Petiole NO3 – , an indicator of recent N status in conventionally-produced vegetables, was measured in the most recently-matured leaves. Petiole NO3 – changes rapidly with growth stage, so the data are graphed by post-transplanting growing degree day to account for phenological differences among fields as a result of slightly different sampling times relative to transplanting.Root RNA was extracted using Trizol reagent according to the manufacturer’s guidelines followed by DNase digestion using RQ1 RNase-free DNase . Total RNA was purified using the RNeasy Plant Mini Kit . RNA concentrations and quality were assessed using the Agilent Nanodrop and the RNA 6000 Nano Assay . Only RNA samples with RNA integrity numbers of at least 7.0 were used for subsequent analyses. These RNA were used for cDNA synthesis for qRT-PCR analysis. cDNA was synthesized from 0.5 μg DNase-treated total RNA using the Superscript III kit . Quantitative real-time RT-PCR was performed as described in previous work , using the primer pairs tested and reported therein and using a Step OnePlus Real-Time PCR system . Seven key genes involved in root N uptake and assimilation that had previously been shown to be responsive to an N pulse in an organic soil were assessed: high-affinity NH4 + transporters AMT1.1 and AMT1.2 ; high-affinity NO3 – transporter NRT2.1 ; nitrite reductase Nii ; cytosolic and plastidic glutamine synthetases GS1 and GS2 ; and NADH-dependent glutamate synthase NADHGOGAT . The tomato actin and ubiquitin genes were used as reference control genes as they did not exhibit differential expression among N treatments in previous field experiments. Relative expression was analyzed according to the ΔΔCT method with multiple reference control genes and using inter-run calibration.Means for variables in all tables, figures,strawberry gutter system and the text are expressed with 95% confidence intervals .

CIs can assist with means comparisons based on the “inference by eye” method. Roughly, 95% CIs that overlap with another mean are not different but when the overlap of intervals is no more than half of one interval arm, then the means are different at p  0.05. Intervals that barely touch are significant at p  0.01 and intervals that are separated by at least half of one interval arm are different at p  0.001. Soil NH4 + and NO3 – showed positive skewing and thus were log transformed prior to calculation of means and CIs. Back-transformed means and 95% CIs are shown for these variables. Fields were clustered based on 28 plant, soil, and microbial variables using the k-means method implemented in R. The optimal number of groups chosen was based on the Calinski-Harabasz criterion and by examining sums of squared error scree plots. F-statistics were calculated for each variable based on their cluster grouping to assess the relative magnitude of the “cluster effect”, i.e. higher F-statistics indicate more differentiation among clusters for a given variable.The 13 organically-managed Roma-type tomato fields spanned a three-fold range of soil C and N and had similar soil texture, soil types, and soil pH . Field numbers are in order of increasing total soil C. Variation in nutrient inputs, including highly-labile secondary inputs indicated diverse and intensive organic management strategies across these farms . The 13 fields encompassed the majority of the variation in the focal landscape , i.e. all but one of the five clusters, or landscape types, identified by GIS and multivariate analysis were represented . Thus, a range of soil characteristics representative of this region was accounted for by the fields sampled. Other characteristics, such as the low number of crop rotation types differed little across the clusters and reflect the intensity of agricultural management in this region.Measures of tomato N sufficiency varied widely across the 13 organic fields, ranging from deficient to luxury N levels.

Total above ground N concentration at mid-season overlapped or fell slightly below the critical N concentration for processing tomatoes in most fields , with N concentrations between 2.5 and 3.5% . Exceptions were fields 1 and 2 which were markedly lower and field 4, which was higher . The same general pattern occurred for the harvest sampling. Petiole NO3 – concentration in four fields overlapped the sufficient concentration, based on published guidelines , while five fell below it, and four rose above it . Petiole NO3 – was especially high in field 4. Petiole NO3 – -N showed a broadly similar pattern to total above ground N concentration, as reflected in the strong linear relationship between them . At the mid-season sampling, shoot δ15N ranged from 4.22 ± 0.65‰ in field 10 to 13.29 ± 1.18‰ in field 6. Fields 3, 4, 6, and 9 had the highest shoot δ15N, all above 12‰, and all but field 3 used seabird guano. Fields 8, 10, 11, and 13 had the lowest shoot δ15N, close to 4‰, and all but field 8 used Chilean nitrate. Mean harvestable fruit yield across all 13 fields was 86.7 ± 7.2 Mg ha-1 and was similar to the overall Yolo County mean 2011 tomato yield , which included both conventional and organic fields . Field 4 had the highest yield overall followed closely by field 9 , and field 1 had the lowest . Nine of 13 fields had means higher than the county average, and six of these fields were significantly higher. There was also substantial variability in tomato above ground biomass and N content at harvest across fields , which largely reflected the pattern of fresh weight yields. For instance, total above ground N ranged from 64 kg N ha-1 in field 1 to 243 kg N ha-1 in field 4 with a mean across all fields of 154 kg ha-1.Expression of cytosolic glutamine synthetase GS1 in roots was more strongly related to indicators of plant-soil N cycling than were the other six key genes involved in root N metabolism . Of the soil variables, GS1 was more strongly related to soil bio-assays for N availability than to inorganic N pools .

Microbial biomass N and PMN were most strongly associated with expression of GS1 in roots, followed by soil NO3 – . Permanganate oxidizable C and MBC, both indicators of labile soil C pools, also had significant associations with GS1 expression in roots, but soil NH4 + did not. Expression of GS1 also was positively associated with shoot N and petiole NO3 – , as was glutamate synthase NADH-GOGAT. Inclusion of GWC as a covariate in multiple linear regression models improved the proportion of explained variation in GS1 expression .PCA of 28 indicators of yield and plant nutrient status, root N metabolism, and soil C and N cycling showed strong relationships among suites of variables, which clearly differentiated fields along the first two principal components . The first principal component explained 28.3% of the variation; on the left side of the biplot are higher values of most variables, including yield, soil bio-assays, expression of root GS1 and NADH-GOGAT, and labile and total soil C and N pools . Soil NH4 + and NO3 – concentrations from all three sampling times as well as AMT1.2 were associated with one another and with positive values along principal component 2, which explained 19.4% of the variation. Total soil C and N were strongly associated with EOC and EON,hydroponic fodder system the soil C:N ratio, and POXC. These variables had negative values along axis 2 and thus contrasted with the pattern of soil inorganic N. Weak loading of AMT1.1, NRT2.1, Nii, and GS2 on the first two principal components reflects the lack of association of expression levels of these genes with bio-geochemical and plant variables. Non-overlapping confidence ellipses for seven out of 13 fields on the PCA biplot indicated distinct N cycling patterns . Fields 1 and 2, with the highest values along axis 1, had low values of all variables included in the analysis. Field 4 had the highest values along axis 2 corresponding with higher soil NH4 + and NO3 – . Fields 10, 11, 12, and 13 were associated with high values of labile and total soil C and N. Overlapping confidence ellipses of fields 3, 5, 6, 7, 8, and 9 close to the origin indicate similar, moderate values of this suite of variables for these fields. Three groups of fields were identified by k-means cluster analysis of the same 28 variables included in the PCA . Group 1 included fields 1 and 2, which had low mean values for yield , the lowest mean soil C and N and soil inorganic N pools , and the lowest mean value of GS1 relative expression in roots. Groups 2 and 3 had similarly higher mean yield , shoot N, and petiole NO3 – than group 1, but these two groups differed substantially in their soil C and N pools. Group 2 had higher soil NH4 + and NO3 – pools as well as root expression of AMT1.2 while group 3 had higher total and labile soil C pools. Expression of GS1 was similar in both groups. Based on the relative magnitude of F-statistics calculated for each variable, soil C and N, EOC, EON, shoot N, and soil NO3 – at transplant and anthesis were most strongly differentiated across the three groups. The high F-statistics of AMT1.2 and GS1 relative to other N metabolism genes indicate that root expression of these genes are most responsive to soil N cycling.This study confirms that working organic farms can produce high yields with tightly-coupled N cycling that minimizes the potential for N losses.

Such farms had the highest soil C and N and used high C:N organic matter inputs coupled with labile N inputs that resulted in high soil biological activity, low soil inorganic N pools, high expression for a root N assimilation gene, adequate plant N, and high yields. Organic systems trials have previously shown crop N deficiencies that lead to less-than-ideal crop productivity; losses of N when Navailability is poorly synchronized with crop N demand; or alternatively, that organic production can reduce N losses. But how working organic farms achieve yields competitive with high-input conventional production with low potential for N losses has not been demonstrated. Elevated expression of a key gene involved in root N assimilation, cytosolic glutamine synthetase GS1, in fields with tightly coupled N cycling confirmed that plant N assimilation was high when plant-soil-microbe N cycling was rapid and inorganic N pools were low, thus showing potential as a novel indicator of N availability to plants. Improving biologically-based farming systems will benefit from research that uses novel tools to uncover innovations happening on farms, especially if the research process helps facilitate knowledge exchange among farmers and researchers.To characterize the substantial variation in crop yield, plant-soil N cycling, and root gene expression across 13 fields growing the same crop on similar soil types, we propose three N cycling scenarios: “tightly-coupled N cycling”, “N surplus”, and “N deficient”. Values of indicator variables suggest differing levels of provisioning, regulating, and supporting ecosystem services in each scenario . Fields in group 3 show evidence of tightly-coupled plant-soil N cycling, a desirable scenario in which crop productivity is supported by adequate N availability but low potential for N loss. Despite consistently low soil NO3 – pools in these fields, well below the critical mid-season level for conventional processing tomatoes in California, total above ground N concentrations were very close to or only slightly below the critical N concentration for processing tomatoes. Tomato yields were also above the county average . This discrepancy between low soil inorganic N pool sizes and adequate tomato N status is due to N pools that were turning over rapidly as a result of efficient N management, high soil microbial activity, and rapid plant N uptake. Composted yard waste inputs with relatively high C:N ratios in concert with limited use of labile organic fertilizers applied during peak plant N demand provided organic matter inputs with a range of N availability. A companion study showed how high potential activities of N-cycling soil enzymes but lower activities of C-cycling enzymes in this set of fields reflect an abundant supply of C but N limitation for the microbial community, thus stimulating production of microbial enzymes to mineralize N. Plant roots can effectively compete with microbes for this mineralized N, especially over time and when plant N demand is high.

The majority of this use is in horticultural crops with California and Florida together accounting for 80% of the 35 million pounds applied each year for preplant fumigation . Many genes are available that potentially could be used to enable alternative weed-control strategies. Horticultural crops are also limited in the numbers of herbicides registered for use. Loss of registration for a few key chemicals could markedly limit grower options, making crop resistance to broad-spectrum herbicides more critical. Resistance to fungal and bacterial diseases would also be desirable, as in some areas extensive use of pesticides is currently undertaken for their control. As for herbicides, it is also difficult to maintain registrations for minor crops grown on smaller acreages, which are primarily horticultural. Biotech strategies are being developed that could provide broader spectrum disease control and reduced dependence upon chemical pesticides . Resistance to viral diseases would be valuable in many horticultural crops, as there are few other options for control, and methods for engineering virus resistance are well established. Tree fruit, nuts and grapes. Research is well under-way to build a robust platform of technologies to utilize genomics in the discovery of useful traits for trees . Transformation technology has been developed and trait evaluation is under way on apple, almond, peach, citrus, walnut, pear, plum, grapevine and persimmon. Good progress has been made in developing resistance to codling moth and fire blight in apple, plum pox virus in plum/Prunus, crown gall and codling moth in walnut, citrus tristeza virus in citrus and Pierce’s disease in grapevine. Engineering of resistance to codling moth in apple to reduce the use of chemical pesticides has advanced to the point of commercial interest in product development. Work is also under way to develop productivity and quality traits such as modified sugar metabolism and ripening in apple and he pivotal year in the history of Hawaii’s papaya industry was 1992.

In May 1992, papaya ring spot virus was discovered in Puna on Hawaii Island, where 95% of Hawaii’s papaya was being grown. Just one month earlier,nft hydroponic system a small field trial to test the resistance of a transgenic papaya line had been started on Oahu Island, where papaya production had previously been devastated by PRSV. The timely commercialization of PRSV resistant transgenic papaya trees has revived Hawaii’s papaya industry and provides an example of the challenges and opportunities for horticultural biotechnology. In 1945, D.D. Jensen made the first report in Hawaii of PRSV, a potyvirus that is transmitted non-persistently by aphids . PRSV was first discovered on Oahu and caused such severe damage that the papaya industry was relocated to Puna in the late 1950s and early 1960s. The papaya industry expanded and prospered inPuna, primarily because PRSV was absent. However, by the 1970s PRSV was found only about 19 miles away in Hilo, and the Hawaii Department of Agriculture took rouging and quarantine actions to prevent its spread to Puna. In 1986, efforts were initiated to develop a virus-resistant transgenic papaya by transforming commercial lines of Hawaiian papaya with the coat protein gene of PRSV from Hawaii. By 1991, the team of Maureen Fitch, Jerry Slightom, Richard Manshardt and Dennis Gonsalves identified a transgenic line that showed resistance under greenhouse inoculations. These plants were micropagated and established in a field trial in Waimanalo on Oahu in April 1992. By December 1992, it was evident that line 55-1 was resistant under field conditions. From the 1992 field trial, two cultivars were developed and designated ‘SunUp’ and ‘Rainbow’. ‘SunUp’ is homozygous for the coat protein gene while ‘Rainbow’ is an Fl hybrid of ‘SunUp’ and the non-transgenic ‘Kapoho’. Unfortunately, by October 1994, PRSV had spread throughout much of Puna, causing HDOA to abandon rouging efforts to slow the spread of PRSV. The race was on to move the transgenic papaya line to commercialization. A 1995 field trial in Puna conclusively showed that ‘SunUp’ and ‘Rainbow’ were resistant under prolonged and heavy disease pressure.

The U.S. Department of Agriculture’s Animal Plant Health Inspection Service deregulated transgenic line 55-1 in November 1996, and the U.S. Environmental Protection Agency deregulated it in August 1997. The consultation process with the U.S. Food and Drug Administration was completed in September 1997. Licenses to commercialize the transgenic papaya were obtained by the Papaya Administrative Committee in Hawaii by April 1998. A celebration was held to mark the debut of the transgenic papaya on May 1, 6 years after PRSV was discovered in Puna and after the first field trial of line 55-1 was initiated. The transgenic fruit is currently sold throughout the United States. In 1992, Puna produced 53 million pounds of papaya, but by 1998 production had dropped to only 26 million pounds as PRSV spread throughout the region. Since then, the transgenic varieties have enabled farmers to reclaim infected areas and in 2001, Puna produced 40 million pounds of papaya. The resistance has held up remarkably well and remains stable after 5 years of extensive plantings. Hawaii also exports papaya to Canada and Japan. The transgenic papaya was recently deregulated in Canada, which is a relatively small market for Hawaii. The main challenge is deregulation of transgenic papaya in Japan, where Hawaii sells about 30% of its papaya. Presently, non-transgenic papaya must also be produced in Hawaii to satisfy the Japanese market, but this is increasingly difficult due to the disease pressure. Exporters face added expenses to guard against the accidental shipment of transgenic papaya to Japan. In December 2000, Japan’s Ministry of Agriculture, Forestry and Fisheries approved line 55-1, and the Ministry of Health, Labor and Welfare is reviewing a recently submitted petition for deregulation. Anticipated approval of transgenic papaya in regulation of self-incompatibility in almond and other Prunus species. Some deployment strategies for transgenic trees are also being developed, such as the use of transgenic trees as “trap crops” to control insects in conventional orchards and the use of transgenic root stocks to control diseases and pests in non-transgenic scion varieties . The latter approach avoids the task of transforming many varieties of a particular tree crop and in the future may be used to regulate quality and productivity traits.Although more difficult technically and therefore not close to market, there are many potential opportunities for enhancing the nutritional value or consumer appeal of horticultural products through biotechnology. In addition to modification of ripening, projects to increase the content of vitamins, minerals or nutraceuticals in horticultural products are in progress .

The development of Golden Rice with enhanced betacarotene in the grain demonstrated the potential for biotechnology to increase nutritional value. Whether such products will have sufficient consumer appeal in fully developed markets to drive their commercialization remains to be seen.Since floricultural and ornamental plants are grown for aesthetic or other nonedible purposes, there may be less potential for public concern about GE varieties than there has been with biotech food crops. Flower color. Several ornamental plants, including carnation, rose and gerbera, have been engineered for modified flower color. Research has focused on the manipulation of either anthocyanins or carotenoids,hydroponic nft system with the intent of creating a wider range of flower colors than occurs naturally, as well as to produce natural dyes for industrial purposes . Florigene is selling Transgenic Moon series carnations engineered for dark violet-purple color around the world. The varieties are developed in Australia and flowers are produced primarily in South America for marketing in the United States and Japan. Floral scent. Putting the scent back into flowers that have “lost” this trait over years of traditional hybridization and selection, or creating new fragrances in plants, has considerable potential and appeal. Research on genes controlling the different biochemical pathways for various floral fragrances is being conducted on wild plants and on crops such as snapdragon, petunia and rose . Plant size. Currently, growth regulating chemicals are applied to ornamental plants to inhibit gibberellic acid synthesis and reduce plant height during crop production. Many newly introduced ornamental species are receiving particular attention via conventional breeding for dwarf plants because their natural habits do not fit into marketing systems requiring compact plants. The manipulation of GA metabolism via biotechnology has the potential to produce ornamental and flowering plants with reduced-height phenotypes . The development of lawn grasses that require significantly less frequent mowing is another obvious application.Leaf life. Engineering of plants to delay leaf senescence is also being pursued in ornamental crops. For years, ornamental breeders have selected new cultivars of plants with more attractive “stay green” phenotypes. Cytokinins are plant hormones well known to delay the loss of chlorophyll in leaves; using biotechnology, targeted expression of genes involved in cytokinin synthesis is now possible. When a gene promoting cytokinin biosynthesis is inserted into plants in conjunction with a regulator that turns the gene on only Fruit and vegetable crops are under constant pressure from pests such as weeds, viruses, fungi, bacteria, insects and nematodes. If not controlled, many of these pests substantially lower yields. Successful agricultural production has depended on the use of pesticides for 100 years, and, yet, losses still occur due to certain pests that are poorly controlled. Some crops incur high costs for hiring laborers to hoe weeds because there are no effective herbicides. In addition, new pests routinely arrive for which effective controls have not yet been developed. Agricultural researchers continuously seek out new methods to control pests, including biological agents, new chemicals and plant resistance through classical breeding. Biotechnology also offers a solution in some situations where traditional methods are ineffective or costly. Numerous researchers around the world are investigating biotechnological solutions to pest problems of horticultural crops.

In 2002, the National Center for Food and Agricultural Policy released a study of current and potential biotechnological approaches to pest management in a wide array of crops . Current plantings. The study identified three varieties of transgenic fruits and vegetables that are currently planted on small acreages in the United States: virus-resistant squash is grown on 5,000 acres in the Southeast, to prevent late-season losses to mosaic viruses; virus-resistant papaya is widely planted in Hawaii ; and insect-resistant sweet corn is planted on a small number of acres and has reduced use of insecticide sprays.Withdrawn varieties. Two transgenic horticultural varieties were available for a short time in the United States but were withdrawn due to marketing concerns. Insect- and virus resistant New Leaf potatoes were planted on 4% of the nation’s acreage in 1999 and were credited with reducing insecticide use. If the transgenic varieties had not been withdrawn due to processor resistance they could have been planted extensively in the Northwest, reducing insecticide use by 1.4 million pounds. In 1999, the U.S. Environmental Protection Agency granted Wisconsin sweet-corn growers emergency permission to spray herbicide-tolerant varieties . The transgenic varieties were not widely planted due to marketing concerns and growers have not reapplied for the use despite continued production losses. Crops currently being tested. Numerous fruits and vegetables have been transformed through genetic engineering and are being tested for their potential role in improving pest management. For example, University of Florida researchers are testing virus resistant tomatoes as a substitute for the extensive insecticide spraying currently utilized to control insects vectoring geminiviruses. In California, herbicide-tolerant processing tomatoes have been tested and have the potential to reduce grower costs by $30 million and replace the use of 4.2 million pounds of fumigants. UC researchers have tested herbicide tolerant lettuce that could reduce herbicide use by 140,000 pounds a year. Herbicide-tolerant strawberries could save Eastern growers several hundred dollars per acre in weed-control costs. Nematode-resistant pineapple is being developed at the University of Hawaii to replace 1.4 million pounds of fumigants. Insect-resistant broccoli developed at Cornell University could improve yields in years of heavy insect pressure. Virus resistant raspberries developed by U.S. Department of Agriculture researchers in the Northwest could help combat bushy dwarf virus, which is present in 80% of Northwest plantings. And transgenic apples resistant to fire blight bacteria have been developed and tested at Cornell University; the transgenic varieties would replace the use of antibiotics, which are used to kill the bacteria on 25% of U.S. apple acreage. Emerging pests. Several research programs are focused on biotechnological approaches to control emerging pest problems.

These studies do not automatically provide information about which, if any, environmental variables are responsible for the pattern. One can test whether patterns of differentiation match an environmental gradient, but this is necessarily a post-hoc interpretation . Association studies use a regression approach to identify loci in which genetic variation is associated with variation in trait values or home environment. Such analyses can be carried out at the individual or population level. Genotype-to-environment association studies identify loci that vary along environmental gradients . An association between an SNP and aridity, for example, suggests that the gene or its regulatory region affects performance in wet vs dry environments. This does not reveal how the locus affects phenotype, and careful interpretation is needed as a result of correlation between climatic variables. Genotype-to-phenotype association studies identify loci correlated with a particular phenotype , but the phenotype may or may not be relevant for fitness in the field. Most association studies in conifers to date have used SNPs in a limited number of candidate genes . This ensures that genes suspected of involvement are surveyed, but limits the ability to identify additional loci. However, with the decreasing cost of sequencing, approaches that generate large numbers of SNPs are increasingly being used for genome-wide association studies . One set of approaches, including RAD-seq and geno typing-by-sequencing, flood and drain tray involves the use of restriction enzymes to cut and sequence a small subset of the genome . This can produce tens of thousands of SNPs with high coverage .

Many of these SNPs will be in noncoding regions, which is good for the potential discovery of regulatory regions, but can limit the number of gene associations detected. Another approach involves the creation of a transcriptome or full genome sequence for a species, and the development of probes for all or most of the putative genes to identify SNPs . This approach can also yield useful gene expression data if multiple tissue types or treatments are included in the development of the transcriptome .Most drought gene expression studies in conifers have focused on pine seedlings, with a few investigating other Pinaceae genera . The direction of expression responses to the environment, including dry conditions, is highly conserved between Pinus contorta and Picea glauca 9 engelmannii, even though average expression levels often differ . It is unclear whether this is true across conifer families. No expression studies have focused on adult drought responses. The methods used to induce drought stress vary. Studies have withheld water for a specified period , until soil moisture reached a threshold or needles wilted , or needle water content declined to a certain level . Some have used chemically induced water stress . Caution must therefore be used in interpreting differences across studies, as these could be methodological artifacts . Genes related to signaling and gene transcription are frequently upregulated in drought-stressed seedlings. Changes in signal cascades must precede changes in their targets, and such expression shifts often occur within the first week of drought stress. Those in the ABA pathway are well represented . In addition to being involved instomatal closure, ABA signaling can affect shoot growth and water uptake . However, there are also ABA-independent pathways in most taxa, which may use leaf water potential as a signal .

Upreg ulation of genes in the ethylene pathway could be related to reduced shoot growth or leaf area . Genes related to protective molecules are also frequently upregulated . Late-embryogenesis-abundant proteins, named for their role in seeds, appear to stabilize proteins and membranes and prevent protein aggregation . Dehydrins, a subgroup of LEAs, often protect against drought stress, although some are induced by other abiotic stresses . Heat shock proteins, detoxification enzymes and genes in the synthesis and transport pathways of osmoprotective carbohydrates and proline may also be upregulated. Genes involved in pathogen or biotic stress defenses are often upregulated during drought stress, but those involved in growth, including cell division and wall construction, are often downregulated . Up or down changes in carbohydrate and lipid metabolism and protein handling pathways are also evident, although these are more difficult to interpret. Aquaporins, which affect membrane water permeability, were found to be upregulated in two studies . When drought-stressed seedlings are re-watered, most gene expression quickly returns to normal. In Pinus taeda, only 76 of the 2445 genes with altered expression during drought were still different after 48 h of recovery . Lorenz et al. found 11 genes upregulated in ‘recovered’ P. taeda seedlings relative to either drought-stressed or well-watered seedlings, including probable cell wall proteins, an aquaporin and a gene involved in vacuole function. These may reflect recovery or repair processes that occur once drought stress is removed.Gene families illustrate the complexity of expression responses to drought. Pinus pinaster has at least eight dehydrin genes, based on expressed sequence tag analyses . Three of five were downregulated during drought, whereas the other two were upregulated . Most dehydrin induction occurred after 20 d of drought , which may be why a similar but shorter study did not reveal the upregulation of dehydrins. Expression can also vary by tissue.

Of seven dehydrins examined in P. abies, drought stress upregulated four in needles, but only two in bark, with one being down regulated in bark . To investigate the link between drought and defense gene expression, Fossdal et al. exposed P. abies seedlings to a pathogen , drought stress or both, and examined the transcription of 14 candidate defense genes. Genes were upregulated more slowly in drought-stressed seedlings than in pathogen inoculated seedlings. The combined treatment led to more rapid and/or higher expression of many defense genes than either alone, which may be related to the synergistic mortality risks posed by biotic and abiotic stressors. Pleiotropic effects for some drought/ defense-related genes are also possible, but none have been identified to date.Multiple provenance studies have identified patterns consistent with local adaptation to drought. Trees from drier climates often exhibit conservative growth strategies , such as slower height or needle growth , less above ground biomass or a shorter growing season . Seedlings from dry environments often also exhibit more root growth and higher drought survival . Provenance trials of Pinus halepensis have shown mixed responses, with low growth and high water use efficiency in dry-sourced populations , but high growth in populations from intermediate-aridity areas , which may be related to growth plasticity. Because of the importance of carbon resources for plants, WUE – the ratio of carbon fixed to water lost – has long been considered to be closely tied to drought tolerance . Measures that integrate over longer time periods, such as the carbon isotope ratio d13C ,nft hydroponics are most frequently used to represent changes in WUE in trees. However, although different measures of WUE are often correlated , they are not interchangeable. For example, carbon discrimination is sensitive to chloroplast carbon concentrations and mesophyll conductance, whereas WUE itself is heavily influenced by evaporative demand, which does not directly affect D . In addition, nitrogen fertilization can increase WUE and decrease D, but does not affect gs or transpiration . Thus, WUE and D do not always co-vary, and caution is needed in the interpretation of d13C as a measure of WUE. Additional caution is warranted when using WUE as an indication of drought tolerance. High WUE may not be adaptive in some dry environments if the use of less water per unit carbon fixed does not result in slower depletion of soil water , or if plants with higher WUE grow faster and thus use more total water. Although a few studies have shown higher d13C for populations from dry sites , others have shown the opposite . There was no difference between three populations of Pinus ponderosa seedlings from varying climates in d13C or instantaneous WUE; the drought adapted populations exhibited greater plasticity in water use . In P. halepensis, however, individuals from more mesic sources showed higher plasticity of WUE than those from drier sources , but dry sources may show higher average WUE . Highly plastic growth and water usage reduce apparent WUE over the whole season compared with consistently moderate to low water usage . Instantaneous measures of WUE can change over a day, whereas integrated measures can differ significantly for a source population grown under different conditions or for the same tree across years . Changes in WUE may thus be a useful indication of drought stress, but, in conifers, radial growth and WUE are often weakly or negatively correlated . In pines, higher WUE usually results from reduced gs and/or reduced leaf area , which can limit photosynthesis and growth . Low gs can also result in higher tissue temperatures, which can be damaging, particularly in seedlings . Drought length and severity can influence measures of relative drought tolerance between populations. In P. ponderosa seedlings,the relative growth rate under moist conditions was positively correlated with previously measured tolerance to severe drought, whereas, under 4-wk drought, the intermediate-drought-tolerant population grew faster .

When Silim et al. examined Picea sitchensis, P. glauca and their hybrids, they found that P. sitchensis and the hybrids had the highest WUE and growth in well-watered conditions, but P. glauca and the hybrids had higher WUE and growth in drought conditions. Similarly, the relative transpiration and photosynthetic rates, WUE and growth of P. halepensis tree provenances differed between near-desert and Mediterranean planting sites . Such shifts in ranking are often a result of plasticity differences between populations. Provenances of P. pinaster from across the species’ range did not vary in cavitation resistance, suggesting uniform selection or lack of genetic variation . In P. halepensis, however, the percentage loss of conductivity differed significantly between provenances, but not between environments . Although plasticity has been observed in xylem wall thickening, time to thickening and number of cells in Picea mariana in drought experiments , cell anatomy studies often focus on only one population, so that the extent of local adaptation is unknown.Genome scans have identified loci in conifers that may be under differential selection across environments . Of 13 candidate genes for drought response in P. pinaster, two showed signs of divergent selection, although only one exhibited a pattern correlated with a climatic gradient; three, including two dehydrins, showed evidence of balancing selection . Prunier et al. examined SNPs from 313 candidate genes in P. mariana and found 16 that exhibited differentiation correlated with precipitation, including a LEA protein and genes in the ubiquitin protein handling pathway. However, differentiation between populations can be driven by processes unrelated to climatic gradients. Conifer populations are likely to violate the assumptions of such tests because they rarely form discrete isolated populations and are often far from demographic equilibrium; mis-specification of population hierarchical structure can lead to high false positive rates . However, newer methods are being developed that avoid frequently violated assumptions and reduce false positives . The approach preferred by recent studies is to directly assess the association of loci with environmental gradients , whilst controlling for population structure . Jaramillo Correa et al. examined the correlation of P. pinaster candidate gene SNP allele frequencies with climate principal component axes, using transcriptome-wide SNPs to control for population structure and demographic history. They identified 18 environmentally associated SNPs, many of which were in genes relating to carbohydrate transport, cell wall construction and photosynthesis. Two surveys of P. taeda examined associations between candidate gene SNPs and environmental gradients. One examined the association of these loci with five climatic PC axes , whereas the other used an aridity index for each county . There was some overlap in function between the loci identified . However, the studies disagreed on whether SNPs associated with climate also tended to be Fst outliers. G2E associations have been detected even over short geograph ical distances, suggesting that selection can drive local adaptation in the presence of high gene flow. Eckert et al. examined Pinus lambertiana populations around Lake Tahoe, an area of c. 35 9 65 km2 , and found 11 genes associated with environmental PCs reflecting differences in water availability. These included genes involved in carbohydrate metabolism and transport and response to biotic stress .

Despite high AR exposure levels , both studies reported very low numbers of animals dying primarily from AR exposure. Nevertheless, AR poisoning may significant impact mortality rates in Californian fisher populations , with increasing prevalence from 2007 to 2014. AR contamination is not limited to mammals. It was also documented in northern spotted owl and barred owl populations, likely through secondary poisoning from predation on contaminated rodents . Despite some limitations due to small sample sizes , these studies draw attention to a potential ecological threat posed by illicit cultivation methods. Far less is known about application of chemicals in legal growing operations, which vary greatly by region and country. While some ARs are illegal or heavily restricted in the United States, various other pest-control methods have been reported for cannabis . In the US, due to the crop’s federally illegal status, no commercially available pesticides have been approved for use on cannabis . In Canada, 25 pesticide and fungicide compounds have been approved for legal use on cannabis.Wang, et al. measured biogenic volatile organic compounds emitted by cannabis plants grown under conditions mimicking greenhouse cultivation. Results suggested BVOC emissions from indoor cultivated cannabis in Colorado could contribute to ozone formation and particulate matter pollution. The authors acknowledged limitations due to small sample sizes,4x8ft rolling benches sub-optimal growing conditions, and a focus on only 4 out of 620 reported cannabis strains.

In a follow-up study, Wang, et al. estimated terpene emissions and regional ozone impacts from indoor cannabis cultivation facilities in Colorado using the Comprehensive Air Quality Model. Results predicted increases in hourly ozone concentrations which may have consequences for regional air quality. This approach was limited by reliance on estimates and assumptions in the absence of data regarding emission capacity of most cannabis strains, number of plants and plant biomass. Nevertheless, preliminary findings indicated that concentrated indoor cannabis cultivation could influence ozone pollution through BVOC emissions from terpenes, particularly in areas where nitrogen oxides are not the limiting factors in ozone formation . Surface- and ground-water pollution from the cannabis industry, including from soil erosion, pesticide and fertilizer in run-off, chemical processing or waste disposal operations, is a likely risk . Nevertheless, we found no peer-reviewed study quantifying the impacts of cannabis cultivation on water quality, although current pilot projects in California are underway. We did find an academic book series and five peer-reviewed publications documenting the effects of pollution from cannabis consumption on water quality. These studies used THC-COOH concentrations in sewage systems, presumably originating from human consumption, as a proxy. Evidence of THC-COOH presence was found in both raw and biologically treated wastewater across major European cities as well as in raw wastewater in the US . Concentrations of chemical compounds derived from cannabis were lower in treated than in raw wastewater. Nevertheless, accumulation of these compounds may contribute to waterway contamination downstream from wastewater effluent discharges in urban areas, although likely to a lesser extent than other illicit drugs . While these studies primarily aim to document urban cannabis consumption, they also point towards potential contamination issues impacting downstream freshwater ecosystems.

Our current understanding of the consequences of wildlife exposure to cannabis-related chemicals remains limited. Parolini, et al. sought to bridge this gap through experimental exposure of zebra mussels to concentrations of cannabis active compounds Δ-9-THC and THC COOH. Results showed that prolonged exposure could contribute to oxidative and genetic damage in the mussels. Still, given the lack of knowledge regarding actual Δ-9-THC and THC COOH concentrations in aquatic ecosystems, and the lack of documentation of the compounds’ effects on mussels or other organisms in the wild, it is difficult to draw broader conclusions about potential environmental risks posed by exposure to active compounds in cannabis for aquatic organisms.Because there are environmental trade-offs across production methods, it is important for policy makers to consider the potential unintended consequences of policy decisions. For example, in California, stringent water-use regulations for outdoor production may incentivize cultivators to turn to alternative indoor production methods. While this shift may alleviate water-stress in sensitive ecosystems, it may also increase the carbon footprint of cannabis by encouraging energy-intensive indoor production. Identifying and understanding trade-offs within and across systems is thus important, and cannabis regulation should be comprehensive in order to prevent impacts from being displaced from one pathway to another. The emerging literature on cannabis and the environment already provides useful insights to guide policy. Still, the majority of studies reviewed here were individual case studies, mostly geographically centered in Northern California. There is a tremendous need for similar studies to be carried out across different biophysical, socioeconomic, historical and cultural contexts, both to confirm the generalizability of these results and to avoid exporting environmental problems from the developed to the developing world. We expect that continued liberalization worldwide will provide expanded geographic scope for this work for years to come, and researchers should be ready to act on this expansion.

Most of the literature reviewed here relies on observational or model-based methodologies . While these approaches provide insights, experimentation is fundamentally needed to understand basic agroecological functions and processes governing cannabis cultivation. Trials quantifying the energy footprints, water use, and nutrient requirements of different cultivation and management methods are also needed to improve the efficiency of production systems. Given increased liberalization trends, we expect to see a normalization of cannabis-related research. Scientists should be encouraged to carry out a range of experiments to bolster scientific capacity to assess the environmental impacts of an expanding cannabis sector. Additionally, as regulations around cannabis cultivation are implemented, long-term studies are needed to understand how these regulations affect cannabis cultivation practices. Cannabis cultivation may lead to additional environmental impacts, which remain scientifically undocumented to our knowledge. For instance, solid waste management of materials originating from cultivation, packaging, or other production processes, will need to be addressed. Life-cycle assessments of the cannabis sector could provide information on how to minimize such waste and more generally increase the efficiency and sustainability of cannabis production processes. Other potential areas for future research include odor pollution risks in communities where increased cannabis production has led to farms being sited near residential areas, cross pollination issues between cannabis and hemp , alternative cannabis farming or transportation efficiency. These topics, and many others, should make the study of cannabis’ environmental impacts a rich field for discovery for many years to come. Traditionally, cannabis has been cultivated remotely and at small scales. Legalization is altering this through cultivation expansion, shifts toward urban areas, and increased size of production facilities , which may in turn affect the environmental impacts of the industry. The intensification of cultivation activities at large-scale facilities may magnify negative impacts. Conversely,flood and drain table economies of scale may increase the efficiency of larger facilities which may have broader capacities to invest in sustainable production processes. Larger facilities are also less likely to be located in remote sensitive areas than historical smaller farms, but may lead to land use trade-offs with other forms of agriculture. Continued diligence by policy makers and consumers is needed to ensure that the move towards industrialization is not a move away from sustainability – and researchers must continue to document shifts in the industry and their environmental impacts. In conjunction with legalization, social and ecological certification schemes could increase environmental performance of the industry. Emerging programs such as Sun and Earth Certification or planned appellation designations in California constitute first steps in this direction. By contributing to consumer awareness and providing incentives for growers to produce in sustainable ways, these programs may pave the way for the development of a more sustainable cannabis sector. In many ways, the question of how to best produce and consume cannabis while protecting the environment echoes larger debates about the environmental impacts of agricultural production in general. Current discourse on the optimal ways to address shifts in the cannabis sector touches upon fundamental sustainability framings such as land sparing vs. land sharing, intensification vs. expansion, technology-driven agriculture vs. agroecology, the role of smallholder farmers vs. industrial-scale facilities. Policy makers working with cannabis have strong interests in developing effective regulations following legalization and are also dealing with regulatory “blank slates”. This may equip them with a novel combination of increased freedom and institutional capacity to test and evaluate the effectiveness of multiple policy approaches. Ultimately, failures and successes of environmental regulations for cannabis may lead to important lessons-learned for agriculture more broadly. This study represents original research on the manufacture of plant-made biologics and plant-made industrial products through application of analytical modeling tools in silico. The main goal of this study was to evaluate unit operations in two plant-made bio-manufacturing processes and estimate the cost of goods of the active ingredient and the impact of those costs on the cost of the final product.

A secondary but equally important goal was to compare the manufacturing cost of plant-produced AI to the cost of the same AI manufactured by predecessor technologies. Much progress has been made towards the development of manufacturing infrastructure for plant-made pharmaceuticals , which typically consist of recombinant proteins applied as vaccine antigens, therapeutic enzymes, or monoclonal antibodies. Progress has also been made in the manufacture of plant-based biologics, biochemicals, and bio-materials for industry, food, and other applications. Significant and industrially relevant advances in gene expression and bio-processing methods have been achieved during the past two decades, as reviewed in several prior studies. Yet, to date, only three PMP products have been approved by regulatory agencies for commercial sale, including an anti-caries antibody , an animal health vaccine , and a therapeutic enzyme to manage a metabolic disorder]. This relative scarcity of PMP products reflects the magnitude of the challenges in creating a new manufacturing industry. The development of the plant-based platform has slowly progressed through a multinational “labor of love” in the absence of the levels of investment originally made by the bio-pharmaceutical industry , which resulted in elevation of fermentation-based systems to their current level of dominance.Interestingly, beginning in 2009, the US Defense Advanced Research Projects Agency’s Blue Angel program made several multi-million dollar investments at various sites with the goals of accelerating the scale up of the PMP infrastructure and assessing production of relevant volumes of pandemic influenza candidate antigens as a model product to test the plant-based platform . This was a shared investment initiative, and as a result of federal and state government and private investments, the expanded PMB manufacturing capacity should now support production of at least several of the many plant-made vaccines, bio-therapeutics, bio-materials, and bio-catalysts that are under development by companies and institutions worldwide . Although capacity expansion helped companies that would manufacture their own or partnered products , these investments also helped expand capacity at PMP contract development and manufacturing organizations such as Kentucky Bio-Processing . This was important to our modeling because the decision to construct a new dedicated manufacturing facility versus contracting services from a CDMO could yield very different cost-of-goods projections. Fundamental to the commercial introduction of PMB products is the availability of an efficient plant-based manufacturing infrastructure that is at a minimum competitive with and ideally superior to traditional animal cell and microbial fermentation systems as well as to extraction from raw materials from natural sources. The cost to manufacture any product is of paramount importance to its market acceptability, availability to those who need it most, and to the profitability of the product for its manufacturer. While plant based technologies are often assumed to offer significant cost advantages relative to cell-based fermentation, such assumptions are based on the lower upstream capital investments required for plant growth, lower cost of media, no adventitious agent removal, and other factors. However, few of these studies have listed engineering process assumptions or analyzed unit operations adequately; reports such as those of Evangelista et al. and Nandi et al. are exceptions. Therefore, results of recent technoeconomic evaluations for PMP/PMB/PMIP have not been widely available in the public literature. To analyze and quantify the cost efficiency of plant based manufacturing, we chose two enzymes representing active ingredients for diverse product classes and derived for each AI the bulk product and per dose or per-unit costs.

DIM BOA-Glc levels on the other hand may not be significantly altered as previously shown for ABA treated plants which is consistent with our results. Fv is highly resistant to MBOA and has the ability to detoxify the compound by actively metabolizing it into N- malonamic acid which is nontoxic. Consistent with Fv detoxification of MBOA, the concentration of MBOA tended to inversely track pathogen bio mass. However, this did not hold true at 2x[CO2] between +H2O and -H2O plants which would suggest additional interactive effects of 2x[CO2] and -H2O on benzoxazinoids metabolism. Further research investigating the effects of multiple climate change factors on maize benzoxazinoids and their derivatives in interaction with a pathogen more sensitive to these defense metabolites will be necessary to fully understand the potential implications of the abiotic stress induced changes in maize benzoxazinoid dominated defenses. Although the fumonisin contamination was significantly higher in infected stems under simultaneous conditions of 2x[CO2] and -H2O, the amount of fumonisin per unit Fv biomass was reduced compared to +Fv maize at 1x[CO2]. These data are consistent with previous results indicating that elevated [CO2] compromises the transcriptional response of many of the 9- and 13-lipoxygenase and their signaling products, which have the potential to stimulate mycotoxin production. Even though the transcript levels of LOX genes were not measured in this study, the metabolite analysis supports this notion. The addition of drought did not ameliorate the effects of elevated [CO2] on the influx of fatty acid substrate needed for oxylipin biosynthesis, nor did it negate the dampened accumulation of the 13-LOX oxylipin JA following Fv infection . Nevertheless, even though host-derived mycotoxin stimu lants are potentially still reduced,stackable planters the even larger amount of Fv biomass on maize at 2x[CO2]- H2O was ample to lead to greater fumonisin levels and could therefore be an agriculturally relevant food safety concern.

Considering that the drought treatment imposed in these experiments was specifically designed to account for the physiological changes in water utilization at 2x[CO2], it is possible that, in comparison to a more moderate drought stress treatment at 1x[CO2], the amount of fumonisin contamination would be higher instead of lower. Numerous reports indicate that drought enhances maize susceptibility to Fv and fumonisin; however, in these experiments at 1x[CO2]-H2O plants displayed both less Fv biomass and fumonisin compared to irrigated plants. Since maize utilizes more water at 1x[CO2] and the degree of drought stress was higher , the percentage of water in the stem tissues was significantly less . Therefore, it is likely that this lower water activity was not conducive to fungal growth and reduced Fv biomass and fumonisin production. While normalizing for soil moisture content would provide additional insight into the effects of [CO2] at variable levels of drought, this is beyond the scope of the current manuscript. The difference in soil water content was a consequence of the plant’s physiological response to elevated [CO2] which was a factor being studied and was thus intentionally not controlled in these experiments. Elevated [CO2] has the potential to ameliorate the severity of drought; therefore, it is essential to understand how these abiotic factors will interact and influence Fv infection in comparison to conditions which will not receive this same benefit of water conservation. Timing of infection will likely also play an important role in fumonisin contamination levels as during episodes of drought maize at 2x [CO2] may also provide a more favorable environment for pathogen growth, allowing for a prolonged period of mycotoxin accumulation. Furthermore, while the chamber based studies provide valuable data in understanding the defense response under controlled conditions of biotic stress, there are multiple limitations including light intensity, breadth of spectral wave length, and hindered root establishment. These factors likely contribute to the abiotic stress imposed and influence resource availability and distribution.

Additional laboratory studies coupled with field based free air gas concentration enrichment experiments are needed to determine the trade offs between the photosynthetic advantage of water conservation and increased susceptibility on maize grain productivity during simultaneous conditions of elevated [CO2] and drought. Nonetheless, given the heightened climate change concerns and the potential consequences of our uncertainties for future agricultural maize production, our findings have provided a foundation for additional research necessary for the development of climate resilient mycotoxin control strategies.Horticultural crops provide 60 percent of total farm revenue in California agriculture, and Cali fornia provides 37 percent of the horticultural crop value in the United States. Clearly, these industries comprise an important part of the agricultural economy. This study provides a detailed statistical profile of California’s horticultural crop industries at the farm level, based on a survey of specialty crop growers that was conducted during the spring of 2002. The Risk Management Agency of the United States Department of Agriculture supported the re search, and the California Office of the National Agricultural Statistics Service helped conduct the survey. Specialty crops, also referred to as horticultural crops, include tree and vine crops, vegetables, and ornamental crops. The statistical profile of California’s horticultural farm industries presented here is the most comprehensive ever undertaken for these industries, drawing on survey data collected from approximately one-third of all horticultural crop producers in the state. Specialty crops are diverse. They differ in their product characteristics, production processes, and market environments. Such heterogeneity extends to risk characteristics of the crops and to the ways farmers cope with various risks. As a preliminary step to development of effective risk management tools, it is important to better understand factors that affect these risks. This report is intended to provide such information to help us understand specialty crop industries, the sources of risk, and behavioral risk responses in these industries.

The following summary of results is organized by topic.About 86 percent of the farms surveyed produced primarily orchard and vine crops, 5 percent produced vegetable crops, and 9 percent produced ornamental crops. About 25 percent of the farms were located in coastal areas, 13 percent in the Sacramento Valley, and 47 percent in the San Joaquin Valley. The remaining 15 percent were in the northern mountain areas, the Sierra Nevada, the Southern coast, and the deserts. Average farm size was 203 acres,stacking pots but the median farm comprised only 34 acres. There were relatively few very large farms and many very small farms. The average number of acres per farm varied substantially among the three crop categories: fruits/nuts, vegetables, and ornamental crops. The average land holding by vegetable growers, 1,106 acres, far exceeded the average of 157 acres for fruits/nuts and 200 acres for ornamental crops. These land figures include land planted to secondary crops . When we examined land planted only in primary crops, our data showed that fruit/nut and vegetable farmers held, on average, about 50 percent of their land in primary crops . However, land for ornamental crops, on average, accounted for only 10 percent of the average 200 acres per farm.Crop diversification has long been recognized as an important risk management tool. Our data showed that crop diversification was much less common for orchard farms than for vegetable farms. About 70 percent of fruit/nut farmers were single-crop growers as opposed to 26 per cent for vegetable farms. The scope of diversification also differed. Fruit/nut farmers predominantly diversified their crops with other varieties of fruits and nuts; only 20 per cent of them used crops other than fruits and nuts for diversification. Vegetable farmers, on the other hand, frequently used other crops for diversification; only one-third of the vegetable farms were diversified among only vegetable crops. Our survey also indicated that primary crop acreage increases with crop diversification for both fruit/ nut and vegetable crops. Farms growing five or more vegetables were, on average, four times larger in vegetable acreage than farms growing a single vegetable crop. In California, 6 percent of specialty crop farmers had some organic or transitional-organic land. In terms of crop category, these farms represented 6 percent of orchard farms, 14 percent of vegetable farms, and 4 percent of ornamental crop farms. Our data showed that these farms also engaged in conventional farming and that they devoted, on average, about one-third of their primary crop lands to organic farming. Judging from acreage assigned to primary crops, the farms were about average in acreage for fruit/nut farms but much smaller than average conventional vegetable farms.Marketing is an important component of risk management. Marketing channels vary by product use . Processing crops are delivered in bulk directly to processing plants, whereas fresh-use crops are sent to operations to be sorted, packaged, cooled , and distributed through marketing channels. California producers were highly specialized in terms of use. Most fruit/nut farms produced mainly for processing use and most vegetable farms produced mainly for fresh use . Only 7 per cent of specialty crop farmers supplied both processing and fresh market outlets.

In processed-use markets, contracts played a major role with contracts with a predetermined price being the most prevalent form. In fresh-use markets, grower/shippers, which combine the packing/shipping business with field production under one ownership, provide a form of vertically integrated business. Our survey showed that grower/shippers accounted for 13 percent of vegetable farmers and 3 percent of orchard farmers and that they mainly supply mass merchandisers . The other fresh-market growers tended to use diverse marketing channels, including selling directly to consumers, marketing through cooperatives and independent shippers, and selling directly to commercial buyers. For fresh vegetable markets, “directly to consumers” was the most commonly used outlet , not by volume of production but by number of farms using this marketing channel.We investigated year-to-year yield variations using yield information for the preceding five years. Taking the average of the five annual yields as an individual’s normal yield, we calculated the percent deviation from the normal yield and then arrived at sample mean deviations for sample categories. Our data indicated that annual yield deviated, on average, 15 percent for fruits/nuts and 8 percent for vegetables over the previous five years. For price and profit fluctuations, we elicited information on the range of the highest fluctuation experienced over the same five year period . For both price and profit, the median of the accumulated distribution fell in the 25–49 percent range for fruits/nuts and the 10–24 percent range for vegetables, indicating that prices as well as profits tend to fluctuate less for vegetables than for fruits/nuts. In response to a list of options as the main cause for the lowest profit, “poor yield,” “low market price due to high domestic production,” and “low market price due to imports” were the three most often cited causes for all crops except ornamentals. They accounted for 70 per cent of the responses for fruit/nut and vegetable farmers. For fruit/nut crops, poor yield was the most cited reason for the lowest profit , but for vegetables, low market price due to high production was cited most , followed by low market price due to imports . This underscores the relative importance of production risks for orchard crops and of market risks for vegetable crops.Two sources of risk, adverse temperature and output price fluctuation, were listed as most important; input price fluctuation, pests, and disease were considered to be moderately important. Crop insurance was a preferred risk management tool for orchard and vineyard farmers, and crop diversification was preferred by vegetable and ornamental crop growers. Diversified marketing was reported to be the second most preferred tool for all three crop categories.As expected, their preferences were closely linked to availability. The most available tools were crop insurance for orchard crops and crop diversification for vegetables and ornamental crops . Orchard and vineyard farmers reported relatively limited availability of other risk management tools.About 53 percent of fruit/nut farmers, 31 percent of vegetable farmers, and 13 percent of ornamental crop farmers said they had purchased crop insurance in the preceding five years and most of those farmers had purchased it for all five years Single-peril insurance is mostly offered by private firms, most commonly for damage from frost, rain, and hail. This insurance was purchased by about 20 percent of fruit/nut farmers and about 10 percent of vegetable farmers.

A constellation of bird cages suspended like lanterns house paper canaries, possibly metaphoric bellwethers for the fortunes of a site, that was after all, was once a mine.Situated 1 km away as the crow flies, but considerably further around the lake shore on foot, the University Gardens comprised projects by nine design schools.Whereas the garden plots allotted to each Master’s Garden were relatively level and orthogonal with a thick bamboo buffer, the University Garden sites exhibited a quite different set of conditions. Set on the banks of one of the Expo’s artificial lakes, half of the garden sites occupy direct water frontage, while those further up-slope benefit from the enhanced overview that comes with elevation. With a greater divergence of lot sizes ranging between 7 500 and 13 000 sq ft, the typically elongated and irregular form of the sites increased the perimeter-to-area ratio of each garden when compared to the more symmetrical Masters’ Garden plots. Furthermore, unlike the Masters’ Gardens, no bamboo buffer was predetermined, so that the individual garden plots were by default directly adjacent and entirely open to neighboring allotments and the surrounding landscape, further amplifying the effect of interface rather than the introversion associated with the Masters’ sites . In the descriptions that follow, I roughly corral the nine University Gardens into four categories; sensory, labyrinthine, representation,blueberry planter and process. As per the masters gardens, I use this draft rubric to explore two University Gardens in detail, two at a more cursory level, and the remaining five in passing.

Three gardens effectively elevate the non-visual senses, so famously repressed as unreliable and deviant under the rationalist hegemony of the all conquering eye of modernity. Employing a multifaceted indulgence of the olfactory senses, the Scent Garden by the University of Toronto is the most legible example . A grove of conifers provides the base-scent, amongst which a survey grid of perforated poles use wind-generated turbines to dispense accent aromas. Finally, a crystal pavilion displays bottled fragrances in the round, acting as a kind of scent-bank for posterity. The Sky Garden by the University of Southern California aims to amplify the sense of touch; not in terms of the haptics of rough and smooth texture, but as a membrane through which the interoceptive senses ascertain temperature . The garden generates extreme microclimates with two mechanical contraptions; at one end a half sphere of adjustable reflective plates creates a solarium effect, while at the other, a complex three-dimensional matrix of overhead wires houses an array of mist emitters forming an artificial cloud. By destabilizing the very ground that we move on, The Net garden by Peking University amplifies the internal feedback mechanism which tracks the relative position of parts of the body to the top of the sensory hierarchy . A field of multi-canted planes clad in flexible expanded mesh of various gauges destabilizes movement, forcing the visitor to recalibrate the habits of bodily calibration and orientation. Despite massively over-engineered safety balustrades that were installed against the designer’s wishes, the garden invokes Shusaku Arakawa and Madeline Gins’ early fractalized Perceptual Landing Sites, where ‘forcing the body off balance forces it to show itself for whom or what it is’.To experience moving over this alien scape requires an investment of effort; as Phillip Ball notes, ‘journeys in fractal land are arduous’, they are ‘noisy and unpredictable’.For Arakawa and Gins, through the act of negotiating the many inclines and declines of the fractalized surface, the perceiver ‘…switches off automatic and onto alert; she realizes that she must, from now on, anticipate the consequences of her every move’.

Over this ‘difficult ground’,visitors become so preoccupied with the immediacies of proprioceptive action that they neglect to maintain sight of the larger picture, leaving themselves vulnerable to disorientation. When moving in such a tactical manner, the distant goal-oriented nature of vision is used less for direction-finding, although the eyes still have a role to play, albeit in a revised capacity.Close vision is body-based in the sense that when it judges distances and textures, it does so not to control or indulge a scene, but to guide the immediacies of movement. Evaluating bodily potential to move between or make contact with a succession of objects, vision effectively becomes a haptic sense; much in the way that bats use their ears to see, the eyes are no longer a device for seeing, but for feeling. In this mode of operation, distant landmarks and sightlines go unnoticed, leaving navigational duties to the habitual nature of proprioception, which is only able to keep the body oriented in the short term. Like the gyroscopes used to track dead-reckoning vectors in ships and airplanes, the error compounded from registering many body-referenced direction changes provides an unreliable account of one’s passage. Indeed, when the visitor steps off The Net and back onto dry land, there is a moment of re-acclimatizing sea legs where we discover that we cannot readily reconcile our point of entry with our exit. Soon—as we look up from our feet and recalibrate distant vision—we re-establish orientation, but while the disjunction lasts, we are, in the words of Arakawa and Gins, ‘more body and less person’. As the only labyrinthine exhibit amongst the University Gardens, the Garden of the Forking Paths by U.C. Berkeley plays on the notion of choice without lucid outcomes . Framed on two sides by a bamboo frame, the garden is entered through a single aperture at the highest corner of the site. Having crossed this threshold,nft channel a critical scene confronts the visitor: the path bifurcates repeatedly, so that one way becomes many, fanning out over the convex landform that runs down to the lake. At eye height, small trees partially obfuscate the view ahead, making the relative value of each fork unclear.

Either side of the path, two flush steel channels are fed with water upwelling from a single source; each time the path bifurcates, the water runnels are also split in concert. At each fork, the visitor must make a choice and then again and again. Further, as the way becomes clearer, paths begin to topographically separate on the vertical axis. Some runnels also separate from pathways, holding level as the path falls away with the lie of the land. One path becomes thirty—some resolving seamlessly at the lake level, others requiring steps to make the transition. At the far corner of the garden, a bonsai tree balances on an elevated but unreachable plane that meets eye level as one descends the adjacent steps. In the collective Chinese imagination, rivers flow from west to east, but the Chan-Ba River, upon whose floodplain the garden is situated, flows in reverse—from east to west . Referencing this site-specific hydrological myth, the garden concept reverses the automated tendency of water to converge, establishing in its place a system of divergent flows of both people and hydrology. Read metaphorically, the bifurcating flows question a worldview in which history converges to form a meta-narrative. Within this familiar order, the tributaries of history, like water, progress downstream converging inextricably towards a single cogent outcome; ‘we say that time flows’, notes Bernard Cache, ‘but we also place ourselves in landscape where … we are already funneling it into a gullet’.Inverting this pattern creates multiplicity rather than resolution; the notion of parallel worlds or stories rather than singular histories. It implies a type of labyrinth with a single entrance and many exits, where each egress is slightly different, invoking perhaps the Borgesian short story in which the Garden of Forking Paths becomes to be understood as ‘an enormous riddle, or parable, …a growing, dizzying net of divergent and parallel times’.But unlike the matrix labyrinth and the single-path labyrinth , the forking labyrinth is never clearly resolved with a critical revelation or a return to the beginning. In the case of the Garden of the Forking Paths, being delivered down to the lake edge is evasive but is reward enough.Unlike the Masters’ Gardens, which focused on the representation of mythical landscapes and nation-states, the two representationally oriented University Gardens attempted the translation of the designers’ home ‘range’ into hilltop garden plots. In the most extreme example, the Pampa Traces garden by Universidad Torcuato de Tella seeks to literally translate an iconic Argentinean landscape to the other side of the world . The Wind Poem garden by University of Hong Kong takes a contrasting approach, viewing the garden as an opposite foil to the restless 24hr lifecycle of their city; the world’s densest.By seeking to embody and amplify dynamic ecological processes, this category included the most polemic garden proposals of the university collection. With waterfront locations, two of the gardens make use of the potentially dynamic interstitial edge of the lake. Seeking scaffoldings for secessional ecologies, the Eco-Plane garden by Columbia University uses a sliding deck while the Thickened Waterfront garden by the Architectural Association employs an enfolded landform of miniature ecotones and peninsulas. Eco-Time garden by Feng Chia University takes a more cybernetic approach wiring up green columns that are designed to dematerialize under the future cloak of verdancy.

To be sure, while processualconcepts are integral to landscape design theory and praxis, it is difficult terrain in a garden expo, given the short window for ‘ecological emergence’ and the singular nature of most visitations; just as geological time is invisible to us in the landscape, so too emergent time is invisible to the Expo visitor.When explicitly interpreted by a designer or commentator, individual garden references become explained. But without such guidance, what do the general public make of these cryptic projects that are so different from the other transparent thematics on offer at the Expo? In this context, is it, as Jane Gillette postulates, ‘very difficult for the garden designer to express complex ideas using only the garden’, and even more ‘difficult for an audience to ‘read’ them’?In this regard, I observe two meta-approaches within the gardens under discussion. Regarding the first, many of the gardens in focus can be defined as theme gardens under Marc Treib’s definition of a theme as ‘perceptually apparent idea’ that has been applied ‘to fashion the garden’s form’. Treib concedes that an ‘obvious concept’ does not necessarily imply significance, but does nevertheless carry a certain ‘underlying assertion of validity’.For example, the labyrinthine-type gardens traded in the stability of a universally accepted theme with which to ground this semiotic transferal between designer and audience. Once the visitor accepts the terms-of-reference that typically come attached to a labyrinth, they appear more open to accepting the garden as a ‘game-board’ and indulging in its idiosyncrasies. Meaning is constructed in a closed/open exchange; while the designer establishes a scaffolding of meaning, the audience seeks to unwittingly deconstruct this edifice by flooding the garden like water or like ants, investigating every interstitial nook for holes and gaps and in the process evolving the dynamic significance of the garden. Here, in answer to Gillette, complex ideas are expressed through the garden, but most importantly they are also received. The great risk associated with themes is their potential for wearing out through overuse, and indeed the labyrinth—although handled with inventive dexterity by those designers who employed it at the Expo—treads this fine line between novelty and cliché. The second meta-approach encompasses garden designs that do not fall so readily under an obviously identifiable concept. To appropriate Treib’s usage of the term, I identify these approaches under the rubric of ‘zeitgeist; they seek to substitute stable but potentially exhausted garden themes with inventions that attempt to capture the essence of a contemporary cultural preoccupation. The ‘processual’ type gardens that I identified as characterizing a number of University Gardens—and to a lesser extent some of the ‘representational’ gardens—fall under this umbrella. Each attempted to build significance around fluid concepts of ecology and process, ideas which are by no means new, but are yet to establish agreed safety lines of communication between author and audience. The result was that zeitgeist gardens had no fallback position and tended to rely on their own self-referential narrative. In these instances semiotic transferal—whether intended or fabricated—was demonstrably absent on the ground, and the limitations of the garden as a conveyor of complex syntax was exposed.

Manipulations to increase soil arsenic availability, fern biomass, and fern arsenic uptake through nutrient application and mycorrhizal fungi inoculation have been investigated to increase phytoextraction rates. In particular, complex relationships between arsenic and phosphorus availability in soil and uptake in P. vittata vis-à-vis fern nutrient deserve fur ther elucidation, including the effects of soil phosphorus relative to supple mental phosphorus . According to the phosphorus starvation theory , arsenic uptake in P. vittata could be a byproduct of nutrient acquisition, especially iron and phosphorus. Phosphate and arsenate are chemical analogues found in soils associated with iron oxides and can be released in the rhizosphere through similar bio-geochemical processes . Furthermore, P. vittata associates with mycorrhizal fungi including Funneliformis spp. . Like hyper accumulators, mycorrhizal fungi evolved under phosphorus starvation conditions, can support plants under nutrient deficiency, drought, and metal stress, and have stress tolerance mechanisms that differ from and are possibly complementary to those of hyper accumulators . Inoculation with mycorrhizal fungi could increase P. vittata biomass , nutrient, water, and possibly arsenic access , with species adapted to local soils important in addition to generalists . Moreover, soil clay content affects nutrient and arsenic availability . Clay-sized particles, the smallest of soil particles, include mineral phases such as iron oxides, aluminum oxides,nft hydroponic and silicate clay minerals that provide a large surface area for adsorption of arsenic, phosphorus, and other nutrients .

Arsenic has been shown to be more plant available and more leachable in soil with lower clay content. Few arsenic phytoextraction studies have investigated arsenic leaching during P. vittata growth or computed arsenic budgets. After P. vittata growth, similar or lower levels of arsenic in leachate were found compared to non-phytoextracted soils. In long-term field studies, greater arsenic depletion has been observed for soil below the fern root zone, compared to surface depths , which could indicate leaching of arse nic below the root zone. It is important to quantify arsenic cycling in the P. vittata-soil-water system during growth, because P. vittata growth processes might lead to spatiotemporally heterogeneous arsenic leaching. Unsaturated flow-through soil column studies with plants are a powerful setup to quantify arsenic cycling in controlled whole plant-soil-water systems that approximate natural systems. With such systems, water balances including transpiration can be calculated, and arsenic transport in soil pore water can be quantified . Combining such soil columns studies with X-ray absorption near edge structure spectroscopy and X-ray fluorescence imaging at the micron scale allows us to investigate rhizosphere processes and relate rhizosphere processes to arsenic cycling at the system scale. We conducted a 22-week soil column study with moderately contaminated soil planted with P. vittata to determine the effects of soil texture , phosphorus application, and fungi inoculation on arsenic cycling in the plant-soil-water system. We used synchrotron-based spectromicroscopy to determine arsenic speciation in bulk and rhizosphere soils and propose mechanisms of arsenic mobilization for plant uptake and/or leaching. We observed greater arsenic uptake and leaching with smaller fern biomass in soil with lower clay content across all treatments, suggesting significant plant growth trade-offs associated with arsenic hyper accumulation and phytoextraction.

Two soils moderately contaminated with arsenic and with contrasting textures were excavated from the 0–30 cm depth in a former railroad right-of-way . Soils were sieved and stored field-moist at 5 °C in sealed containers under oxic conditions. Soil pH was measured on triplicate samples and cation exchange capacity and percent organic matter were measured on duplicate samples as described previously . Bulk density was measured in triplicate on intact cores of a known volume from which the volume of the fraction >2 mm was subtracted, following a modified protocol for rocky soils . Porosity was calculated from bulk density . Soil texture was measured in triplicate with the hydrometer method . In both soils, clay minerals identified with X-ray diffraction following USGS protocols included nontronite, trioctahedral montmorillonite, and/or vermiculite.Experiments with novel unsaturated flow-through soil columns with plants were performed in triplicate to estimate arsenic uptake by P. vittata, arsenic desorption and transport in soil, and arsenic leaching. We compared control columns without amendments with columns fertilized with phosphorus, and columns inoculated with the fungus F. mosseae . Phosphorus was mixed into soil before packing columns. Fungal inoculant was applied to each hole at transplanting. Column experiments without plants were also performed, with one replicate per soil per treatment for control and phosphorus treatments. However, because fungal inoculant was not expected to survive in the absence of ferns, unplanted column experiments with fungal inoculation were not performed. For the remainder of the manuscript, any mention of columns in text refers to planted columns, unless specifically noted. Soil columns were made of acrylic tubes . At the bottom of each column a 0.2 μm filter and glass fiber filter separated soil from the effluent port, which was filled with quartz sand with glass wool on either end. To ensure reproducibility between columns, they were all packed similarly at bulk density representative of field conditions .

Soil was well-mixed be fore weighing into 20 increments of equal mass appropriate for packing into a 2 cm depth. Soil was packed to a total depth of 40 cm using a custom-made 3-pronged device to apply downward force over a small surface area to avoid formation of layers. P. vittata ferns with bare roots and 3–7 fronds 10–20 cm in length were planted with roots 0–10 cm deep in columns. Pore water samplers and tensiometers were filled with degassed synthetic rain and inserted horizontally at depth with the tip 7.5 cm inside planted columns.A synthetic rain solution that served as column influent was made following rain composition of the field site adjusted to current rainfall pH . Globally, rainfall pH ranges from 3.5 to 8.0 so pH 5.33 was considered widely applicable. The synthetic rain was kept in the dark during experiments. The influent solution was supplied by a single port at the rhizome, and was eluted with a peristaltic pump through planted columns for 22 weeks at a constant flow rate equivalent to maximum daily rainfall . The flow rate was chosen to approximate conditions encountered in the field, made constant to simplify experiment design, and is representative of high rainfall climates and irrigated environments. A conservative bromide tracer was eluted through unplanted control columns for half a pore volume and the synthetic rain was eluted for 2 pore volumes at the same flow rate as used in the planted columns. The column experiments were conducted in a greenhouse with a 16-h photoperiod.Pore water samples were extracted from planted columns approximately every 2 weeks using acid-washed syringes maintained at 4 °C during collection. Pore water samples were immediately filtered under anoxic conditions, sub-sampled for analyses,blueberry pot size and stored at −18 °C in the dark until analysis. Planted column effluent was collected weekly into polyethylene bottles pre-acidified with concentrated HNO3 . After collection, effluent was adjusted to 2% HNO3, filtered , and stored for analysis. Unplanted column effluent was col lected every 228 min using a fraction collector. After collection, samples for bromide analysis were reserved and samples for arsenic analysis were combined on a weekly basis and treated the same as the planted column effluent. Pinnae samples were collected at 11 and 21 weeks. At the end of the experiments, all above ground biomass was removed 2 cm above the rhizome and separated based on development of sori and tissue senescence .

Each soil column was sliced at designated depths . The entire rhizome was removed, though not the entire root mass. All root and soil samples were immediately stored at 4 °C.Above ground biomass was washed 3 times in deionized water to remove soil particles and dust . Rhizomes and root samples for elemental analysis were washed in deionized water until clean of soil. Fronds, rhizomes, and root samples for elemental analysis were dried for 24 h or until constant mass was achieved. Whole root samples for microprobe and DNA sequencing were flash-frozen in liquid nitrogen within 6 h of harvesting and stored at −80 °C until analyses. Additional root samples were dried, processed, and maintained under anoxic conditions for X-ray absorption spectroscopy . Soil samples were either air-dried for elemental analysis or dried, processed, and maintained under anoxic conditions for XAS.Dry plant biomass was measured on pinnae, frond, and rhizome samples. Pinnae sample biomass served as a proxy for whole plant biomass during growth. Plant tissue and soil samples were digested following a modified EPA 3050B protocol described previously . Total arsenic, phos phorus, and iron concentrations of soil and fern digests were determined using inductively coupled plasma optical emission spectroscopy . The detection limit was 20 μg/L for each analyte. Extractable soil nutrient concentrations were measured as described previously . Total arsenic concentrations in effluent and pore water samples were analyzed using either ICP-OES or, if concentrations were less than 50 μg/L, hydride generation-ICP-OES after addition of 0.8 mL of 40% KI/8% ascorbic acid to 4 mL sample and 2.7 mL 1.1 M HCl . In pore water, arsenic concentrations were determined using HG-ICP-OES in samples buffered at pH 5.0 through addition of 2.5 mL 0.5 M disodium citrate to 2.5 mL sample, dissolved organic carbon concentrations were analyzed using an O-I-Analytical analyzer, and pH was determined using a Denver Instruments meter with pH/ATC Sartorius ATC combination electrode. Bromide concentrations were measured using a Dionex Ion Chromatograph with a detection limit of 2.4 mg/L. Samples of roots with rhizospheric soil, of soil aggregates, and of bulk soil were prepared for XAS, and measurements were collected as described pre viously . Briefly, whole roots and root or aggregate thin sections were mounted onto a Peltier cooling stage for X-ray micro probe analysis, X-ray flfluorescence mapping and X-ray absorption near-edge structure , at the Advanced Light Source XFM beamline 10.3.2 , Lawrence Berkeley National Laboratory . Bulk X-ray XANES spectra were collected at Stanford Syn chrotron Radiation Laboratory beamline 7.3 on bulk powdered samples mounted on filters. Least-square linear combination fitting was performed with custom LabVIEW software and XAS databases of iron and arsenic compounds available at beamline 10.3.2. Details of the spectromicroscopy measurements are available in the Supplemental Information.Transport and desorption of total arsenic within the soil columns was modeled using a solute transfer model through unsaturated soil amended with a retardation factor to account for the desorption of arsenic from soil. In unsaturated soils, the transport of solutes occurs in two phases: a mobile phase where water flows through the porous medium and an immobile phase in which water is stagnant within dead-end pores or between soil particles. Briefly, bromide tracer breakthrough curves collected from unplanted columns were used to constrain transport parameters and to check for non-ideal flow conditions in the columns. Adsorption constants were fit that best described the total arsenic desorption from each of the four unplanted soil columns. Changes in arsenic concentrations in the solid phase , where Cs_m and Cs_im are the arsenic concentration within the mobile and immobile solid phase , respectively, were derived from isotherm equations, . Since the binding sites in the soil are non-uniform, the Freundlich isotherm was used in the model as it represents multiple types of binding sites. Full details are available in the Supplemental Information.Statistical analysis was performed in R . Analyses of covariance were performed on linear models to analyze main effects of explanatory variables including time , depth , soil, and treatment on response variables including fern arsenic concentrations, biomass, arsenic accumulation, arsenic uptake rate, and changes in soil arsenic concentrations during phytoextraction. Models were selected based on AIC criteria using the MuMIn package . Two-way interactions were included when those models were most highly ranked. Regression summaries were used to compare effects of explanatory variables, and differences in means were determined with Tukey’s Honestly Significant Difference test on means normalized to initial soil arsenic concentration where appropriate. A paired t-test was used to determine the mass balance across all columns, com paring mass arsenic leached per column to mass arsenic accumulated per fern. Effects of soil and treatment on the AMF assemblage were determined using a permutational multivariate analysis of variance with pairwise comparisons.

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,flood tray and pepper.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 divinylethers participate in the plant response to EP observed in solanaceous plants is unresolved, although ongoing research in our laboratory will address this issue. The search for a traditional PRR for EP in plant cells analogous to those for other MAMPs, although intriguing, may not be productive given other mechanisms for rapid uptake of PUFA by plant cells and their entry into oxylipin metabolism.β-linked glucose polysaccharides are the most abundant compo nent of Phytophthora cell walls, comprising more than 80% of the wall dry weight . These include insoluble β-1→4-linked and β-1→3, β-1→6-linked glucans, with the latter by far the more abundant of these polymers. In addition to the abundance of glucose, compositional analyses of cell walls also reveal minor amounts of mannose and glucosamine, as well as protein and lipid similar to levels found in cell walls of fungi. In addition to the insoluble glucans, solu ble β-1→3-linked glucans are present at various developmental stages in the oomycete life cycle. For example these can be found in the germination fluids of cystospores as well as other stages, and during synthesis and remodeling of the wall during growth, thus making them potentially available at the host–pathogen interface during infection .

Laminarans are linear β-1→3-linked glucans that provide the dominant storage carbohydrate in Phytophthora and other oomycetes, as well as other stramenopiles . The β-1→3, β-1→6-linked glucans present a very complex array of possible structures, some with well-established activity in modulating plant innate immunity. The most prominent example is the elicitor activity associated with glucans isolated from cultures and cell walls of the soybean pathogen P. sojae . Albersheim et al. showed that these were potent inducers of the flflavonoid phytoalexin, glyceollin, and related defense reactions in soybean cotyledons. β- glucan oligosaccharide fractions of varying complexity had elicitor activity suggesting a model whereby cell wall fragments released during infection provide the physiological triggers of the plant defense response. The smallest active fragment following partial acid hydrolysis of P. sojae cell walls was purified and shown to be a hexa -D-glucitol. This oligosaccharide and its corresponding unreduced hepta-β-glucoside elicited at concentrations between 10−7 to 10−9 M . Subsequent work by Michael Hahn and coworkers further defined the branched β-1→3, β-1→6 structural motif essential to maximally induce phytoalexin accumulation and found that the hepta-β-glucoside specifically bound to soy bean membranes with high affinity . These investigators provided strong evidence that the binding activity was associated with a membrane protein or glycoprotein. Subsequent efforts by other laboratories identified hydrophobic membrane proteins that bind β-glucans with high affinity from soybean and other legumes . Reconsti tution of the soybean homolog in lipid vesicles strongly bound the hepta-β-glucoside , which could be displaced by glucans with different degrees of polymerization in competitive binding assays. The elicitor activity and high affinity binding of the hepta-β- glucoside and related β-glucans are limited to members of the Fabaceae . Biochemical purification and additional studies indicate the binding proteins from legumes constitute a family of proteins of different sizes , with different carbohydrate active domains, one that binds β-glucans and another with glucanase activity capable of releasing elicitor-active fragments from Phytophthora cell walls .

What would further strengthen the case for these as physiological receptors for β-glucan-triggered immune responses in soybean is evidence that the binding speci- fificity for diverse oligoglucosides matches their bio-activity as elicitors. To our knowledge corresponding knock-out or knock down genetic experiments within legumes to corroborate receptor function have not been reported, although the soybean protein expressed in tomato confers binding of the hepta-β-glucoside .β-1→3-glucans also figure prominently as immune modulators in the potato – P. infestans interaction,arandanos planta although the story here is complicated by their reported action as both enhancers and suppressors of elicitor activity. However, this differential activity has not been reconciled with the degree of biochemical resolution as was done with P. sojae glucans to unambiguously assign enhancer or suppressor activity to the various oligoglucosides within the active fractions. Doke and Tomiyama using a potato protoplast assay showed that water soluble, anionic and non-anionic β-glucans suppressed the elicitor activity of a crude hyphal wall fraction from P. infestans. They suggested a degree of race-specificity in that glucans from compatible races of the pathogen were more active than those of incompatible races in suppressing the HR and ROS induced by the hyphal wall elicitor. The suppressive glucans were partially characterized and shown to have a DP of 17–23 glucose units with β-1→3 and β-1→6 link ages, and were present in the fluids of germinating cystospores . The purified hepta-β-glucoside from P. sojae was neither active as an elicitor nor as a suppressor in potato. A subsequent study showed that water soluble glucans from spore germination fluids of P. capsici have similar effect in suppressing elicitor-induced cell death in pepper and tomato cell suspensions . Race specificity attributed to the glucans in the context of HR suppression is difficult to reconcile with the contemporary paradigm of effector-triggered immunity and resistance -gene action . The model for β-glucans as suppressors is further complicated by their enhancement of EP elicitor activity. β-glucans, although lacking inherent elicitor activity in potato, can strongly enhance the activity of EP. Several lines of evidence suggest the combined action of eliciting and non-eliciting components provide a maximal defense response. Initial evidence came from reconstitution experiments whereby highly elicitor-active, solubilized cell wall fractions were hydrolyzed in base-borohydride, leaving polysaccharides intact but hydrolyzing any esterified fatty acids, which were then removed by solvent extraction. This resulted in complete loss of elicitor activity, which was restored by addition of AA and EPA to the base hydrolyzed wall fractions at their levels initially present . Subsequent fractionation, partial purification and analysis showed that the enhancers were indeed β-1→3-glucans . Preisig and Ku´c further demonstrated that the glucans provide a 10–100 fold enhancement of the activity of AA concentrations that alone are below the thresh old for induction of phytoalexins and related responses. The glucans also revealed elicitor activity of other EPs, particularly 5-eicosatrienoic acids. The most active β-glucan fractions had similar DP as the suppressor glucans, and were then found to suppress the HR induced by incompatible races of P. infestans, suggesting that the enhancers and suppressors could be the same. These classic experiments indicate that members of the Solanaceae have an intriguing system for perceiving specific β- glucans and EP to coordinate a strong resistance response.

The activity of these glucans in modulating immunity in potato, in particular, suggests a receptor-mediated process subject to attenuation by competing ligands as observed in legumes. For example, the suppressive action of the β-glucans against the HR induced by pathogen inoculum or the crude hyphal wall elicitor may have resulted from similarities in oligosaccharin motifs that compete for a putative MAMP receptor. Algal polysaccharides, such as the storage β-glucans laminarin and carrageenan, activate defense responses in some plants, although sulfated carrageenans appear to be far more active than laminarins as elicitors . However, in potato, laminarin neither elicits nor suppresses, providing a negative control treatment in the studies of the more complex β-1→3-linked glucans. Although considerably less active than the β-glucans, N, N’- diacetyl-D-chitobiose, the hapten for the potato lectin, inhibited the HR induced by incompatible races of P. infestans in potato and modestly enhanced the elicitor activ ity of AA . Although other carbohydrates may modulate the plant immune response to some degree, the exceptionally strong biological activity of the oomycete oligosac charins indicates considerable structural specificity in their action.Protection against fungi in vertebrates involves both innate and adaptive immunity . Innate anti-fungal immunity is primarily mediated by diverse pattern-recognition receptors associated with phagocytes, which upon activation ingest and kill or degrade the invading microbe. Carbohydrates associated with the fungal cell wall, in particular, are well positioned to be recognized by these receptors. The adaptive and highly specific immune response to the invader is then engaged following generation of cytokines and chemokines along with the presentation of microbial antigens to lymphocytes. There are multiple pattern-recognition receptors for β-glucans in phagocytes and the molecular details for some of these inter actions have been characterized . These include the transmembrane dectin-1, a natural killer cell-receptor-like C-type lectin found on macrophages, neutrophils and dendritic cells, which specifically recognizes β-1→3- and β-1→6-linked glucans as well as intact yeast cells . Zymosan, a complex cell wall preparation from Saccharomyces cerevisiea used to promote inflammation in experimental models, also stimulates dectin-1 and macrophage activation. Of particular interest in relation to the topic of this review is that zymosan induces cytosolic phospholipase A2 in macrophages that releases AA for conversion into pro-inflammatory prostaglandins and leukotrienes . Intriguing here is the apparent cross-kingdom conservation whereby β-glucans operate in concert with AA metabolites and other signals to orchestrate an innate immune response. The extent to which this analogy and underlying mechanisms trans late to plant–oomycete interactions remains to be determined. Arabidopsis and Solanum species have proteins with C-type lectin motifs with some homology to dectin-1. However, they appear to be rare in plants and their functions are unresolved .Stories and rumors have circulated for years about biotechnology projects in horticulture being shelved because of intellectual property conflicts. In a typical situation, a plant scientist at a university agricultural-experiment station or a smaller seed firm has developed a remarkable new variety using the cutting-edge scientific tools of plant biotechnology. Then, as they or the nursery or the growers’ association with whom they work take the next steps to develop and release the new variety to commercial growers, their efforts are quickly and quietly shut down by a letter from an attorney.

Our Thai rice population belongs to the indica group . While its size is relatively small compared human studies, GWAS with similar population size has been effective in Arabidopsis and rice. Indeed, diversified composition of our population, lack of strong sub-population structure, and its homozygosity facilitated GWAS.Altogether, GWAS using this Thai rice population lev eraged more than 110,000 SNPs to identify 448 SNPs associated with salt tolerance, which were located in 200 loci in the rice genome. As presented in Table 3, 73% of candidate genes from association mapping associated with salt stress were located within salinity tolerance QTLs identified in bi-parental segregating populations. Functional annotation of the 200 identified genes revealed a number of plausible candidates. The gene annotations we employed relay on the presence of a protein domain or of a homolog with a known functionin rice and other crop species such as maize or sorghum, as well as Arabidopsis. Two chromosomes contained the highest number of reported salt QTLs overlapping with 7 of our candidate loci: chromosome 1, which included 16 QTLs and chromosome 2, which included 10 QTLs. The nature of the candidate genes indi cates that different molecular and cellular strategies have evolved to favor survival during salt stress. Several genes belong to the receptor kinase family , encoding signaling factors during environmental stresses. LOC_Os01g18850, one of candidate genes detected by GWA mapping of UFG trait, encodes SQUAMOSA promoter binding protein-like transcription factor , a plant specific TF, whose function was suggested to affect a broad range growth and development processes, including flower development and 19 SPL genes were identified in rice. The role of SPL gene in salt stress response has been studied by Mao et al. . The 31 SPL genes were identified in maize and the expression profiles of SPLs revealed that most SPL genes were induced under salt stress condition.

This enzyme, common to bacteria,grow table hydroponic plants and humans, shares a common catalytic center, a heme with an iron coordinated to the thiolate of a conserved cysteine. They oxidize disparate substrates through activation of molecular oxygen. The plant P450 gene superfamily plays crucial roles in plant metabolic processes. Narusaka et al. analyzed the expression of 49 Arabidopsis P450 genes under various stresses, including salt stress, and found that 29 P450- genes were induced by various stresses. In the CYP709B subfamily of P450, a cyp709b3 Arabidopsis mutant showed sensitivity to salt stress during germination and high salt-damage at the seedling stage. In rice, Tamiru et al. reported that a P450 gene, OsDSS1 located on chromosome 3 was involved in growth and drought stress responses. Compared to WT, the dss1 rice mutant exhibited improved recovery after germination under drought stress. Additionally, ectopic expression of the P450 gene PtCYP714A3 from Populus trichocarpa was studied in rice. Transgenic rice expressing PtCYP714A3 was semi dwarf with improved tolerance to salt and osmotic stress, resulting in higher survival rates than WT. Interestingly, several novel candidate loci with 144 significant SNPs identified from this GWA mapping were found on chromosome 10, in which no salt QTL was reported. This represents the highest density of significant SNPs found in the same LD block . Interestingly, seven of these SNP-associated genes encoded F-box domain containing proteins . Previous studies have reported the role of F-box proteins in regulating various abiotic stress responses in Arabidop sis, wheat and rice. A conserved N-terminal F-box domain , is a component of the multi-subunit of ubiquitin E3 ligase, an enzyme in the last step of the ubiquitination pathway. The rice gen ome harbors more than 600 F-box genes whose divergence is consistent with adaptive roles and regulation of 25 of these genes responds to salinity stress. Rice seed lings over expressing F-box protein gene, MAIF1 reduced inhibition of root growth and tolerance under salt stress compared with WT.

Salt induced the expression of OsMsr9, a novel rice putative F-box containing protein, especially in the panicle. Over expression of OsMsr9 increased root length, shoot length and survival rate under salt stress. Moreover, SNP with the lowest p-value found on chromosome 12 of GWA mapping of UFG was located in LOC_Os12g36630, which was annotated as a universal stress protein domain containing protein. In fact, USP genes are widely distributed across many organisms including plant, which encode a protein containing the 140–160 highly conserved residues of the Universal Stress Protein A domain . These genes were reported as environmental stress-responsive genes and played role in the ability of plant to respond to the stresses. To date, there are no report on the role of USP genes in salt-treated rice. However, in the study on OsUsp1 in rice under oxygen deficiency condition, it was found that OsUsp1 expression was strongly induced within 1 h of submergence and it played a role in ethylene-mediated stress adaptation in rice. Furthermore, the role of the USP protein in enhancing oxidative stress has been reported in the plant model Arabidopsis. They found that the over-expression of AtUSP conferred a strong tolerance to oxidative stress, primarily via its chaperone function.Fresh and minimally processed fruit and vegetables contribute significantly to the burden of food borne illness in the U.S.. While introduction of pathogens can occur at any step from production through consumption, preharvest contamination of leafy greens has been identified in a number of recent outbreaks. Recurrent outbreaks and product recalls associated with leafy greens and the isolation of Salmonella or Escherichia coli O157:H7 from implicated product suggest that specific plant bacteria interactions or environmental conditions might favor the introduction and persistence of these pathogens in the production environment. To evaluate the risks of preharvest contamination of leafy greens, the ability of E. coli O157:H7 to colonize or survive on plants has been assessed under both laboratory and field conditions.

On plants in growth chambers, E. coli O157:H7 was found to grow and survive under specific conditions on lettuce seedlings and mature plants. Growth of E. coli O157:H7 was generally associated with the phyllosphere,growing strawberries in pots or the aerial portions of plants. Endophytic growth has also been demonstrated, but mainly for plants in hydroponic solution. Young lettuce leaves were found to sustain higher populations of the pathogen than older leaves. Although controlled conditions in the laboratory provide an opportunity to study virulent strains of human pathogens, such investigations do not fully mimic the biotic conditions plants are exposed to in the field. Shortly after application of E. coli O157:H7 onto lettuce in production regions, culturable populations of the pathogen rapidly declined, regardless of the season or location . However, E. coli O157:H7 can apparently survive, at least in low quantities, on field-grown plants, and viable cells were found on a fraction of lettuce plants for several weeks after inoculation. Studies of E. coli O157:H7 survival on plants in the field have generally relied on colony-based assessments coupled with enrichment methods to detect the presence/absence of the pathogen. Such approaches do not permit measurements of total cell amounts or the detection of individual cells that are viable but injured and no longer recovered on standard laboratory growth medium. These sub-lethally injured cells may still be infectious or capable of resuming growth under favorable conditions. The latter possibility is relevant to leaf surface habitats, which are characterized as stressful environments for microorganisms and are subject to fluctuating temperatures and moisture levels as well as other environmental insults. Therefore, culture-independent methods, such as fluorescence based cell detection or genetic approaches, might be complementary for measurements of pathogen presence and survival on plants. In particular, combining membrane-impermeable, DNA intercalating compounds, such as propidium monoazide , with quantitative real-time PCR might offer novel opportunities to sensitively and selectively detect pathogens among a background of other microorganisms in environmental samples. PMA efficiently enters cells with compromised but not intact membranes, and, upon photo activation, PMA is irreversibly cross linked to DNA and prevents PCR amplification of target sequences. In this study, we developed a PMA real-time PCR assay to quantify both viable and total E. coli O157:H7 recovered from lettuce plants in the laboratory and field; these results were compared with quantities of this organism determined by culture methods. We also used this approach to design plant inoculation and incubation conditions in the laboratory that more closely mimic field environments and could be used to study the plant associated behaviors of virulent outbreak-associated strains of the pathogen.

Accurate measurements of pathogen amounts and viability are important for risk assessments and control mechanisms aimed at preventing outbreaks of food borne illness. This study was the first to apply a culture-independent method to quantify the number of E. coli O157:H7 cells on plants inoculated with the pathogen in the field. We also compared the utility of the method for quantifying virulent and avirulent pathogen survival on laboratory-grown lettuce. The first step in the development of culture-independent methods for bacterial enumeration is to ensure that the technique is optimized for the specific organism and environmental conditions of interest. While numerous assessment methods are available, we selected PMA combined with real-time PCR because this technique offered the possibility of rapid enumeration of viable cells in a variety of environments. For E. coli O157:H7 ATCC 700728, we first identified appropriate real-time PCR primers, PMA concentrations, and PMA incubation times with a goal of selective and sensitive detection of viable E. coli O157:H7 cells in the presence of high levels of dead E. coli O157:H7 cells. Among the results was the confirmation that PMA-mediated PCR-inhibition is incomplete when the product size is less than 200 bp. Although our tests on PMA real-time PCR revealed some potential short-comings with the method, most notably the interference of high quantities of inactivated cells and the relatively high numbers of cells required for detection, these parameters were within the range found in other PCR-based studies. E. coli O157:H7 strains ATCC 700728 and EC4045 were inoculated onto leaves of potted lettuce plants and incubated in a growth chamber under temperature and low humidity conditions that favored the decline of cell numbers in trends similar to those recorded in field trials performed in the Salinas Valley. This approach differs from most other laboratory studies that examined plants exposed to high moisture and temperatures supportive of pathogen growth. Within 2 days after inoculation on lettuce plants, the number of E. coli O157:H7 ATCC 700728 cells able to form a colony on TSA declined 1000-fold when the pathogen was initially applied at a level of 6 log CFU per plant or per leaf using an aerosolized spray and declined .105 -fold when either strain was applied directly in 2-ml drops. The numbers of culturable cells remained similar for the subsequent 5 days, suggesting that the initial events after contact with the plant are crucial for determining the survival of the organism on lettuce. In contrast with plate counts, real-time PCR targeting of the E. coli O157:H7 ATCC 700278 and EC4045 inoculants showed that the genomic DNA of the organisms remained on the lettuce in quantities nearly equivalent to the inoculum concentrations over the duration of the 7-day experiment. This finding confirmed that both strains adhered to the leaf surfaces and the recovery method was sufficient to remove the majority of the cell inoculants from the plant. Moreover, the results indicated the presence of high numbers of intact E coli O157:H7 cells on lettuce even when very few were able to form colonies on TSA. Exposure of E. coli O157:H7 suspensions to PMA prior to real time PCR quantification facilitated the detection of cells with an intact cell membrane, and these cells were likely viable. Application of this method to E. coli ATCC 700728 and EC4045 recovered from growth chamber lettuce indicated that a significant fraction of the pathogen inoculants were viable, even though colony-based assessments indicated otherwise. This finding was particularly evident for E. coli O157:H7 inoculated onto leaves by direct drop inoculation. While an average of 5 log CFU per leaf were viable as measured by PMA real-time PCR, less than 1 log CFU per leaf were enumerated by culturing. These results are supported by previous studies reporting that E. coli O157:H7 enters into a viable but not culturable state on lettuce leaves incubated at cold temperatures. Similar outcomes were reported for other E. coli including E. coli O104:H4 in water or after exposure to toxic concentrations of copper.

The chief consumers of Colombian marijuana were still North American citizens, and the dealers continued to design new clandestine mechanisms for exporting their product. Marijuana crops occupied an area of between 10,000 and 12,000 hectares between 1988 and 1989, and there was certainly nodecrease in exports. The government had had some success in 1986, but the phenomenon persisted, so much so that by 1988 Colombia had become, once more, the world’s major marijuana producer — followed by Mexico and the United States itself. Sixth, in the second half of the Barco administration it became urgent to establish priorities in actions designed to counter the ever-expanding drug trade. On the one hand, the country’s budget limitations imposed a rational use of the meager resources available. Besides, aid from the United States had been reduced from US$11,553,000 in 1987 to US$9,767,000 in 1988. On the other hand, it became important to decide on priorities, given the dimensions of the problem. Priority had to be given to those tasks which were both feasible and most urgent. The Liberal Party government continued to insist that eradication was the prime tactical mechanism to be used against the production and exportation of illegal drugs. Nonetheless spraying was reduced, despite urgings from Washington. In 1990, seeing that the policy of manual and aerial fumigation offered no practical results, eradication began to occupy a less important place in Colombian anti-drug policy. Emphasis was placed more and more on “the drug war”; that is to say, on military action and extradition. Between August 1989 and August 1990, President Barco’s government extradited a number of Colombian citizens to the United States, and for this paid a huge price in terms of violent upheaval within Colombia.

Perhaps this explains why Washington did not put greater pressure on the Colombian government to implement a more aggressive eradication policy. In any case,flood table reduced pressure from the United States again gave the authorities in Bogotá a margin of maneuverability in which to define certain internal aspects of their anti-drug strategy. This margin was due, also, to the fact that the efforts being made by Colombia to counteract the drug trade had been acknowledged internationally — and in particular by Europe. The administration of President César Gaviria inherited from its predecessors very poor results as far as the policy of eradication was concerned: experimental fumigation during the Turbay regime, massive fumigation in the period of Belisario Betancur and sporadic fumigation by the Barco government — all of them ineffective, clumsy and deplorable. Colombia had tried paraquat and then glyphosate in an attempt to stay the advance of marijuana, and garlon-4 against the coca plantations. The merchandise was different in each case, the legal herbicides used were different also, but the results were very similar: organized drug traffickers in Colombia had efficiently diversified the production and the processing of illegal drugs, while successive governments were combating them with actions that did not seriously affect the illegal trade itself nor the increasing power of Colombia’s drug lords. It seemed difficult to overlook these antecedents; yet President Gaviria and his team did not appear to have learnt any lessons from the experience of previous administrations. In the final months of the Barco administration, chemical fumigation had ceased, and yet this fact did not seem to have an adverse effect on relations between Bogotá and Washington. In a routine manner, those US officials in charge of international anti-narcotic policy would suggest a return to fumigation, but they did not do so peremptorily, nor were these suggestion accompanied by any strong threats.

Manual eradication of coca was being carried on, and the number of hectares dedicated to marijuana crops had decreased; so it seemed unnecessary to give pride of place to fumigation in the narco-diplomacy between Colombia and the United States. What altered this situation dramatically was the discovery that Colombia was becoming — albeit incipiently — an important zone for the growing of poppies.In 1984, small plantations were destroyed in Tolima and Meta. In 1986, invasions and confiscations were carried out by government officials, but they were relatively insignificant. The first 2,297 grams of heroin were seized in that same year; and in 1988 two laboratories for processing heroin and morphine were discovered, one in Bogotá and the other in Barranquilla.18 In September 1991 the weekly magazine Semana published a long report on the sudden appearance of poppies on the national scene, quoting official sources that claimed to have discovered 2,000 hectares of what was called “the curséd flower.”At the end of that same year, the National Security Agency spoke of some 2,500 hectares where poppies were being grown,and the Anti-narcotics Police pointed out that the year had seen an unprecedented increase in the number of poppy plantations in the main Colombian mountain ranges within the jurisdiction of a number of departments such as Huila, Tolima, Cauca, Boyacá, Cundinamarca, Caquetá, Antioquia, Caldas, Meta, Nariño, Risaralda and Santander, to name only the most important ones.”The government responded to these alarming facts by reporting the manual eradication of 1,406 hectares of poppies, the seizure of 17 kilograms of morphine and 30 kilograms of opium, and the destruction of five laboratories for morphine-processing in Neiva.It is worth mentioning, also, that as from May 1991, Colombian heroin began to be identified and seized in the United States.It was hoped that having eradicated 56 percent of the plantations reported in 1991, the question of poppy crops would not acquire alarming proportions. Nonetheless, in March of that year, the director of the Anti-narcotic Police, General Rosso José Serrano, stated that poppy production might very well soon expand to occupy 10,000 hectares of Colombia’s soil.A month later, press reports spoke of a possible 20,000 hectares already in existence.

A study made under the auspices of the National Council for Defense and National Security claimed that approximately 20,000 hectares of poppy plantations did indeed exist and were located in seventeen different departments within Colombia.In January 1992 the CNE had authorized both manual and aerial fumigation,macetas redondas with glyphosate, of another 2,900 hectares of poppies.It seems that this decision was not the result of any special pressure from Washington, even though there did exist a powerful incentive to avoid negative reactions in Washington after the Colombian government had refused to accept US$2.8 million in official US aid to set up an anti-narcotics unit in the army similar to that which already existed in the Police Force.Washington could hardly justify an unusually strong protest against Bogotá’s behavior regarding the control of poppy fields, since Colombia was not even a medium-sized producer of heroin.Officials from DEA and the US Embassy in Bogotá urged the Colombian government to spray the poppy fields, and they were pleased to see that their wishes were carried out. They also helped legitimize the use of glyphosate by circulating scientific papers and opinions by United States experts in favor of the substance.Nevertheless, the decision to spray the poppy fields with that particular chemical seems to have been taken by President Gaviria’s government on discovering, to its surprise, the proportions of the Colombian poppy/heroin phenomenon, rather than because of any imposition from Washington. From 1992 onwards little effort was made to eradicate coca and marijuana plantations. 944 hectares of coca were destroyed in 1992, and 100 of marijuana; and in 1993, 846 hectares of coca were destroyed, and 138 hectares of marijuana.31 This reduction in efforts to eradicate was due, in part, to two notions which predominated among bureaucrats and experts since the Betancur regime. On the one hand, that Colombia was not an important coca producer, but rather was the main scenario for the processing of cocaine and of its exportation to the principal markets for consumption abroad. And secondly, that the United States was effectively replacing imports thanks to the development of its own national variety of marijuana which was satisfying the home market and causing a considerable decline in the number of Colombian marijuana planters. Such reasoning was only partially correct. In the nineties, for example, Colombia coca growing not only increased, but the quality of the leaf improved considerably. On top of that, severe frosts in the United States occasionally affected marijuana crops there, and that, together with repressive measures against marijuana-growing in Mexico, tended to explain why, from time to time, there was a resurgence of the marijuana business in Colombia. Colombia’s infrastructure made it easy to plant more coca and marijuana whenever attractive market conditions justified doing so.

It was unrealistic, therefore, to think that Colombia had to overcome serious problems in order to produce illegal substances. In February 1994, on being informed by US sources that coca plantations covered an area of 39,700 hectares,the Colombian government ordered aerial spraying. At the same time, marijuana crops had increased from 2,000 hectares in 1991 to 5,000 hectares in 1993.33 The poppy boom kept growing throughout 1993. A new report from the National Council for Defense and National Security showed that the poppy business was flourishing in eighteen departments.12,864 hectares of poppies had been destroyed in 1992 . In 1993, 9,821 hectares were eradicated, but in 1994 poppy fields were still proliferating. During the year 1994, 5.314 hectares were eradicated .However, according to US estimates, poppy fields that year did not fall below 20,000 hectares, an estimate that was never denied by the Colombian authorities.The illegal heroin trade of the eighties and nineties appeared to follow a similar pattern to that of the marijuana business in the sixties and seventies. In the case of marijuana, the production triangle in this hemisphere had been made up of Mexico, Jamaica and Colombia. When repression took its toll in one country, especially due to the use of herbicides, the business moved to another, although it always returned to the spot where initially larger amounts had been planted. Something similar occurred with poppies between Mexico, Guatemala and Colombia. The original problem of illegal crops was never overcome, nor were the authorities able to dismount the equipment and infrastructure which enabled such plantations and laboratories to stay in business in the above-mentioned countries. By attacking temporarily, and in an isolated fashion an illegal crop, public anti-drug policy automatically attacks the weakest and least decisive link in the vast and complex chain of illegal drug dealing, and at the same time has the worst possible negative effect from a social viewpoint on small farmers and Indian populations, while affecting hardly at all the area of organized crime financed by the drug traffickers. The Gaviria administration had decided to deal with the drug problem by placing its emphasis on a policy of submission and making clear that it differentiated between drug trafficking and narco-terrorism. President Gaviria stated that “while narco-terrorism is our problem, drug trafficking is an international phenomenon.” Nonetheless, in the case of poppy growing, Gaviria did what former governments had done in their attacks on coca and marijuana fields. The results of his efforts were insignificant and ephemeral, as were those of his predecessors. When a government acts on the basis of punishment alone and without offering incentives, believing that it is indulging in a technically-approved and non-harmful type of fumigation, it finishes up contributing to environmental damage and to a greater social breakdown in the zones where the plantations are grown. Occasional voices were raised to criticize chemical fumigation. But discussions on the subject assumed an elitist, moral tone: on one side were the “good, hard-line, intelligent people” uncontaminated by drug traffic, and on the other “the softies, the badies, the dumb idiots” who were either mouthpieces of the traffickers or were unconsciously letting themselves be used by them. In February 1992, Colombia’s Justice Minister made a comment which illustrates this point: he claimed that “a cloak of complicity has been thrown over things by those who object to herbicide fumigation for environmental reasons, while all the time playing into the hands of the drug traffickers.”At no time was there a lobby sufficiently coherent, serious and affirmative to combat the government’s determination to keep on fumigating. The executive did not receive substantial criticism nor impediments to the actions it carried out through legislation and the judiciary.