Ecological intensification of cereal production systems: Yield potential, soil quality, and precision agriculture

Wheat (Triticum aestivum L.), rice (Oryza sativa L.), and maize (Zea mays L.) provide about two-thirds of all energy in human diets, and four major cropping systems in which these cereals are grown represent the foundation of human food supply. Yield per unit time and land has increased markedly during the past 30 years in these systems, a result of intensified crop management involving improved germplasm, greater inputs of fertilizer, production of two or more crops per year on the same piece of land, and irrigation. Meeting future food demand while minimizing expansion of cultivated area primarily will depend on continued intensification of these same four systems. The manner in which further intensification is achieved, however, will differ markedly from the past because the exploitable gap between average farm yields and genetic yield potential is closing. At present, the rate of increase in yield potential is much less than the expected increase in demand. Hence, average farm yields must reach 70–80% of the yield potential ceiling within 30 years in each of these major cereal systems. Achieving consistent production at these high levels without causing environmental damage requires improvements in soil quality and precise management of all production factors in time and space. The scope of the scientific challenge related to these objectives is discussed. It is concluded that major scientific breakthroughs must occur in basic plant physiology, ecophysiology, agroecology, and soil science to achieve the ecological intensification that is needed to meet the expected increase in food demand.

The Respiratory Burst and Electrolyte Leakage Induced by Sulfhydryl Blockers in Egeria densa Leaves Are Associated with H2O2 Production and Are Dependent on Ca2+ Influx1

In leaves of Egeria densa Planchon, N-ethylmaleimide (NEM) and other sulfhydryl-binding reagents induce a temporary increase in nonmitochondrial respiration (ΔQO2) that is inhibited by diphenylene iodonium and quinacrine, two known inhibitors of the plasma membrane NADPH oxidase, and are associated with a relevant increase in electrolyte leakage (M. Bellando, S. Sacco, F. Albergoni, P. Rocco, M.T. Marré [1997] Bot Acta 110: 388–394). In this paper we report data indicating further analogies between the oxidative burst induced by sulfhydryl blockers in E. densa and that induced by pathogen-derived elicitors in animal and plant cells: (a) NEM- and Ag+-induced ΔQO2 was associated with H2O2 production and both effects depended on the presence of external Ca2+; (b) Ca2+ influx was markedly increased by treatment with NEM; (c) the Ca2+ channel blocker LaCl3 inhibited ΔQO2, electrolyte release, and membrane depolarization induced by the sulfhydryl reagents; and (d) LaCl3 also inhibited electrolyte leakage induced by the direct infiltration of the leaves with H2O2. These results suggest a model in which the interaction of sulfhydryl blockers with sulfhydryl groups of cell components would primarily induce an increase in the Ca2+ cytosolic concentration, followed by membrane depolarization and activation of a plasma membrane NADPH oxidase. This latter effect, producing active oxygen species, might further influence plasma membrane permeability, leading to the massive release of electrolytes from the tissue.

Use of a New Tetrazolium-Based Assay to Study the Production of Superoxide Radicals by Tobacco Cell Cultures Challenged with Avirulent Zoospores of Phytophthora parasitica var nicotianae1

The relationship between the production of reactive oxygen species and the hypersensitive response (HR) of tobacco (Nicotiana tabacum L.) toward an incompatible race of the Oomycete Phytophthora parasitica var nicotianae has been investigated. A new assay for superoxide radical (O2−) production based on reduction of the tetrazolium dye sodium,3′-(1-[phenylamino-carbonyl]-3,4-tetrazolium)-bis(4-methoxy-6-nitro) benzene-sulfonic acid hydrate (XTT) has enabled the quantitative estimation of perhydroxyl/superoxide radical acid-base pair (HO2·/O2−) production during the resistant response. Tobacco suspension cells were inoculated with zoospores from compatible or incompatible races of the pathogen. Subsequent HO2·/O2− production was monitored by following the formation of XTT formazan. In the incompatible interaction only, HO2·/O2− was produced in a minor burst between 0 and 2 h and then in a major burst between 8 and 10 h postinoculation. During this second burst, rates of XTT reduction equivalent to a radical flux of 9.9 × 10−15 mol min−1 cell−1 were observed. The HO2·/O2− scavengers O2− dismutase and Mn(III)desferal each inhibited dye reduction. An HR was observed in challenged, resistant cells immediately following the second burst of radical production. Both scavengers inhibited the HR when added prior to the occurrence of either radical burst, indicating that O2− production is a necessary precursor to the HR.

Cyanogenesis in Cassava1 : The Role of Hydroxynitrile Lyase in Root Cyanide Production

In the cyanogenic crop cassava (Manihot esculenta, Crantz), the final step in cyanide production is the conversion of acetone cyanohydrin, the deglycosylation product of linamarin, to cyanide plus acetone. This process occurs spontaneously at pH greater than 5.0 or enzymatically and is catalyzed by hydroxynitrile lyase (HNL). Recently, it has been demonstrated that acetone cyanohydrin is present in poorly processed cassava root food products. Since it has generally been assumed that HNL is present in all cassava tissues, we reinvestigated the enzymatic properties and tissue-specific distribution of HNL in cassava. We report the development of a rapid two-step purification protocol for cassava HNL, which yields an enzyme that is catalytically more efficient than previously reported (Hughes, J., Carvalho, F., and Hughes, M. [1994] Arch Biochem Biophys 311: 496–502). Analyses of the distribution of HNL activity and protein indicate that the accumulation of acetone cyanohydrin in roots is due to the absence of HNL, not to inhibition of the enzyme. Furthermore, the absence of HNL in roots and stems is associated with very low steady-state HNL transcript levels. It is proposed that the lack of HNL in cassava roots accounts for the high acetone cyanohydrin levels in poorly processed cassava food products.

Controlled Cytokinin Production in Transgenic Tobacco Using a Copper-Inducible Promoter

The cytokinin group of plant hormones regulates aspects of plant growth and development, including the release of lateral buds from apical dominance and the delay of senescence. In this work the native promoter of a cytokinin synthase gene (ipt) was removed and replaced with a Cu-controllable promoter. Tobacco (Nicotiana tabacum L. cv tabacum) transformed with this Cu-inducible ipt gene (Cu-ipt) was morphologically identical to controls under noninductive conditions in almost all lines produced. However, three lines grew in an altered state, which is indicative of cytokinin overproduction and was confirmed by a full cytokinin analysis of one of these lines. The in vitro treatment of morphologically normal Cu-ipt transformants with Cu2+ resulted in delayed leaf senescence and an increase in cytokinin concentration in the one line analyzed. In vivo, inductive conditions resulted in a significant release of lateral buds from apical dominance. The morphological changes seen during these experiments may reflect the spatial aspect of control exerted by this gene expression system, namely expression from the root tissue only. These results confirmed that endogenous cytokinin concentrations in tobacco transformants can be temporally and spatially controlled by the induction of ipt gene expression through the Cu-controllable gene-expression system.

Floral Scent Production in Clarkia breweri1 : III. Enzymatic Synthesis and Emission of Benzenoid Esters

The fragrance of Clarkia breweri (Onagraceae), a California annual plant, includes three benzenoid esters: benzylacetate, benzylbenzoate, and methylsalicylate. Here we report that petal tissue was responsible for the benzylacetate and methylsalicylate emission, whereas the pistil was the main source of benzylbenzoate. The activities of two novel enzymes, acetyl-coenzyme A:benzylalcohol acetyltransferase (BEAT), which catalyzes the acetyl esterification of benzylalcohol, and S-adenosyl-l-methionine:salicylic acid carboxyl methyltransferase, which catalyzes the methyl esterification of salicylic acid, were also highest in petal tissue and absent in leaves. In addition, the activity of both enzymes in the various floral organs was developmentally and differentially regulated. S-Adenosyl-l-methionine:salicylic acid carboxyl methyltransferase activity in petals peaked in mature buds and declined during the next few days after anthesis, and it showed a strong, positive correlation with the emission of methylsalicylate. The levels of BEAT activity and benzylacetate emission in petals also increased in parallel as the buds matured and the flowers opened, but as emission began to decline on the 2nd d after anthesis, BEAT activity continued to increase and remained high until the end of the lifespan of the flower.

Phytochrome B and the Regulation of Circadian Ethylene Production in Sorghum1

The sorghum (Sorghum bicolor L. Moench) cultivar 58M, which contains the null mutant phytochrome B gene, shows reduced photoperiodic sensitivity and exhibits a shade-avoidance phenotype. Ethylene production by seedlings of wild-type and phytochrome B mutant cultivars was monitored every 3 h, and both cultivars were found to produce ethylene in a circadian rhythm, with peak production occurring during the day. The phytochrome B mutant produces rhythmic peaks of ethylene with approximately 10 times the amplitude of the wild-type counterpart with the same period and diurnal timing. The source of the mutant's additional ethylene is the shoot. The diurnal rhythm can be produced with either light or temperature cycles; however, both light and temperature cycles are required for circadian entrainment. The temperature signal overrides the light signal in the production of diurnal rhythms, because seedlings grown under thermoperiods reversed with the photoperiod produced ethylene peaks during the warm nights. To examine the effect of extreme shading on ethylene production, seedlings were grown under dim, far-red-enriched light. This treatment duplicated the phytochrome B mutant's shade-avoidance phenotype in the wild type and caused the wild type to produce ethylene peaks similar to those observed in the mutant. The results confirm that phytochrome B is not required for proper function of circadian timing, but it may be involved in modulating physiological rhythms driven by the biological clock oscillator.

Transgenic male-sterile plant induced by an unedited atp9 gene is restored to fertility by inhibiting its expression with antisense RNA.

We have previously shown that the expression of an unedited atp9 chimeric gene correlated with male-sterile phenotype in transgenic tobacco plant. To study the relationship between the expression of chimeric gene and the male-sterile trait, hemizygous and homozygous transgenic tobacco lines expressing the antisense atp9 RNA were constructed. The antisense producing plants were crossed with a homozygous male-sterile line, and the F1 progeny was analyzed. The offspring from crosses between homozygous lines produced only male-fertile plants, suggesting that the expression antisense atp9 RNA abolishes the effect of the unedited chimeric gene. In fact, the plants restored to male fertility showed a dramatic reduction of the unedited atp9 transcript levels, resulting in normal flower development and seed production. These results support our previous observation that the expression of unedited atp9 gene can induce male sterility.

Assembly and movement of a plant virus carrying a green fluorescent protein overcoat.

Potato virus X (PVX) is a filamentous plant virus infecting many members of the family Solanaceae. A modified form of PVX, PVX.GFP-CP which expressed a chimeric gene encoding a fusion between the 27-kDa Aequorea victoria green fluorescent protein and the amino terminus of the 25-kDa PVX coat protein, assembled into virions and moved both locally and systemically. The PVX.GFP-CP virions were over twice the diameter of wild-type PVX virions. Assembly of PVX.GFP-CP virions required the presence of free coat protein subunits in addition to the fusion protein subunits. PVX.GFP-CP virions accumulated as paracrystalline arrays in infected cells similar to those seen in cells infected with wild-type PVX The formation of virions carrying large superficial fusions illustrates a novel approach for production of high levels of foreign proteins in plants. Aggregates of PVX.GFP-CP particles were fluorescent, emitting green light when excited with ultraviolet light and could be imaged using confocal laser scanning microscopy. The detection of virus particles in infected tissue demonstrates the potential of fusions between the green fluorescent protein and virus coat protein for the non-invasive study of virus multiplication and spread.

The sigma factor sigma s affects antibiotic production and biological control activity of Pseudomonas fluorescens Pf-5.

Pseudomonas fluorescens Pf-5, a rhizosphere-inhabiting bacterium that suppresses several soilborne pathogens of plants, produces the antibiotics pyrrolnitrin, pyoluteorin, and 2,4-diacetylphloroglucinol. A gene necessary for pyrrolnitrin production by Pf-5 was identified as rpoS, which encodes the stationary-phase sigma factor sigma s. Several pleiotropic effects of an rpoS mutation in Escherichia coli also were observed in an RpoS- mutant of Pf-5. These included sensitivities of stationary-phase cells to stresses imposed by hydrogen peroxide or high salt concentration. A plasmid containing the cloned wild-type rpoS gene restored pyrrolnitrin production and stress tolerance to the RpoS- mutant of Pf-5. The RpoS- mutant overproduced pyoluteorin and 2,4-diacetyl-phloroglucinol, two antibiotics that inhibit growth of the phytopathogenic fungus Pythium ultimum, and was superior to the wild type in suppression of seedling damping-off of cucumber caused by Pythium ultimum. When inoculated onto cucumber seed at high cell densities, the RpoS- mutant did not survive as well as the wild-type strain on surfaces of developing seedlings. Other stationary-phase-specific phenotypes of Pf-5, such as the production of cyanide and extracellular protease(s) were expressed by the RpoS- mutant, suggesting that sigma s is only one of the sigma factors required for the transcription of genes in stationary-phase cells of P. fluorescens. These results indicate that a sigma factor encoded by rpoS influences antibiotic production, biological control activity, and survival of P. fluorescens on plant surfaces.

Induction, modification, and transduction of the salicylic acid signal in plant defense responses.

Studies in our laboratory as well as others strongly suggest that salicylic acid (SA) plays an important signaling role in plant defense against pathogens. We have found that increases in endogenous SA levels correlates with both resistance of tobacco to infection with tobacco mosaic virus and induction of defense-related genes such as that encoding pathogenesis-related protein 1 (PR-1). Some of this newly synthesized SA was conjugated to glucose to form SA beta-glucoside. A cell wall-associated beta-glucosidase activity that releases SA from this glucoside has been identified, suggesting that SA beta-glucoside serves as an inactive storage form of SA. By purifying a soluble SA-binding protein and isolating its encoding cDNA from tobacco, we have been able to further characterize the mechanism of SA signaling. This protein is a catalase, and binding of SA and its biologically active analogues inhibited catalase's ability to convert H2O2 to O2 and H2O. The resulting elevated levels of cellular H2O2 appeared to induce PR-1 gene expression, perhaps by acting as a second messenger. Additionally, transgenic tobacco expressing an antisense copy of the catalase gene and exhibiting depressed levels of catalase also showed constitutive expression of PR-1 genes. To further dissect the SA signaling pathway, we have tested several abiotic inducers of PR gene expression and disease resistance for their ability to stimulate SA production. Levels of SA and its glucoside rose following application of all of the inducers except 2,6-dichloroisonicotinic acid. 2,6-Dichloroisonicotinic acid was found to bind catalase directly and inhibit its enzymatic activity. Thus, it appears that many compounds that induce PR gene expression and disease resistance in plants inactivate catalases directly or indirectly.

Immunogenicity of transgenic plant-derived hepatitis B surface antigen.

The focus of the Children's Vaccine Initiative is to encourage the discovery of technology that will make vaccines more readily available to developing countries. Our strategy has been to genetically engineer plants so that they can be used as inexpensive alternatives to fermentation systems for production of subunit antigens. In this paper we report on the immunological response elicited in vivo by using recombinant hepatitis B surface antigen (rHBsAg) purified from transgenic tobacco leaves. The anti-hepatitis B response to the tobacco-derived rHBsAg was qualitatively similar to that obtained by immunizing mice with yeast-derived rHBsAg (commercial vaccine). Additionally, T cells obtained from mice primed with the tobacco-derived rHBsAg could be stimulated in vitro by the tobacco-derived rHBsAg, yeast-derived rHBsAg, and by a synthetic peptide that represents part of the a determinant located in the S region (139-147) of HBsAg. Further support for the integrity of the T-cell epitope of the tobacco-derived rHBsAg was obtained by testing the ability of the primed T cells to proliferate in vitro after stimulation with a monoclonal anti-idiotype and an anti-idiotype-derived peptide, both of which mimic the group-specific a determinant of HBsAg. In total, we have conclusively demonstrated that both B- and T-cell epitopes of HBsAg are preserved when the antigen is expressed in a transgenic plant.

Free living astigmatid mites (Astigmatina): new taxa, rearing and use for mesostigmatid (Mesostigmata) predatory mite production

The cohort Astigmatina is divided in two major groups: Psoroptidia, composed mainly by feather and fur mites, and Non-psoroptidia, a dominant component of the acarofauna in ephemeral habitats. In these environments Astigmatina usually are saprophages or feed on fungi or bacteria. Astigmatina protonymphs undergo a complete reorganization of the body structure leading to the production of heteromorphic deutonymphs, generally specialized for dispersion through phoresy using arthropods and vertebrates as phoronts. Although most Astigmatina occur in natural environments, some species live in anthropic environments, such as food deposits, where some of them became pests; some Astigmatina infest subterraneous plant organs. Despite their economic and ecological importance, studies on the diversity and taxonomy of Astigmatina in Brazil have been rare over the last decades. The general objective of this thesis was to collaborate to the knowledge of the diversity and to evaluate the potential practical uses of these mites in Brazil. For this, new genera and species were described, method for rearing dust mites was studied and the efficiency of Astigmatina as prey for edaphic predators was evaluated. A new species of Thyreophagus (Astigmatina: Acaridae) was described based on specimens collected in Brazil, the association of three other species of this genus with stored food was reviewed and a key to all species of this genus was prepared. The genus Neotropacarus (Astigmatina: Acaridae), commonly found on plant leaves, was reviewed with the redescription of two species and description of new species collected in Brazil and from the Philippines. Two new genera and seven new species of Acaridae associated with the bee family Apidae was described and a key to Acaridae genera in subfamily Horstiinae was prepared. Several species of Astigmatina were evaluated as prey for predatory mites Stratiolaelaps scimitus (Womersley) (Mesostigmata: Laelapidae) and Protogamasellopsis zaheri Abo-Shnaf, Castilho and Moraes (Mesostigmata: Rhodacaridae), which oviposited on all evaluated astigmatids, with Tyrophagus putrescentiae (Schrank) and Aleuroglyphus ovatus (Tropeau) (Acaridae) being the most suitable prey. Seven foods and two development period, 30 and 60 days, after the introduction of 400 females of two important dust mite species, Blomia tropicalis van Bronswijk, de Cock e Oshima and Dermatophagoides pteronyssinus (Trouessart) were evaluate. With the most suitable foods, the population growth were higher than 20.2 and 15.3 for B. tropicalis and D. pteronyssinus, respectively.

A Regulatory Compliance Plan for Decommissioning a Sodium Phosphate Manufacturing Plant

Sodium phosphates are a class of chemicals that have been widely employed in commercial and consumer applications. However, declining use of these chemicals due to environmental concerns has lead to restructuring within the industry that has caused, and is likely to continue to cause, reduction of sodium phosphate production capacity. Closure of a sodium phosphate manufacturing plant necessitates decommissioning and decontamination activities that are subject to a variety of federal, state, and local regulations. A compliance plan was developed to provide a blueprint for ensuring that all federal regulatory requirements are met, however, site dependent state and local requirements were excluded. The compliance plan provides a framework that addresses project team formation and project planning, regulatory requirements, identification of affected processing equipment, plant pre-shutdown activities, waste stream identification and waste management facilities, safety, training, and emergency preparedness planning, and project decommissioning remedial actions. This regulatory compliance plan will enable sodium phosphate plant operators to complete decontamination and decommissioning work in a timely, efficient, compliant, and cost effective manner.

Biodiesel production by transesterification of sludge in supercritical conditions

In this study wastewater treatment plant (WWTP) sludge was subjected to a reactive pyrolysis treatment to produce a high quality pyro-oil. Sludge was treated in supercritical conditions in the presence of methanol using hexane as cosolvent in a high pressure lab-autoclave. The variables affecting the pyro-oil yield and the product quality, such as mass ratio of alcohol to sludge, presence of cosolvent and temperature, were investigated. It was found that the use of a non-polar cosolvent (hexane) presents advantages in the production of high quality pyro-oil from sludge: increase of the non-polar pyro-oil yield and a considerable reduction of the amount of methanol needed to carry out the transesterification of fatty acids present in the sludge.