wage. The 75th percentile marks the highest earnings that a non-supervisory worker could normally expect to achieve — 75% of workers reported earning less than this amount and 25% earned more.

Five hotpots were found in chloroplast genome of Veroniceae , and two universal marker, trnH-psbA and matK were identified, respectively. Then highly variable regions were selected as potential molecular markers for Fritillaria, including ycf1, which was also selected in this study. Sequences of these variable regions founded in this study could be regarded as potential molecular markers for species identification and evolutionary studies and have been shown to be valuable for studies in other groups.Oligonucleotide repeats play an important role for generating indels, inversion and substitutions. Repeat sequences in the chloroplast genome could provide valuable information for understanding not only the sequence divergence but the evolutionary history of the plants. We have detected five types of large repeats in the seven Pulsatilla cp genomes. Among them, the most common repeat types are forward and palindromic repeats, followed by reverse repeats, and only little complement repeats were found in Pulsatilla cp genomes . Most of the repeats were short, ranging from 30–49 bp . We also identified multiple microsatellite repeats, also known as simple sequence repeats or short tandem repeats. Due to their codominant inheritance and high variability, SSRs are robust and effective markers for species identification and population genetic analyses. Most of the mononucleotide repeats were composed of A/T. The other microsatellites types were also dominated by AT/TA, with very little G/C . In this study, plentiful microsatellite loci were found through the comparative analysis of Pulsatilla cp genome sequences. In total,raspberry grow in pots we detected six types of microsatellite based on the comparison of seven Pulsatilla cp genomes . Each Pulsatilla cp genome had 69–87 microsatellites. The lengths of repeat motifs of these microsatellites ranged from 10 to 21 bp .

Among the four structural regions in the cp genomes, most of the repeats and microsatellites were distributed in LSC, followed by SSC, and fewest in IRa/IRb , which were also reported in other studies in angiosperms. These SSRs and repeat sequences are uncorrelated with genome size and phylogenetic position of the species, but will provide important information for further studies of phylogenetic reconstruction and infra- and inter-specifc genetic diversity.Chloroplast genomes have been widely used and have made significant contributions to phylogeny reconstruction at different taxonomic levels in plants. To better clarify the evolutionary relationships within Pulsatilla, we used each data set to construct phylogenetic trees using the ML analytical methods. We also construct phylogenetic trees with those eight highly variable regions using the ML, MP analytical methods. All tree topology structures were identical. Therefore, here we presented the phylogenetic studies using the ML tree with the support values from the MP analyses recorded at the corresponding nodes . The phylogenetic tree based on all data sets from the complete plastid genome sequences yielded the same topology. The phylogenetic tree based on chloroplast genome differed from that of the DNA barcode combination rbcL+matK+trnH-psbA, but with higher support values. The phylogenetic trees based on data from complete plastid genome sequences showed that the species of Pulsatilla formed a monophyletic group which in turn includes two strongly supported clades. One clade comprised P. alpina and P. occidentalis, members of subg. Preonanthus. The other comprised two subclades: members of P. hirsutissima, P. ludoviciana, P. multifdi, P. patens and P. vernalis, and species of P. chinensis, P. dahurica, P. grandis and P. pratensis. All the species of the two subclades are members of the subg. Pulsatilla. These results were congruent with our former results based on universal markers.

In phylogenetic analyses, compared to the combination of barcodes, the full chloroplast genome sequence data formed distinct clades with high bootstrap support, improving the inadequate resolution of barcodes combination. The LSC regions and coding regions have the same topology structures with robust support. However, sequencing of genomic DNA is still expensive. It is necessary to utilize variation within chloroplast regions for rapid species-specific assay. Here we found that phylogenetic inference based on highly variable regions yielded a tree with the same topology as the one recovered based on complete chloroplast genome sequences, demonstrating the high utility of hot spots of variability for species identification and phylogenetic analysis. More samples and laboratory works are needed in the future to increase the number of these variable regions available for study.High frequency irrigation systems involve fastidious planning and complex designs, so that timely and accurate additions of water and fertilizer can result in sustainable irrigation. At the same time these production systems are becoming more intensive, in an effort to optimise the return on expensive and scarce resources such as water and nutrients. Advanced fertigation systems combine drip irrigation and fertilizer application to deliver water and nutrients directly to the roots of crops, with the aim of synchronising the applications with crop demands , and maintaining the desired concentration and distribution of ions and water in the soil . Hence a clear understanding of water dynamics in the soil is important for the design, operation, and management of irrigation and fertigation under drip irrigation . However, there is a need to evaluate the performance of these systems, because considerable localised leaching canoccurnear the driplines, evenunderdeficitirrigation conditions .

The loss of nutrients, particularly nitrogen, from irrigation systems can be expensive and pose a serious threat to receiving water bodies . Citrus is one of the important horticultural crops being grown under advanced fertigation systems in Australia. Fertigation delivers nutrients in a soluble form with irrigation water directly into the root-zone, thus providing ideal conditions for rapid uptake of water and nutrients. Scholberg et al. demonstrated that more frequent applications of a dilute N solution to citrus seedlings doubled nitrogen uptake efficiency compared with less frequent applications of a more concentrated nutrient solution. Delivery of N through fertigation reduces N losses in the soil-plant system by ammonia volatilisation and nitrate leaching . However, poor irrigation management, i.e., an application of water in excess of crop requirements, plus the storage capacity of the soil within the rooting depth, can contribute to leaching of water and nutrients below the root zone. Therefore, optimal irrigation scheduling is important to maximise the uptake efficiencies of water and nutrients . Most of the citrus production along the Murray River corridor is on sandy soils, which are highly vulnerable to rapid leaching of water and nutrients. Nitrogen is the key limiting nutrient and is therefore a main component of fertigation. An increasing use of nitrogenous fertilizers and their subsequent leaching as nitrate from the root zone of cropping systems is recognised as a potential source of groundwater contamination,30 planter pot because the harvested crop seldom takes up more than 25–70% of the total applied fertilizer . Several researchers have reported substantial leaching of applied N under citrus cultivation in field conditions . Similarly, in lysimeter experiments, Boaretto et al. showed 36% recovery of applied nitrogen by orange trees, while Jiang and Xia reported N leaching of 70% of the initial N value, and found denitrification and leaching to be the main processes for the loss of N. These studies suggest that knowledge of the nitrogen balance in cropping systems is essential for designing and managing drip irrigation systems and achieving high efficiency of N fertilizer use, thereby limiting the export of this nutrient as a pollutant to downstream water systems. Quantifying water and nitrogen losses below the root zone is highly challenging due to uncertainties associated with estimating drainage fluxes and solute concentrations in the leachate, even under well-controlled experimental conditions . Moreover, direct field measurements of simultaneous migration of water and nitrogen under drip irrigation is laborious, time-consuming and expensive . Hence simulation models have become valuable research tools for studying the complex and interactive processes of water and solute transport through the soil profile, as well as the effects of management practices on crop yields and on the environment . In fact, models have proved to be particularly useful for describing and predicting transport processes, simulating conditions which are economically or technically impossible to carry out in field experiments . Several models have been developed to simulate flow and transport processes, nutrient uptake and biological transformations of nutrients in the soil .

HYDRUS 2D/3D has been used extensively for evaluating the effects of soil hydraulic properties, soil layering, dripper discharge rates, irrigation frequency and quality, timing of nutrient applications on wetting patterns and solute distribution because it has the capability to analyse water flow and nutrient transport in multiple spatial dimensions . In the absence of experimental data we can use multidimensional models solving water flow and nutrient transport equations to evaluate the multi-dimensional aspect of nitrate movement under fertigation . However, earlier simulation studies have reported contradictory results on nitrate distribution in soils. For example, Cote et al. reported that nitrate application at the beginning of an irrigation cycle reduced the risk of leaching compared to fertigation at the end of the irrigation cycle. On the other hand, Hanson et al. reported that fertigation at the end of an irrigation cycle resulted in a higher nitrogen use efficiency compared to fertigation at the beginning or middle of an irrigation cycle. These studies very well outlined the importance of numerical modelling in the design and management of irrigation and fertigation systems, especially when there is a lack of experimental data on nutrient transport in soils. However, there is still a need to verify the fate of nitrate in soils with horticultural crops and modern irrigation systems. Therefore, a lysimeter was established to observe water movement and drainage under drip irrigated navel orange, and to calibrate the HYDRUS 2D/3D model against collected experimental data. The model was then used, in the absence of experimental data on nitrate, to develop various modelling scenarios to assess the fate of nitrate for different irrigation and fertigation schemes.The study was conducted on a weighing lysimeter assembled and installed at the Loxton Research Centre of the South Australian Research and Development Institute. The lysimeter consisted of a PVC tank located on 1.2 m × 1.2 m pallet scales fitted with 4 × 1 tonne load-cells, and connected to a computerised logging system which logged readings hourly. A specially designed drainage system placed at the bottom of the lysimeter consisted of radially running drainage pipes, which were connected to a pair of parallel pipes, which facilitated a rapid exit of drainage water from the lysimeter . These pipes were covered in a drainage sock and buried in a 25-cmlayer of coarse washed river sand at the base of the lysimeter, which ensured easy flushing of water through the drainage pipe. A layer of geo-textile material was placed over the top of the sand layer to prevent roots growing down into it, as this layer was intended to be only a drainage layer. A healthy young citrus tree was excavated from an orchard at the Loxton Research Centre and transplanted into the lysimeter. A soil profile approximately 85 cm deep was transferred to the tank with the tree and saturated to remove air pockets and to facilitate settling. The final soil surface was around 10 cm below the rim of the tank. Soil samples were collected from0 to 20, 20 to 40, 40 to 60, 60 to 85, and 85 to 110 cm depths to measure bulk density and to carry out particle size analysis. Two months after transplanting, the lysimeter was installed amongst existing trees in the orchard. Measurements were initiated after about six months, in order to enable the plant to adjust to the lysimeter conditions. The lysimeter was equipped with Sentek® EnviroSCAN® logging capacitance soil water sensors installed adjacent to the drip line at depths of 10, 20, 40, 60, and 80 cm to measure changes in the volumetric soil water content.The experimental site was approximately 240 m from an established weather station, which measured air temperature, relative humidity, wind speed , rainfall, and net radiation.Irrigation was applied using 3 pressure compensated emitters with a discharge rate of 4 L h−1. Emitters were located on a circle 25 cm away from the tree trunk at an equal distance from each other . The irrigation schedule was based on the average reference evapotranspiration during the last 10 years at the site, multiplied by the crop coefficient taken from Sluggett .