The cuticles of tomato and soybean leaves are much thinner, and therefore foliar absorption will be more rapid. Here, our findings indicate that a relatively high percentage of total Zn in the leaves from foliar applications would be bound to the cell wall rather than be present in a soluble, intercellular form. Zn mainly exists in a bound form in the cytoplasm, as well as in other cellular compartments, to avoid uncontrolled Zn2+ binding to nontarget sites. Various studies have also shown that the abundance of negatively charged sites in the cell wall limits the translocation of positively charged Zn2+ . For example, the major component of pectin in cell walls is polygalacturonic acid, which has a high binding capacity for Zn2+.Our previous research also shown that most of the Zn was deposited in the cell walls of apple leaves in coordination with different stages of development, with the nature of Zn binding being dependent on tissue age, as old and mature leaves exhibited higher proportions of Zn in the cell wall than young leaves. In general, there is low potential for remobilization of foliar-absorbed nutrients until the potential binding sites for that element within the leaf are saturated. Therefore, we hypothesized that there would be very limited utilization of Zn applied to the adaxial leaf surface because of the highly hydrophobic cuticular wax layer that would limit Zn penetration,dutch bucket wholesale as well as having a higher capability for cationic Zn fixation.

Previous studies have suggested that there is high diversity among trichome structure and function, which can hinder or promote water penetration. In the present study, we found that a relatively high trichome density resulted in a high degree of hydrophobicity , which was not consistent with the results of several studies that reported that trichomes may participate actively in the absorption of water and foliar-applied nutrient solutions. These differences may be related to the surface roughness provided by the high density, chemical composition, and structure of trichomes on apple leaves. The high density of nonglandular trichomes creates the hydrophobic characteristic of apple abaxial leaf surfaces to a certain degree, and repulsion of fertilizer drops by abaxial leaf trichomes could hinder the penetration of liquid Zn solutions. Our findings were similar to the results of Fernandez et al., who used Holm oak as a model to assess the capability of abaxial surfaces versus adaxial surfaces to absorb surface-deposited water drops. Another study by Li et al. also demonstrated that trichomes are not part of the primary pathway through which foliar-applied Zn moves across the leaf surface, even though Zn was found to accumulate around the base of trichomes. On the other hand, the possible contribution of stomata to the penetration of leaf-applied chemicals has been a matter of controversy for many decades, and it is still not fully understood whether stomata allow the penetration of foliar-applied solutes. Nonetheless, several studies show a clear effect of stomata in promoting foliar solute penetration, even in the absence of surfactants. However, the underlying mechanism is not understood, although it is hypothesized that penetration of solutes through stomata is restricted by their morphological and physical properties. Our study provides further evidence that supports the relevance of stomatal nutrient absorption, as indicated by the increased penetration rates from plant surfaces whose stomata were present and open.

Information about nutrient interactions will guide fertilization practices and optimize the efficiencies of fertilization strategies. The levels of two different elements for each pixel in an XRF data set can often provide important chemical information.Here, we analyzed the ionic changes that occurred in leaves in response to foliar Zn application to reveal the interactions between and homeostasis of Zn and other metals. Induction of element accumulation was observed after foliar Zn penetration for all the tested nutrients . One of the possible reasons for this promotional effect may be the important role of Zn in the constituents of enzymes involved in photosynthesis. The concentration of water-soluble Zn in leaves was found to be closely correlated with the levels of chlorophyll. The positive influence of foliar Zn fertilizer on photosynthesis and chlorophyll synthesis may help increase mineral nutrient absorption and accumulation in functional mature leaves. A reduction in this accumulation was observed for Mn and Fe along with a further increase in Zn. This might be due to the possible Zn toxic reaction in the treated leaves because locally toxic levels of Zn may occur at the point of fertilizer application. Excess Zn may cause uncontrolled displacement of essential cofactor metal cations such as Mn2+ and Fe2+. For example, it was reported that exposure to high Zn can subsequently inhibit PSII activity by replacing Mn in thylakoid membranes. The underlying mechanisms of the above effects remain uncertain, and further research is needed to uncover the complex interaction between foliar Zn status and plant nutrient responses. The increased spatial correlation of Zn versus P along with the penetration of foliar Zn fertilizer , together with the presence of Zn-phytic acid in the treated leaves , suggested that P may play an important role in the complexation of Zn, potentially in response to toxic concentrations of Zn.

The formation of Zn-phytic acid in vegetative tissues was reported to occur under two circumstances: within some hyperaccumulator species or under conditions in which plants are exposed to high exogenous Zn concentrations, the binding of Zn to phytic acid helps plants limit Zn mobility and reduces toxicity. Our results were consistent with the previous findings showing an increased proportion of Zn-phytate in wheat leaves after Zn-EDTA and ZnSO4 applications. Agronomic strategies aim to deliver phyto available Zn via the application of Zn fertilizers to leaves. Knowledge of the ability and mechanism of Zn to penetrate the leaf surface from the site of application and the factors that are associated with penetration efficiency is critical for the development of strategies for Zn bio-fortification of crops. In the present study, the high spatial resolution and direct imaging capability of XRF was valuable for distinguishing differences in Zn penetration of epidermal cells,blueberry grow pot and these techniques provided a powerful strategy for investigating foliar microelement applications to plants with a high level of sensitivity. By the use of micro- and nano-XRF techniques, our results provide direct visual evidence for Zn penetration of the leaf surface. Furthermore, we provide new insights that can help in the development of Zn bio-fortification approaches in fruit crops. To further increase the Zn content in food crops, future studies need to focus on elucidating the pathway by which Zn penetrates plant leaves, the subcellular compart mentation of Zn, and the specific formulation of Zn foliar fertilizers.Educational theorists have recently begun to call for more immersion of trainee scientists and medical practitioners in the humanities, particular through the study of history as part of their educational program. In a widely circulated analysis that first appeared in an August 2014 Inside Higher Education supplement, Elizabeth H. Simmons suggests that “to fully prepare for careers in science, it is essential that students grasp how the impetus for scientific work arises from the world in which the scientist lives, often responds to problems the scientist has personally encountered, and ultimately impacts that society and those problems in its turn.” Every nascent scientist, according to Simmons, “should read, think, and write about how science and society have impacted one another across cultural and temporal context” because “ethical concepts absorbed” in such study will help them “hew more closely to the scientific ideal of seeking the truth.”Since C.P. Snow’s famous 1959 Rede Lecture lamented the gap between the “Two Cultures” of the sciences and humanities, academic initiatives such as Stanford University’s Science, Society, and Technology program have been founded to assert the wider societal impact of the natural sciences.Yet far fewer programs and courses have been designed to show how scientific endeavors might benefit from the study of the humanities, particularly history.

The newest version of the Medical School Admissions Test now encompasses questions on the psychological, social, and biological determinants of behavior to ensure that admitted medical students are “prepared to study the sociocultural and behavioral aspects of health.” But as Simmons notes, while “pre-medical and engineering students are being required to learn about issues linking science and culture, most students in science fields are still not pushed to learn about the human context of their major disciplines.”As Dean of the College of Liberal Arts at Michigan State University, Simmons may well know that her state’s own educational system incorporates a project with important implications for the nexus between the humanities and the sciences. The Decolonizing the Diet project at Northern Michigan University has begun to show how the study of early American history – particularly Native American history – can inform public policy and healthcare paradigms, while also impacting the agenda of cutting edge research in the biological, nutritional, and ecological sciences. In the project, students and local community members have learned how the move away from their ancestral nutritional principles has been detrimental to the health of Native Americans and the wider ecology of the Great Lakes region of North America. Aware of disproportionate instances of diabetes, heart-disease, depression, fertility problems, and chronic inflammatory conditions, project members have studied and then simulated pre-European contact Native American diets, using historical and anthropological methods. They have even begun to produce medically-relevant data on improvements in health and well-being after their nutritional intervention. A similar project, the American Indian Health and Diet Project , has been inspired by the work of Devon Abbott Mihesuah, a Choctaw historian and writer. Through her teaching and writing, Mihesuah has used the study of history, anthropology, and literature to inform contemporary health and nutritional practices – coming a little closer to the diets that many Native American communities consumed prior to European contact.The Decolonizing the Diet project is currently restricted to the Great Lakes region, where the authors of this article reside and work. As a professor of history and a researcher in biological science, we aim to show how a model course in the University of Minnesota system might offer a new pedagogical paradigm for the study of ancestral health principles. The course builds on the aims of the existing projects detailed above. But it also synthesizes their methods and objectives with the latest peer-reviewed research in evolutionary health and nutrition, bringing about new insights and research paradigms relating to the link between nutrition, metabolic health, and immunity to infectious disease. Some recently founded nutritional programs have aimed to educate public-sector workers in order to prevent a mismatch between their optimal nutrition, as understood from within an evolutionary health framework, and their current eating habits.This article demonstrates how the same public health agenda might be explored from within a large public research university system, led by a joint effort between professors in the humanities and teachers and researchers in the biological sciences. The 1862 Morrill Land-Grant Acts defined the American public research university’s ongoing mission “to promote the liberal and practical education of the industrial classes in the several pursuits and professions in life.”From the 1950s scientists used the land-grant system to ally their scientific and nutritional research with new policies that came to be promoted by the federal government. Within the University of Minnesota system, for example, Ancel Keys famously sought a new research agenda in order to question the health benefits of fats from animals, fish, and dairy. Contrary to those who highlighted the problematic medical implications of foods that were readily converted into glucose, as well as the potential inflammatory effects of polyunsaturated fats, Keys and others used their land-grant mandate to promote research that supported the government of the day in its stated desire to increase agricultural output in soy, wheat, corn, and seed oils.Scholars, scientists, public policy analysts, and journalists have begun to suggest that the resulting federally recommended food-pyramid has worsened certain aspects of American public health, particularly through the associated growth of metabolic disorders such as diabetes, and even in the prevalence of certain forms of heart disease .This article examines the present state of historical research in early American studies in order to assess whether similarly problematic associations and health markers appeared among Native American communities many decades – and even centuries – before the development of the problematic modern American food pyramid.

Gene Ontology enrichment was performed on the male and female differentially expressed genes showing at least twofold expression differences between sexes. Gene Ontology enrichment for the male-upregulated genes showed a significant over representation of 76 terms, including pollen and anther development, male gamete development, the terpenoid biosynthesis pathway, and cytokinin metabolism . The male-upregulated gene set showed an under representation for 71 terms, including terms relating to transcription and RNA regulation, splicing, and modification . Among the female-upregulated genes, there was an over representation of RNA transcription and metabolism and shoot and organ development , and an under representation of 47 terms, including cell metabolism and biosynthesis .WGCNA network analysis was performed to explore pathways that may be involved in floral sex dimorphism and development.In total, 17,953 genes were assigned to modules, accounting for 49.8% of all expressed genes and alternative transcripts. The remaining unassigned genes were placed in the “gray” module. Three modules accounted for the majority of assigned genes: the “purple”, “cyan”, and “brown” modules. The “purple” module was strongly correlated to the female sex and captured 30.2% of all female-expressed genes , this module likely represents the genes involved in primary and secondary female sex dimorphism. In the “purple” module, 17 GO terms were enriched,fodder system including RNA and nucleic acid metabolism and regulation, photosynthesis, and phenylpropanoid metabolism . This is consistent with the female differential expression of the majority of genes in this module and supports the contention that this module may be responsible for female sex-dimorphic traits.

The cyan module was the most male-correlated and included 45.5% of all male-upregulated genes likely representing the genes in primary and secondary male sex dimorphism pathways. The “cyan” module contained 230 significantly enriched GO terms, notably multiple terms related to pollen development . The “brown” module consisted mostly of genes not showing differential expression, and probably represents gene pathways involved in basic cell and biological processes.A total of 1,381,813 cis-eQTL and 811,499 trans-eQTL were identified after accounting forcovariates. Notably, there appears to be an eQTL “hotspot” on chr07, to which the expression levels of 550 genes are associated The exceptional number of genome-wide eQTL at this locus suggests that it may have a major role in regulating sex dimorphic gene expression in catkin tissue. Expression levels of 2127 genes were found to be associated with polymorphisms in the SDR, of which 1686 were trans . To identify top-level regulatory and intermediate pathway genes, 2127 genes with eQTL in the SDR were a subset for genes with involvement in secondary metabolism , hormone signaling , RNA splicing and regulation , or transcription . Of the 96 genome-wide transcription factors found to have SDR eQTL, 70 were differentially expressed in either males or females. Because these transcription factors include genes relating to floral development, phenylpropanoid production, and cytokinin signaling, they are candidates for top-level regulatory genes that may regulate further downstream expression. However, confirmation of such roles will require further investigation using methods such as ChIPSeq and DAP-Seq. Fourteen MADS-box and floral development genes, 15 phenylpropanoid pathway genes, and five terpenoid pathway genes were also found to have eQTL in the SDR , representing candidates for intermediate pathway genes directly responsible for dimorphisms in floral morphology, pigmentation, and volatile and secondary metabolite profiles.We identified eleven genes that are strong candidates as master regulators of sex using the following criteria: presence on chr15W and absence from chr15Z, a significant log2 M:F < − 1, presence in the femalecorrelated “purple” WGCNA module, and gene annotation either consistent with a possible floral sex dimorphism pathway or of unknown function.

Genes meeting all four of these criteria are expected to be present only in females and have expression levels and module membership that would implicate them in sex dimorphism. Four copies of ARR17, a truncated AGO4 gene, DRB1, GATA15, a CCHC zinc finger nuclease, and three genes coding hypothetical proteins met these criteria and were identified as candidate master regulator genes .Total RNA-Seq and small RNA-Seq captured the unique sex-specific transcriptomic profiles during catkin development, after floral meristem differentiation and prior to maturation of any stamens or pistils. Within a single maturing catkin, there are hundreds of individual flowers across a range of developmental stages, resulting in pooled expression data from across floral development time points as well as tissue types . In addition to the primary sex dimorphism genes responsible for anther and carpel development, this enables the identification of secondary sex dimorphisms, such as genes involved in pigmentation, volatile emission, and differences in catkin phenology, which can also inform differences in vegetative emergence and secondary metabolites. By using network analysis and incorporating genomic data through eQTL, we can hypothesize how the SDR may regulate differential gene expression in catkins. Nearly two-thirds of all expressed genes in the floral tissue exhibited differential expression between males and females. This number is due in part to the large sample size of 159 individuals, whereby there is enough statistical power to detect even slight differences in expression. Nevertheless, over 21% of the expressed genes showed at least twofold expression differences between sexes, providing evidence of global expression differences, which would require robust transcriptional regulation, ultimately leading back to the sex-determinant genes in the SDR. These genes provide important clues about the regulation of sex determination in this species and the molecular mechanism responsible for diecy and floral sex dimorphism, as described in more detail below.Four copies of ARR17, a type A cytokinin-response regulator, in the SDR, show high levels of expression in female S. purpurea: Sapur.15WG073500, Sapur.15WG073900, Sapur.15WG074000, and Sapur.15WG075200.

Two additional copies of ARR17 are present on chr19 but are not differentially expressed. The cytokinin signaling pathway has been proposed as a common pathway for sex determination in angiosperms. Cytokinin-response regulators serve as feminizing factors in Actinidia, where they are master regulators, and Diospyros, where they act as top-level regulators downstream of the SDR. There is recent evidence implicating ARR17 as the master regulator of sex in the closely related genus Populus, where it may function as a feminizing factor whose expression is suppressed in males by small RNAs. The presence of two complete copies of ARR17 on S. purpurea chr19, expressed in both males and females, suggests that the dosage of ARR17 may play a role in sex determination in willow. Interestingly, these findings suggest a different mechanism for ARR17 than the leading model for sex determination in Populus proposed by Müller et al.. They proposed that functional ARR17 in P. alba is a feminizing factor, and in XY species, ARR17 is silenced by inverted repeats on the Y chromosome through the RNA-directed DNA methylation pathway, leading to a male phenotype. To confirm this, they silenced the ARR17 gene in an early-flowering female line and observed male flowers in tissue culture. We found no evidence of an ARR17 RNA interference mechanism in S. purpurea catkins. Salix purpurea has a similar truncated inverted repeat of ARR17 on chr15Z, but we did not observe small RNAs mapping to the ARR17 genes and their proximal regions, nor to the ARR17 homologs on Salix chr19. There were also no differential methylated regions in the putative promoter regions of any of the ARR17 genes, and S. purpurea males show expression of the ARR17 copies on chr19, whereas in P. trichocarpa males, there is no ARR17 expression. Furthermore, Carlson et al. did not find that ARR17 was differentially expressed in shoot tips containing floral primordia, indicating that this mechanism is not present at an earlier floral development stage either. Taken together, these results suggest that the RNAinterference mechanism of ARR17 may be absent in S. purpurea. The observation of ARR17 expression in both male and female S. purpurea, combined with a lack of small RNA loci in these same regions,fodder system for sale demonstrates that the Salix sex-determination mechanism is likely different from the model proposed by Müller et al.. Instead, our data suggest that if ARR17 is a master regulator in S. purpurea, it likely involves a unique mechanism, possibly through gene dosage, such that a threshold of ARR17 expression must be reached to activate a switch from male-to-female development. Alternatively, there may be another feature in the S. purpurea SDR that is suppressing this silencing mechanism, one such candidate is the adjacent AGO4 homolog described below.A single copy of an Arabidopsis AGO4 homolog, Sapur.15WG074400, is present within the ARR17-inverted repeat region of the chr15W SDR that exhibits a log2 M:F expression of −7.94, and has a cis-eQTL in the SDR. AGO4 is a component of the RNA-induced silencing complex in the RNA-dependent DNA methylation pathway, where it binds small RNAs and silences mRNA. In the bisulfite sequencing data, nearly three times as many regions showed increased methylation in males compared with females , supporting that methylation activity is down regulated in females and may have a role in mediating sex dimorphisms . The SDR AGO4 gene appears to be truncated to only 79 amino acids in length compared with five other catkin expressed AGO4 homologs in S. purpurea, which are 893–922 amino acids, and has multiple indels and substitutions when aligned .

The most similar AGO4 paralog to Sapur.15WG074400 by MUSCLE multiple-sequence alignment is Sapur.008G00580 which has a nearly seven fold greater expression in males . We speculate it is possible that the truncated version of AGO4 is interfering with expression of the full-length Sapur.008G00580 in males by a long noncoding RNA. This could have wide-ranging effects on sexually dimorphic gene expression and could explain the decreased genome-wide methylation observed in females. The findings from the bisulfite-sequencing data indicate that methylation is globally reduced in females. We hypothesize that the Sapur.15WG074400 could be competing for binding of siRNAs with a full-length AGO4 and sequestering male-specific RDDM in females. These global methylation differences could be responsible for sex determination, such as in Melandrium album where demethylation of male plants results in monoecy, with no effect on female plants. Such a mechanism could also explain ARR17 expression levels, and why no small RNAs were observed mapping to ARR17 in Salix, despite evidence for this mechanism in Populus.A female-expressed homolog of GATA15, Sapur.15WG 062800, is located in the W-specific region of the SDR and shows a cis-eQTL association with polymorphisms on Chr15. GATA15 is a transcriptional regulator that binds GAT or GATA motifs in gene promoters and is involved in cell differentiation, morphogenesis, and development. This is consistent with the GO enrichment analysis of female-expressed genes, which contains many significant terms related to morphogenesis and development. Furthermore, chr15 GATA15 was found by Carlson et al. to be differentially expressed in F1 S. purpurea shoot tips containing floral primordia, suggesting that this may be the earliest cue for floral sex differentiation, which would implicate it as a master regulator gene. While functional genomics data are required to elucidate its precise function, its expression in females both during floral differentiation and catkin emergence suggests that it could be directly involved in gynecium development.Four genes were identified that fit the criteria for candidate master regulator genes, but whose functions are not known or whose annotations are insuffificient for further analysis. These included Sapur.15WG068800, a CCHC-type zinc finger, and three hypothetical proteins: Sapur.15WG075300, Sapur.15WG074900, and Sapur.15 WG075700. While there is mounting evidence pointing toward ARR17 as the master regulator in Populus spp, the evidence for different expression profiles of ARR17 in Salix, as well as the presence of additional candidate genes, suggests that the mechanism may be more complicated or altogether different in Salix. Nevertheless, expression data from the ARR17 homologs in S. purpurea do support a possible role in sex determination, either as a single gene master regulator or part of a two-gene system in conjunction with another master regulator, and would provide further evidence to support cytokinin response as a common mechanism for diecy in angiosperms, assuggested by Montalvão et al. Further functional genomics studies will be necessary to elucidate the precise functions of candidate master regulators and their role in sex determination.Among the floral development genes with eQTL in the SDR were homologs of AGL11 and AGL32, AGL29 and AGL30, and AGL6, as well as TOC1, WOX1, RGA, and CONSTANS. While differential expression of MADS-box genes is expected in floral tissues, their association with the SDR through eQTL, even after accounting for sex as a covariate, suggests that the SDR may have a direct role in controlling expression of these genes and subsequent primary sex dimorphisms.

The interaction between K and Mg was found to be significant with the addition of Mg causing a significant increase in the N uptake at each level of K though the magnitude of increase diminished as levels of Mg increased. As the level of Mg application increased without K addition the N uptake was found to decrease. Addition of increased levels of K increased the uptake of phosphorus. The application of high rates of Mg decreases the favorable effect of K on P uptake and hence Mg is seen to have an antagonistic effect on P uptake. This reduction of P uptake in spite of increased P availability might be due to the interference of sulphate ions on the absorption of P by the plant. Increasing the rate of K application significantly increased the K uptake. As the level of Mg application increased the K uptake also showed a general increase, contrary to the reports that K and Mg are antagonistic. Thus Mg might have diminished only the solution concentration of K in soil and not the uptake. At higher levels of K, a significant increase in K uptake was noticed as Mg levels were increased. Thus a positive interaction existed between K and Mg on the uptake of K. This increase in K in the tissue with increasing Mg might be because of the decreased dry matter production associated with high Mg. Calcium uptake was significantly reduced with increasing K levels especially in the absence of Mg. Similar results were reported by Fageria who found an antagonistic effect of K application on Ca uptake.

When applied with high levels of Mg, though higher levels of K caused a reduction in the Ca uptake,hydroponic dutch buckets no significant decrease was generally obtained. Addition of increasing levels of Mg remarkably decreased the Ca uptake. The high Mg levels must have hindered the absorption of Ca by the plant due to the action of the Ca- Mg antagonistic effect. Such antagonism of Mg on the uptake of Ca was also reported by Kumar et al. . It is also seen that the antagonistic effect of increased levels of Mg occurred only in the absence of K. The decrease in tissue concentration and uptake of Ca with increasing concentration of Mg is presumably due to the replacement of Ca by Mg for the neutralization of negative charges within the vacuole and on the exchange sites in the apoplast of the plant cell . Though Mg uptake increases with its level of application, the presence of K especially at higher levels was found to decrease uptake by the crop. This may be because of an induced reduction in availability of Mg to the crop by the excess application of K, leading to less absorption of Mg by the plants. Thus, at higher Mg levels, by supplying additional excess levels of K, the antagonism of additional K is great enough to repress Mg absorption regardless of the Mg level. Thus at low soil K and sufficient exchangeable Mg levels, uptake of Mg is not hindered. The California strawberry industry produces about 85% of the strawberries grown in the United States, on 37,000 acres, with a value of $1.5 billion in 2008 . To control soil borne diseases and weeds, California strawberry fields have long been fumigated with methyl bromide plus chloropicrin . However, methyl bromide is being phased out as an ozone-depleting substance under the Montreal Protocol , an international treaty. Currently, some California strawberries can still be treated with methyl bromide under a critical-use exemption, subject to annual review by parties to the Montreal Protocol.

Alternative fumigants permitted for use in California strawberries are 1,3-dichloropropene , chloropicrin and, as of December 2010, methyl iodide. About 81% of California strawberries are grown in soils that were previously treated with chloropicrin , while 30% are also fumigated with 1,3-D and 43% with methyl bromide . Since soil treatments began in the 1960s, entire fields have been covered with polyethylene film to hold in the fumigant at concentrations needed to kill soil pests . More recently, a sizable portion of strawberry acreage has been treated with fumigants applied to beds via the drip irrigation system . The major alternatives to methyl bromide, 1,3-D and chloropicrin, are heavily regulated. The transition away from methyl bromide to alternatives has been complicated by regulations aimed at protecting workers and others from exposure to fumigants. In California, 1,3-D use per 36-square-mile township is limited to 90,250 pounds, called a “township cap,” which severely limits its availability in key strawberry production areas . The recent critical use nomination for strawberry indicates that “township caps currently limit the use of 1,3-D on 40% to 62% of total strawberry land” . In other words, methyl bromide use continues in California because restrictions on alternative fumigants leave few options. Among the reasons that fumigants are so heavily regulated in California is that they are classified as volatile organic compounds . Alternative fumigants such as 1,3-D are released into the air and, after reacting with nitrogen oxides, can convert to form ground-level ozone — a harmful air pollutant . Regulations have been developed to reduce the contribution of fumigants to ozone formation, which, for example, has seriously affected the use of fumigants in Ventura County, a key strawberry production area.impermeable film can greatly reduce fumigant emissions and enhance their distribution in soil, in comparison with conventional polyethylene films or uncovered soil .

VIF differs from traditional high-density polyethylene tarps in that it has additional gasimpermeable layers, such as nylon or polyaminides, between the polyethylene layers . Fumigant concentrations of 1,3-D and chloropicrin were higher under VIF than low-density polyethylene tarp, 1 to 4 days after drip fumigation . The improved retention of fumigants under VIF also provides more opportunity for them to degrade in the soil rather than be released into the atmosphere . A number of researchers have found that VIF as a tarp can reduce the amount of 1,3-D plus chloropicrin needed for effective soil disinfestations by 50% . Santos et al. found that reducing methyl bromide plus chloropicrin rates by one-half under VIF controlled nuts edge similarly to the full rate of 350 pounds per acre applied under standard films. A relatively new barrier, totally impermeable film , has been shown to apply easily and retain fumigant better than VIF . TIF is a five layer film with two thin ethylene vinyl alcohol layers embedded in three layers of standard polyethylene film . Our studies evaluated the compatibility of TIF and standard films with the two major fumigant application methods for strawberry, broadcast fumigation and chemigation. The primary objective was to compare fumigant retention under TIF and standard film. Secondary objectives were to measure the effects on strawberry fruit yield and weed control.We compared the retention of methyl bromide plus chloropicrin under TIF and standard films at a commercial farm near Salinas in 2007. The soil was a Chualar sandy loam. Methyl bromide 57% plus chloropicrin 43% and 1,3-D 61% plus chloropicrin 35% , both at 350 pounds per acre, were applied by a commercial applicator on Oct. 15, 2007. As the fumigant was applied,bato bucket it was immediately tarped by 13-foot-wide standard film  or 13-foot-wide TIF . The plots were 280 feet long and 33 feet wide to allow for three passes, each 11 feet wide, of the application tractor. The films were 13 feet wide overall with 1 foot on the leading edge used to anchor the film in the soil and 1 foot on the trailing edge used to glue to the leading edge of the previous pass, creating a 1-foot overlap. Hence, the applied film is like rows of overlapped roofing shingles. The proprietary glue used by the commercial applicator adhered to the TIF film and held it in place without incident. Each treatment was replicated two times and arranged in a randomized complete block design. Fumigant concentrations under the tarp were monitored with a MiniRae VOC meter at 3, 27, 51, 76, 97, 120 and 166 hours after application. The MiniRae VOC meter uses a photo ionization detector to measure the concentrations of volatile compounds such as fumigants. Fumigant samples were taken from airspace between the soil surface and the tarp at three random locations near the center of the plots. The film was cut and removed 192 hours after application. The field was then prepared for strawberry planting by the installation of 52-inch-wide raised beds with two drip irrigation lines per bed. ‘Albion’ strawberry was transplanted on Nov. 11, 2007. Strawberry fruit were harvested from two 40-plant sample stations per plot from April 18 to Sept. 1, 2008, and fruit were sorted into marketable and cull fruit by a trained crew. Weeds were sampled from two 125-square-foot sample areas on Feb. 15, April 28 and July 8, 2008.In 2008, we evaluated TIF at the USDA Agricultural Research Service farm on Spence Road near Salinas. The soil was a Chualar sandy loam. We injected 1,3-D 35% plus chloropicrin 60% plus an emulsifier 5%  through the drip irrigation system on Oct. 21, 2008, at 50, 100, 200, 300 and 400 pounds per acre, under both standard and TIF film . Briefly, the fumigants were injected in a closed system directly from nitrogen pressurized cylinders and metered into irrigation water with a flow meter . A static mixing device was installed at the point of injection to mix fumigants with irrigation water before distribution via the drip irrigation system.An emulsifiable formulation of methyl bromide 57% plus chloropicrin 43% was applied on Oct. 29, 2008, at 350 pounds per acre, also through the drip irrigation system. Each treatment was replicated four times, and the trial was arranged in a randomized complete block design.

Plot sizes were a single 52-inch-wide by 75-foot-long bed. Fumigant concentrations under the tarp were sampled at one location near the plot center with a MiniRae VOC meter as described above, at 3, 8, 24, 48, 72, 96, 144, 192, 240 and 336 hours after application. The MiniRae meter was calibrated with known concentrations of 1,3-D and chloropicrin prior to each sampling. The plastic films were left on the beds for the length of the strawberry season. Before transplanting strawberries, planting holes were punched in the bed, and ‘Albion’ strawberry was transplanted by hand into all plots on Nov. 24, 2008. Visual crop injury was estimated on Jan. 6, 2009, using a scale of 0 = safe to 10 = dead. On March 10, 2009, diameters were measured on 20 plants per plot. Fruit were harvested from 50 sample stations in each plot once or twice weekly as needed from March 30 until Oct. 30, 2009. Fruit were graded as described in the 2007 trial. Weed densities were measured in 2007 and 2008. In 2008, nylon bags containing yellow nuts edge tubers and weed seeds were buried in each plot before the fumigant application, at a depth of 6 inches. These species were evaluated because they represent a range of susceptibility to fumigants from difficult , to intermediate , to easy . Little mallow and chickweed are common in strawberry. Weed seeds were retrieved 2 weeks after the methyl bromide plus chloropicrin application, and their viability was determined. The yellow nuts edge was planted in potting soil and placed in an illuminated growth chamber at 85°F for 4 weeks. Weed seed viability was determined using tetrazolium assays. Weed density ratings were measured in 125-square-foot sample areas on the bed tops, on Feb. 15, April 28 and July 8, 2008 , and Dec. 11, 2008, and Feb. 3 and March 17, 2009 .The data was subjected to analysis of variance in SAS v. 9.1 , and Duncan’s multiple range test was used for mean separation for all data at the 5% significance level. Weed seed and yellow nuts edge tuber survival data were analyzed to evaluate the effects of fumigant rate, film, and the interaction between rate and film. Linear contrasts were used to compare weed seed survival under the TIF and standard films using SAS PROC GLM. To determine if there was any correlation between strawberry fruit yield and fumigant concentrations, the 2008 data was tested using the SAS PROC CORR routine.

The first is based on the possibility that the bio-stimulant contains within it, previously unrecognized molecules that are the sole and discrete cause of the observed improvement in plant productivity. This concept emphasizes both the need for clear demonstration of plant productivity benefits and the unknown nature of the mode of action. Thus, a bio-stimulant could be defined as “a formulated product that improves plant productivity by a mechanism of action that is not the sole consequence of the presence of known essential plant nutrients, plant hormones, plant growth regulators or plant protective compounds.” By this definition, once the primary biological mechanism of bio-stimulant function has been identified it should henceforth, be subject to classification on the basis of that functional component. The majority of bio-stimulants in use today are complex mixtures of chemicals derived from a biological process or extraction of biological materials. The complexity of these mixtures is often considered to be essential to the performance of the bio-stimulant, and bio-stimulants may have properties of the whole, that cannot be fully elucidated by knowing the characteristics of the separate components or their combinations. This theory of complexity or “emergence” was described by Mayr ,planting gutter who argued that in many biological systems “the properties of the whole cannot be fully elucidated by knowing the characteristics of the separate components or their combinations.” “The term emergence describes the onset of novel properties that arise when a certain level of structural complexity is formed from components of lower complexity.

In the last few decades, emergence has been discussed in a number of different research fields, such as cybernetics, theory of complexity, artificial intelligence, non-linear dynamics, information theory, and social systems organization” . “Emergence” and “emergent properties” are thus closely related with the notion of the “systems biology” . Emergence was described by Johnson as “unexpected behaviors that stem from interaction between the components of an application and their environment,” “there is, however, considerable disagreement about the nature of ‘emergent properties.’ Others refer to emergent properties when an application exhibits behaviors that cannot be identified through functional decomposition. In other words, the system is more than the sum of its component parts” . Thus, a bio-stimulant could also be defined as “a formulated product of biological origin that improves plant productivity as a consequence of the emergent properties of its constituents.” To our knowledge, however, there have been no clear demonstrations that any bio-stimulant exhibits truly emergent properties. This is not however a unique challenge and all “biological systems are extremely complex and have emergent properties that cannot explained, or even predicted, by studying their individual parts” . Emergent properties have been demonstrated in the networks of biological signaling pathways ; in system-level study of traditional Chinese medicine , and in microbial communities . To adequately explain the biological complexity present in plants and their interactions with the environment, Lüttge and Bertolli et al. emphasize that classic reductionist biology/chemistry is indeed insufficient. While the two theoretical definitions provided in this section share a requirement that the mode of action is unknown, they differ in the core assumption that bio-stimulant function is a consequence of the discrete components in the bio-stimulant or as a consequence of the “emergent” properties of the bio-stimulant as a whole.

Each of these definitions is also incomplete in that it is certainly possible that a bio-stimulant may contain several molecules that act synergistically while not being truly “emergent,” and it is indeed possible and indeed likely, that even if a bio-stimulant is demonstrated to have emergent properties, that not all components of that bio-stimulant are required for that property to be expressed. We propose, therefore, a definition of a bio-stimulant that integrates these two concepts. Thus, a bio-stimulant is defined here as: “a formulated product of biological origin that improves plant productivity as a consequence of the novel, or emergent properties of the complex of constituents, and not as a sole consequence of the presence of known essential plant nutrients, plant growth regulators, or plant protective compounds.” Consistent with this definition, the ultimate identification of a novel molecule within a bio-stimulant that is found to be wholly responsible for the biological function of that bio-stimulant, would necessitate the classification of the bio-stimulant according to the discovered function. A review of the history of bio-stimulants and related products provides insight into the diversity of these products and the development of this field of study. The evolution of bio-stimulant classifications as described by various authors is presented in the Table 2. To the best of our knowledge, one of the first attempts to categorize bio-stimulants was provided by Filatov when 4 groupings of biogenic stimulants were suggested. Karnok compiled a list of 59 materials presenting in 15 bio-stimulants; Ikrina and Kolbin systematized patent literature and specified 9 categories of natural raw materials used to derive bio-stimulants; Basak suggested that bio-stimulants could be grouped on the basis of single or multi-component formulations and classified on the origin of the active ingredient, and the mode of action of the active ingredient. Du Jardin developed a scientific rationale of classification considering 8 categories of bio-stimulants and subsequently reduced this list to 7 categories .

Du Jardin was explicit in his exclusion of microorganisms from his categorization primarily to avoid conflict with existing categorization of microorganisms as bio-pesticides and sources of plant hormones. Later Bulgari et al. proposed a bio-stimulant classification on the basis of their mode of action rather than on their composition. Many bio-stimulant products have been classified into completely divergent groups and categories of function, use, and type of activity . For example, humate based products are often described as soil health amendments while plant growth promoting rhizobacteria could be categorized as bio-fertilizers, phytostimulators, and bio-pesticides . Du Jardin has proposed that bio-fertilisers are a subcategory of bio-stimulants. Seaweed extracts have been considered as bio-fertilizers and microorganisms have also been described as bio-fertilizers . Some inorganic elements or small molecules that are not known to be essential may also be classified as bio-stimulants if evidence of plant growth promotion is available . Thao and Yamakawa , for example, consider phosphites to be bio-stimulants since plant response to phosphites frequently cannot be explained as a consequence of the known anti-fungal function of these molecules. While the categorization of bio-stimulants by their origin does not, a priori, provide information on their mode of action this categorization may still be a useful tool to aid in the process of discovery and facilitate comparison between similar products. Registration of products used in agriculture is crucial to ensure their practical, safe and legitimate application. In the absence of a sound definition of bio-stimulants as a discrete group of products , the registration procedure and subsequent classification regime is untenable and this inevitably creates a barrier to trade and development. Various countries, states, and administrative regions have developed different categories for registration of potential bio-stimulants including terminology such as plant conditioners, “other fertilizers,” supplements, soil improvers, gutter berries plant strengtheners, fitofortificants, etc. . In many jurisdictions regulatory practices require an itemized description and identification of substances in all commercial product classifications while in others the registration of non-fully identified substances is allowed if those products are considered of complex composition. There is even a proposal for complex bio-stimulants to not specify the chemical name and note as “None” with the definition that “this product is a complex mixture of chemical substances” . If we accept the concept that a bio-stimulant is a product of clear benefit but unknown mode of action, then it can only be regulated by its safety and proof of efficacy. For example, in pharmacology it has been suggested that “the demand to demonstrate the mode of action of each single component in a phytopharmaceutical may not be obligatory any more” . The complex multi-component nature of many bio-stimulants clearly complicates discovery of their modes/mechanisms of action, production, registration and use. What is clearly needed however, is a regulatory mechanism to ensure that the products are “generally recognized as safe,” have “a positive benefit on crop productivity” and are discrete from exisiting categories of products. The task of identifying function and agronomic utility can then be pursued independently and will be driven by the marketplace imperative for product quality and consistency. Coordinating national legislation within this framework will become critical for the optimization of bio-stimulants and trade between different countries. The possible place of bio-stimulants in the regulatory system of pesticides and agrochemicals is illustrated in Figure 1.We have conducted an exhaustive analysis of the literature and categorized the majority of the reported bio-stimulants by origin . Microorganisms are widely used for the production of bio-stimulants and may be derived from bacteria, yeasts, and fungi. These preparations may include living and/or nonliving microorganisms and their metabolites. The concept of microorganism-based preparations as bio-stimulants is described by Xavier and Boyetchko , Sofo et al. , Colla et al. , Matyjaszczyk , and Ravensberg . different species of algae, mostly seaweeds, are also commonly used for producing bio-stimulants.

Seaweed-based preparations as bio-stimulants are described in reviews by Crouch and van Staden , Khan et al. , Craigie , Sharma et al. ; and experimental papers by Goatley and Schmidt , Jannin et al. , Billard et al. , Aremu et al. . Raw materials for bio-stimulants are also commonly based on higher plant parts including seeds, leaves, and roots and exudates from families Amaryllidaceae, Brassicacae, Ericaceae, Fabaceae, Fagaceae, Moringaceae, Plantaginaceae, Poaceae, Rosaceae, Solanaceae, Theaceae, Vitaceae, among others . bio-stimulants may also be based on protein hydrolysates and amino acids of animal origin including wastes and by-products , and insect derived chitin and chitosan derivatives . Humate-based raw materials are widely used to derive bio-stimulants and have been reviewed by Sanders et al. , Kelting et al. , Ertani et al. , and Jannin et al. . A final category of bio-stimulants includes those derived from extracts of food waste or industrial waste streams, composts and compost extracts, manures, vermicompost, aquaculture residues and waste streams, and sewage treatments among others. Because of the diversity of source materials and extraction technologies, the mode of action of these products is not easily determined. The technologies used in the production and preparation of bio-stimulants are highly diverse and include cultivation, extraction, fermentation, processing and purification, hydrolysis, and high-pressure cell rupture treatment . In some instances, a bio-stimulant product may also contain mixes of components derived from different sources and production methods. Frequently the rationale for utilizing extracts rather than raw biomass is a consequence of the need for a standardized manufacturing process to produce a uniform commercial product . For many products, the production processes are driven by process and marketing demands and are not the result of a targeted strategy to optimize the biological efficacy of the commercial product. While the ultimate composition and possible function of commercial bio-stimulant products may be partially determined by both the source of raw material and the process by which it is prepared , there may be manufacturing processes and product treatments utilized that result in compounds that are not present in the initial material. An example of this is the multitude of commercial seaweed extracts, often derived from the same species, that are rarely equivalent . Commercial bio-stimulant manufactured from similar sources are usually marketed as equivalent products, but may differ considerably in composition and thereby in efficiency . Many manufacturers do not reveal the technology of bio-stimulant production since that is a commercial secret .A diversity of substances contained in raw materials is used for the production of bio-stimulants. Whereas, primary metabolites are contained in most preparations de facto, the presence of secondary metabolites is more specific and depends to a large extent on the raw material used . Primary metabolites include amino acids, sugars, nucleotides, and lipids . Secondary metabolites are formed from different primary metabolic pathways, including glycolysis, the tricarboxylic acid cycle , aliphatic amino acids , the pentosephosphate and shikimate pathways which are primarily the source of aromatic AA and phenolic compounds , terpenoids/isoprenoids, nitrogen-containing compounds , sulfur-containing compounds. Frequently, bio-stimulants are shown to have a multicomponent composition and may include plant hormones or hormone-like substances, amino acids, betaines, peptides, proteins, sugars , aminopolysaccharides, lipids, vitamins, nucleotides or nucleosides, humic substances, beneficial elements, phenolic compounds, furostanol glycosides, sterols, etc. .

Several companies in the United States and the EU have developed recombinant ACE2 and ACE2-Fc fusion proteins for preclinical and clinical testing, although all these products are currently produced in mammalian cell lines . The impact of plant-specific complex glycans on the ability of ACE2-Fc to bind the RBD has been studied using molecular dynamic simulations and illustrates the important role that glycosylation may play in the interaction between the S protein and ACE2 .Griffithsin is a lectin that binds high-mannose glycans, and is currently undergoing clinical development as an antiviral against HIV-1. However, it also binds many other viruses that are pathogenic in humans, including HSV , HCV , Nipah virus , Ebola virus, and coronaviruses including SARS-CoV and MERS , and as recently determined, also SARSCoV-2. A clinical product in development by University of Louisville is currently manufactured in N. benthamiana by Kentucky Bio-processing using a TMV vector. The API is also undergoing preclinical development as a nasal spray for use as a non-vaccine prophylactic against coronaviruses, with clinical evaluation planned for 2020 . This candidate PMP antiviral could be deployed under the EUA pathway if found effective in controlled clinical studies. Griffithsin is an interesting example of a product that is ideally matched to plant-based manufacturing because it is naturally produced by a marine alga. Griffithsin has been expressed with limited success in E. coli and tobacco chloroplasts, ebb flow table but better results have been achieved by transient expression in N. benthamiana using A. tumefaciens infiltration or TMV vectors, with expression levels of up to 1 g kg−1 fresh mass and recoveries of up to 90% .

A TEA model of griffithsin manufactured in plants at initial commercial launch volumes for use in HIV microbicides revealed that process was readily scalable and could provide the needed market volumes of the lectin within an acceptable range of costs, even for cost-constrained markets . The manufacturing process was also assessed for environmental, health, and safety impact and found to have a highly favorable environmental output index with negligible risks to health and safety.In addition to COVID-19 PCR tests, which detect the presence of SARS-CoV-2 RNA, there is a critical need for protein-based diagnostic reagents that test for the presence of viral proteins and thus report a current infection, as well as serological testing for SARS-CoV-2 antibodies that would indicate prior exposure, recovery, and possibly protection from subsequent infection. The most common formats for these tests are the ELISA and lateral flow assay. The design and quality of the binding reagents , along with other test conditions such as sample quality, play a key role in establishing the test specificity and selectivity, which determine the proportion of false positive and false negative results. Although the recombinant protein mass needed for diagnostic testing is relatively small , the number of tests needed for the global population is massive, given that many individuals will need multiple and/or frequent tests. For example, 8 billion tests would require a total of ~2.5 kg purified recombinant protein, which is not an insurmountable target. However, although the production of soluble trimeric full-length S protein by transient transfection in HEK293 cells has been improved by process optimization, current titers are only ~5 mg L−1 after 92 h . Given a theoretical recovery of 50% during purification, a fermentation volume of 1,000 m3 would be required to meet the demand for 2.5 kg of this product.

Furthermore, to our knowledge, the transient transfection of mammalian cells has only been scaled up to ~0.1 m3 . The transient expression of such protein-based diagnostic reagents in plants could increase productivity while offering lower costs and more flexibility to meet fluctuating demands or the need for variant products. Furthermore, diagnostic reagents can include purification tags with no safety restrictions, and quality criteria are less stringent compared to an injectable vaccine or therapeutic. Several companies have risen to the challenge of producing such reagents in plants, including Diamante , Leaf Expression Systems , and a collaborative venture between PlantForm, Cape Bio Pharms, Inno-3B, and Microbix.Resilience is the state of preparedness of a system, defining its ability to withstand unexpected, disastrous events , and to preserve critical functionality while responding quickly so that normal functionality can be restored . The concept was popularized by the 2011 Fukushima nuclear accident but received little attention in the pharmaceutical sector until COVID-19. Of the 277 publications retrieved from the National Library of Medicine22 on July 9th 2020 using the search terms “resilience” and “pandemic,” 82 were evenly distributed between 2002 and 2019 and 195 were published between January and July 2020. Resilience can be analyzed by defining up to five stages of a resilient system under stress, namely prevent, prepare, protect, respond, and recover . Here, prevent includes all measures to avoid the problem all together. In the context of COVID-19, this may have involved the banning of bush meat from markets in densely populated areas . The prepare stage summarizes activities that build capacities to protect a system and pre-empt a disruptive event. In a pandemic scenario, this can include stockpiling personal protective equipment but also ensuring the availability of rapid-response bio-pharmaceutical manufacturing capacity. The protect and respond stages involve measures that limit the loss of system functionality and minimize the time until it starts to recover, respectively. In terms of a disease outbreak, the former can consist of quarantining infected persons, especially in the healthcare sector, to avoid super-spreaders and maintain healthcare system operability . The response measures may include passive strategies such as the adjustment of legislation, including social distancing and public testing regimes, or active steps such as the development of vaccines and therapeutics . Finally, the recover phase is characterized by regained functionality, for example by reducing the protect and response measures that limit system functionality, such as production lockdown. Ultimately, this can result in an increased overall system functionality at the end of a resilience cycle and before the start of the next “iteration” .

For example, a system such as society can be better prepared for a pandemic situation due to increased pharmaceutical production capacity or platforms like plants. From our perspective, the production of recombinant proteins in plants could support the engineering of increased resilience primarily during the prepare and respond stages and, to a lesser extent, during the prevent and recover stages . During the prepare stage, it is important to build sufficient global production capacity for recombinant proteins to mount a rapid and scalable response to a pandemic. These capacities can then be used during the response stage to produce appropriate quantities of recombinant protein for diagnostic , prophylactic , or therapeutic purposes as discussed above. The speed of the plant system will reduce the time taken to launch the response and recovery stages,hydroponic grow table and the higher the production capacity, the more system functionality can be maintained. The same capacities can also be used for the large-scale production of vaccines in transgenic plants if the corresponding pathogen has conserved antigens. This would support the prevent stage by ensuring a large portion of the global population can be supplied with safe and low-cost vaccines, for example, to avoid recurrent outbreaks of the disease. Similarly, existing agricultural capacities may be re-directed to pharmaceutical production as recently discussed . There will be indirect benefits during the recover phase because the speed of plant-based production systems will allow the earlier implementation of measures that bring system functionality back to normal, or at least to a “new or next normal.” Therefore, we conclude that plant-based production systems can contribute substantially to the resilience of public healthcare systems in the context of an emergency pandemic.The cost of pharmaceuticals is increasing in the United States at the global rate of inflation, and a large part of the world’s population cannot afford the cost of medicines produced in developed nations23 . Technical advances that reduce the costs of production and help to ensure that medicines remain accessible, especially to developing nations, are, therefore, welcome. Healthcare in the developing world is tied directly to social and political will, or the extent of government engagement in the execution of healthcare agendas and policies . Specifically, community-based bodies are the primary enforcers of government programs and policies to improve the health of the local population . Planning for the expansion of a bio-pharmaceutical manufacturing program to ensure that sufficient product will be available to satisfy the projected market demand should ideally begin during the early stages of product development. Efficient planning facilitates reductions in the cost and time of the overall development process to shorten the time to market, enabling faster recouping of the R&D investment and subsequent profitability. In addition to the cost of the API, the final product form , the length and complexity of the clinical program for any given indication , and the course of therapy have a major impact on cost. The cost of a pharmaceutical product, therefore, depends on multiple economic factors that ultimately shape how a product’s sales price is determined . Product-dependent costs and pricing are common to all products regardless of platform. Plant-based systems offer several options in terms of equipment and the scheduling of upstream production and DSP, including their integration and synchronization .The efficiency of this translation has a substantial impact on costs, particularly if processes are frozen during early clinical development and must be changed at a subsequent stage. Process-dependent costs begin with production of the API.

The manufacturing costs for PMPs are determined by upstream production and downstream recovery and purification costs. The cost of bio-pharmaceutical manufacturing depends mostly on protein accumulation levels, the overall process yield, and the production scale. Techno-economic assessment models for the manufacture of bio-pharmaceuticals are rarely presented in detail, but analysis of the small number of available PMP studies has shown that the production of bio-pharmaceuticals in plants can be economically more attractive than in other platforms . A simplified TEA model was recently proposed for the manufacture of mAbs using different systems, and this can be applied to any production platform, at least in principle, by focusing on the universal factors that determine the cost and efficiency of bulk drug manufacturing .Minimal processing may be sufficient for oral vaccines and some environmental detection applications and can thus help to limit process development time and production costs . However, most APIs produced in plants are subject to the same stringent regulation as other biologics, even in an emergency pandemic scenario . It is, therefore, important to balance production costs with potential delays in approval that can result from the use of certain process steps or techniques. For example, flocculants can reduce consumables costs during clarification by 50% , but the flocculants that have been tested are not yet approved for use in pharmaceutical manufacturing. Similarly, elastin-like peptides and other fusion tags can reduce the number of unit operations in a purification process, streamlining development and production, but only a few are approved for clinical applications . At an early pandemic response stage, speed is likely to be more important than cost, and production will, therefore, rely on well characterized unit operations that avoid the need for process additives such as flocculants. Single-use equipment is also likely to be favored under these circumstances, because although more expensive than permanent stainless-steel equipment, it is also more flexible and there is no need for cleaning or cleaning validation between batches or campaigns, allowing rapid switching to new product variants if required. As the situation matures , a shift toward cost-saving operations and multi-use equipment would be more beneficial.An important question is whether current countermeasure production capacity is sufficient to meet the needs for COVID-19 therapeutics, vaccines, and diagnostics. For example, a recent report from the Duke Margolis Center for Health Policy24 estimated that ~22 million doses of therapeutic mAbs would be required to meet demand in the United States alone , assuming one dose per patient and using rates of infection estimated in June 2020. The current demand for non-COVID-19 mAbs in the United States is >50 million doses per year27, so COVID-19 has triggered a 44% increase in demand in terms of doses. Although the mAb doses required for pre-exposure and post-exposure COVID-19 treatment will not be known until the completion of clinical trials, it is likely to be 1–10 g per patient based on the dose ranges being tested and experience from other disease outbreaks such as Ebola .

Two clones remained negative after challenges and a third had a low percentage infection. The ultimate evaluation will be in the field where they are exposed to repeated natural infection by the insect vector. Trees of the three clones were planted in November 2007 amongst old trees with greening symptoms. This report is on their current status regarding greening infection as well as growth, production and fruit quality.The objective was to evaluate soil-applied SAR inducers or transgenic enhancement of SAR expression for effect on HLB disease progress in citrus trees challenged by graft-inoculation or psyllid-mediated infection. One yr-old Hamlin sweet orange trees were planted in May 2009 and treated as follows: 1) non-treated check , 2) foliar insecticide to control psyllids, 3) soil-applied imidacloprid/thiamethoxam to induce SAR, 4) soil-applied IMID/THIA plus foliar insecticides, 5) graft-inoculated UTC, 6) graft-inoculated with IMID/THIA. A randomized block design was used with 5 repetitions of 10 tree plots. In 2010, the SAR inducer acibenzolar-S-methyl which does not control psyllids was substituted in treatments 3, 4 and 6. At 24 months after treatments were established in a field site, 105 trees were PCR+ in the trial. Higher numbers of PCR+ trees occurred in the UTC, the UTC with graft inoculation, and the IMID/THIA/ASM with graft-inoculation. A lower number of PCR+ trees occurred in the treatments with SAR inducers, foliar insecticides, and foliar insecticide plus SAR inducers,vertical farming racks but no effect of SAR treatment on rate of HLB disease progress was detected. Transgenics of Duncan grapefruit and Hamlin trees were constructed that expressed the NPR1 gene from Arabidopsis , a key positive regulator of SAR.

Over-expression of AtNPR1 in transgenic lines of these susceptible hosts reduced citrus canker lesions. Resistance to Xanthomonas citri subsp. citri infection was related to expression levels of AtNPR1. Two lines each of ‘Duncan’ grapefruit and Hamlin with the highest expression of AtNPR1 were screened in a greenhouse containing HLB-infected plants and psyllids. Rate of infection progress over 12 months was similar for transgenic lines and non-transgenic controls. Again, an effect of SAR on HLB disease progress was not-detected which confirmed the ineffectiveness of enhanced SAR for HLB disease control. Trioza erytreae is in itself a minor pest of citrus, but its significance is attributed to its ability to vector the citrus African Greening disease pathogen, Candidatus Liberibacter africanus. The population fluctuation of the citrus triozid is correlated to the flushing rhythm of the citrus host, but seasonal fluctuation in infection of populations carrying the Greening pathogen have been difficult to monitor in the past due to limited detection methodologies. This study explored this fluctuation in infectivity in an orchard entirely infected with African greening by using PCR to test individual triozids caught on sticky traps placed weekly in the orchard. Two season’s data are presented from a small sour orange orchard in the Nelspruit district in South Africa. The triozid population fluctuations correlated to previous findings of population peaks following the citrus flush cycles and responses to specific climatic influences. Fluctuations in the percentage infectivity of the T. erytreae populations were observed, with infectivity peaking at or just after the citrus flush seasons, but with peaks in infectivity differing to population peaks.Over the last decade the plant disease Huanglongbing has emerged as a primary threat to citrus production worldwide. HLB is associated with infection by one of a group of phloem-limited bacteria that are readily transmitted by the Asian citrus psyllid, Diaphorina citri.

However, the temporal progression of infection, time to symptom expression , and how they relate to transmissibility remains unresolved. We graft inoculated sweet orange trees with C. Liberibacter asiaticus, then at different times after inoculation, we inspected plants for HLB symptoms, measured bacterial infection levels in plants, and measured acquisition by psyllid adults that were confined on the trees. Plant infection levels increased rapidly over time, saturating at uniformly high levels near 200 days after inoculation – the same time at which all infected trees first showed disease symptoms. Pathogen acquisition by vectors was positively associated with plant infection level and time since inoculation, with acquisition occurring as early as the first introduction 60 days after inoculation. These results suggest that there is ample potential for psyllids to acquire the pathogen from trees during the asymptomatic phase of infection. If so, this could limit the effectiveness of tree rouging as a disease management tool and would likely explain the rapid spread observed for this disease in the field. The diseases Huanglongbing and citrus tristeza are both phloem limited and have significant economic impact on citrus production wherever they are found. Studies of host resistance have indicated that Poncirus trifoliata has tolerance/resistance to both diseases, suggesting there may be some common factors in the two kinds of resistance. We have conducted studies of host gene expression changes that occur in response to infection to gain further insight. Controlled inoculation by grafting infected budwood was used to infect potted greenhouse plants of Cleopatra mandarin , US-897 , and US-942 with CTV and with Candidatus Liberibacter asiaticus, the pathogen associated with HLB. Stem and leaf tissues was collected at 10, 20, and 30 weeks after inoculation. DNA-free RNA was subjected to real-time one-step SYBR Green RT-PCR analysis and relative gene expression was determined using the 2–ΔΔCt -method. Differences in gene expression between mockinoculated and Citrus tristeza virus -inoculated plants were generally not prominent. Differences in gene expression between mock-inoculated and Lasinoculated plants were most pronounced in susceptible Cleopatra plants and at the later stages of infection. Transcripts for a constitutive disease resistance protein were induced in response to Las in susceptible plants, but showed higher expression levels independent of infection in the tolerant genotypes.

A gene for a Myb-like transcriptional regulator family protein which is associated with resistance to some phytoplasma, responded strongly to Las, but also responded to CTV in tolerant plants. Transcript levels for other genes, such as a 2-oxoglutarate and Fe- dependant oxygenase and a plastidic glucose transporter , were considerably higher in US-897 and US-942 plants compared with Cleopatra plants independent of infection with either pathogen. It is hypothesized that these genes play a role in the resistance or tolerance of trifoliate orange and its hybrids to HLB and CTV.The Asian citrus psyllid is fairly well established in all counties in southern California and is spreading up the coast into Ventura and Santa Barbara Counties, but is not established in the major citrus production area of the Central Valley. The current status of Huanglongbing in California remains as only one backyard tree in Los Angeles County. This tree was identified as being infected with Candidatus Liberibacter asiaticus in March 2012 and removed. Despite continued surveys in that area,vertical rack system no additional trees have been found positive for HLBassociated pathogens.As a means of early diagnosis, adult and nymph ACP are collected and tested to determine whether HLB-associated bacteria are present in a localized area. The current approved diagnostic method for HLB in the United States is q-PCR; however, as demonstrated in Florida, this method may detect HLB-associated bacteria too late for the implementation of an effective eradication strategy. Hence, the Citrus Research Board has made the development of early HLB diagnostic methods their highest research priority. Methods being pursued involve the detection of molecules or compounds unique to Liberibacter infection within the host plant including volatile organic compounds , proteins secreted by the bacteria, small RNAs, and metabolomic compounds. These methods are very promising and are being tested against the approved q-PCR assay in time-course experiments conducted within a containment facility on the University of California, Davis campus. In addition, the Jet Propulsion Laboratories/NASA will monitor the reflectance properties of leaves for changes during the course of an infection. If this method is successful, JPL will further evaluate whether a large scale survey can be conducted by air or satellite. These new methods are also being field tested in Los Angeles County in the neighborhood where the CLas-infected tree was found in 2012 and in Texas in the vicinity of previous detections of HLB-positive trees. Candidatus Liberibacter is a genus of plant entophyte gram-negative bacteria in the Rhizobiaceae family vectored by psyllids. Most of the members of the genus are associated with damaging plant diseases and are subject to phytosanitary regulation. Ca. L. asiaticus, Ca. L. africanus and Ca. L. americanus are associated with Huanglongbing, one of the most destructive diseases of citrus. The disease is present in different countries of Asia, America and Africa but the three pathogens and their vectors Diaphorina citri and Trioza erytreae have a more complex distribution. The disease continues to spread rapidly and is a threat to the Mediterranean Basin countries, where the disease has not been reported. Early detection of the citrus Liberibacter in host plants and vector insects is essential to prevent its introduction and spread and for HLB control and management in countries where the disease is present. Diagnostic methods have to cope with the uneven distribution of the bacterium in plant tissues associated with irregular vector transmission, low bacterial titer during early stages of infection in host plants and vector insects and require differentiation of the individual species. By using the InCheck Platform , which combines multiplex PCR and microarray hybridisation on a single chip, we are developing a diagnostic assay to identify and discriminate among the species of Liberibacter in plant DNA extracts. The design of the probes on the microarray module for the identification and discrimination was based on the sequences of the following species: Ca. L. asiaticus, Ca. L. africanus, Ca. L. americanus, Ca. L. solanacearum, Ca. L. europeus, Ca. L. crescens and outgroup species within the Rhizobiaceae family.

The theory of natural spread of psorosis by insects in Argentina was stated by Rodríguez Pujol and Beñatena, researchers from the Concordia Experiment Station of the National Institute of Agricultural Technology , in 1965. In 1966, Rodríguez Pujol mentioned natural spread of psorosis in seedlings in the citrus region along the Uruguay River in Argentina. Timmer and Beñatena reported 0.1 – 1% of the root stock seedlings in the nursery were rogued because of psorosis. Timmer and Garnsey observed in Texas natural spread of Citrus ringspot virus in grapefruit at a rate less than 2%. Beñatena and Portillo showed psorosis transmission from diseased plants to healthy citrus seedlings by Toxoptera citricidus and T. aurantii winged individuals, by wingless adults of Toxoptera spp., by colonies of Toxoptera spp. and Aphis citricola, with a long incubation period and erratic symptom appearance. Since then, there have been advances in the study of the causal agent, diagnostic methods, and possible transgenic lines tolerant and /or resistant to CPsV but little has been mentioned about the natural spread of this virus. This work confirms the aerial spread by insects of CPsV in the field, both in a plot of certified citrus plants free of CPsV and in trials for testing aerial and/or soil spread of the virus in the Uruguay River citrus region in Argentina.Citrus psorosis virus is the causal agent of Psorosis, an important disease causing significant economic losses in Argentina and Uruguay. CPsV is the type member of the genus Ophiovirus, family Ophioviridae. Its genome has three singlestranded RNAs of negative polarity, encapsidated with a coat protein . As there is no natural resistance to Psorosis, transgenic Pineapple sweet orange plants were obtained expressing an intron-RNA hairpin from the cp gene . This RNA transcript induces the post-transcriptional gene silencing mechanism, degrading all RNA with cp gene sequences, thus preventing the progress of the infection. When ihpCP citrus trees were CPsV-challenged by graft-transmission, at least two SwO lines did not show any symptoms and CPsV was not detected by molecular and serological analysis. The hallmark of the PTGS is the presence of small RNAs , which were detected in the transgenic tissues indicating that resistance is PTGS-mediated. Our interest is to evaluate whether ihpCP is able to transfer this feature of resistance to susceptible grafted-tissues. For that, non-transgenic buds were grafted on the two resistant ihpCP lines used as rootstocks, which had previously been inoculated with CPsV. In another assay, a susceptible CP-line, expressing the complete CP-mRNA, was grafted on the same two ihpCP resistant rootstocks. Both, the non-transgenic and transgenic CP were susceptible. New flushes showed characteristic symptoms and TAS-ELISA and qRT-PCR analysis were positive.

My research tests for the possibility that foreign investors are drawn to so-called “pollution havens”—regions in which environmental regulations are either less severe or not well enforced. This research focuses on the manufacturing sectors in four countries: Mexico, Venezuela, Ivory Coast and Morocco. In the late 1980s, the share of foreign assets in manufacturing varied from 38 percent in Ivory Coast to seven percent in Venezuela. Foreign investment accounted for about 15 percent of total assets in Moroccan manufacturing, and ten percent in Mexico. The analysis of pollution havens and foreign investors is divided into three parts. First, we examine whether foreign investors in these four developing countries are attracted toward “dirtier” sectors, defined as product groups where either pollution emissions are high or pollution abatement costs are high . Second, we then compare the energy efficiency of domestic enterprises and multinationals in these same countries. This allows us to see whether foreign investors played an important role in improving the environment by using more energy-efficient technology as well as cleaner sources of energy. Finally, we examine whether the pattern of outward U.S. foreign investment is skewed toward sectors with high pollution abatement costs. We first analyzed whether the pattern of foreign investment in the four developing countries provides any evidence of pollution havens. Is there more foreign investment in sectors with high pollution abatement costs or high emissions, after controlling for other factors that affect the pattern of foreign investment? This means that we need to control for such factors as the degree of competition,dutch bucket hydroponic the size of the domestic market, domestic wages and other factors that make the market attractive.

Once we control for these factors, we then analyze whether there is more foreign investment in sectors where pollution abatement costs in the United States are high, using annual, sector-level data drawn from U.S. surveys on pollution abatement costs among manufacturing plants. The results suggest that pollution abatement costs in the U.S. have no impact on the pattern of foreign investment in these four countries. In other words, there is no more investment in Mexico in petroleum refining or cement, sectors with high abatement costs, than there is in other sectors. As a follow-up, we used emissions data from the U.S. toxic release inventory to analyze whether or not there is more foreign investment in these four developing countries in areas of manufacturing where U.S. pollution emissions are high. As measures of emissions, we use total particulates , a measure of air pollution; biological oxygen demand , which is a broad measure of water pollution; and total toxic releases . Total particulates, which capture small and large dust particles, are closely related to phenomena such as the London smog and to air pollution in cities with emissions from fuel and diesel oil combustion, energy-intensive processes such as steel and cement, two-stroke engines, coal use, and burning of wood and residues. Analysis in the World Bank and elsewhere indicates that the release of particulates is the main air pollution problem in many third world cities . Biological Oxygen Demand indicates how discharges to water bodies deplete their oxygen levels, and is widely accepted as a broad measure of water pollution. Total toxic releases is an unweighted sum of releases of the 320 compounds in the U.S. EPA’s toxic chemical release inventory. All of these measures are by weight. Emissions are divided by the total output of the firm, measured in monetary terms, to arrive at sector-specific emission intensities for the three pollutants. Table 1 reports the correlations between these three measures of emission intensity and U.S. pollution abatement costs . Correlation coefficients are a measure of the statistical relationship between two variables. They range from –1.0 to 1.0 .

A correlation coefficient near zero implies little relationship between the two variables. Table 1 shows that, in a comparison among different manufacturing sub-sectors within the United States, there is no significant correlation between air pollution, water pollution and toxicity. Thus, although these three measures of pollution are very broadly defined, there is no general tendency that a sector which pollutes in one medium also pollutes in another medium. However, Table 1 does report a statistically significant correlation between abatement costs and toxic releases. Industries which on average have high abatement costs typically also emit toxic substances. If we use these alternative measures of pollution intensity in our analysis, we again find no relationship between high emissions in the U.S. and the pattern of foreign investment abroad. The second part of the study examines the behavior of foreign and domestic enterprises in these four host countries. We find that multinational firms are significantly more efficient in their use of energy than domestic plants. In addition, multinationals tend to use cleaner types of energy, such as electricity and natural gas. Even if we take into account the fact that multinational enterprises are typically younger than domestic firms, we still find that multinational firms of the same vintage are more energy efficient. To the extent that energy use is a good proxy for pollution emissions, this suggests that multinationals in developing countries tend to use cleaner technologies than domestic firms. To test whether energy use is a good proxy for emissions, we explore the relationship between energy use and toxicity across sectors, using U.S. data. We show that even in the U.S., where respectable air pollution control programs have been in place for more than 20 years, and the choice of fuels and electricity is highly varied, there is a strong statistical relationship between air pollution coefficients and energy use. As in Table 1, we use three measures of emissions: particulates, which measure air pollution; BOD, which measures water pollution; and toxics.

We report the rank correlations between those emissions and six different factor inputs in Table 2: the share of unskilled labor in total value of shipments, the skilled labor share, capital share, manufactured input shares, raw material input shares, and the share of energy inputs in total output. Energy use is highly correlated with different measures of emissions. The correlation between energy use and particulates is .58; between toxics and energy use the correlation varies between 0.52 and 0.55. The correlation with BOD is lower at 0.22,dutch buckets system though also significantly different from zero. Table 2 also shows that the correlation between pollution and energy use is much higher than for other factor inputs. Yet even if energy intensity could provide a good proxy for emissions across industries, energy intensity may not be a good proxy for differences in emissions between plants within the same industry. To investigate this issue, we used a cross section of U.S. manufacturing firms to examine the relationship between different types of factor inputs and plant-specific emissions, one industry at a time. The strength of the relationship between energy use and emissions varies with the type of industry. Specifically, particulates are highly correlated with energy use at the plant level for only four industries: chemicals, petroleum refining, lumber and wood products, and non-electrical machinery. Two of the most polluting activities in manufacturing—chemicals and petroleum refining—are included in these four sectors. Consequently, we repeated the analysis, restricting ourselves to only those four sectors where energy use serves as a reasonable proxy for emissions. We find that in those sectors, foreign firms are indeed more efficient in their use of energy.The third part of the study shifts the focus from developing countries to the United States. We examined the pattern of outbound U.S. foreign investment, asking whether, after controlling for other factors, there is any indication that most foreign investment originating in the United States and relocating abroad is located in pollution-intensive sectors. We find some evidence in support of this hypothesis, but the results are too weak to make strong claims.The Citrus Variety Improvement Program of INTA, in Argentina was established 29 years ago as an industry insurance policy. Since 1984, the Concordia Experiment Station has maintained citrus mother trees tested for freedom from disease and trueness to type. The objective is to secure propagating material of commercial varieties for industry needs. Varieties are selected from research projects and/or introductions and are subject to quarantine controls and a sanitation process. The selected trees are shoot-tip grafted and indexed for tristeza, psorosis, exocorThis, cachexia, citrus variegated chlorosis, canker and huanglongbing. Two hundred and eighteen scion and rootstock varieties are maintained and evaluated annually for their agronomic characteristics and sanitary status by visual observation and biological, immunochemical and/or molecular diagnostics. Data obtained on origin, botanical characteristics, agronomic performance, sanitary status and availability of propagation material is recorded. The information is available at INTA’s website . Basic material is exported to citrus producing countries and sent to germplasm banks on request. The isolation, annual evaluations, pest monitoring and disease diagnosis guarantee the quality of the material offered by the program to the Argentine citrus industry.Lemon cultivars recovered by shoot-tip grafting were compared with the original 30-40 year old nucellar clones located in a germplasm block in Tucuman, Argentina. Cultivars evaluated were Frost Eureka, Limoneira 8A Lisbon, Feminello Santa Theresa, and Genoa EEAT. These were grafted on Poncirus trifoliata Flying Dragon, except for Frost Eureka which was grafted on 79 AC Cleopatra mandarin x Swingle citrumelo [Citrus reshni xdue to its incompatibility with trifoliate rootstock. The selected clones were naturally infected with CTV, an endemic disease, and Frost Eureka was also infected with one viroid .

Trees propagated with STG budwood were indexed and confirmed to be free of CTV, psorosis, and citrus viroids before planting. A field trial was planted in November 2007, with four randomized blocks per treatment and four replicate trees per block. Tree height, canopy volume, trunk circumference, fruit yield and quality were evaluated. CTV infection was monitored annually in spring of 2009, 2010 and 2011, and thereafter in spring and fall of 2012 and 2013. Direct immuno printing ELISA with 3DF1+3CA5 monoclonal antibodies was used to detect CTV in field samples. In 2009, Limoneira 8A had the highest incidence of CTV infected plants followed by Genoa EEAT while Feminello Santa Theresa remained CTV free. In 2013, 100% of STG lemon lines were infected with CTV. Cumulative yield after four harvests of Limoneira 8A, Feminello Santa Theresa, and Genoa EEAT was no different from trees budded from their respective mother trees. In contrast, the STG line of Frost Eureka yielded 80% more fruit than the source tree with the viroid. In conclusion, CTV did not substantially affect the growth and yield of the lemon cultivars tested, while the viroid in Frost Eureka considerably reduced tree productivity.Shoot-Tip Grafting is an important part of a comprehensive certification program. Reduction in the time for certification to an average of two years is possible. Components of a highly successful STGing program include good scheduling, dedicated STGing personnel, a comprehensive testing program, a reliable source of seed, and a clean area for working including specialized tools and equipment. Proficiency in both the bud stick technique and the leaf-stripping technique will give flexibility in scheduling and allow faster completion times.Laboratory tests on young STGs should be used for a early indication of failure; follow up testing will determine if elimination of pathogens was successful. Precision trimming starts with the use of Gelrite for a solid base of STG set up, allowing the STGs to be evaluated in a horizontal position with a dissection microscope. Detailed notes are taken weekly and micro-trimming can prevent the selection of root sprouts by accident. Most STGs begin sprouting at two to three weeks. Small STGs grafted into root stocks will grow faster in the greenhouse and be ahead of STGs grown for a longer period in culture tubes in the laboratory. Increased sucrose can speed the development of some selections, but the increased number of root sprouts and associated trimming can be disadvantageous. The transfer to the greenhouse may be a problem area; it is best if personnel involved with STGing do the grafting and follow-up. Kinkoji is used for grafting of STGs as it is easy to work with year around and has increased our success rate.

Together they establish water use objectives and reporting standards for indoor and outdoor residential and commercial use; require SWRCB and CDWR to adopt long-term standards for efficient water use; update urban water management plans to include the reliability of the water supplies and strategies for meeting current and future water needs; require urban water suppliers to conduct a water supply and demand assessment and make water shortage contingency plans available to customers; and require water suppliers to declare emergency measures to ensure sufficient water for human consumption, sanitation and fire protection. AB 2371 was enacted on Sept. 28, 2018. It continues to enforce many current landscape water conservation practices in and out of drought, including hydrozoning, water budgeting, storm water collection, use of recycled water and irrigation equipment maintenance. In addition, it requires the Contractors State License Board to update the C-27 landscape contractors’ exam as needed to include questions on new and emerging landscape irrigation efficiency practices; allows potential purchasers of housing units containing in-ground landscape irrigation systems to require irrigation system inspections; and requires the formation of a working group to examine and consider updating current consumer information on landscape water use. It also requires CDWR, following a public hearing every 3 years,hydroponic nft channel to update MWELO or determine that an update is not needed and consider revising and updating the WUCOLS database.

UC continues to play a major role in providing objective information to policymakers as they formulate and update legislation on water conservation in commercial and residential landscapes. UC also continues to advance the science to conserve water and help ensure that legislative targets are met. Due to continued improvements in the efficiency of sprinkler and drip irrigation systems , ETAF was further reduced in 2015 from 0.7 to 0.55 for residential landscapes and from 0.7 to 0.45 for commercial landscapes . Conservation on this scale will rely heavily on implementing best practices that decrease water loss, identifying new species of drought-resistant landscape plants and improving irrigation system performance. In practice, many irrigation systems fall far short of the irrigation efficiencies used in the current MWELO. Bijoor et al. found that smart irrigation systems were more effective at reducing water loss than irrigation systems operated by conventional timers and that the difference exceeded water savings realized from selecting a warm-season turf species over a cool-season species. Reid and Oki continue to screen a wide variety of landscape plants for their drought resistance to expand the palette of California-friendly landscape plants. Work by their team has already led to the identification of hundreds of drought-resistant plants included in MWELO. UC ANR Specialist Amir Haghverdi is leading a project to further define water requirements of turf under deficit irrigation and reclaimed water regimes; evaluate the performance of soil moisture–sensing and ET-based smart landscape irrigation technologies on water use effectiveness under deficit irrigation; and monitor turf responses to multiple levels of water stress using multi-spectral and thermal remote-sensing techniques.

David Fujino and the Western Center for Agricultural Equipment established the Smart Landscape project at UC Davis in collaboration with more than 20 organizations and companies. Smart Landscape provides workshops and on-site training for landscapers and students on new water-saving technologies. Several UC faculty, specialists and advisors are involved in various projects throughout the state alone and in cooperation with the U.S. Forest Service and other groups to determine the long-term durability of a wide variety of underused landscape trees under warmer, drier conditions due to climate change and urban heat islands.Horticulture likely originated 20,000years ago. There are over 100 species of horticultural crops, consisting of diverse fruits, vegetables, and tubers, many of which are of high economic value with enormous production volume worldwide. The amounts of fruits, vegetables, and tubers produced in 2018 were 868, 1089, and 832 million tons respectively , and the increased demand from a growing, and afuent global population, is predicted to drive further expansion of horticultural output. Horticultural crops not only provide basic calories , but also, are among the most crucial sources of fiber, organic acids, micro- and macro minerals, vitamins, and antioxidants in human diets. Healthy attributes, and a wide range of tastes, textures, and favors make horticultural crops attractive. Starch is critical to human society given its versatile uses. Starch is the dominant energy source in the human diet, providing over 50% of our daily caloric needs. In the food industry, starch is widely used as a thickener, stabilizer, lipid replacer, defoaming agent, gelling agent, emulsifer, and dietary fiber, and in the pharmaceutical industry, starch is used as an excipient for drug delivery . In addition to these diverse uses, starch is an excellent renewable material for making ethanol biofuels and degradable ‘bioplastic’ products. Starch is almost ubiquitous in higher plants, including horticultural crops, in ways that may or may not be noticed.

For instance, potato, sweet potato, yam, and cassava are starchy, but spinach, lettuce, and ripe tomatoes, berries, and citrus are not, yet starch is likely to be important to the growth, development and fitness of all of these crops, as they are in better studied models. The widely accepted view is that starch accumulates either in a transitory state, or for long-term storage starch. Transitory starch follows a diurnal pattern: it is synthesized and accumulated directly from the products of photosynthesis in the leaf and in the stem during the daytime, and is then degraded into sugars as an energy source for the following night. In comparison, storage starch is defined as that located in perennating organs such as seeds, grain, embryos and tubers , where it provides sustenance for the next generation during germination and sprouting in sexual and asexual propagated crops, respectively. A third class of starch: ‘transitory-storage starch’ has been proposed. It describes starch that is accumulated and degraded during development in the storage organ. Transitory-storage starch is a feature of many species including horticultural crops of economic value such as tomato, banana, kiwi, strawberry, nectarine, and apple fruit . Starch accumulates as semi-crystalline, water insoluble granules that vary in diameter from 1 to 100μm depending on species. Starch is organized into two glucan polymers: amylose and amylopectin. Amylose and amylopectin consist primarily of linear chains of glucoses joined by α-1,4-glycosidic bonds. In amylopectin,vertical farming racks the α-1,4-glucan chains are branched more frequently through α-1,6-glycosidic bonds, compared to amylose. The branching of the amylopectin chains is such that chains of diferent lengths are produced: short, medium and long chains, and the frequency with which each fraction occurs influences starch functionality. Side chains of amylopectin form clusters around branching points, and two adjacent chains make up a double helix. These physical features of amylopectin polymers leads to a semi-crystalline granule; amylose with a randomly coiled conformation, fills the matrices within the granule. Amylopectin and amylose account for around 25 and 75% of the starch in major heterotrophic storage organs, respectively, while the starch in leaf tissues is approximately 5 – 10% amylose.Amylose and amylopectin are synthesized by the coordinate action of a group of four key enzymes. The core starch biosynthetic enzymes include ADP-glucose pyrophosphorylases , starch synthases , starch branching enzymes , and de-branching enzymes , of which there are many isoforms. In brief, AGPases initiate the frst step of starch biosynthesis by catalyzing the formation of ADP-glucose. SSs elongate the glucan chains in amylose and amylopectin; SBEs branch the glucan chains, while the DBEs shorten and modify the starch chains which enable a higher-order semicrystalline structure to form. SBEs, the focus of this review, hydrolyze α-1,4-linked glucan chains, and attach the newly-created ‘free’ chain to another glucan chain within the starch granule, via an α-1,6-linkage. Through this action, SBEs largely determine the proportion of the relatively unbranched amylose to the highly-branched amylopectin. Two major classes of SBEs are bio-functionally known: SBE1 and SBE2 , and they vary in terms of their substrate selectivity, whereas the function of SBE3 awaits verifcation across a broader set of species. SBE1 preferentially branches ‘amylose-like’ long glucan chains as the substrate, while SBE2 prefers a more branched substrate.SBEs are the key players in the regulation of the amylose-to-amylopectin proportion in plants. However, their functions in many harvested horticultural crops have been under-investigated, although evidence points to the importance of starch in determining the post harvest quality of these crops.

We aimed to develop a better understanding of the role of SBEs in fruits, tubers, and leafy greens in physiological processes by exploring SBE sequence relationships, expression, and starch phenotypes in diverse crops.SBEs have three classes of isozymes including two functional SBE classes and one putative class 3 SBE . SBE1 isoforms appeared earlier than SBE2 and SBE3 in the viridiplantae, but plant SBE1 and SBE2 are more homologous to each other, than to SBE3. SBEs have been identified and relatively well-characterized in cereal crops, tubers, and Arabidopsis thaliana over the last two decades ,but, as mentioned, little attention has been paid to the diverse group of species that are classified as horticultural crops. Within each class of SBE, the cereals grouped together, while most non-cereals formed another cluster . This pattern is due to the divergence of monocots from dicots around 200 million years ago. In contrast to the presence of ‘a’ and ‘b’ sub-isoforms of SBE2 in cereal crops, horticultural plant species generally have one SBE2 isoform. It was also observed that not all species have a known or predicted class 3 isoform. The SBE sequences contained within diverse organs, i.e., fruits, tubers, roots, and leafy vegetables , clusThered together based on their respective plant families. Te class 1 SBE is absent in Arabidopsis thaliana, and so it was not surprising that this SBE class is not present in the Brassicaceae. However, the class 1 SBE is also absent in apple , and European olive , but these species all have two class 2 SBE isoforms . In addition, banana contains at least four types of SBE2, and transcripts corresponding to these SBE2s have been identifed, indicating that they are expressed.Starch Branching Enzymes belong to the α-amylase family of enzymes, specifically the glycoside hydrolase family 13 super family, with multiple isoforms encoded by diferent genes . The overall structure of the SBE polypeptide is highly conserved: all SBEs possess a central α-amylase catalytic domain , and an NH2- terminus, and a carboxyl- terminus. The SBE NH2-terminus contains two conserved domains: a chloroplast transit peptide for plastid-targeting, and a CBM48 domain for binding to starch. The C-terminus contains the residues that determine substrate preference and catalytic activity. The central region of the enzyme contains the “A” catalytic domain, that is made up of 8–barrels. Notably, the class 3 SBE may not directly participate in starch biosynthesis in Arabidopsis, but it has a demonstrated function in mediating cesium toxicity of photosynthesis. However, the role of SBE3 is unlikely to be conserved. In potato, StSBE3 has a unique coiled-coil motif which is absent in the AtSBE3 polypeptide . Notably, the CBM48 domain is also deficient in AtSBE3 . It is possible that the StSBE3 may interact and complex with other starch biosynthetic enzymes through its coiled-coil domain, in a similar way to the SS4-PTST2 interaction in Arabidopsis, the GBSS-PTST1 interaction in rice or the SBE containing protein complexes in cereal endosperm , rendering an assistant function in starch biosynthesis. This species-specific mode of action of SBE3 may reveal a novel function of SBEs generally. Indeed, although all SBEs are predicted to form complexes with starch phosphorylases , the starch synthases and isoamylase , interactions with other proteins show differences depending on the species and SBE isoform.Four conserved regions critical for catalysis, named Regions 1-4 , are found within the catalytic A-domain . Regions 1-3 are directly involved in catalysis, while Region 4 is involved in direct substrate binding. SBE1 and 2 have largely invariant residues, but the residues in the SBE3 isoform of many species have substitutions at these sites. Post-transcriptional phosphorylation of the SBE-protein complexes formed with other starch biosynthetic enzymes has been found in cereal crops and in cassava, while experimental evidence of this regulation in the majority of horticultural crops is absent. SBE1 and SBE3 have fewer possible phosphorylation amino acid sites than SBE2 .

Human pathogen–plant models should be developed for the purpose of breeding efforts to enhance food safety based on enteric pathogen strain– plant commodity variety pairs identified from prominent or recurring foodborne illness outbreaks. At the same time, plant genetic resources that may facilitate genome-wide association studies should not be excluded. Furthermore, the use of human pathogens in routine assays requires highly trained personnel and laboratory/greenhouse bio-safety conditions according to NIH guidelines, in addition to considerable costs associated with the handling of microbial hazards in contained facilities. These approaches will require collaborative efforts among food safety experts, plant–microbe interaction biologists, microbiologists, and crop breeders for successful advancements in the field. Roots are not only vital for anchorage and for acquisition of water and nutrients from the soil, but are also engaged in complex physical and chemical interactions with the soil. Plant roots release approximately 11–40% of their photosynthetically fixed carbon, commonly known as root exudates, into the soil . Root exudates and mucilage act as nutrient sources and as signaling molecules for soil microorganisms, thus shaping the microbial community in the immediate vicinity of the root system . In turn, microbial processes promote plant growth by aiding in nutrient acquisition, plant growth hormone production and bio-control of plant pathogens . The physicochemical characteristics of the surrounding soil are also affected by interactions between roots and the microbial community. This interplay between the different rhizosphere components is affected by spatio-temporal processes,hydroponic grow system which culminates in dynamic feedback loops that maintain the complex rhizosphere environment with physical, chemical and biological gradients that are distinct from the bulk soil .

Understanding these intricate rhizosphere relationships is vital in devising strategies to increase plant productivity and comprehend localized bio-geochemical processes. In many rhizosphere studies, the use of pots and containers is predominant as it allows the plants to be cultivated under controlled conditions and at low cost. Compared to field studies, growth of plants in defined spaces also offers advantages in ease of handling, monitoring and sampling . Much of what we know of the rhizosphere microbiome has resulted from such pot-grown plants. However, since the rhizosphere and roots are still out of view in the soil, destructive sampling of the root is required prior to analysis. Destructive sampling may result in the loss of three-dimensional spatial information on rhizosphere processes over time, which is increasingly being recognized as a critical parameter. On the other hand, soil free techniques such as hydroponics and aeroponics can provide visual access to the rhizosphere circumventing the need for destructive sampling. Other alternatives are gel-based substrates which can maintain rhizosphere transparency as well as the 3D architecture of roots and have been applied successfully in high throughput imaging, phenotyping and trait mapping platforms . Nonetheless, the root phenotype and traits of plants grown under soil-free conditions are known to differ from those of soil-grown plants . These soil substitutes do not also accurately simulate the heterogeneous nature of soil aggregates, thus complicating extrapolations for field relevance. Sophisticated imaging approaches such as magnetic resonance imaging and X-ray computer tomography can be used to analyze root systems in the soil with minimal disturbance but they are low throughput, expensive and may not be easily accessible . It is apparent that structural changes in design caThered to solving specific challenges in the rhizosphere are indeed necessary.

To overcome these challenges relating to the rhizosphere in soil, specialized plant growth chamber systems have been designed, and successful implementation has led to multiple variations of similar designs. These specialized systems often have a visible rhizosphere which enables coupling with other technologies thereby increasing the breadth of experimental techniques applicable to the rhizosphere system. This review discusses representative growth chamber systems designed to study major rhizosphere processes and interactions in soil.Specifically, the reviewed growth systems are selected based on the following criteria: the growth chamber is amenable for use with soil/soil-like substrates and therefore, hydroponics, aeroponics and agar/gel-based systems are not discussed except in microfluidic-based platforms, it is built with the intention to maintain growth of the plant and has architectural features distinct from conventional pots, and lastly it is able to be set up in a laboratory; i.e., field measurement systems and observation platforms are not included. For instance, a minirhizotron, consisting of a camera mounted in a glass tube submerged in the soil which provides non-destructive root imaging over time will not be discussed as it is out of the scope of this review. Through our assessment of lab-based chamber systems, we identify unique advantages and challenges associated with each system . We hope that future fabrication designs can benefit and improve on designs that work well. Lastly, we offer our perspectives on areas in which technological advances are needed to fill current knowledge gaps. In studying rhizosphere processes, the myriad of complex interactions among members of the rhizosphere are often dissected to two interacting variables such as root-and-soil or root-and-microbes, etc. Each of these interactions inherently operates under distinct parameters and requires specifically designed platforms to effectively answer different research questions. This review is structured in a way that first describes each rhizosphere process briefly and then reports on the specific growth chamber systems designed to facilitate experiments for answering related research questions.

The major rhizosphere processes discussed below include root system architecture, physicochemical gradients in the soil, exudation patterns by the roots and interactions between roots and nematodes, fungi or bacteria.Root system architecture encompasses structural features that provide spatial configuration such as root length, width, spread and number and is an important rhizosphere parameter in regulating soil porosity, and nutrient and water uptake efficiency by plants . Plants have been observed to “sense” and direct root growth toward nutrient sources in soil, and the RSA of a plant exhibits great malleability in response to environmental stimuli which in turn, influences microbial communities . For instance, bean plants grew deeper roots under drought conditions to enhance water foraging capabilities while low phosphate conditions stimulated the formation of dense lateral roots involved in P uptake from upper soil layers . Given that most soils are heterogenous, understanding the RSA of plants becomes critical in improving resource use efficiency and agricultural yields . Often, RSA in pot-grown plants is investigated by excising the roots via mechanical means such as root washing or blowing with compressed air . These methods are, however,ebb and flow trays time-consuming, cause inevitable damage of fine root hairs and result in loss of spatial and temporal information . An appealing alternative for studying RSA is the use of rhizotrons. Rhizotrons were initially constructed as underground facilities designed for viewing and measuring roots in the field . In the lab, the rhizotron implies a chamber constructed using two vertical sheets with at least one or both of the sheets being transparent and/or removable . This allows repeated visual inspections of individual roots; a feature unachievable with destructive sampling. In some cases, the word “rhizobox” is used for a similar set up although this was first introduced in as compartmentalized systems to separate the root and soil compartments . Rhizotrons/rhizoboxes are often constructed with PVC or acrylic materials and come in many sizes to accommodate different plants with soil or soil-less substrates . Root growth and morphology in the rhizotron can be tracked by a variety of methods ranging from manual tracing onto a plastic sheet, using handheld or flat bed scanners to fully automated time-lapse imaging camera systems .Data can be subsequently analyzed with a wide range of software packages . Affordable and robust RSA imaging platforms using rhizotrons have also been developed for increased accessibility in low-income countries . The versatile construction of a rhizotron design for RSA studies has inspired many variations. For instance, ara-rhizotrons were designed to enable the study of 3D canopy competition with simultaneous root growth observation in an Arabidopsis plant population . The horizontal and radial design of HorhizotronTM and mini-Horhizotron consisting of transparent quadrants attached to a central chamber were developed to study lateral growth of roots in a semi-3D space and to perform post-transplant assessment . The separated quadrants can also be used with different soil substrates simultaneously to study substrate effects on root growth . A rhizotron fitted with water-tight gasket seals has also been used successfully to investigate the RSA of plants under water-logged conditions . Despite the continuous real-time visual read-out, most rhizotron designs suffer from inevitable loss of information from roots occluded by soil particles. The GLO-Roots system overcomes this by imaging from both sides of the rhizotron while using bio-luminescent roots to create higher contrast against the soil, enabling quantitative studies on RSA . Following advances in engineering and device fabrication, more rhizotron variants adapted to specific plant growth conditions can be envisioned.In a typical topsoil, approximately half is composed of solid minerals and organic matter while the rest is a fluctuating composition of water and gas filled spaces influenced by environmental conditions and uptake/release of solutes from plants .

Changes in gaseous and hydrologic parameters, such as ions, O2 and moisture among others, create a spatially complex environment that influences microbial communities and overall plant health. These physicochemical fluxes are heterogeneously distributed along roots and vary with root types and zones . Often, they exist as gradients in the rhizosphere , thus emphasizing the need for non-destructive sampling in order to accurately capture processes occurring at biologically relevant times and scales. Rhizotron chambers with a visually accessible rhizosphere allows in situ and continuous mapping of these gradients in the soil through the use of different types of imaging methods. For instance, photo luminescence-based optical sensors enable in situ, repeated detection of small molecule analytes in addition to pH , O2 and NH4 . Methods like zymography to detect enzyme activity and diffusive gradients in thin film can be used to map solute concentrations in the soil down to sub-mm scales with high spatial resolution more realistically than traditional destructive approaches. For example, transport and distribution of water in the rhizosphere soil has been imaged on both 2D and 3D planes by coupling a rhizotron with neutron radiography and tomography, respectively and showed varying moisture gradients along the root system with higher water uptake at the rhizosphere compared to bulk soil. On the other hand, if the rhizotron slabs are thin enough , even simple imaging solutions based on light transmission can be set up to capture water uptake by roots in sand . Despite trade-offs in method sensitivity between these two studies, a rhizotron set up is critical in both designs and illustrates its adaptability to multiple equipment.Roots exude a substantial amount of photo synthetically fixed organic carbon into the soil consisting of a wide variety of compounds such as sugars, organic acids, and primary and secondary metabolites . Together with mucilage and border cells , root exudates provide a major source of nutrients for the rhizosphere microbiome . Root exudation is regulated under genetic control as well as in response to environmental conditions in the soil such as nutrient limitations or increase in toxicity . Exudate patterns are also recognized as one of the strongest drivers shaping the rhizosphere microbiome . As a central player in the rhizosphere ecosystem, it is imperative to understand root exudation patterns to unravel subsequent impacts to the surrounding soil and microbial community. Improvements in analytical instrumentation have made it possible to move from targeted to untargeted explorations with mass spectrometry to create root exudate fingerprints in its entire complexity . Regardless, the impact of such techniques relies partly on our exudate sampling techniques. Detection of exudates in real-time is difficult due to rapid biotransformation and sorption to the soil matrix. As such, common collection methods rely on root washing in hydroponic systems to overcome complications in the soil matrix and preserve native exudation profiles. However, a comparison between a soil-based collection method and hydroponic methods showed varied responses particularly in amino acid exudation although the underlying cause was not elucidated . It is possible that the differing growth conditions between hydroponics and soil, which include differences in gas concentrations, mechanical impedance and microbial spatial composition, can elicit differing root exudation responses to the same environmental stimuli.

Historically, from 1950-2002, alfalfa and cotton have been among California’s top commodities in terms of total value . In 1950 cotton was ranked third in terms of value of production in California with a value of $202 million. By 2001, cotton had slipped to the eighth most valuable commodity in California in value of production. The trend has been downward during the period 1950-2002. Hay was ranked fifth in 1950 in California with a value of production of $121 million. In 2001, hay was ranked seventh in value of production just ahead of cotton. Models are developed for California alfalfa and cotton acreage, production, and consumption. Both single equation and systems of equations are estimated. The data consist of 33 annual observations from 1970 to 2002. In some models, there were slightly fewer observations due to lags in the specifications. A brief description of the alfalfa market is given prior to reporting the estimations of the models. In addition, some issues related to the nature of the data are discussed. Alfalfa hay acreage in California has averaged about a million acres per year during the past 30 years . Alfalfa contributes about 85 percent of the value of all hay production in California. Alfalfa is influenced by profitability of alternative annual crops such as cotton, tomatoes, trees, and vines. The demand for alfalfa hay is determined to a large degree by the size of the state’s dairy herd, which consumes about 70 percent of the supply. Horses consume about 20 percent. Alfalfa is a perennial crop with a three to five-year economic life. Since it is a water intensive crop,grow table its profitability is strongly influenced by water and water costs. In addition, alfalfa is important in crop rotations because of its beneficial effects on the soil .

Alfalfa production in California has been increasing annually since the mid nineties . It reached a peak in 2002 at 8.1 million tons. The increase in production has been primarily due to the upward trend in yields and not to increases in acreage. Alfalfa real grower price in California, using a 1983/84 base, has exhibited a downward trend since the early eighties . In 2002 the real grower price was about $60 per ton. A three-equation system for alfalfa was developed and estimated. Iterative three stage least squares are used to estimate a model consisting of acreage, production, and demand relationships for alfalfa. We assume that the market for alfalfa is in equilibrium, that is, that quantity demanded is equal to production. We further assume that stocks are included in the demand for alfalfa. Thus, the three endogenous variables are: acreage, production, and alfalfa price. The estimators will be asymptotically efficient given that the model is specified correctly. The gain in efficiency is due to taking into account the correlation across equations. And three-stage least squares will purge the correlation that exist between endogenous variables on the right hand side of the equations in the model with the error terms. Cotton is the most important field crop gown in California. Growers in California grow two types of cotton: Upland, or Acala and Pima. Upland cotton makes up about 70 to 75 percent of the California cotton market and is the higher-quality cotton. Upland has a worldwide reputation as the premium medium staple cotton, with consistently high fiber strength useful in many apparel fabric applications. Export markets are important, attracting as much as 80 percent of California’s annual cotton production in some years making it California’s second highest export crop . Historically, California cotton, in terms of value of production, was the third highest ranking crop in California in 1950 below cattle and calves and dairy products.

In 2001 cotton was ranked the eighth highest valued crop below milk and cream, grapes, nursery products, cattle and calves, lettuce, oranges, and hay . There has been a downward trend in cotton acreage and production in California since 1979. California growers produced 3.4 million bales of cotton on l.6 million acres in 1979. In 2002 they produced about 2 million bales of cotton on 700,000 acres . Cotton yields have experienced an upward trend since 1979 . Nominal producers’ prices in California for cotton exhibit an upward trend since the 1970s, but real producers’ prices in California has exhibit a downward trend since the mid seventies . Recently the World Trade Organization ruled against U.S. cotton subsidies. U.S. cotton subsidies totaled about $10 billion in 2002 and the WTO ruled that the subsidies created an unfair competition for Brazil, which filed the complaint. California producers received about $1.2 billion in subsidies in 2002. California cotton is not as subsidized as cotton in other states, such as Texas, because subsidies are based on price and California’s higher-quality cotton is more expensive . Acreage, production, and demand equations are estimated for California cotton. Single equation and system of equations models are developed and estimated. In this report we aggregated the different cotton varieties. Disaggregated models of cotton were also estimated because of changes in the cotton industry and to allow for different impacts for subsidized and unsubsidized varieties. The number of observations in the disaggregated models present in the next section are limited due to the relatively recent introduction of Pima in California. The estimated models indicate that the short-run own-price elasticity of alfalfa acreage is inelastic but more elastic when ample water is available. By applying water marginally through out the growing period, a producer can obtain more cuttings of alfalfa. Alfalfa yields are also responsive to increases in prices. The own price elasticity of yields is 0.08 and highly significant.

Alfalfa yields are negatively related to the previous year’s cotton price. Production is positively related to own price with an estimated elasticity of 0.44 and significant. Production was negatively related to risk with an elasticity of risk equal to -0.75. Demand for alfalfa is a derived demand and is positively related to the number of cows and milk price support and negatively related to its own price. The estimated own-price elasticity of cotton acreage is 0.53 and highly significant. Cotton acreage decreases with an increase in risk in growing cotton and as price of alfalfa increases. The short-run own-price elasticity of cotton production is 0.497 and the long-run estimate is 0.503. The own-price elasticity of cotton demand is – 0.684. Rayon is a substitute for cotton. The empirical results support the fact that alfalfa and cotton are rotating crops in California. In recent years there has been an increase in Pima acreage relative to the traditional Upland variety in California. Upland cotton prices have a positive impact on acres planted to Upland. When Pima prices increase, the acres planted to Upland decrease. A similar situation applies to Pima acreage. That is, an increase in Upland prices causes a decrease in Pima acreage. Thus, the empirical results support that hypothesis that relative prices of Upland and Pima have a significant impact on the adoption of the two varieties. Future research needs to focus on the collection of more data related to the consumption of California cotton and alfalfa, stocks and inventories, and interstate trade of alfalfa between California and Oregon and Nevada. California is one of the major producers of rice in the US. The other most important states are Arkansas, Louisiana, Mississippi, Missouri and Texas. The market in California appears to be fully integrated with the southern states, as suggested by an empirical check of the law of one price. This conclusion is hardly surprising,growing lettuce hydroponically given that rice is a storable and easily transportable commodity. Figure 1 illustrates the law of one price between California and Arkansas. A simple ordinary least squares regression of California rice price on Arkansas price gives an R2 of 0.939 and an estimated slope coefficient between 0.80 and 0.94 . Moreover, a simple cointegration test suggests the absence of unit roots in the disturbances. Thus, California price and Arkansas rice prices move together over the long run. Market integration suggests that a US level model can be useful to describe California rice production. In this study, however, we present both national and state models. Prior to reporting the estimated production function for rice, a brief discussion of some aberrations of the rice market will be explained. Around 1976-77 there was a price collapse that caused producers to rotate to other crops or not plant rice at all. This lead to decreases in rice production. In the early eighties rice prices collapsed again and this caused many growers to forfeit their crop to the government because the price was below the value of the government loan. This was not only the case with rice, but other program crops such as wheat and corn. In an attempt to reduce acreage and sell off the rice that the government had claimed, the government implemented the 50/92 plan. Subsides were directly linked to production. Thus, if a grower did not produce he was not paid. The 50/92 program allowed the grower to produce on 50% of his acreage and receive 92% of the subsidies that he would receive if he had produced on 100% of his land. This reduced production allowed the government to reduce the stocks of commodities that they had to claim in 1981-82. The 50/92 program ran until about 1988. Since then subsidies have been decoupled from production to prevent problems like this from happening again. The 50/92 program was popular in the south, especially in Texas where their production was lower and they had low fixed costs of land, but in California it was only widely used for a few years.

Policy variables are incorporated into some of the models below. The estimated US price expectation elasticity of supply is 0.45 which is also inelastic but is not significant. The estimated coefficient of Thailand price of rice is 0.27 with a t-ratio of about two. The index for price support is positive but not significant. There is also a positive and significant time trend in the supply of rice. According to the estimated price coefficient , the elasticity of demand of US rice implies that an increase of 1% in price results in a decrease of 0.36% change in the quantity demanded. As the price of Thailand rice increases, the demand for US rice increases, but again the estimated coefficient is not significant. The income elasticity is 0.33 and the estimated coefficient of Japanese income is 0.34 as expected. Both coefficients are not significant, however. Rice producers in California and throughout the United States respond positively to increases in rice prices. The short-run price elasticity of production, based on a partial adjustment model, for the US was estimated to be 0.23. When policy variables were included in the production equation the price elasticity dropped to 0.18 .The production equation was an aggregated one. For a disaggregated approach that estimates how rice producers respond to different support programs, see McDonald and Sumner. The estimated own-price elasticity of demand for rice was found to be inelastic for a SUR system. The income elasticity for rice was estimated to be 0.74 in a single-equation demand function . US rice producers export less when the US price increases . They export more when the Thailand rice price increases since Thailand is a major competitor in the world market. California is the second leading producer of fresh tomatoes in the US, after Florida. Figures 1-3 compares fresh tomatoes planted acreage, production and nominal price for US, Florida and California. California accounts for about 95 percent of the area harvested for processing tomatoes in the United States—up from 79 percent in 1980 and 87 percent in 1990. The other major producers are Texas, Utah, Illinois, Virginia, and Delaware and Florida. In Figure 1, total U.S. fresh tomato acreage has declined over the period 1960 to 2002, but acreage in California and Florida has remained steady. The declined in acreage has come from the states of Texas, Utah, Illinois, Virginia, and Delaware . Figures 4-6 illustrates the trends for California and US planted acreage, production and nominal prices for processed tomatoes.Tomato growing is based on grower-processor contract agreements. The majority of production is traded this way with the spot market playing a marginal role. Most initial processing is by firms that manufacture tomato paste, a raw ingredient.