Horticulture

The combination of these approaches is useful for the identification of genes and traits under selection in natural settings, but so far few studies have taken this approach. Eckert et al. tested the association of SNPs with five phenotypic traits and 11 environmental variables across 10 P. lambertiana populations around Lake Tahoe. This study identified six genes associated with phenotypic traits , and 31 associated with environmental PCs. Two genes were associated with both a trait and an environmental axis, including a glucose transport protein associated with d13C and environmental variables linked to water availability. A study focusing on multiple drought response traits and a larger number of SNPs might be able to identify more genes that have variants associated with both environmental gradients and drought tolerance traits. Some traits and processes involved in drought response have been better studied at the genetic level than others . Provenance studies have indicated that differences in stomatal control and shoot growth are often involved in local adaptation to drought, and all other study types have identified the genes likely to be involved . However, although root growth has also been identified as important by provenance studies,nft growing system root-growth-related genes have not been identified.

Conversely, although genes related to resistance traits, such as changes in carbohydrate metabolism, and protective and pathogen defense molecule production, have been identified in expression or association studies, these traits have been largely ignored in provenance studies. Finally, xylem traits, including refilling ability, have not been the focus of any genetic study type.Tree improvement programs that aim to increase growth potential and stress resistance face the challenges of long generation times, the need for large-scale field experiments and the late expression of traits such as wood density . Genomic selection, already routinely used in livestock breeding, has been proposed as a method of speeding up this process by using marker-predicted breeding values for phenotypes of interest . This approach is suitable for species with low LD and for traits with complex genetic architectures as it uses thousands of markers with effects that are estimated simultaneously . As with traditional phenotypic selection, accuracy is likely to be greatest when tests are carried out in environments similar to the target environment, because of the high likelihood of genotype 9 environment interactions . Several recent studies have demonstrated the potential of genomic selection approaches for traits of interest to forestry. Resende et al. carried out an early evaluation of genomic selection in P. taeda, making use of clonally replicated individuals grown on four sites and genotyped at 4825 SNPs. They found that the accuracy of prediction models within sites ranged from 0.63 to 0.75 for diameter and height, and estimated that the breeding cycle could be speeded up by 50% with this method.

Gamal El-Dien et al. used GBS to genotype over 1000 interior spruce trees over three sites that had been phenotyped for yield and wood attributes, and found that the incorporation of genomic information produced more accurate heritability estimates. Genomic estimated breeding values were most accurate when data from multiple sites were used to fit the model. Of even more relevance to selection for drought tolerance, Jaramillo-Correa et al. identified 18 SNPs associated with climatic PC axes in P. pinaster, and found that the frequency of locally advantageous alleles at these loci correlated with population level survival rates in a common garden at the hot/dry end of the species range. Together with the growth trait analyses, these results suggest that association techniques could be applied to predict breeding values for overall drought tolerance or particular drought tolerance traits even though only some of the loci involved have been identified. There is evidence of significant potential for selection approaches to improve drought responses in conifers. Provenance studies have shown evidence of genetic differentiation between populations in drought responses, and genome scan and G2E associations are finding evidence of natural selection on within-species genetic variation. Second, heritabilities for drought tolerance traits, when these have been examined, tend to be moderate to high. The calculation of heritability requires pedigree information: parent– offspring or sibling and half-sib comparisons. Narrow-sense heritability is the fraction of the variance in a trait attributable to additive genetic variation, as opposed to environmental and nonadditive genetic variation. Because heritability depends on both genetic variation in the population assessed and the degree of variation caused by the environment, estimates are not transferable between situations.

In P. pinaster, estimates of d13C narrow-sense heritability ranged from 0.17 to 0.41, depending on how many individuals of what populations were assessed in what sites; and ring width and height growth rates were also moderately heritable . In the same species, heritability of P50 was 0.44, but this was driven more strongly by low levels of other sources of variation rather than high additive genetic variation . Across species, measured heritabilities for d13C range from the very high 0.7 for Araucaria cunninghamii to < 0.1 for P. taeda . Managers of wild forests are often focused on ensuring the resilience and function of the ecosystem rather than productivity. G2E and G2P association studies may help to identify seed sources that could be ‘pre-adapted’ to projected conditions for replanting in wildlands. However, wild trees face a range of challenges, including disease and competition, as well as drought . Stand structure and soil properties may also directly affect how trees experience drought stress. Studies that integrate stand level processes with genetic testing can further bridge gaps between genetic experiments and forest-scale management. Restoration projects could be used as experiments to test genomic predictions of survival and growth in a given environment, as well as the effects of genetic composition and diversity of the planted population on restoration success.Common garden, gene expression and genetic association studies all have different strengths and weaknesses, and none alone will answer the question of how genetic differences affect drought tolerance . As described previously, a combination of different types of association study may help to identify loci that are under selection in the wild and the traits they influence. Similarly, gene expression studies could easily be combined with common garden studies of adults or seedlings to address whether differences in drought responses between populations or genotypes are a result of differences in gene sequences, gene expression patterns or both.Many studies to date have focused on WUE, often using d13C as a proxy. As discussed above, however, WUE is a ratio of changes in photosynthesis and transpiration, which can both vary, and higher WUE may or may not be associated with greater survival or growth in dry conditions. Moreover, different measures of WUE are not entirely consistent. We therefore recommend that future studies use survival and/or growth during and following drought as the metric of overall ‘drought tolerance’,vertical hydroponic nft system and measure photosynthesis and water loss separately if these are processes of interest. The time involved in the measurement of traits for hundreds or thousands of individuals has encouraged the focus on easily measured d13C, but much progress has been made in high-throughput phenotyping techniques . For instance, thermal and long wave infrared sensors can measure leaf temperature or stomatal conductance, near and short-wave infrared sensors can measure leaf water content, and fluorescence sensors can measure chlorophyll content and photosystem efficiency .There are several traits and processes that have been suggested to be important for drought response by physiological studies, but about which there is little genetic information .

Genetic studies frequently identify genes related to carbohydrate metabolism and transport as having altered expression or allele frequencies depending on water availability. It is difficult to make sense of these patterns because the link between these metabolic changes and tree function and survival during drought is still unclear. We also know relatively little about which species can refill cavitated xylem, under what circumstances and by what mechanisms. Thus, it is difficult to determine whether any genes identified by expression or G2E studies are involved in this process. Similarly, how roots and root growth respond to changes in water availability, and what genes are involved in these responses, remain poorly understood. Although the measurement of root architecture can be complex, high-throughput methods are being developed for this as well .Most experimental studies, including those looking at gene expression, have focused on seedlings. There may be important differences in how different life stages respond to drought.

For instance, Pinus nigra ssp. laricio adults have been observed to follow an isohydric strategy, whereas seedlings in a glasshouse experiment did not . Although it is more complicated to impose drought treatments on adults, drought experiments have been carried out on adult trees using networks of rain shields/ gutters to intercept precipitation and direct it away from the trees . This water can also be re-directed to other plots to create ‘well watered’ treatments. For the most part, these studies have been carried out on natural populations. However, if they were coupled with provenance study plantings, one could test for population or genotypic differences in adult drought response. Likewise, apart from some long-term provenance studies , most experiments span a few days to a few months. In order to investigate drought resilience and legacy effects, more multi-year studies are needed.The length and intensity of drought can affect which trait combinations result in greater fitness. In Section IV.1, we mentioned the great diversity of methods used to induce or measure drought stress treatments in gene expression studies. The same diversity is found in G2P and provenance studies as well. There is a need to assess: whether environmental treatments roughly match the range of conditions in the environments in which the target species does or might grow; how environmental treatments relate to plant stress measures ; and whether traits, responses or genotypes associated with drought tolerance in the glasshouse or laboratory predict performance in the field. In addition, studies testing longer term drought treatments are lacking, as are those that explicitly test variable combinations of drought length and severity. Future work should address these gaps.In most of the genetic studies cited above, a relatively high proportion of the genes expressed or linked to phenotypes or environmental gradients of interest either have unknown or poorly defined functions. Behringer et al. , for instance, found that, of the 832 transcripts analyzed for gene ontology, 538 either had no database hits or could not be assigned to a biological process. Although this could be partly addressed with further studies intraditional model organisms, such as Arabidopsis, analysis of loblolly pine and Norway and Sitka spruce genome sequences suggests that there could be thousands of conifer-specific gene families . This shortcoming must be addressed by further development of model systems in conifers.Unsurprisingly, the conifer taxa that have received the most attention in terms of drought tolerance studies or genomic studies are those that are of high economic value, especially those that are frequently grown in plantations.This means that most drought tolerance genetics studies have been carried out on pines , with a modest representation of spruce and Douglas-fir. Although this focus is understandable, this means that a number of ecologically or economically significant taxa have been left out, notably the Cupressaceae . Many Juniperus and New World Cupressus and Calocedrus species are impressively drought tolerant. In the well-studied pinyon–juniper woodlands of the American Southwest, anisohydric juniper tends to exhibit lower mortality than Pinus edulis during severe drought . However, not all pine species exhibit isohydric behavior . Thus, the genes involved in drought response could differ substantially between families, genera or species.In this study, known negative regulators of the drought signaling pathway were knocked down using RNAi to generate increasingly drought tolerant plants for both Arabidopsis thaliana and Brassica napus. Previous studies have shown that knocking out Clade A Type 2C protein phosphatases , which are negative regulators in the drought signaling pathway, resulted in enhanced drought tolerance, but has come at the cost of decreased growth in non-drought conditions. As a mechanism for enhancing drought tolerance without compromising yield in well-watered conditions, RNAi was utilized to knockdown PP2Cs instead of knocking them out. Therefore, we generated stress inducible and constitutive knockdowns of PP2Cs in A.thaliana and stress-inducible knockdowns of PP2Cs in B.napus.

The region of Araraquara has the largest insect Las+ population in summer, while Bebedouro has the smallest, during the whole year. The Asian citrus psyllid is the only known vector of the bacterium, ‘Candidatus’ Liberibacter asiaticus , that causes huanglongbing or citrus greening disease. This insect acquires CLas from an infected citrus tree while feeding as a nymph. Transmission to uninfected trees occurs when infected adults emerge and fly to, and feed on, uninfected trees. Our current understanding of the CLas-psyllid interaction suggests that adults become competent for transmission only after the bacterium moves from the insect gut into the hemolymph and eventually to the salivary glands. We hypothesize that specific molecular interactions between the bacterium and gut epithelial cell membranes are necessary to initiate the movement of the bacterium throughout the psyllid’s body. Furthermore, we hypothesize that if we could block these specific molecular interactions, we may block the bacterium from reaching the psyllid’s salivary glands and therefore block the psyllid’s ability to transmit this bacterium to healthy trees. We tested these hypotheses by first developing an assay to screen and identify psyllid gut membrane binding peptides from a combinatorial peptide library and then feeding selected peptides to psyllids and determining their ability to reduce movement of CLas from the gut to the salivary glands. A total of 7 peptides were identified that bind psyllid gut preparations that demonstrated gut-binding after oral uptake.

These peptides were detected bound to gut epithelial cells in adults that had emerged from nymphs that were fed on the peptides 4 days earlier. Using qPCR assays,ebb and flow table we demonstrated that oral delivery of a combination of three of these peptides apparently inhibited movement of the bacterium into the psyllid’s salivary glands. These results support our above stated hypotheses and demonstrate a potentially new strategy for preventing the spread of citrus greening disease. Research on development of solutions to citrus greening has been hampered by the inability to culture ‘Candidatus’ Liberibacter asiaticus , the bacterium associated with this disease. Further complicating the matter, this bacterium resides within the phloem of the plant and targeting it with bactericides requires penetration into the plant so that the bactericide can reach the bacterium. To overcome these obstacles we developed a rapid screen for both bactericidal activity against CLas and phloem mobility within the citrus phloem In this assay, single CLas+ leaves from Valencia are removed from CLas+ infected citrus, indexed for CLas titer by Q-PCR and Q-RT-PCR of petiole clippings. The leaves are then placed in liquid solutions for 6 days, after which, the CLas titer is determined in both the petiole and leaf midrib. Using this assay we have demonstrated that streptomycin and penicillin are active against the CLas bacterium but that streptomycin bactericidal activity moves more readily into the leaf phloem. We have also used this assay to identify two new antimicrobial molecules more active at reducing leaf midrib CLas than the standard antibiotics when compared at the same concentration .

Using both Q-PCR and Q-RT-PCR we demonstrated that reductions in CLas16S rRNA abundance was more pronounced than reductions in the cognate DNA sequence. Therefore analysis of 16S rRNA is a more sensitive analysis of perturbations in CLas status in the plant. This assay allows concomitant analysis of bactericide activity and plant mobility and is currently being used to screen potential antimicrobial molecules for use in fighting citrus greening. RNA interference , mediated by double-stranded RNA is a gene suppression method that triggers sequence specific mRNA degradation. In citrus, transgenic expression of antisense RNA or hairpin RNA can be used to generate plants with specific genes silenced. However, this technique involves lengthy procedures because of slow growth and long juvenility of citrus trees. Here we set out to develop a quick and convenient method to silence genes in established trees. In one test, commercially synthetized dsRNA was applied weekly to potted Hamlin sweet oranges grown in artificial rooting medium. A 170 base pair dsRNA homologous to citrus phytoene desaturase was able to significantly reduce the expression of the target transcript, at a dose of 200 µg per plant. Photo-bleaching and reduced chlorophyll content, from suppression of CiPDS, were observed in 1 tree at a dose of 200 µg and 2 trees at a dose of 2 mg per plant. In another test, citrus Terminal Flower 1 that negatively regulates flower initiation was targeted in an attempt to induce early flowering. Persian lime plants grown in soil had dsRNA applied as drench weekly, in combination with cold and drought treatment to induce flowering. After 11 treatments, flowers were observed from two individual plants treated with 200 µg dsRNA-CiTFL1, but not from the control treatment .

In summary, initial results indicated root application of dsRNA is capable of triggering gene silencing in citrus. We hypothesize that the magnitude and consistency of the silencing effect can be further improved through optimized application protocols. With appropriate targets identified, we hope that RNAi may be used in citrus plantings to alter physiological/developmental plant responses to improve tolerance to Candidatus Liberibacter, or mitigate negative effects caused by infection.Pathogen-associated molecular patterns -triggered immunity is an important component of the inducible defense system of plants. Citrus species possess varying levels of responsiveness to PAMP flg22 derived from the flagellum of Xanthomonas citri ssp. citri , the bacterial causal agent of citrus canker. Higher sensitivity to Xcc-flg22 is mediated by robust expression of the plant cell surface receptor gene FLS2 and confers an increased level of canker resistance. In this study, we explored the induction of PTI by chitin, another PAMP that is a structural component of insect stylets and exoskeleton, and studied its effect on the feeding behavior of Asian citrus psyllid , the vector of Huanglongbing. The results showed that infiltration of chitin solution into ‘Sun Chu Sha’ mandarin leaves induced expression after 1h of defense genes including WRKY22, GST1, SGT1, RAR1, PAL1, NDR1 and EDS1, a set of genes previously shown to be induced by Xcc-flg22 in the canker-resistant ‘Sun Chu Sha’, but not in canker-susceptible varieties. However, the chitin-induced gene expression was not detected at 18h after infiltration. ACP feeding behavior was monitored using electrical penetration graph to study how induction of PTI by chitin modifies the number and duration of xylem and phloem feeding bouts. When adult ACP started feeding at 1h after infiltration, reduced xylem and phloem feeding were observed in chitin-treated leaves compared with untreated controls. However, this difference was smaller when psyllids started feeding at 18h after infiltration. In summary, our study shows that chitin treatment induced a transient PTI that can affect ACP phloem ingestion,flood table and this inducible defense will be further studied for its utilization on selection and generation of resistant citrus materials. Citrus production has plummeted in Florida by more than 50% in the last 10 years, although many Florida growers still maintain yields in spite of huanglongbing by increasing inputs of insecticides and nutrients. Consequently, focus is shifting to health and profitability of the next generation of trees. Area wide Asian citrus psyllid management and new planting systems are needed to maintain tree health and bring new blocks into rapid production. Systemic insecticides applied to the soil supplemented by foliar sprays provide some protection against ACP but have proven insufficient to adequately control HLB infection during critical the first 3 years after planting. Planting into a bed covered with metalized polyethylene film offers an additional layer of protection by warding off incoming ACP adults through flight disruption. Lack of similar effects with white mulch reflecting only visible light indicates that reflected light in the UV range is required. Use of plastic mulch is a novel practice in citrus production requiring delivery of water, nutrients and systemic pesticides by drip irrigation. Therefore, adoption of this practice to commercial production is necessarily a gradual process. Nevertheless, some large scale trials have been conducted on commercial farms and interest in growing as results come in and technology improves. The metalized mulch must be of sufficient thickness and protected with a clear coat to last 3 years.

Ground must be well prepared and film mechanically installed as a tight 125-145 cm wide strip, higher in the center to shed water and sprays. Drip irrigation can be provided by one or two relatively inexpensive drip tubes placed under the mulch or punch-in emitters at the base of each tree. Trees are typically planted through 15 cm holes cut in the plastic using a post-hole digger. Oil containing sprays should be avoided to protect the polyethylene from undo degradation. Metalized mulch consistently reduces ACP populations and HLB incidence compared to whiteface mulch or bare ground. In addition, the mulch plus drip irrigation system increases soil moisture, reduces weed pressure, and accelerates growth. Chemigation has proved more efficient than soil drenches for delivering systemic insecticide once the root system is established. Yield after 3.5 years in a large scale replicated experiment was improved by 44% from trees planted on UV reflective mulch and treated with insecticides compared to insecticides alone and brix improved by 5%. Thus, metalized mulch can improve growing conditions and augment current insecticide based ACP control measures for young trees. The potential to more than compensate for additional costs by savings of water, fertilizer and herbicides, shortening time to crop profitability and increasing yields has inspired interest in using this technology to protect young citrus plantings threatened by HLB. Huanglongbing is severely impacting Florida citrus. The sweet oranges and grapefruit dominating Florida production appear to be among the most HLB-susceptible citrus cultivars. Transition to resistant or tolerant cultivars will be essential to sustain the Florida citrus industry, with solutions needed in the short and long term. Evidence mounts that useful resistance/tolerance to HLB is present in cultivated citrus and greater resistance may be drawn from more distant members of the gene pool, and both approaches are targeted by the USDA citrus breeding program. Potentially useful HLB- tolerance is apparent in several mandarin hybrids.After six years of growth following almost immediate infection, ‘SugarBelle/Sour Orange’ and ‘Tango/Kuharske’ had the largest trunk diameters, good canopy density, and were progressing into fairly normal bearing. In a planting of seedlings from 85 diverse gene bank accessions there is a strong association between tolerance and pedigrees including citron. Hybridization is ongoing to combine diverse sources of tolerance. There is evidence of resistance to HLB in several Citrus crosscompatible genera. In a study of diverse scions on both unifoliate and trifoliate root stocks, root stock had little effect but scions with Poncirus in their pedigrees had significantly lower populations of the HLB bacterium. Strong resistance and even immunity should be possible using genetic engineering and USDA efforts are also underway to produce HLB-resistant scions using biotechnology. Programmed cell death or apoptosis is a plant defense response that can be triggered upon pathogen infection, and some triggers, such as Reactive Oxygen Species , can activate or prime plant defenses over long distances. There are also some anti-apoptosis proteins in plants which dampen plant defense responses. By suppressing expression of these anti-apoptosis proteins, PCD should occur in a more rapid manner and with augmented defense responses upon pathogen infection. Huanglongbing , caused by Ca. Liberibacter asiaticus , threatens citrus production worldwide. Las grows strictly inside living citrus cells. Citrus cells detect Las, but fail to trigger adequate native defense systems or PCD until the bacteria have multiplied to very high levels. To help accomplish this defense suppression, Las secretes both a functional peroxiredoxin and a peroxidase directly into the citrus cell cytoplasm that significantly dampen both the ROS response and signaling pathway. We have developed a citrus gene silencing strategy to suppress a brake on the natural citrus defense reaction to Las. Five silencing constructs were used to transform Carrizo root stocks. Average silencing efficiencies of the transformed root stocks ranged from 77.30% to 82.35%. Using a Las flagellin protein fragment applied at 10 µM as a proxy for Las infection, expression levels of three citrus defense response genes NDR1, PR1 and EDS1 were significantly higher in silenced lines than in NT controls. Challenge inoculations with Las were then performed by approach grafting selected transgenic Carrizo lines representing all 5 silencing constructs to mature Las-infected nontransgenic citrus scions.

The original smooth-cylinder trap model did not catch many psyllids, <10%, even in high density population exposure; Psyllid trap efficacy also requires correct field positioning ; One design accidentally had a rougher texture on the outside, and it caught more psyllids than similar traps that were smooth; Newer traps were then developed with intentional projections on the outside; Our best trap design so far has yielded a catch rate of 30-40% ACP, compared to psyllid-capture on yellow sticky traps, as determined by a limited number of controlled field trials; 3D-print designs can be modified rapidly from results of field testing, which is currently underway in FL, TX, and CA; 3D-printed traps can also incorporate species cues for light, sound, and odor attractants to overcome field positioning effects. The Asian citrus psyllid is known to obtain nutritional and defensive benefits from its complement of endosymbiotic bacteria. The most abundant of its associated endosymbionts, ‘Candidatus Profftella armatura,’hydroponic nft system has been shown to devote approximately 15% of its predicted genome to polyketide biosynthesis. The interaction between the ACP and Profftella is an ideal target to combat the psyllid since this symbiont is found in all populations of the ACP surveyed thus far and in no other insect.

A dual proteomic-metabolic analysis indicated that the complement of polyketides produced by Profftella plays a significant role in the insect host response to infection with ‘Candidatus Liberibacter asiaticus’ , the bacterial pathogen responsible for Huanglongbing . The primary mechanism of this defense is via production of the cytotoxic polyketide diaphorin, estimated to be produced at 1.3 ± 0.3 mg per 1,000 adult insects. ACP populations reared on CLas-infected citrus trees show significant upregulation of Profftella proteins involved in polyketide biosynthesis as well as quantitative differences in abundance of diaphorin and a related polyketide compared to ACP reared on healthy trees. In this work, we aim to verify previous structural characterizations of diaphorin and probe its potential interactions with receptors in the ACP to gain a better understanding of the interplay between Profftella, diaphorin, and CLas within the insect. We extracted approximately 6,000 adult ACP and verified the presence of diaphorin in the extract using infusion mass spectrometry. Diaphorin is further enriched from the extract using preparative High Performance Liquid Chromatography . Nuclear magnetic resonance is used to evaluate the structure of diaphorin isolated within the fractions, and LC-high resolution mass spectrometry is used to confirm stability of the compound. Future plans include immobilization of purified diaphorin on a solid support resin for use in an affinity purification strategy to identify interacting insect proteins.

This will allow a better understanding of the specific role and localization of diaphorin within the insect, as well as provide insight into the systemic response to CLas acquisition with an aim toward potential control strategies. Citrus Huanglongbing or greening is a devastating bacterial disease that has destroyed millions of trees and is associated with phloem-residing ‘Candidatus Liberibacter asiaticus’ in Florida. In this study, we evaluated the control effect of different antimicrobials including oxytetracycline, streptomycin, and plant defense inducers via trunk injection. Spatiotemporal dynamics of oxytetracycline in planta was evaluated. Las-infected ‘Hamlin’ sweet orange trees on ‘Swingle’ citrumelo root stock at the early stage of decline were treated with oxytetracycline hydrochloride using trunk injection with varying number of injection ports. Spatiotemporal distribution of OTC and dynamics of Las populations were monitored by HPLC method and qPCR assay, respectively. Uniform distribution of OTC throughout tree canopies and root systems was achieved 2 days post injection. High levels of OTC were maintained in leaf and root for at least 1 month and moderate OTC persisted for more than 9 months. Reduction of Las populations in root systems and leaves of OTC-treated trees were over 95% and 99% between 2 and 28 DPI. Conditions of trees receiving antimicrobial treatment were improved, fruit yield was increased, and juice acidity was lowered than water-injected control even though their differences were not statistically significant during the test period. Our study demonstrated that trunk injection of OTC could be used as an effective measure for integrated management of citrus HLB. The Citrus Genome Database is being developed as a one-stop resource for citrus genetics, genomics, and breeding research.

In this presentation we highlight CGD features that provide the citrus community with data and tools to combat Huanglongbing . The database has been redesigned and features a streamlined user interface that allows for quick access to data and tools and is also mobile friendly. It contains curated citrus genetic marker, map, and QTL data, genome data for clementine and sweet orange, as well as annotated reference transcriptomes generated by analysis of published RNA-Seq and EST datasets. The database also has the most up-to-date version of CitrusCyc v3.0 which includes metabolic maps for the C. clementina and C. sinensis genomes. Tools such as BLAST for searches against the genome sequences, GBrowse/JBrowse for viewing genomes, and CMap/TripalMap for viewing and comparing genetic map data are also available on CGD. In addition to the tools to view and search published data in CGD, the Breeding Information Management System allows breeders to upload their data via the web, or directly from the Field Book app, to a private account. The private data than can be analyzed in conjunction with the public data. In addition to citrus data, the genomes of the different ‘Candidatus Liberibacter’ species and psyllid vector are available in CGD in JBrowse and the sequence data is searchable with the BLAST tool. As a community resource, CGD is being developed based on user feedback with the goal of providing the data and tools that will enable citrus crop improvement. CGD is supported by USDA-NRSP10, NSF-PGRP, and USDA-SCRI. First report of delivery of two synthesized oligo nucleotide products into plants. Subsequent feeding on treated plants by insects resulted in ingestion and systemic movement of the two gene-based targeting products: FANA_ASO, _; and a PPMO . Topical sprays onto citrus leaves, root absorption, and tree trunk injections, resulted in cell delivery of the molecules which were designed to: Asian citrus psyllid; Plant pathogenic bacteria of citrus, Candidatus Liberibacter asiaticus , Psyllid endosymbionts, and Diaprepes abbreviatus, weevil.Treatments reduced CLas bacteria within infected citrus trees,nft channel reduced Wolbachia in cell cultures, the insects, while resulting in increased insect mortality. This is the first evidence for successful delivery of FANA_ASOs and PPMO into plants, and use of these molecules for plant delivered strategies to reduced plant pathogens in citrus, and to manage insect pests. Molecules were shown to move systemically through plant tissues as visualized with Confocal microscopy and spectrophotometry.Adult insects showed systemic movement through hemolymph and organs: supra and sub esophageal ganglions, fatbodies, nerves, and alimentary tract. Results suggest a role for these products in the reduction of plant pathogens like CLas, associated with Citrus greening disease; and reduction of insect vectors of pathogens on citrus and other agricultural crops.Two circular prophage genomes have previously been described integrated in Ca. Liberibacter asiaticus strain UF506 genome, and all Florida Las strains have both prophages. Either one or rarely, both, prophages were reported absent in certain recently sequenced Chinese and Japanese Las strains. The SC1 lytic cycle, marked by upregulation of several late genes including a functionally lethal holin , is activated when Las is in planta, but not when infecting the Asian citrus psyllid host. The holin promoter was cloned into the wide host range vector pUFR071, structurally replacing the native lacZ promoter to drive the beta-glucuronidase reporter gene. The holin promoter exhibited very strong and constitutive expression of the GUS reporter gene in Liberibacter crescens , a culturable proxy for Las. We previously reported the presence of an unidentified, heat labile, protease-sensitive repressor in aqueous psyllid extracts that quenches the expression of the holin:GUS reporter. Tandem LCMSMS analyses were used to identify ca. 25 peptide fingerprints in the DNA-binding protein eluate captured by the holin promoter DNA immobilized on magnetic DynaBeads.

A putative repressor protein was identified that was unique to the Wolbachia bacterial endosymbiont found in psyllids and absent in Wolbachia found in the fruit fly . This putative Wolbachia repressor gene was cloned in an expression vector and used for in vitro cell-free protein synthesis. The in vitro translated Wolbachia protein partially repressed the holin::GUS reporter in a dose-independent manner, as compared to the aqueous extract from the psyllid, thus indicating that complete suppression of holin promoter requires an additional partner. Heat-inactivated psyllid extract was unable to enhance the Wolbachia repressor- induced suppression of holin promoter activity, confirming the heat labile proteinaceous nature of the additional psyllid-sourced partner. A conspicuous feature of plant defense reactions in response to an attempted pathogen invasion is the engagement of an oxidative burst. We have previously shown that an SC2 prophage-encoded peroxidase in Ca. Liberibacter asiaticus strain UF506 represents an important lysogenic conversion gene whose expression may increase bacterial fitness and delay symptom development in the host plant. However, absence of either one or both prophages in certain recently sequenced Chinese and Japanese Las strains, prompted a review of H2O2-detoxification mechanisms conserved amongst Liberibacters. Two putative peroxiredoxin genes have been annotated on the Las chromosome, which are conserved and identical among all the sequenced Las strains. The BCP-like peroxiredoxin is predicted to be nonclassically secreted. The expression levels of Las PR2 in both citrus and psyllid were equivalent. Notably, however, the expression of the predicted secreted BCP-like peroxiredoxin was similar to that of PR2 in citrus but nearly undetectable in psyllids, reminiscent of the low level of SC2_gp095 peroxidase expression in psyllids. Despite carrying a native ortholog, Lcr transformed with the BCP-like peroxiredoxin showed a significant increase in tolerance to 100 µM H2O2, as compared to a marginal 4-5 fold tolerance provided by either the PR2 peroxiredoxin or SC2 peroxidase. Most importantly, BCP-like peroxiredoxin augmented Lcr tolerance to 50 µM tert-butyl hydroperoxide by nearly 1000 fold. We hypothesize that both the Las peroxiredoxins may be implicated in alleviating the oxidative burden originating from physiological electron transport reactions. However, the extracellular BCP-like peroxiredoxin may be essential for Las survival in an oxidative host cell environment through a) direct detoxification of host-generated H2O2, b) limiting the catastrophic chain reaction spread of a lipid peroxidation event initiated by H2O2 permeating the bacterial cell membrane, and c) most importantly, akin to the secreted SC2_gp095 peroxidase, dampening the systemic propagation of H2O2-mediated defense signaling in the host plant following an initial colonization event. Las BCP-like peroxiredoxin may represent a critical secreted effector conserved across all pathogenic Liberibacter species that functions to suppress host symptoms, a tactic used by most biotrophic plant pathogens. Detection of HLB-associated Candidatus Liberibacter asiaticus by real time PCR has been widely used in citrus industry since its development a decade ago. However, the methodology is not adaptable to quantitation by digital droplet PCR since it uses a multicopy mitochondrial gene as an internal control. Since there are hundreds of copies of COX gene per cell, inefficient amplification of the low copy target gene can occur. In this study, we sequenced a single copy nuclear gene, malate dehydrogenase from over 100 accessions including several citrus and citrus relatives in the subfamily Aurantioideae. The plant samples selected are known to be hosts of both HLB and the psyllid vector. Primers and probe were developed using conserved regions of MDH gene for Taqman® based qPCR and also for ddPCR analyses. We conducted qPCR assays using various dilutions of HLB infected plant samples to detect CLas in duplex reactions using two different internal controls – COX and MDH in separate reactions. ddPCR assays were conducted using a BioRad QX200 droplet digital PCR System. The advantages of using MDH as an internal control gene in both qPCR and ddPCR will be discussed. Since confirmatory tests to detect the presence CLas cannot be done with plant samples having low bacterial titers, additional confirmatory assays by ddPCR of other regions of CLas genome were also developed. Detection of CLas in samples with low titers is critical for both prevention and management of HLB, especially during early stages of the epidemic, and to slow down the spread of disease. Testing of psyllid vectors for the presence of huanglongbing-associated Liberibacters is known to be an effective method for early detection of HLB in citrus groves.

In a second replicated long-term field trial with ‘Valencia’ scion severely affected by HLB, highest yields over four harvests were with ‘US-942’ and ‘US-802’ root stocks. In both trials, some other root stocks that are commonly used in commercial production performed poorly, probably indicating less tolerance of those root stocks to HLB. Similarities and differences between the two trials will be discussed, as well as the value of different metrics for assessment of relative field tolerance of root stocks to infection by Las. The implications of these observations for the choice of root stocks to be used in commercial plantings will also be discussed. Interactions between plants and their associated microbial communities are complex, dynamic, and of varying impact. These interactions include plant-microbe and microbe-microbe relationships for each member of the community and can vary from positive to negative on each species. The structure of these communities can alter plant defense against other microbes and herbivores, plant productivity, and uptake of nutrients. Given these impacts, it is of interest to characterize the variation in microbial communities associated with crops facing major disease. The most devastating disease of Citrus is Huanglongbing caused by infection with at least one of Candidatus Liberibacter asiaticus, C. L. americanus, or C. L. africanus. Current methods of disease control involve disease vector suppression through conventional and bio-control methods, detection of infected trees, and inoculum removal.

Despite the success in slowing disease spread,growing strawberrries hydroponically these methods haven’t stopped the spread of disease. Methods to inhibit pathogen invasion may involve manipulations of microbial communities. In preparation for manipulative experiments, the microbial community of Citrus was characterized using 16S amplicon sequencing. Samples were collected from multiple locations within Florida, Texas, and California and consisted of trees cultivated using conventional and organic methods. Leaf midribs, fibrous roots, and soil were collected from trees infected with and trees with no detectable C. L. asiaticus. Results are discussed in terms of their impact on future microbial community manipulation experiments as well as their impact on disease detection. Liberibacter infection and tissue specificity. Several of these transcripts were selected as bait to detect protein-protein interactions using the yeast-2 hybrid system. Results indicate that transcripts involved in the immune response, endocytosis, and cytoskeleton assembly are important to Ca. Liberibacter infection of the psyllid host and/or in psyllid-mediated Ca. Liberibacter transmission. Ca. Liberibacter asiaticus is the obligate, fastidious bacterial pathogen that causes citrus greening disease, or Huanglongbing. CLas is transmitted in a circulative, propagative manner by Asian citrus psyllid Diaphorina citri Kuwayama adults. Identification and functional characterization of effectors involved in invasion of the psyllid gut and its presumed entry into the salivary glands was investigated using transcriptomic, proteomic, yeast-2 hybrid and coimmunoprecipitation analyses. In silico annotation and differential expression analysis of contigs from ACP nymphs and adults, and adult midgut and salivary gland tissues identified transcripts and proteins with altered expression in response to CLas infection. Several differentially expressed transcripts were selected and used as bait for Y2H detection of protein-protein interactions.

Those positive by Y2H were subjected to verification by bait to prey co-transformation and CoIP. Proteins positive in one or both assays were tested in ‘knock down’ experiments using dsRNA to induced RNA-interference and quantified by qPCR. Candidates of the greatest interest have been those with a predicted role in virulence and invasion, that if disrupted by RNAi could abate CLas accumulation in and exit from the gut, circulation in hemolymph and systemic infection, and acquisition e.g. in salivary glands. Collectively, the results suggest a model for invasion in which CLas- and prophage-encoded effectors transform the endocytic host pathway into a ‘pathogen-mediated phagocytic scenario’ utilizing membrane ruffling in CLas interactions with the gut, leading to bacterial exit into the hemocoel and systemic invasion of other psyllid host tissues and organs. Ca. Liberibacter asiaticus and solanacterum are the causal agents of Huanglongbing and Zebra chip disease of citrus and solanaceous crops, respectively. Bacterial effectors that interact with psyllid vector proteins during invasion of and exit from the gut, and that facilitate entry into the salivary glands were identified using functional genomics, proteomics, yeast-2 hybrid , co-immuno precipitation , and electron microscopic analyses. In silico annotation and differential expression studies of psyllid nymphs and adults, and midgut and salivary glands identified transcripts and proteins with altered expression in response to Ca. Liberibacter infection. Differentially expressed transcripts were used as bait in Y2H studies to corroborate protein-protein interactions. Those positive by Y2H were subjected to verification by bait to prey co-transformation and Co-IP. Electron micrographs of the PoP midgut revealed the presence of CLso in the ventricular lumen, apical and basal epithelial cytosol, and in the filter chamber periventricular space.

CLso were also prevalent in salivary gland pericellular spaces and head epidermal cell cytosol. Collectively, the results suggest a model for invasion in which Ca. Liberibacter- and prophage-encoded effectors manipulate the endocytic host pathway into a ‘pathogen-mediated phagocytosis scenario’ leading to bacterial exit from the gut into the hemocoel, and systemic invasion of other psyllid host tissues and organs. Beyond the devastating effects of HLB on tree health and crop size are the secondary impacts of preharvest fruit drop and the quality degrading flavor profile of symptomatic fruit. A precise number for the increasing percentage of the smaller, symptomatic fruit being sent to processors for juice extraction is not readily available. But inspection of data provided by NASS does indicate the number of fruits per box has been trending higher over the last five seasons. As previously noted increasing disease severity will increase the percentage of symptomatic fruit being used for juice extraction. In a 2015 survey of growers, production managers and other associated occupations 81% of the responders reported that 80% – 100% of their acreage was infected and the average percent of infected trees was 90%. As more and more fruit become symptomatic their detrimental effect on juice quality will become more and more pronounced. Beyond a blending ratio of 3:1 off-flavors in the product become discernible. The incidence of pre-harvest fruit drop also has been increasing with a recently reported 10% – 20% rise. Enabling a technology to utilize these two groups of HLB damaged fruit and recover lost value would be beneficial to the citrus industry during this transition period before resistant trees are available, especially if that technology also could provide increased value for all juice extracted biomass. Here we report on a pilot-scale continuous process to release and recover multiple value added co-products from culled fruit, preharvest dropped fruit and juice extracted biomass. Using steam-explosion followed by a simple water wash we have been able to recover pectic hydrocolloids, bioactive phenolics and fermentable sugars.Pectic hydrocolloids are functional polysaccharides that can be used to modify the rheology of water and as a hydration control or ion capture agent. Citrus phenolics contain a number of compounds that possess bioactivity against chronic human illnesses. Sugars can serve as a feed stock for a variety of synthetic options as well as an energy source in varied fermentation schemes. Limonene has a variety of established commercial applications. Summing these potential co-products could promote the dropped or flavor degrading fruits as an alternative value added resource and recover increased value from juice extracted biomass vs. a traditional feed mill operation.Huanglongbing constitutes the most destructive disease of citrus worldwide,rolling bench yet no established efficient management measures exist for it. Brassinosteroids, a family of plant steroidal compounds, are essential for plant growth, development and stress tolerance. As a possible control strategy for HLB, epibrassinolide was applied to as a foliar spray to citrus plants infected with the causal agent of HLB, ‘Candidatus Liberibacter asiaticus’. The bacterial titers were reduced after treatment with epibrassinolide under both greenhouse and field conditions but were stronger in the greenhouse. Known defense genes were induced in leaves by epibrassinolide. With the SuperSAGE technology combined with next generation sequencing, induction of genes known to be associated with defense response to bacteria and hormone transduction pathways were identified.

The results demonstrate that epibrassinolide may provide a useful tool for the management of HLB. Tamarixia radiateestablished and spread throughout Florida following first release in 1999 for control of the Asian citrus psyllid Diaphorina citri . Nevertheless, persistence of the pest and continued losses from citrus greening disease have created the need for more effective biological control, including mass rearing and augmentative release of T. radiata. However, information is lacking on ways to manipulate host density to optimize rearing efficiency. Three release rates of D. citri per shoot, and four host: T. radiate ratios were evaluated. Results indicated that 20 psyllid adults per flush provided optimal host density without excessive stickiness from honeydew. Sixty T. radiate females per cage of approximately 4,800 nymphs provided most economical use of wasps and hosts. However, only 60% of the hosts were utilized for progeny production under these conditions, possibly due to competition between females mediated through deterrent host marking. In order to determine the existence of a host mark on parasitized hosts, a three-day-old T. radiate females was released in a petri dish with six parasitized hosts and six clean hosts randomly distributed on an orange jasmine shoot. The number of hosts probed and parasitized in each host category was noted. To evaluate volatility of the host mark, 10 parasitized 4th instar D. citri nymphs and 10 clean nymphs were randomly assigned to one of the arms of the T- maize olfactometer. Ten T. radiate females were released at the releasing chamber to make a choice of the two odor sources. Results showed that T. radiate females probed and parasitized significantly more on the clean hosts versus parasitized hosts but showed no a preference for either of the odor sources. Thus, T. radiate females were able to discriminate between clean hosts and host parasitized by herself by perceiving a non-volatile host mark. Future research will focus on determining whether the mark also has a deterrent effect on nearby hosts. The ultimate goal is to improve mass rearing efficiency and biocontrol effectiveness in the field. Candidatus Liberibacter asiaticus is the bacteria associated with the economically-important citrus disease, Huanglongbing . Understanding the pathogenicity of CLas and its effects on the citrus host is challenging, as CLas has yet to be cultured. To understand the impact of CLas infection on host leaf metabolism, Parent Washington Navel orange Osbeck greenhouse plants were graft-inoculated with CLas+ or CLas- budwood, and leaf samples were collected longitudinally until tree death. Samples were analyzed with metabolomics , transcriptomics , and proteomics to identify CLas-associated changes in the plant. Changes in metabolite content could be observed as soon as 6 weeks post-infection, before symptom development. Further changes in the metabolome, transcriptome, and proteome were observed throughout the study, with the most drastic differences noted at the last time point as the trees were nearing death. CLas affected both primary and secondary metabolism, including photosynthetic processes, cell wall synthesis, and stress responses. These data show that early changes in tree metabolism occur in asymptomatic plants with CLas infection, and shed light on how CLas impacts the citrus host throughout the disease. The concept of growing degree-day , which integrates the rate of development of an organism as a linear function of air temperature to which it is exposed, has been widely used in studies of crop and insect phenology for scheduling agricultural practices. In citrus, however, these studies have focused on the reproductive development and on the physicochemical characteristics of the fruits but not on vegetative shoot ontogeny. This is important because vegetative shoots are strictly linked to the biological performance of Diaphorina citri. Although the thermal requirements of the psyllid have been established in growth chambers, little is known about the thermal requirements for the psyllid in natural environments or about the cumulative heat units for the emission and development of new shoots, taking into consideration their ontogeny. To assess this, experiments were carried out in growth chambers using 2.5 year-old potted ‘Valencia’ orange grafted on ‘Swingle’ citrumelo. The plants were pruned 20–30 cm above the grafting line and evaluated for several flushing cycles. Daily assessments of shoot size, new leaf emergence and leaf area were carried out on a single shoot per plant.

Local governments readily looked to these studies to justify new programs and initiatives to support efforts to address food access issues locally. And yet, some researchers were more concerned with the conceptual basis of the term ‘food desert,’ which failed to account for the ways that poor communities have managed to cope in spite of grocery stores or supermarkets. For example, some argued that areas defined as food deserts in fact are replete with smaller food retailers, which contribute to food security and may present future opportunities to do so . Others—particularly the mass media—also questioned the still inconclusive connection between the development of supermarkets and improved health outcomes.Overall, food desert research made a strong public health case for developing new grocery stores in under served communities, despite sometimes conflicting, and inconclusive evidence. As community-based food projects continued to expand through the early 2000s, a new generation of food activists sought to reframe the community food security movement to encompass social justice, human rights, and economic self-reliance through food systems . Inspired by international peasant farmers and environmental justice movements, “food justice” activists approached food access issues through programs and entrepreneurial activities focused on engaging disenfranchised populations in directly revitalizing their communities.

As such, ‘food justice’ is more squarely premised in “ensuring the benefits and risks of where,hydroponic growing what and how food is grown and produced, transported and distributed and accessed and eaten are shared fairly .” Frequently cited examples of the distinctive “food justice approach” include People’s Grocery in West Oakland and Growing Power in Milwaukee. People’s Grocery was established in 2002 with the goal of “build[ing] a local food system that improves the health and economy of West Oakland” . Its foundational programs comprised a youth-run community garden, nutrition education programs, Community-Supported-Agriculture, and a mobile grocery market—all pursued with the ultimate goal of developing a grocery store in West Oakland. Although this program was discontinued in 2009, a for-profit subsidiary of People’s Grocery is developing plans and securing financing for a new grocery store in West Oakland. Established in 1993 by former professional basketball player, Will Allen, Growing Power is an organization that whose mission is “to grow food, to grow minds and to grow community” . Initially focused on vermiculture and aquaponics, the organization has expanded to include a community center, grocery store, and fourteen greenhouses in both Milwaukee and Chicago. These models of “food justice” have been readily replicated across the country, with a distinct focus on connecting food production and retail to serve broader community goals. Organizations such as People’s Grocery also define their work explicitly as a response to food deserts. Alongside of these activities, Philadelphia-based CDFI, The Reinvestment Fund initiated the Pennsylvania Fresh Food Financing Initiative , the first statewide financing program specifically designed to attract supermarkets and grocery stores to under served urban and rural communities. The program was established in 2004 through a public-private partnership between non-profit organization, The Food Trust and The Reinvestment Fund following dissemination of a 2001 report “Food For Every Child: The Need for More Supermarkets in Philadelphia” confirmed the findings of other studies of food deserts.

As a response to the findings, the Philadelphia City Council convened a task force, which recommended the creation of a statewide initiative focused on incentivizing grocery store development. Eventually in 2004, state representatives and then Governor Ed Rendell enacted an economic stimulus package, which allocated $30 million for the creation of the FFFI. The Reinvestment Fund then leveraged another $120 million through the New Markets Tax Credits program and private banks . Since its inception, the program has offered grants of up to $250,000 and loans ranging from $25,000 to $7.5 million primarily to independent, locally owned businesses including green grocers, food cooperatives, and full-service supermarkets. These grants and loans primarily support predevelopment, acquisition, equipment, construction costs, start-up costs, and employee recruitment/training . As of June 2010, the FFFI financed 88 grocery stores comprising $73.2 million in loans and $12.1 million in grants . Overall, community food security movements combined with the growing body of research on food deserts represented the beginnings of a strong public health agenda around the development of grocery stores in low-income communities and communities of color.And yet the limitations of this early research began to exhibit the very complexity of food access issues in low-income communities of color understated by the simplicity of the term food deserts. Still the emergent public health voice around food deserts served to reinvigorate old discussions about the urban grocery gap to new ends. Accordingly, the “metaphor” of the ‘food desert’ provoked both a community-based and local government response, which marked the beginnings of an expanded set of social and economic goals around grocery development as evident in today’s context. By the late 2000s, food deserts captured the attention of researchers, journalists, policymakers, the grocery industry, and the general public. food deserts would enter into federal policy agendas provoking a new set of research and local accounts calling the basis of food deserts into question. And yet, despite debates over the characterization of supermarkets as a proxy for “healthy food access,” grocery stores would form the basis for the federal program devoted entirely to the subsidization of grocery stores in under served areas.

The momentum around food deserts in late 1990s to mid 2000s reached a critical point with the 2009 USDA-sponsored national survey, “Access to Affordable and Nutritious Food: Measuring and Understanding Food Deserts and Their Consequences.” Congress commissioned the study as part of the 2008 Food, Conservation, and Energy Act . In contrast to previous studies, this study was the first ever, national assessment of food deserts . Over the course of one-year, the USDA worked in collaboration with the National Poverty Center at the University of Michigan and the Institute of Medicine of the National Academies for U.S. Census data analysis, interviews, and focus groups. Furthermore, the study found that further research was necessarily “to conclusively determine whether some areas with limited access have inadequate access” . Despite the mixed findings, the authors strongly recommended policies and programs to expand healthy food access in under served communities. This includes: 1) incentive programs to attract new stores and improving existing ones , 2) community-based programs such as corner store conversion, 3) transportation improvements to make supermarket access easier and more affordable, 4) policies that specify healthy food offerings fore retailers authorized to participate in the SNAP program, and 5) the integration of grocery planning with housing and community development. Months prior to the release of the report, Oakland-based think tank, PolicyLink, The Reinvestment Fund, and The Food Trust began working with the Office of the White House to develop a national version of the Pennsylvania FFFI. The release of the USDA study was further affirmation of their efforts. In months following, a series of initiatives laid the groundwork for a comprehensive federal program to address food deserts nationally. The USDA study would provide the evidence base for a “Food Environment Atlas,” an online national food desert locator, designed to identify counties with limited supermarket access. The USDA created the tool specifically to locate priority areas for future interventions. The development of this online tool coincided with the instatement of Let’s Move,mobile vertical farm national childhood obesity prevention campaign launched by First Lady Michelle Obama in early February 2010. In the year that followed, HFFI administered its first cycle of grants to sixteen organizations across the country for initiatives ranging from CDC-chain supermarket partnerships, farmers markets, and business incubation programs . The HFFI inspired newly elected Chicago mayor, Rahm Emanuel convene a ‘Food Desert Summit’ to meet with major grocery retailers to discuss a plan for eliminating food deserts. Soon following in July 2011, the First Lady hosted her own summit with major food retailers, where she secured commitments for store expansion from Supervalu,Walgreens, Walmart, and regional retailers. Together, the retailers agreed to newly develop or expand 1500 stores over a five-year period. Additionally, the First Lady’s summit resulted in the creation of California-based counterpart of the Pennsylvania FFFI, the California Fresh Works Fund. A project of statewide health foundation The California Endowment, the $200 million public-private partnership was created to finance a variety of “healthy food retailing” initiatives . Several subsequent studies further reinforced these critiques. A controversial longitudinal study in the Archives of Internal Medicine questioned the connection between food environments and diet quality . Over a 15-year period, researchers monitored eating habits of thousands of residents in four cities. The study found that proximity to fast food retailers increases the risk for poor dietary quality and obesity .

And yet, proximity to supermarkets did not impact fruit and vegetable consumption . Ultimately, the researchers conclude that supermarkets may not be enough to change unhealthy diets. And yet, a string of subsequent studies similarly refuted the idea that obesity could be combatted through grocery development. A 2012 study by the Public Policy Institute of California found that poor neighborhoods were not all food deserts. While some tended to have a higher concentration of liquor stores and fast food restaurants , others also tended to have a larger concentration of grocery retailers compared to suburban areas . In line with the previous study, this was among research that suggested no correlation between supermarket proximity and dietary patterns . These studies were subject to their own critiques, in part due to the differences in the study populations—be it low income men , children , or young adults . Despite competing claims regarding the efficacy of supermarkets in addressing obesity, grocery retailers have been reentering urban areas for the past decade, with more incentives than ever. Several retailers have created “urban format” stores, such as Walmart Neighborhood Market, Fresh and Easy , and Tesco Express. Community-based organizations have continued to seek out alternatives to these types of stores be it through farmers markets and/or cooperatives. Accurate and high-throughput protein quantification is fundamental to proteomic studies . To provide the highest quantification accuracy when comparing samples one needs to minimize differences introduced in the processing of samples and acquiring the data. This can be best achieved through the introduction of stable isotopes into samples that allow samples to be mixed and then analyzed in the mass spectrometer. The application of metabolic labeling, which uses stable-isotope labeled amino acids in cell culture or 15N nitrogen-containing salts into the whole cell or organism in vivo, enables relative quantifications of proteins on a global scale. In such a quantitative experiment, one sample is labeled with the natural abundance , and the other with a stable isotope of low natural abundance during growth. The samples are mixed, processed, and analyzed by the mass spectrometer. Chemically identical peptides from these light- and heavy-labeled mixed samples co-elute by chromatography into the mass spectrometer, which can distinguish between the light and heavy peptides based on their mass difference, and thereby quantify the difference in peptide, and hence protein abundance between the samples. An alternative stable isotope-based strategy is to chemically tag peptides after enzymatic digestion; the most popular reagents for this strategy are isobaric tagging reagents Tandem Mass Tags . The TMT isobaric tagging reagents allow comparison of a larger number of samples, but the labeling is done at the peptide level after sample digestion and then samples are mixed. In contrast, the metabolic labeling is introduced into the proteins during growth, thus samples can be combined at the beginning, minimizing variations introduced by sample processing that can compromise quantification accuracy . Although SILAC has been widely used in animal cell lines and has been the gold standard for MS-based proteomics quantification , 15N-labeling based quantitative applications are still quite limited in plants despite it being cheaper . This could be due to the complexity of the data analysis. SILAC pairs are easily identifiable because they have well-defined mass differences as typically only lysine and arginine are labeled. In contrast, in 15N labeling, each amino acid in the expressed proteins is labeled, and therefore, the mass difference in 15N pairs varies depending on the number of nitrogen atoms in their composition.

Accordingly, many of the Master’s gardens employed thematics associated with the labyrinth and the grotto; of hiding and revealing, of voyeurism, exotica, minutiae, and narrative . In the descriptions that follow, I roughly corral the nine Masters’ Gardens into three categories; labyrinths, rooms, and representative gardens. Using this draft rubric, I explore three Masters Gardens in detail, three at a more cursory level, and the remaining three in passing. My choices in this regard certainly reflect a hierarchy of my experiential and theoretical impressions of particular gardens.Of the labyrinthine-type gardens, the Maze Garden by Martha Schwartz Partners is the most overtly fabricated . Comprising a set of high walls incised transversely by equally spaced arched passages, the garden presents itself as an open labyrinth. In this regard the sensation of moving amongst the array of walls and apertures is reminiscent of Eisenman’s Holocaust Memorial design, where the object is perhaps to test Walter Benjamin’s edict that we must have approached and left a place by all four cardinal directions to truly know it.But this effect is quickly subverted; firstly, the grove of willow trees that supplies a canopy over the garden is forever offered but seemingly never substantiated as the trees themselves remain evasively encapsulated within chambers between wall sets. Secondly, the alignments of the garden walls perform a cunning rotation which serves to subtly disorient the visitor and provide niches for actuating the third effect: self-reflection.

At the entrance to the labyrinth, a freestanding wall is clad on one side with dark mirrors. Like a magician showing the audience an empty hat before drawing out a rabbit,vertical grow rack these mirrors purport to be as they appear; a harmless arena for mass self-vanity.Moving into the walled matrix, we are habituated to more dark mirrors at the end of each elongated space. Further still, as the geometry rotates and tapers, mirrored chambers are increasingly encountered, some small, some large. One threshold further discloses a penultimate roofed cavern. Here, a grove of willows is finally revealed, albeit through deeply hued glass which serves to obfuscate the demarcation between the real and the represented. But this is not the final revelation, which is delivered as one follows the cavern around to the exit. From this privileged position of hindsight, we are placed behind the looking glass and discover that the mirrors throughout the garden are one-way glass. At the largest interfaces—attracted like moths to a lamp—entire groups of people gaze innocently at their reflections. In the smallest chambers, young couples make use of their mistakenly private niches and engage in intimate embraces, unaware of the public viewing gallery beyond the glass. As unwitting participants in a social experiment, visitors have been lured into carefully orchestrated traps using human vanity as bait.In this context, the disorienting walled maze and hall of mirrors trade light-heartedly in the excitement that we derive from ‘getting lost, of finding a way back, and ultimately a way out or to a goal’. However, mazes operate through turning pleasure seamlessly into confusion so beneath this playfulness lays a disturbing metaphor in the infernal prospect of there being no goal or exit. Within this context, the open labyrinth model upon which the Maze Garden is based reflects an increasingly complex contemporary life-path where we expect a maze to harbor choices, divergences and dead ends. In contrast—as if tracking a fatalistic passage through life—labyrinths were historically manifested more as a single route spiraling inward to a central goal and then back outward to reconnect with the point of origin. The Garden of Bridges by West 8 takes this motif of the single path and entangles it as a Gordian Knot . In the place of walls, the garden uses the same bamboo employed in the frame to inundate the site.

Into this dense forest is incised a single narrow trail, which continually twists and turns back, affording occasional glimpses of other people elsewhere on the same path. In instances where the path loops over itself, implanted bright red arching bridges enable grade-separated passage. When immersed in the bamboo everything is close at hand and body based; as Robert Harbison observes, we allow plants to confine us in ways that would be unpalatable in stone.Gaston Bachelard conveyed the sense of immensity that the forest imparts, noting that this bodily impression openly contradicts geographical reality. 18 Similarly, Yi-Fu Tuan concurs that even if small, the forest gives the appearance of being limitless when we are lost within it. At just 10 000 sq ft, The Garden of Bridges is saturated with this effect, with the notable digression of occasional respite in the form of the bridges. As we climb up and emerge above the canopy with a clear overview, vision takes over as we strive to take our bearings by reasserting general orientation amongst the landmarks of the Expo site before descending back down into the thicket. The deceit is that the bridges represent false ‘pyramids of reason’ within the labyrinthine forest, since the path below is always obscured, meaning that the overview is useful neither for reconciling one’s journey thus far, nor reconnoitering the future route. We can see our companion at the top of another red bridge, but cannot tell whether to head forwards or backwards to get to them. Instead, we rely on a combination of dead-reckoning recall and faith in the universal consistency of Cartesian space; earlier, we walked ahead of our accomplice, so therefore, they cannot be in front of us now. On a single path, to avoid getting lost no matter how geometrically complex, we need only to keep a record of where we came from in differentiation to where we are headed—a tough ask when the empirical world of entangled bamboo and bridges says otherwise. Where the Garden of Bridges fills the frame to generate the effect of the thicket, the Big Dig garden by Topotek 1 uses the opposite tactic of a frame opened on one side and an empty field to present the surface of the garden plot in its entirety to passers-by .

A parabolic hole at the centre of the site is calibrated so that the bottom is never revealed; the void becomes in effect vertiginous and infinite. The illusion of tunneling through the mantle toward the other side of the world is manifested instead as a collaged soundscape, so that sounds of other cities and landscapes emanate from the depths. The effect invokes Buckminster Fuller’s observation of changes in perceptions of spatial relations as a result of the aerial warfare of WWII, on which he noted that ‘the world has been surprising itself by coming in its own back doors and down its own chimneys from every unlooked-for direction’. This act of tunneling takes perceptions that originate from afar and incises them into the composition of local space, producing as Brian Massumi notes, ‘a fusional tension between the close at hand and the far removed’. In the ultimate local-global exchange, ‘as the distant cuts in,vertical planting tower the local folds out’.The garden is reminiscent of Bernard Lassus’s 1970s bottomless Well design concepts, which proposed a deep vertical shaft into which stones could be thrown to infinity. These speculations explored the psychological space of depth; not in terms of the abyss that reveals the feared absence of foundations,but rather, as a refuge for the imagination in a world without uncharted spaces for the mind. As Lassus notes, with the complete mapping of the surface of the earth in the age of exploration erased the terrestrial or horizontal frontier. A replacement frontier came in the conquest of the ‘immeasurable verticals’, culminating in the first moon landing. As a counterbalance to these giddy heights, Lassus invests poetically in a depth beneath the surface that we tread on, also ‘immeasurable, vertical and obscure’.Stephen Bann interprets this to mean that garden design should involve the ‘poetic creation of depths’. Whether indirectly invoked or actually constructed, Bann sees a landscape of depths as the creative balance to ‘an increasingly strong preoccupation with the vertical dimension’. It is as filling the holes left by archaeology will re-balance the cosmically distant with the terrestrially deep, stabilizing landscape’s regular field of operations, the surface itself. Topotek’s scheme trades not in poetic depth as a counterbalance as per Lassus but rather in didactic depth. For Lassus ‘dropped pebbles travel forever’,thus fabricating the illusion of the terra incognita that the world lost when it ‘closed the map’. For Topotek’s ReinCano on the other hand, the pebble metaphorically returns, with compound interest, from the other side of the noisy and full world. Indeed, as Bann notes ‘the last white patches have vanished from the map of the world’ because ‘China has finally lost its monstrous otherness and become a Mecca for tourists’.Fittingly located in China, the Big Dig is like the last tiny residue pin-hole of this filled up map. But it is a false holdout, since—like an intrepid climber who scales a Swiss mountain only to discover a restaurant perched on the summit—others are already present, having taken a convenient shortcut.

Unlike Lassus, in this hole there is no depth, only more surface, since every hole has a lining; ‘the hole lining is the hole’. Indeed, if we ignore the inconvenient balustrading around the hole and focus on the surface, the parabolic fall-away becomes a Pierce’s Puzzle, which confounds what is the hole and what is not. Are we— like the caged fox that decides it is free while the rest of the world is incarcerated—actually already in the hole without realizing it?The room-type gardens fall into two sub-categories; cloister-like rooms operating as islands within the garden, and open plan rooms spanning the width of the garden. Passages Garden by Terragram falls into the latter category, using the wall as a spatial calibration device . A stone path that leads in to the garden cuts through several walls at oblique angles before disintegrating down into a body of water. Ahead, a low aperture in a bright orange wall allows glimpses of the paving stones re-integrating from the water to reform the path. Water seeps from a fissure that runs the length of the battered back retaining wall, refreshing the pond and heightening the sensation of being in a situation that is in tension and flux. With the impassable aperture preventing egress to the recomposed path, visitors strike out across an expanse of white river stones peppered with diminutive pine trees. Here, at an opening in the other end of the orange wall, a single smooth rock levitates precariously at eye level, the gravity field of its copious weight appearing to deflect the overhead beam. In Chinese garden tradition, stones hold a special meaning and allure; like a talisman, this rock the place to which visitors are drawn, to paw at the marbled surface, realign it around its pivot, and to contemplate its meaning. The circular cloister at the heart the Botanist’s Garden by Gross.Max operates as a retreat deep within the garden . Constructed from tightly stacked roof tiles, the wall forming a Hortus Contemplationis is battered at an angle reminiscent of the ancient city walls encasing Xi’an. This geometry, combined with the bulk of the wall neutralizes the cacophony of the Expo. The garden planting seems innocuous at first, but in time a second layer of delicate exotic botanica comes in to focus. Whereas elsewhere in the Expo, monocultures of floral displays are measured in hectares, within this botanical heterotopia, species are indulged one specimen at a time. Of a similar scale, the Quadrangle Garden by Atelier DYJG employs the repetition of four rooms that have been deflected from rectangles. However, unlike the Botanist’s Garden cloister, which by virtue of the mass of the wall is oriented wholly internally and vertically, complex apertures in the walls of the quadrangle rooms provide for a kaleidoscope of fragmentary glimpses of scenes in the surrounding garden.Of the three gardens that overtly represented other landscapes, two embodied more mythical landscape types while the other embodied a nation-state. On the latter, whereas many of the non-architected exhibits at the Expo attempted to represent a particular province, the Landscape Garden by Mosbach Paysagistes gathered all of China into its representational net.

Studies of PTI have focused on the bacterial peptides flagellin and EF-Tu and their action in Arabidopsis. These peptides are perceived by PRRs, receptor-like kinases that are crucial for perception of flagellin/EF-Tu and activation of PTI. However, unlike flagellin and EF-Tu, many of the historical elicitors that stimulate well-characterized defense responses in plants have not been sufficiently investigated to resolve their modes of action . The elicitors AA and EPA conform to the definition of MAMPs: they are not present in higher plants, are essential components in oomycete cells, are largely absent from other classes of microbes, and elicit similar defense responses in plant species where they have been studied . Eicosapolyenoic acid elicitor activity in plants was first discovered in the interaction between Phytophthora infestans and potato. Mycelial extracts of P. infestans induced sesquiterpenoid phytoalexins, lignin deposition and cell death in potato tissue in a reaction similar to a HR to incompatible races of the pathogen. Purification and analysis of all active fractions in these extracts identified AA and EPA, without exception, either free or esterified to other molecules .

Elicitation was specific to AA and EPA. Treatment with 15 other fatty acids, including LA and ALA ,mobile vertical grow racks the primary unsaturated fatty acids found in higher plants , as well as structurally similar eicosatrienoic acid and arachidonyl alcohol, did not elicit defense responses. Treatment of tuber disks with AA also protected them from subsequent P. infestans infection .Eicosapolyenoic acids induce systemic resistance in potato as well as in other plant species to various pathogens. Although the mechanisms remain unresolved, EP have been shown to elicit SA, JA, and ET in different experimental systems. Colonization of avocado seedling roots by P. cinnamomi was reduced in roots treated with AA prior to inoculation . Pearl millet seedlings were protected to a greater degree against infection by the downy mildew pathogen, Sclerospora graminicola, following seed treatment with AA or EPA, in contrast to seedlings emerging from seeds treated with LA, ALA, DHA or water . EP elicit SAR or SAR-like responses in tobacco, potato, and tomato. Treatment of lower leaves of tobacco plants with AA induced local and SAR to TMV . EP treatment of the lower leaves of potato plants protected the upper leaves from infection by P. infestans, a systemic resistance that developed within 5 days of the inducing treatment . Plants treated with LA, ALA, or oleic acid displayed partial protection but not to the level of EP-treated plants. AA also induced resistance in potato leaves to the early blight pathogen, Alternaria solani, with levels of SA and a PR1-like protein elevated in the AA-treated leaves . AA-treatment of tomato leaves induced localized accumulation of transcripts for P4 , a PR-1 family member and SAR marker in tomato , but did not induce expression of the proteinase inhibitor gene PI-2. The latter is strongly induced by wounding and JA treatment and serves as a marker for JA-mediated resistance in tomato . Although the studies in tobacco, potato, and tomato indicate that EP-induced resistance may operate through SA, recent research suggests EP action is more complex .

Treatment of tomato and Arabidopsis leaves with AA increased JA levels, reduced SA levels, and increased resistance to Botrytis cinerea. Arabidopsis plants transformed to produce small amounts of EPs were less susceptible to P. capsici, B. cinerea, and feeding by aphids. However, these plants were more susceptible to Pseudomonas syringae pv. tomato . The EP plants had constitutively elevated levels of JA and JAmarker gene expression and reduced levels of SA and SA-marker gene expression relative to wild-type plants. The differential effect of EP on disease and pest outcomes corresponds to EP’s impact on SA and JA defense signaling, and this effect is dependent upon JA as demonstrated with a JA-deficient aos mutant line . Salicylic acid and JA can be mutually antagonistic , making it difficult to reconcile these different findings. AA treatment elicits ET production in both pepper and potato , and ET can modulate SA- and JA-defense networks . The different experimental outcomes may result in part from differences in EP concentrations used in the various studies. Higher concentrations of EP can induce an intense, localized necrosis at the site of application, particularly in solanaceous plants. This strong phenotype could trigger or result from phytohormone changes different from those induced by low concentrations. Also, it is possible that all three phytohormones are important in establishing EP-induced resistance through a process of transitional signaling . A study in potato indicates that both SA and JA are important in PTI responses , and a study of PTI in Arabidopsis using signal allocation analysis of mutants deficient in ET, SA, and JA signaling indicated that PTI depends on synergy among ET, SA, and JA . Further research is needed to fully elucidate the interactions among SA, JA, and ET in their involvement in EP-induced resistance and defense responses.Eicosapolyenoic acids have been useful in dissecting aspects of secondary metabolism in plants, with a focus on sesquiterpenoid phytoalexins in solanaceous plants. However, EP elicits production of defense metabolites in other plant families as well. The isoflavanoid phytoalexins phaseollin and coumestrol accumulate in leaves of French bean following infiltration with AA . Phenol-2,4-bis , a defense compound in avocado, is induced in roots treated with AA as well as with SA .

Among solanaceous plants EP elicit sesquiterpenoid phytoalexin synthesis in thornapple, eggplant, chili pepper, green pepper, potato, and tomato . In potato tuber, AA elicits sesquiterpenoid phytoalexin biosynthesis with strong expression of sesquiterpene cyclase, a committed step in the pathway. Concurrent with this is a complete suppression of wound-induced squalene synthase and steroid glycoalkaloid accumulation . HMGR catalyzes the first step in the synthesis of stress-induced isoprenoids from mevalonate in potato. Three isoforms of HMGR are differentially induced by wounding and AA treatment , and a similar expression pattern of the corresponding HMGR isoforms occurs in tomato .In addition to potato, EP have been shown to elicit PCD, characteristic of the HR, in other plant species. Pearl millet seedlings treated with AA displayed a HR similar to that induced by the oomycete, S. graminicola, the causal agent of downy mildew. Following treatment with AA, the HR developed more quickly in pearl millet seedlings with genotypes rated as resistant versus susceptible to S. graminicola . Tomato protoplasts treated with AA underwent PCD with characteristic DNA fragmentation and laddering, while LA and ALA treatment had no PCD-inducing effects . In both potato and pepper, AA was found to induce ROS in a similar manner. AA treatment of potato tuber disks elicited a biphasic oxidative burst peaking at 1 and 6–9 h after treatment and increased expression of StRBOHB, a homolog of gp91, which encodes a subunit within the neutrophil NADPH oxidase complex . As in potato, treatment of pepper fruit with AA elicited an immediate, rapid ROS burst. When DPI, an inhibitor of NADPH-dependent oxidases, was applied to the fruit prior to application of AA, ROS generation decreased as the concentration of DPI was increased .The mode of action of EP in PTI is unresolved, although the structural requirements of EP as elicitors are well characterized. These include at least a 20 carbon backbone with all cis-1,4-pentadiene unsaturation beginning at the 5 position and at least four double bonds in the chain . While this specificity could provide evidence for involvement of a receptor that recognizes these structural features,vertical garden growing previous studies of EP in potato indicate that initial perception by plant cells may be quite different than other MAMPs. Initial recognition of AA and EPA may occur by specific disruption of host membrane integrity and/or perturbation of oxylipin metabolism, with the possibility that plant cells produce novel oxylipins from EP . Studies in potato showed that U-14C radiolabeled AA was quickly incorporated into neutral lipids and polar lipids . A small fraction, ∼2–5% of the AA, was oxidized . Also, sporangia of P. infestans readily incorporated exogenous 14C-AA into phospholipids , diglycerides and TGs. By 12–14 h after inoculation, microautoradiographic studies revealed that the radioactivity from sporangia was released into the epidermal and palisade mesophyll cells adaxial to the inoculated leaf surface and distant from fungal structures . Plant phospholipases are activated following attack by pathogens or treatment of plants with elicitors . This could create an opportunity for any EP incorporated into plant lipids during infection to be released and accessible to plant oxylipin enzymes. Research in potato and tomato indicates that the 9-LOX pathway may play an important role in EP action. The first step in the enzymatic formation of phyto-oxylipins involves the action of LOX .

Plant LOXs act on PUFA containing a cis– pentadiene system, inserting an oxygen molecule to form hydroperoxy fatty acids. These are further metabolized to various oxylipin families by members of CYP74 cytochrome P450s: AOSs, HPLs, and DESs, or by less well-characterized POX or PXG and EASs . The importance of LOX, in particular a 9-LOX2, in EP elicitor activity is supported by fatty acid structure-activity requirements and studies of LOX expression. The carboxyl function of EP is critical, a feature consistent with the substrate requirement of plant LOXs . A 5 double bond at the beginning of a methylene-interrupted series with at least four double bonds provides the highest elicitor activity . AA stimulates LOX expression in potato and tomato , with 5-HPETE a principal LOX product formed after treatment of tissue with AA . Expression of pLOX1, a potato LOX gene now identified as a 9-LOX type 1 , was strongly induced in AA-treated and P. infestans-inoculated potato tuber disks and leaves , as was a tomato LOX in AA-treated tomato leaves . LA-treatment did not induce pLOX1 expression or LOX activity. Heat treatment of tuber disks inactivates enzyme activity and abolishes HPETE formation following AA treatment , and EP-induced responses are strongly diminished when LOX activity is inhibited or absent . Nonetheless, definitive experiments with LOX knock-out/knock-down or over expression lines to critically test specific LOX isoforms in EP action have not been reported. While it has been proposed and is quite likely that the 9-oxylipin pathway metabolites of AA may directly act as signal molecules to activate defense responses , AA and/or its metabolites may also induce expression and activity of oxylipin pathway enzymes to form biologically active metabolites from the plant LA and ALA pools. Studies during the past 15 years in solanaceous plants point to the importance of 9-LOX and the 9-oxylipin pathway in defense, and have demonstrated that the 9-LOXs from potato, tobacco, and pepper can utilize AA as a substrate. Many of these studies have investigated defense responses against oomycete pathogens or used elicitor preparations from oomycetes likely containing EP . 9-hydroperoxy fatty acids can be utilized by downstream oxylipin pathway enzymes to form other compounds that have been found to function in defense. In particular, DESs are induced in response to elicitors and pathogen attack in several solanaceous species including potato, tobacco, and pepper . DESs are CYP74D P450s that produce the divinyl ethers CA from 9-HPOD and CnA from 9-HPOT. Recent experiments indicate that treatment of tomato roots with EP induces resistance against P. capsici. Hydroponically grown tomato plants whose roots were treated with EP and subsequently inoculated with P. capsici experience significantly less rot and collapse at the crowns than plants whose roots were treated with H2O, LA, or ALA, indicating that exposure of tomato roots to EP prior to inoculation with P. capsici reduces susceptibility of the plants to P. capsici . Further experiments demonstrate that roots and crowns display significantly increased lignification responses following root treatment with AA and EPA and subsequent inoculation with P. capsici compared to roots treated with H2O, LA, and ALA. AA-treatment of tomato roots elicits increased expression of 9-LOX and 9-DES genes in tomato roots compared to control treatments . Expression of 9-DES is also increased following inoculation of roots with P. capsici . In conclusion, although EP action in plants is complicated, evidence supports an important role for LOX and likely a 9-oxylipin pathway in the initiation of plant responses. Furthermore, in Arabidopsis an intact JA pathway is required for AA activity, implicating a 13-LOX. Whether DES and divinyl ethers participate in the plant response to EP observed in solanaceous plants is unresolved, although ongoing research in our laboratory will address this issue.

Although we did not directly measure selection in this study, we used the large body of work on drought adaptation to infer the adaptive value of specific trait combinations. We predicted that due to the drought escape strategy conferred by derived loss of function mutations at FRI, accessions with these alleles would inhabit environments with consistently wetter growing seasons, relative to accessions with functional FRI alleles. To confirm the allelic association with drought, we generated a climate envelope for both FRI allele classes . Functional alleles tend to be present in areas with lower growing season precipitation than non-functional alleles. We have demonstrated that lines that diverged only at FRI exhibit altered positions along an adaptive phenotypic correlation. Scarcelli et al. found antagonism between the floral morphology traits affected by FRI, and we cannot rule out that a portion of FRI’s pleiotropic gene action is maladaptive. However, analyses presented here demonstrate a strong adaptive role of the physiological and phenological phenotypic correlations conferred by FRI. Given our results, it is not surprising that FRI is associated with strong population genetic signatures of diversifying selection. Studies demonstrating historical selection on FRI invoke the timing of flowering as the phenotype under selection. Our results indicate that the observed signature of selection is not only an effect of FT variation, but may also be due to upstream physiological effects.Stress caused by salinity is one of the most serious environmental factors, which inhibits plant growth and decreases crop productivity worldwide. Primary effects occurring at the beginning of salt stress include retarded cell division and expansion, stomata closure and photosynthesis reduction. During long-term exposure to salt stress, accumulation of salt ions in plant aerial parts via the transpiration stream leads to ionic stress. To adaptively respond and survive under salinity, plants require changes of various cellular, physiological and metabolic mechanisms, which are controlled by the regulated expression of specific stress-related genes through cascades of complex regulatory networks. Rice , one of the world’s most important cereal crops, is classified as a salinity sensitive crop. An electrical conductivity of ~ 6 dS m− 1 would result in more than 50% reduction in yield of many rice varieties. Therefore, plant breeders are continuously improving salt tolerant rice cultivars to increase yield productivity. However, salt tolerance is a multi-genic trait, which underlying mechanisms are controlled by many genes and affected by the environment. Breeding efforts for developing salt tolerant rice have been limited because the salt tolerance mechanisms and the genes that control them are not completely understood.

To fill the knowledge gap between genotypes and phenotypes of the salt stress response in rice,vertical planters for vegetables forward and reverse genetics have been performed to identify salt-responsive loci/genes such as genetic mapping of quantitative trait loci using cross population; screening of mutants generated by chemical- or irradiation-induced mutagenesis; and transgenic approach. To identify salt-responsive genes using cross population, a number of mapping studies have identified QTLs of physiological traits related to salinity tolerance in economic crops such as soybean, barley and rice. Although QTL mapping is a powerful and popular method to tag the salt tolerance region in plants, the examination of the variation is one of the limitation because QTL mapping can identify only allelic diversity that segregates between the parents of a particular F2 cross or within recombinant inbred lines and the mapping resolution is limited by the amount of the genetic recombination event occurring in the mapping populations. Moreover, the genotyping by SSR markers, which is usually based on polymerase chain reaction , is limiting to examining the kinds of variations, and laborious and time-consuming when high-density genotyping is needed for a large number of individuals. Over the past several years, next generation sequencing has been used to rapidly generate a large amount of accurate genomic data, providing a powerful approach for functional genomics and molecular breeding studies, including the genome-wide association study. GWAS, which is the analysis of the statistical association between genetic variants and traits on the whole genome scale in a large number of individuals within an organism, has been employed to identify causal genetic variability for target traits, including those in Arabidopsis and crop species. Compared with the QTL linkage mapping method, GWAS provides high resolution mapping using single nucleotide polymorphisms as genetic markers. GWAS in rice was performed for agronomic traits such as tiller number, grain width, grain length and spikelet number in the indica subspecies based on SNPs identified by whole-genome sequencing. In another report, the genetic architecture of rice chlorophyll content at the heading stage was revealed by GWAS.

Forty-six significant loci were identified and Ghd7 was highlighted as a major locus for the natural variation of the chlorophyll content. GWAS also revealed three QTLs located on chromosomes 3, 6 and 12 associated with the responsiveness of yield-determination traits under field condition. Application of GWAS for causative gene identification has been reported in rice responding to abiotic stresses such as aluminum, boron, cold, drought and salt stresses. On salt stress, there are several GWA studies in rice with different growing stages and traits. Shi et al.studies GWAS on germination stage of salt-treated rice using ~ 6000,000 SNPs, 11 loci containing 22 significant SNPs responsible for stress-susceptibility indices of the vigor index and germination time were identified. The strongest association region for germination time was detected on chromosome 1, near salt-tolerance QTL controlling Na+ uptake and K+ concentration. At tillering stage, GWAS was performed on rice exposed to short- , medium- and longterm salt stress based on ~ 200,000 SNPs. Around 1200 candidate genes associated with growth parameters, and Na+ and K+ content were identified.For salt treated rice at reproductive stage, only a study of Kumar et al. were reported. Based on 6000 SNPs, it was shown that 20 loci were associated with the Na+ /K+ ratio, and 44 loci were associated with other traits. Twelve association mappings with Na+ /K+ were located on chromosome 1 where Saltol, a major QTL that controls shoot Na+ /K+ homeostasis in rice at the seedling stage, is located. However, GWAS has not been applied for the analysis of photosynthetic and yield-related traits in rice exposed to salt stress at the flowering stage, which is a highly salt-sensitive stage. Additionally, no rice accession from Thailand where a large collection of diverse rice germplasms can provide new allelic diversity for salt tolerance, were analyzed by GWAS. The objectives of this research were to investigate and cluster Thai and Asian rice accessions based on physiological responses and yield-related traits under the salt-stress condition at the flowering stage and to perform GWAS for these traits to identify regions/genes responsible for salt tolerance.

The association panel consisted of a diverse collection of 190 rice cultivars including both standard salt-tolerant and salt-sensitive varieties. The rice accessions in this study were kindly provided by the Pathum Thani Rice Research Center . The experiment was designed with a randomized complete block design with four replications. According to the limitation of the time-consuming process of data collection, the experiment was performed in three separate sets of experiments. The standard salt tolerant and salt-sensitive cultivars were included in every experimental set. Twenty-one day old seedlings were cultivated using a hydroponic system with WP No. 2 nutrient solution and transplanted into pots containing soil and maintained until harvest. At heading stage in the flowering phase of each accession, water on the soil surface was drained before salt stress treatment. Rice plants were then watered with 900 mL of 150 mM NaCl solution to reach the desired final soil electrical conductivity of 8–9 dS m− 1 and treated for 9 days. For the control condition, rice plants were treated by tap water for the same period. To recover,vertical farming technology tap water was used to wash out salt ions in the soil every day until the soil EC was lower than 2 dS m− 1 ; this condition was maintained until harvest to collect yield-related traits. These experiments were conducted in the greenhouse facility at the Nakhon Ratchasima Rice Research Center, Rice Department, Ministry of Agriculture and Cooperatives. The air temperature was maximum at 32 °C with natural light and minimum at 21.1 °C during the night.

The average relative humidity was 72.5%. The short-sequence reads from the Illumina Genome Analyzer were grouped into the correct categories using the pipeline created by Missirian et al.. The rice reference genome was downloaded from the database , and indexed by SAMtools. Raw reads were aligned against the reference genome using the Burrow-Wheeler Aligner. Variants were called using genome analysis toolkit. Variants were filtered if they fitted the following criteria: to be called heterozygous, minimum coverage and minimum percentage of each of the two observed major base calls were 5 and 20, respectively and minimum total coverage was 10; for a position to be called homozygous, minimum coverage was 6 or 3 if positions with the minimum coverage of 6 were present in at least 10 accessions. SNP density was visualized using R ‘CMplot’ .We evaluated photosynthetic parameters and cell membrane stability on 104 rice accessions individually at the flowering stage after salt stress for 3, 6 and 9 days and analyzed yield-related traits at harvesting time. The mean values and frequency distributions of all parameters of each accession are shown in Additional file 2: Table S2 and Additional file 3: Figure S1. The highest reduction of phenotypic traits was observed at day 9 after salt stress: photosynthetic rate, PN ; stomatal conductance, gs ; transpiration rate, E , and cell membrane stability, CMS when compared with the control condition . However, we found that the mean values of intercellular CO2 concentration, Ci increased about 6% at day 9 after salt stress treatment. For yield-related traits, on average, number of tillers per plant, TIL; number of panicles per plant, PAN; number of filled grains per plant, FG decreased by 19, 11 and 26%, respectively, whereas number of unfilled grains per plant, UFG increased by 10% . To determine substantial genotypic variation in salt-stress responses, relative phenotypic values were calculated by the salt stability index of each rice accession [ × 100] . These parameters tended to decrease when plants were exposed to salt stress, except Ci , which tended to increase under salt stress. The variations of phenotypic traits were found in all parameters and were pronounced, particularly in the case of PN, FG and UFG . The relationships of the salt stability index of all parameters were determined by Pearson’s correlation r . We found a strong positive correlation between PN and gs, or E . PN also had a positive correlation with CMS, though weaker, at days 6 and 9 after salt treatment. Conversely, a strong negative correlation between PN and Ci was found. As expected for yield-related traits, the strongest positive correlation was observed between TIL, PAN and UFG. In addition, the relationship between photosynthetic performance and yield-related traits were observed. TIL was negatively correlated with gs at days 3 and 6; and with E at day 3. Similarly, PAN was negatively correlated with gs at day 3 as well as PN. Following the same trend, UFG was negatively correlated with gs or E at days 3 after salt treatment, and with Ci both at days 3 and 6. . In an opposite trend, a positive correlation was found between FG and gs at day 6. At day 9, no correlation was observed between photosynthetic parameters and yield-related traits.The list of rice accessions used for exome sequencing is shown in Additional file 1: Table S1. In total, 190 rice accessions were used for exome-sequencing, with the capture probes designed to cover about 50 Mb of the nucleotide target covering all 12 chromosomes of rice. SNPs that showed a minor allele frequency of < 5% of our population were removed to decrease overestimation of the effect of low-MAF SNPs. Therefore, the resultant number of 112,565 SNPs , which were high-quality SNPs genotyped across this population, was subsequently used for GWAS.EINGENSOFT was implemented for population structure analysis, which was based on PCA. Using SNPs identified by exome sequencing, two main sub-populations were delineated , consisting of five accessions in the first group and 185 accessions in the second group, respectively. The rice accessions in the first group included ‘Ai Tai’, ‘Jao Haw’, ‘Beu Saw Mi’, ‘E-Puang’ and ‘Leung Tah Young’ rice, which were grouped as upland rice .

Karl Hardy, in his excellent essay on the historical-colonial roots of “utopia” and the tentative possibilities of its use in Indigenous politics, cites research that Thomas More’s Utopia may have been the first instance of the Roman word colonia in the English language. Hardy also points out that More’s utopians are colonists themselves, who conquer lands near their island when necessary, and kill or “civilize” others when they are deemed to be using the land improperly . Early fictional utopias, the Christian utopias of the pre-Enlightenment especially, were steeped in colonial tropes: an explorer must sail a trans-oceanic ship to arrive at utopia, will sometimes encounter savages, and these explorers ostensibly seek riches of the new frontier.The colonial roots of utopia are well-outlined by Hardy, and scholars of science fiction show the ways this history still informs utopian fiction, for example in the ways the language of the frontier is used to describe cyberspace, and the ways aliens are racialized in narratives of space travel. The dystopian works I study are also primarily works of Indigenous Futurism. Grace Dillon defines the genre as work that “enclose ‘reservation realisms’ in a fiction that sometimes fuses Indigenous sciences with the latest scientific theories available in public discourse, and sometimes undercuts the western limitations of science altogether” .

These works estrange sf itself because they are often situated in an apocalypse that has already occurred — the colonial apocalypse — but they project a hopeful,vertical agriculture decolonized future from these ruins. Thus they are concerned with how historical colonialism continues into the present and permeates North American societies and cultures, as they interact with each other. Indigenous science and sustainability are common themes for Indigenous futurisms, hence they receive special attention in Chapter 2. Spiritual and physical healing at an individual and societal level — emphasizing balance and harmony with the environment — is also a predominant theme, and is especially reflected here in Chapter 4. All of the texts of this study are rich grounds for both Settler Colonial and Utopian Studies readings, but they work together on more than just a literary level. While they may make strange bedfellows, both Utopian and Postcolonial Studies have Marxist roots. Reading texts through a utopian and postcolonial lens makes up for what I see as two blind spots in utopian studies. First, contemporary utopian theory is centered around Western concepts of human rights and, while it gestures toward intersectionality, has to date not adequately grappled with just how contingent utopia must be in a global context. It is a commonplace that classical Marxism itself famously does not fully account for colonialism in its understanding of class struggle, a fact that postcolonial theorists since Fanon have sought to remedy. We can see this reflected in the context of utopian studies in Levitas’ Utopia as Method: her conclusion briefly cites global inequality as a major problem and advocates for international redistribution of wealth, but both the philosophy on which she draws and her concrete ideas for utopia as method are dependent on familiarity with the language and ideas of democratic socialism, a language that often does not account for Europe and the United States’ accumulated wealth through colonial dispossession, and as such is applicable to non-Western societies in a limited way, one that does not fully account for cultural difference. This understanding helps account for the ways that even sf from the political left, such as the works I study in Chapter 1, has not fully engaged with the inextricability of colonialism from class and race struggle.

Eurocentrism leads to another problem of utopian studies I also attempt to address: if we wish to decolonize the concept of utopia, we must engage with the violence of colonial oppression, and the likelihood that true decolonization in the form of the overthrow of capitalism globally will be likely violent. Power holds many tools of violence to protect its wealth , and anticolonial nonviolent resistance is routinely met with violence. Postcolonial studies, especially Settler Colonial Theory, also helps address this problem. SCT, which takes Frantz Fanon’s The Wretched of the Earth as a founding text, grapples with the bare fact that “decolonization is always a violent event” , and that nonviolence is typically called for by the colonizer when the colonized rebel against violence and dispossession, revealing the colonizer’s belief that only the colonizer has the only right to violence. While the fact of violence leaves everyone uneasy, and is a difficult subject even in Postcolonial Studies, it cannot be ignored simply because it is not part of the relatively peaceful utopian vision of the future. One way I suggest working through this uneasiness is to separate “violence” as harm to people and “vandalism” as harm to property. Though typically seen as synonymous, vandalism tends to be the form of resistance that the authors I study here are most concerned with, one that harms capitalist systems rather than individuals. Indeed, many of the texts I study will suggest that “utopia as method” — as a contingent, emergent practice that imagines and works toward radical change — will sometimes and in some places involve violent resistance, questioning the dichotomy between utopia as “peaceful” and dystopia as inherently violent . All of these texts have implications for activism that I will explore, within the context of my own subjectivity as a White scholar of European descent. In this spirit I echo Wolfe’s conclusion in Traces of History: “It is not my place to instruct colonized people on how to resist their condition, let alone to impersonate their agency … I have tried to offer an analysis, in the hope that it may prove useful” — useful to scholars who consider themselves activists and allies of colonized peoples, and who must grapple with these unsettling questions. In adding Postcolonial Science and Technology Studies and discussions of Indigenous Science to the mix in some chapters, especially Chapters 2 and 4 dealing with agricultural sciences and bio-piracy, respectively, I historicize the ways that colonization used/uses Western science and technology — to further utopian projects of colonization and globalization, and in doing so I take up the challenge postcolonial STS makes in decentering Western science and technology.

STS theorists observe the slippage between science fiction and science fact; some, like Colin Milburn, go so far as to suggest that science fiction is a sort of intellectual workbench of science . However, Western science fiction and the real-world projects which it reveals and reflects upon do not have a monopoly on techno-optimism. Instead, the texts I study puttechno-optimism to work in an anticolonial way, by subverting not-yet-existing Western technologies and by imagining indigenous technological innovations. The contention that postcolonial sf uses technology and/or utopian ideas more broadly as means of anticolonial subversion is not new. However, in my view, the subfield of Postcolonial SFS has an unfortunate tendency to situate colonialism in the past that can be corrected through a concerted engagement with Settler Colonialism. Patricia Kerslake, for example, in her work Science Fiction and Empire, not only considers colonialism a historical event but suggests that sf’s concerns with empire are not Western at all, but rather “the tendency we all share” to dominance and oppression. Ralph Pordzik, another problematic example,vertical farming aeroponics looks productively at third world sf in his book, The Quest for Postcolonial Utopia: A Comparative Introduction to the Utopian Novel in the New English Literatures. However, as Eric Smith notes, he subsumes his study in multiculturalism to the point of diminishing productive differences between different postcolonial geographies. Thus, an SCT-based study of dystopian speculative fiction and works of Indigenous Futurism can help correct this oversight and bring the discussion into our contemporary moment of increasing environmental instability and socio-economic upheaval. This study rethinks utopia against the anti-utopian projects of the settler colonizer, projects that began centuries ago and continue to this day. Some of these projects include: colonial attempts to increase the efficiency of land through new farming technologies, such as bioengineering; the use of genetics research on Indigenous peoples to further medical advancements that will not be accessible to research subjects; and using technologies of surveillance and security to maintain the borders of the settler colonial utopia. I discuss these and more in the following chapter outline, but first I must note that all of these technologies are forms of colonial violence in themselves, and are also protected with the use of violent and deadly technologies. Technologies of colonial violence loom large over the works I study, and also loom over every chapter.Without a doubt, fumigation has been one of the most controversial aspects of the process of eradicating illegal crops in Colombia. The toxicity of the herbicides used and the importance that fumigation acquired in the international strategy of the United States against drugs, have caused this subject to become an essentially polemical one.

The present article takes an historical look at the matter and describes and explains the fumigation policy imposed by the US on Colombia as part of a drug diplomacy which has characterized relations between the two countries over the past twenty-five years. Before beginning this chronology, however, it would be worthwhile to take a look at the basis for the US policy in this regard. Fumigation with herbicides corresponds to a rationale based on five premises: An implicit refusal to accept the notion that every demand produces a supply. When it come to repression, therefore, the emphasis is placed on growing, producing, processing, transporting and trading in drugs, rather than on the centers where drugs are consumed or on the places where there is the greatest margin of profit for the international illegal trade in drugs.It is assumed that, in terms of results and resources, strong repressive measures taken at the centers which provide drugs constitute the most effective way of combating the drug trade. It is assumed that punitive strategies designed and executed by those states in which the demand exists, and in those where the supply has originated, are pertinent when it comes to attacking a highly lucrative illegal trade that arises and evolves in a non-state situation and which is in the hands of powerful groups within society. It is assumed that, for the consumer countries, a greater and more effective eradication of illegal crops will lead to three results: fewer stimulants of this kind will be available on the market, they will be more highly priced, and they will be less pure. As a consequence of this triple assumption, it is supposed that there will be a decrease in urban criminality associated with drug dealing and a decrease in consumption. It is assumed that greater and more efficient eradication of illegal crops in the producing countries will lead, among other things, to a reduction in the value of illegal crops in the zones of production, a weakening of the drug traffickers’ power, a containing of violence generated by drug traffic affecting the more vulnerable sections of the population linked to these illegal plantations, and a decrease in the environmental damage caused by illegal plantations in soil which is fragile and extremely valuable. During the administration of President Julio César Turbay Ayala , Washington began putting pressure on the Colombian government to use chemicals for eradicating marijuana crops, especially in La Guajira on Colombia’s Caribbean coast. By 1978, Colombia had become the number one producer and exporter of marijuana to the United States. Of the 10,000 tons introduced into the US during that year, between 60 and 65 percent was supplied by Colombian traffickers. At the time it was estimated that Colombia had between 25,000 and 30,000 hectares planted with marijuana.During the administration of President Jimmy Carter , interdiction anderadication were the two keynotes governing international anti-drug policy. The attempt to manually eradicate marijuana plantations was no longer seen by Washington as sufficient. Congress and the White House began to agree that herbicides should be used to put an end to plantations not only of marijuana but also of poppies . In Latin America, herbicides had already been used in Mexico and in Jamaica. In the mid-seventies, Operación Condor in Mexico attempted to destroy marijuana plantations and was presented as a resounding success in the fight against drugs.

Harvesting activity moves to the coastal plains in the second quarter of April June, as lemons and oranges are harvested in southern California and vegetable crops are thinned and then harvested in the Salinas Valley of northern California. June marks the second highest month of employment on the state’s farms, as workers harvest strawberries and vegetables as well as early tree fruits, including cherries and apricots; melons and table grapes are harvested in the desert areas. Other workers thin peaches, plums, and nectarines, remove leaves in some vineyards, and thin large acreage crops such as cotton. Farm employment peaks in September, during the third quarter, reflecting the harvests of crops from Valencia oranges to tomatoes to tree fruits in the Central Valley of the state. However, the single largest labor-intensive harvest involves raisin grapes—some 40,000 to 50,000 workers have been hired to cut bunches of 20 to 25 pounds of green grapes and lay them on paper trays to dry into raisins. The workers typically receive $0.20 a tray, and the contractor who assembles them into crews of 30 to 40, and acts as their employer, receives another $0.05 a tray. During September, there is something of an early morning traffic jam, as vans ferry workers to fields and orchards,stackable flower pots and employers wanting to wait as long as possible to harvest to raise the sugar content of their grapes worry that not enough workers will show up.

During the fourth quarter, harvesting activities slow, and after the last grapes, as well as olives and kiwi fruit are harvested in October, most seasonal farm and food processing workers are laid off. Most workers remain in the areas in which they have worked—most workers are not migrants who follow the ripening crops—but many were born in Mexico, and some return to Mexico with their families for the months of December and January. If workers were willing to follow the ripening crops, and to switch between citrus and grapes, they could harvest work for 6 to 8 months a year.But few workers migrate from one area to another, and few switch crops within an area. In the mid-1960s, when migrancy was at its peak, a careful survey of farm workers found that only 30 percent migrated from one of California’s six major farming regions to another . A 1981 survey of Tulare county farm workers found only 20 percent had to establish a temporary residence away from their usual home because a farm job took them beyond commuting distance , and surveys of California farm workers in the 1990s found that fewer than 12 percent followed the crops . A 2000-01 survey of 300 farm workers found 19 percent who moved in the previous two years to find farm work; fewer than 25 percent planned to move in the current year to find a farm job . There are many reasons why most farm workers stay in one area of California: the harvesting of many fruits and vegetables has been stretched out for marketing and processing reasons; the availability of unemployment insurance makes migration less necessary; and some farm workers with children who are not likely to follow them into the fields realize that migrancy makes it very difficult for children to obtain the education needed to succeed in the U.S.

An easy test of the degree of follow-the-crop migrancy is to check turnover in a farm labor center.If follow-the-crop migrants filled the center, workers and families would be constantly arriving and departing, as they moved on to another job in a distant area. In fact, most migrant centers fill as soon as they open, and keep the same tenants for the season: workers know that they can obtain services for themselves and their children, especially in the state-run centers, and it is very hard to find alternative housing if the family packed up and sought another job in the manner of John Steinbeck’s Joad family.Until the 1940s, it was common for the wives of field workers to be employed in the packing houses that canned, froze or dried fruits and vegetables. However, after unions pushed packing-house wages to twice field worker levels in the 1950s and 1960s, packing-house jobs became preferred to field worker jobs, often representing a first rung up the American job ladder for field workers. About 40,000 workers are employed in the preserved fruits and vegetables subsection of the state’s manufacturing industry, down from 50,000 in the early 1990s.In the case of some vegetables and melons,  non-farm packing and processing jobs have been turned into farm worker jobs by field packing, having workers in the field put broccoli or cantaloupes directly intocartons rather than having the crop picked by field workers and packed by  non-farm workers in packing houses.4 In other cases, farm jobs have become  non-farm jobs, as when the cutting and packing of lettuce in the field is replaced by fewer workers simply cutting lettuce, and when there are more  non-farm jobs in packing plants as lettuce is cut and bagged: bagged lettuce uses almost 40 percent of U.S. lettuce.Employment on California farms was expected to drop sharply in the 1960s, as the end of the Bracero program, which brought Mexicans to work in U.S. fields between 1942 and 1964, was followed by sharply rising wages and unionization—the United Farm Workers union won a 40 percent wage increase in its first table grape contract in 1966. Processing tomatoes provides an example of the sharp drop in farm worker employment as a result of labor-saving mechanization.In 1960, a peak 45,000 workers, 80 percent Braceros, hand picked 2.2 million tons from 130,000 acres of the processing tomatoes used to make ketchup. In 2000, about 5,000 workers were employed to sort 11 million tons of tomatoes from 350,000 acres that were picked by machines.

The keys to tomato harvest mechanization included cooperation between scientists and between farmers, government, and processors. Plant scientists developed smaller tomatoes more uniform in size that ripened at the same time, and were firm enough so that the stalk could be cut, and the tomatoes shaken off, without damage. Engineers developed a machine to cut the plant, shake off the tomatoes, and use electronic eyes to distinguish red and green tomatoes and discard the green ones . Processors agreed to accept tomatoes in 12.5 ton truck mounted tubs rather than 60- pound lugs, and the government established grading stations at which random samples were taken to determine the quality and price. The cost of mechanizing the tomato harvest was relatively small—less than $1 million—and the estimated rate of return was hundreds of percent.5 The rapid diffusion of tomato harvesting machines in California—none were harvested by machine in 1960, and all were harvested by machine by 1970—was expected to usher in an era of machines replacing men on farms,flower pots for sale economists and engineers boldly predicted that, by 2000, there would be practically no jobs left for unskilled seasonal farm workers by 2000 .6 However, the cooperation between researchers, farmers, processors, and the government that transformed the processing tomato industry in the 1960s proved to be the exception, not the rule. Farmers remained very interested in and supportive of mechanization research during the 1970s, when there were hundreds of public and private efforts to develop uniformly ripening crops and machines to harvest them, but interest waned in the late 1970s due to rising illegal immigration and a lawsuit. Mexico devalued the peso in 1976, and in 1977, for the first time, apprehensions of unauthorized Mexicans in the U.S. first topped 1 million. Apprehensions remained at about 1 million a year until after 1982, when another peso devaluation caused them to jump by 25 percent, and the rising number of unauthorized Mexicans, many of whom were from rural Mexico and sought jobs on U.S. farms, guaranteeing an ample supply of hand workers. Meanwhile, the UFW and California Rural Legal Assistance in 1979 filed a lawsuit against the University of California , charging that efforts to develop labor-saving machines were an unlawful expenditure of public funds because they displaced small farmers and farm workers . The suit asked that UC mechanization research be halted and a fund was created to assist small farmers and farm workers equal in size to what UC earned from royalties and patents on agricultural innovations .The suit was eventually settled by establishing a committee to review research priorities, but public and private support for mechanization research decreased, and scientists and engineers moved on to other issues. Most labor-saving research today is conducted by the private sector, and most of it is far less visible than machines replacing 90 percent of the hand harvesters, as in tomato processing. Precision planting and improved herbicides have dramatically reduced the need for thinning and hoeing labor. Many farmers have planted dwarf trees to increase yields, which can also reduce harvest labor needs. Much of today’s mechanization is motivated as much for non-labor reasons as to save on labor costs. For example, drip irrigation systems reduce the need for water as well as irrigator labor, and a machine harvesting wine grapes at night results in higher-quality grapes and uses less labor.

In the 19th century, U.S. agriculture in general and California agriculture in particular were considered land-abundant and labor short, which led to labor shortages that were compounded in California by the dominance of large and specialized farms. California began producing fruits in the 1870s, when the completion of the transcontinental railroad and falling interest rates encouraged a shift from grazing cattle and growing grain without irrigation to labor-intensive, irrigated fruit and vegetable farming. The expectation was that large farms, many derived from Spanish and Mexican land grants, would be broken up into family-sized units and sold to farmers arriving on the railroad, because only with a family-farm system would there be enough workers for labor-intensive agriculture . However, large farms were not broken up into family-sized units because new workers were available to be seasonal farm workers. Some 12,000 Chinese workers had been imported to help build the railroad through the Sierra Nevada mountains and, when they were laid off in 1870, they were kept out of urban jobs by anti-Chinese movements . Chinese workers were paid low wages only when they were needed which helped to raise land prices, and made it hard for family farmers to buy land and get started in farming, and gave landowners an incentive to keep the door open to immigrants. However, anti-Chinese sentiment eventually led to a halt to Chinese immigration in 1883, but a new source of immigrant workers was found, in Japan. Japanese immigration was stopped in 1907, and workers were imported from present-day India and Pakistan until World War I. There was little immigration during World War I, when Mexico was experiencing a civil war. The U.S. government was trying to restrict immigration from Europe, imposing head taxes and literacy tests on new arrivals in 1917, but western farmers won an exemption for Mexican farm workers coming to the United States for up to one year, beginning the U.S.-government-approved recruitment of Mexican farm workers. There were many problems with this first Bracero program, and government approved recruitment was halted in 1921, but Mexicans continued to arrive and travel around California seeking farm work. Many Mexicans were sent back to Mexico during the Great Depression, and the source of farm workers in the mid-1930s shifted to the Midwest, where many of the Okies and Arkies who lost their farms during the so-called Dust Bowl moved to California, expecting to become small family farmers. The gaps between farmers and farm workers in California led to some of the most enduring American literature, including John Steinbeck’s 1939 novel, The Grapes of Wrath. Okies and Arkies continued to be the mainstays of the seasonal harvest work force in the 1940s, when “fruit tramps” migrated from farm to farm, but their children often went in to the military during World War II, or found jobs in wartime factories, and California farmers asked the federal government to once again approve the recruitment of Mexican Bracero workers. The federal government agreed, and the first of a series of Bracero agreements was signed in 1942; almost 5 million Mexican workers were admitted over the next 22 years—many individuals returned year after year, so that only 1 to 2 million Mexicans gained experience working on U.S. farms.