Lyophilized Leaf Tissue Research Articles

Bt-toxin susceptibility and hormesis-like response in the invasive southern armyworm (Spodoptera eridania)

Bacillus thuringiensis (Bt) varieties of soybean and cotton have been widely adopted in South America, but defoliation by the southern armyworm Spodoptera eridania (Lepidoptera: Noctuidae) has increased in these crops. This issue may be linked to the low inherent susceptibility of the larvae to some Bt toxins. However, this hypothesis was not yet tested, nor was the potential sublethal effects of the toxins on larval development of southern armyworm. Here we determined the response of southern armyworm larvae exposed to Bt toxins representative of those used in soybean and cotton cultivars. In concentration-response bioassays, whereas a low median lethal concentration (LC50) value was found for Cry2Aa (11 ng/cm2), higher LC50 values were found for Cry1F (>3000 ng/cm2) and Cry1Ac (>10000 ng/cm2). Larval growth of the southern armyworm showed a hormesis-like response when the larvae were reared on artificial diet containing increasing Cry1Ac concentrations. Such enhanced larval growth was also observed when fresh foliage of Cry1Ac soybean or its lyophilized tissue in an artificial diet were fed to the larvae. Although the foliage of the dual-toxin Bt cotton producing Cry1Ac + Cry2Ab killed all larvae, its lyophilized leaf tissue caused no significant larval mortality when diluted 25 times. These results indicate that southern armyworm larvae have low inherent susceptibility to Cry1Ac and Cry1F toxins, while the Cry1Ac Bt toxin can enhance its larval growth. In addition, Cry1Ac + Cry2Ab Bt cotton may not meet the high-dose condition for the southern armyworm. These findings have important implications for pest management of the southern armyworm, which is discussed.

A hyper-thermostable \u03b1-amylase from Pyrococcus furiosus accumulates in Nicotiana tabacum as functional aggregates

BackgroundAlpha amylase hydrolyzes α-bonds of polysaccharides such as starch and produces malto-oligosaccharides. Its starch saccharification applications make it an essential enzyme in the textile, food and brewing industries. Commercially available α-amylase is mostly produced from Bacillus or Aspergillus. A hyper-thermostable and Ca 2++ independent α-amylase from Pyrococcus furiosus (PFA) expressed in E.coli forms insoluble inclusion bodies and thus is not feasible for industrial applications.ResultsWe expressed PFA in Nicotiana tabacum and found that plant-produced PFA forms functional aggregates with an accumulation level up to 3.4 g/kg FW (fresh weight) in field conditions. The aggregates are functional without requiring refolding and therefore have potential to be applied as homogenized plant tissue without extraction or purification. PFA can also be extracted from plant tissue upon dissolution in a mild reducing buffer containing SDS. Like the enzyme produced in P. furiosus and in E. coli, plant produced PFA preserves hyper-thermophilicity and hyper-thermostability and has a long shelf life when stored in lyophilized leaf tissue. With tobacco’s large biomass and high yield, hyper-thermostable α-amylase was produced at a scale of 42 kg per hectare.ConclusionsTobacco may be a suitable bioreactor for industrial production of active hyperthermostable alpha amylase.

Assessing the potential for interaction between the insecticidal activity of two genetically engineered cotton events combined by conventional breeding: An example with COT102 × MON 15985

Bollgard® III was developed by combining cotton events COT102 and MON 15985 through conventional breeding to improve efficacy against lepidopteran feeding damage. COT102 produces the Vip3Aa19 protein and MON 15985 produces the Cry1Ac and Cry2Ab2 proteins. COT102 × MON 15985 has also been bred with Roundup Ready Flex® cotton (MON 88913) that confers glyphosate tolerance. This study evaluated the activity of COT102 and MON 15985 and the combined activity of COT102 and MON 15985 against the cotton bollworm (CBW, Helicoverpa zea). COT102, MON 15985, COT102 × MON 15985 and COT102 × MON 15985 × MON 88913 have comparable Vip3Aa19 and/or Cry1Ac, Cry2Ab2 protein expression levels as determined by enzyme-linked immunosorbent assay. CBW demonstrated concentration-dependent growth inhibition after 7-days of feeding on lyophilized leaf tissue derived from COT102, MON 15985, COT102 × MON 15985 and COT102 × MON 15985 × MON 88913 incorporated into an artificial diet. Observed EC50 values for COT102 × MON 15985 and COT102 × MON 15985 × MON 88913 were comparable (≤4% deviation) with the predicted EC50 value under the assumption of additivity using the combined activity of COT102 and MON 15985. No interaction in biological activity between COT102 and MON 15985 is consistent with results from competition and ligand blotting assays that demonstrated that Vip3Aa does not inhibit the binding of either Cry1Ac or Cry2Ab2 and vice versa. The results from this study demonstrate that the activity of COT102 × MON 15985 against CBW is consistent with predictions of additivity.

Genetic diversity of turmeric germplasm (Curcuma longa; Zingiberaceae) identified by microsatellite markers

Turmeric (Curcuma longa) is a triploid, vegetatively propagated crop introduced early during the colonization of Brazil. Turmeric rhizomes are ground into a powder used as a natural dye in the food industry, although recent research suggests a greater potential for the development of drugs and cosmetics. In Brazil, little is known about the genetic variability available for crop improvement. We examined the genetic diversity among turmeric accessions from a Brazilian germplasm collection comprising 39 accessions collected from the States of Goiás, Mato Grosso do Sul, Minas Gerais, São Paulo, and Pará. For comparison, 18 additional genotypes were analyzed, including samples from India and Puerto Rico. Total DNA was extracted from lyophilized leaf tissue and genetic analysis was performed using 17 microsatellite markers (single-sequence repeats). Shannon-Weiner indexes ranged from 0.017 (Minas Gerais) to 0.316 (São Paulo). Analyses of molecular variance (AMOVA) demonstrated major differences between countries (63.4%) and that most of the genetic diversity in Brazil is found within states (75.3%). Genotypes from São Paulo State were the most divergent and potentially useful for crop improvement. Structure analysis indicated two main groups of accessions. These results can help target future collecting efforts for introduction of new materials needed to develop more productive and better adapted cultivars.

Expression and assembly of ApxIIA toxin of Actinobacillus pleuropneumoniae fused with the enterotoxigenic E. coli heat-labile toxin B subunit in transgenic tobacco

Actinobacillus pleuropneumoniae is a major etiological agent of swine pleuropneumonia, a highly contagious respiratory infection that causes severe economic losses in the swine production industry. ApxIIA, one of the virulence factors in A. pleuropneumoniae, is considered a candidate for the development of a vaccine against this bacterial infection. In this study, a fusion gene consisting of enterotoxigenic Escherichia coli heat-labile enterotoxin B subunit (LTB) and an ApxIIA fragment (amino acids 619–801) (ApxIIA619–801) was fused to the endoplasmic reticulum (ER) retention signal (SEKDEL) and introduced into a plant expression vector under the control of a ubiquitin promoter. The plant expression vector was transformed into tobacco Nicotiana tabacum L. cv. MD609 using an Agrobacterium-mediated transformation procedure. The integration and transcription of the LTB fusion gene were confirmed by genomic DNA PCR amplification and Northern blot analysis, respectively. The synthesis and assembly of LTB fusion protein were evidenced by Western blot analysis and the GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). A quantitative ELISA used to measure the amount of LTB fusion protein produced in the transgenic plant revealed high levels of the fusion protein, up to 30 μg g−1, in lyophilized leaf tissue. These results demonstrate the feasibility of using a transgenic plant to express the LTB-ApxIIA619–801 fusion protein as a first step towards producing plant-based edible vaccines to elicit immune responses against A. pleuropneumoniae via an oral mucosal delivery system.

Insufficient evidence for the discovery of transgenes in Mexican landraces

In the recently published article ‘Transgenes in Mexican maize: molecular evidence and methodological considerations for GMO detection in landrace populations’ by Pineyro-Nelson et al. (2009), the authors report the finding of transgenes in Oaxacan maize landraces and make several claims to which we feel compelled to respond. The authors maintain that testing of leaf tissues in general, and in particular at Genetic ID (GID), is inclined to false negative results and that the presence of metabolites in landraces could lead to the reporting of false negatives. Much of the data reported by Pineyro-Nelson et al. is from end point PCR analyses of leaf material from single plants. We suggest and provide evidence that, for a study of such importance, conclusions must be drawn from real time qPCR analyses. Genetically any given plant should be either non-transgenic or transgenic, therefore for leaf tissue of a single transgenic plant, a GMO level close to 100% is expected. In their study, the authors chose to classify leaf samples as transgenic despite GMO levels of 0.1%. We contend that results such as these are incorrectly interpreted as positive and are more likely to be indicative of contamination in the laboratory. The authors claim that analysis of lyophilized leaf tissue can lead to reporting of false negative results. Real time PCR analysis of dried leaf tissue samples from three transgenic plants (MON810, NK603, Bt11) verified that transgenic corn can be detected in such samples. Dried leaves were stored at GID for 3 years at room temperature prior to testing. DNA was extracted from 10 mg of leaf material, and the transgenic events were successfully detected using endpoint PCR. Subsequent analysis of the DNA by real time qPCR confirmed the expected GMO value of 100% for all three of the transgenic plants for the 35S promotor, NOS terminator and the specific event. Identical results were obtained from a MON810 dried leaf tissue positive control which the authors sent to us in a blinded fashion in 2005 (i.e. 100% GMO value for the 35S promoter target gene and correct identification of the MON810 event).

Fall Armyworm (Lepidoptera: Noctuidae) and Southwestern Corn Borer (Lepidoptera: Crambidae) Leaf Feeding Damage and Its Effect on Larval Growth on Diets Prepared from Lyophilized Corn Leaves

Fall armyworm, Spodoptera frugiperda (J.E. Smith), and Southwestern corn borer, Diatraea grandiosella Dyar, are economically important pests of maize (Zea mays L.) in the southeastern United States. These insects attack plants in both the vegetative and reproductive stage so f growth. Plant resistance is widely considered a desirable means for reducing losses to both insects, and corn germplasm lines with resistance to leaf feeding damage have been developed and released. Fall armyworm and southwestern corn borer larvae feeding on resistant genotypes grow more slowly than those feeding on susceptible genotypes. The objectives of the investigation were to evaluate 20 single cross maize hybrids for leaf feeding damage by southwest- ern corn borer and fall armyworm, to compare larval growth on laboratory diets prepared from lyophilized leaf tissue of single cross hybrids with different levels of resistance to leaf feeding, and to determine whether larval growth differed between diets prepared from leaf tissue collected from plants previously infested with southwestern corn borer or fall armyworm and non- infested plants. Both fall armyworm and southwestern corn borer larvae weighed significantly less when fed on laboratory diets prepared from lyophilized leaf tissue of resistant single cross hybrids than susceptible hybrids. When tissue from either resistant or susceptible plants that had been infested with either insect in the field was used in bioassays, larval weights were further reduced. It appears that both constitutive and induced defensive mechanisms may be operating.

Differential Growth of Fall Armyworm Larvae (Lepidoptera: Noctuidae) Reared on Three Phenotypic Regions of Whorl Leaves from a Resistant and a Susceptible Maize Hybrid

Two laboratory bioassays were conducted to determine the effect of feeding selected whorl leaf regions of a resistant and a susceptible maize, Zea mays L., hybrid on fall armyworm, Spodoptera frugiperda (J. E. Smith) growth. In one bioassay, larvae were fed fresh excised whorl leaf tissue and in the other, they were fed reconstituted diets containing ground lyophilized leaf tissue from three phenotypic leaf regions of both hybrids. Results of the two bioassays were similar. Differences in larval weights were found for those larvae fed tissue from leaf regions within and across hybrids. The largest differences among treatments were found within the resistant hybrid, thus showing that regions of the same whorl leaf differ in suitability of food source for larval growth.

Factors Associated with Resistance to Fall Armyworm (Lepidoptera: Noctuidae) and Southwestern Corn Borer (Lepidoptera: Crambidae) in Corn at Different Vegetative Stages

Corn, Zen mays L., germplasm with resistance to leaf feeding by southwestern corn borer, Diatraea grandiose/la Dyar, and fall armyworm, Spodoptera frugiperda (J. E. Smith), has been released. Resistant genotypes sustain less leaf-feeding damage than susceptible genotypes. Larvae that feed on plants of resistant genotypes or on tissue collected from these plants !!;row more slowly than those that feed on susceptible genotypes. Both resistant and susceptible genotypes sustain less damage as the plants mature. This investigation was undertaken to determine the associations between various chemical and physical characteristics of resistant and susceptible corn hybrids and leaf-feeding damage and larval growth on hybrids infested at different vagetative stages of growth. Leaf feeding was heavier on plants infested 28-35 d after planting than older plants. Larvae from the field weighed more when plants were infested between 28 and 35 d. Larvae fed on lyophilized leaf tissue in laboratory bioassays were heaviest when whorl tissue was collected from plants 28-35 d after planting. Larvae fed on tissue of resistant hybrids weighed less than those fed on tissue of susceptible hybrids. Resistant hybrids completed the transition from juvenile to adult earlier than susceptible hybrids. The 7th leaf was the 1st adult leaf of the resistant hybrids, but the 10th or 11th was the 1st adult leaf of susceptible hybrids. Leaves of susceptible hybrids were less tough than leaves of resistant hybrids, and toughness increased with plant age. Relative amounts of hemicellulose were higher in resistant genotypes and in older plants of all genotypes. Hemicellulose and protein levels appear to be associated with fall armyworm resistance, but not southwestern corn borer resistance. Relative amounts of cellulose were negatively correlated with leaf feeding by both insects. Relative amounts of ash, fat, and lignin did not appear to be associated with resistance. Further investigations of resistant and susceptible genotypes at different vegetative stages of growth should be useful in identifying factors associated with resistance to fall armyworm and southwestern corn borer.

Raffinose oligosaccharide concentrations measured in individual cell and tissue types in Cucumis melo L. leaves: implications for phloem loading.

Raffinose, stachyose, and galactinol are synthesized in intermediary cells (specialized companion cells) of the minor-vein phloem of cucurbits. To better understand the role of these carbohydrates and the regulation of their synthesis and transport, we measured the concentrations of each of the components of the raffinose oligosaccharide synthetic pathway in mesophyll and sieve element-intermediary cell complexes (SE-ICCs) in the leaves of melon (Cucumis melo L. cv. Hale's Best Jumbo). These concentrations are consistent with a polymer-trapping mechanism for phloem loading, with sucrose diffusing from mesophyll into intermediary cells and being made into raffinose and stachyose, which are too large to diffuse back to the mesophyll. To determine carbohydrate concentrations, we developed a method involving microdissected tissues. Blind endings of areoles, and mesophyll surrounding these veins, were separately removed from lyophilized leaf tissue. Carbohydrates were quantitated by high-performance liquid chromatography with pulsed amperometric detection. A small amount of mesophyll remained attached to the blind endings; the carbohydrate contribution of these cells to the vein sample was eliminated by subtraction, based on the amount of chlorophyll. Volumes of cells and subcellular compartments were calculated by morphometric analysis and were used to calculate carbohydrate concentrations. Assuming no subcellular compartmentation, the additive concentration of sugars in the SE-ICCs of minor veins is about 600 mM. Stachyose and raffinose concentrations are about 330 mM and 70 mM, respectively, in SE-ICCs; concentrations of these sugars are much lower in mesophyll (0.2 and 0.1 mM). This is consistent with the view that stachyose and raffinose are unable to pass through the plasmodesmata between intermediary cells and bundle-sheath cells. Sucrose levels appear to be higher in the SE-ICC (about 130mM) than in the mesophyll (about 10 mM), but if compartmentation is taken into account the gradient for sucrose is probably downhill from mesophyll to intermediary cells. Flux through plasmodesmata between the bundle sheath and intermediary cells was calculated and was found to be within the range of values of flux through plasmodesmata reported in the literature.

Combining ability in maize for fall armyworm and southwestern corn borer resistance based on a laboratory bioassay for larval growth

The fall armyworm, Spodoptera frugiperda (J. E. Smith), and southwestern corn borer, Diatraea grandiosella Dyar, are major insect pests of maize, Zea mays L., in the southern USA. Both insects feed extensively on leaves of plants in the whorl stage of growth. A diallel cross of seven inbred lines with different levels of susceptibility to leaf feeding damage in the field was evaluated in a laboratory bioassay for fall armyworm and southwestern corn borer larval growth. Diets were prepared from lyophilized leaf tissue of field-grown plants of the inbred lines and their 21 F1 hybrids. One inbred line, Tx601, exhibited heavy leaf damage in field tests but showed moderate resistance in the laboratory bioassay. Both general and specific combining ability were highly significant sources of variation in the inheritance of fall armyworm and south-western corn borer larval growth in the laboratory bioassay. Tx601 showed excellent general combining ability for reduced larval growth of both species.

Growth of Fall Armyworm (Lepidoptera: Noctuidae) Larvae on Resistant and Susceptible Corn

Growth of larvae of fall armyworm, Spodoptera frugiperda (J. E. Smith), was evaluated in a laboratory bioassay on diets containing lyophilized leaf tissue of resistant and susceptible inbred lines of corn, Zea mays L. Diets were prepared by adding lyophilized tissue of each of six inbred lines alone and in pairs to distilled water, agar, gentamicin sulfate, sorbic acid, and ascorbic acid. Larvae were fed the test diets for 10 d and then weighed. Larvae that were fed the diets containing leaf tissue of the resistant plants weighed 52% less than those fed diets containing only susceptible plant tissue. Growth of larvae on diets containing mixtures of leaf tissue from different inbred lines revealed that the variation among diets was accounted for by the average performance of different lines.

Partial characterization of guard-cell phospho enolpyruvate carboxylase: Kinetic datum collection in real time from single-cell activities

Maximum velocity and K m(PEP·Mg) of phospho enolpyruvate carboxylase (PEPC) from stomatal guard cells of Vicia faba L. were determined as a function of pH, presence of malate, and physiological state of guard cells. The biochemical rationale for these measurements is that (a) massive proton extrusion from guard cells, the primary event that drives stomatal movements, has been speculated to alkalinize the cell; (b) guard-cell malate concentration increases severalfold on stomatal opening, and malate, generally an inhibitor of PEPC's, affects the oligomeric state of some PEPC's; and (c) the apparent in vivo activity of guard-cell PEPC is greatly enhanced during stomatal opening, compared with that of other physiological states of these cells. As there are precedents for cell-specific expression of particular forms of PEPC and for labile reversible, posttranslational modifications (which are manifested kinetically as distinct physiological-state isoforms), individual assays were initiated on the addition of a single stomatal complex directly to a microdroplet of assay cocktail. The stomatal complexes (each of which comprises a pair of guard cells having a mass of 6 × 10 −9 g) were dissected from lyophilized leaf tissue that had been freeze-quenched either before, during, or after a treatment to open stomata. V max at pH 7.0 was not significantly different from that at pH 8.5. Neither V max nor K m(PEP·Mg) was distinguished on the basis of the physiological state of the tissue from which the enzyme was extracted. However, K m(PEP·Mg) was >4 × lower at pH 8.5 than at pH 7.0. Malate inhibition was competitive at both pH's, but inhibition was >3 × greater at the lower pH. These data indicate that the combined effects of pH and malate over the range studied can produce changes in enzyme velocity of approximately 24-fold. Thus, the results are consistent with an interpretation that guard-cell PEPC is regulated by the cytoplasmic chemical environment and not by alternations between physiological-state isoforms.