Cloning and Characterization of a Cytolytic and Mosquito Larvicidal δ-Endotoxin from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> subsp. <Emphasis Type="Italic">darmstadiensis</Emphasis>

The cytolytic δ-endotoxin gene from Bacillus thuringiensis subsp. darmstadiensis was amplified from genomic DNA by PCR by using primers designed from the sequence of cyt2Aa1 gene of B. thuringiensis subsp. kyushuensis. DNA sequence analysis revealed an open reading frame translating to a 259-amino acid sequence. The cloned gene was designated cyt2Aa2. This gene was highly expressed in Escherichia coli as inclusion bodies that could be solubilized in 50 mM Na₂CO₃, pH 10.5. Activation of the solubilized protoxin by proteinase K (1% wt/wt, proteinase K/protoxin) yielded the active fragment of about 23 kDa. Cyt2Aa2 showed high hemolytic activity against sheep erythrocytes (hemolytic end- point 0.25 μg/ml) and was toxic to Culex quinquefasciatus and Aedes aegypti larvae (LC₅₀ 0.5–1.0 μg/ml). Received: 27 March 2002 / Accepted: 30 April 2002     

Selective Feeding of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> (Hübner) and <Emphasis Type="Italic">Spodoptera litura</Emphasis> (Fabricius) on Meridic Diet with <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Toxins

Laboratory experiments were conducted to evaluate the behavior of Helicoverpa armigera (Hübner) and Spodoptera litura (Fabricius) larvae on meridic diet with different concentrations of Bt spray formulation Delfin or isolated Cry1Ac protein or the foliage and bolls from transgenic cotton, Bollgard hybrid RCH-317 Bt. Both insect species selectively fed on nontreated diet compared with the diet treated with Delfin. While H. armigera exhibited concentration response with Cry1Ac, this protein did not affect S. litura larvae. In general Helicoverpa selected diet with low concentrations (EC₂₀ and EC₅₀ levels) of Cry1Ac compared with higher concentrations of Cry1Ac. In order to develop appropriate management strategies, a thorough understanding of the behavioral mechanisms leading to the responses of insects to the proteins in transgenic varieties is required. Thus, based on results of the insects fed individually on the leaf discs or bolls from transgenic cotton plants alone or under choice situation with meridic diet revealed that H. armigera larvae preferred meridic diet to transgenic leaves or bolls expressing Cry1Ac protein. H. armigera larvae preferred meridic diet to plant material; more than 70% larvae were seen on the meridic diet, and average larval weight gain was in the range of 121.7–130.5 mg. However, in case of S. litura the larvae showed no significant discrimination between meridic diet and the leaf discs. In fact more than 60% larvae preferred leaf discs for feeding, though Cry1Ac expression in leaf discs was in the range of 0.9–2.18 μg/g. Thus differences in behavioral response could potentially impact the level of efficacy of crop cultivars that have been genetically engineered to produce these proteins.     

Interaction of Two <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> δ-Endotoxins with the Digestive System of <Emphasis Type="Italic">Lygus hesperus</Emphasis>

The active-toxin form of Cry1Ac (65 kDa) or Cry2Ab was fed to a non-susceptible insect, Lygus hesperus, in an artificial diet. Biochemical and immunocytochemical methods were used to determine the distribution of ingested toxin. The toxins did not elicit a feeding deterrent response. Cry1Ac and Cry2Ab were ingested; small amounts were absorbed into the hemolymph as holoproteins, but most was excreted. SDS-PAGE analysis of Cry1Ac and Cry2Ab incubations with salivary gland homogenate showed a small decrease in the molecular weight of the active toxins. Proteolytic processing of the toxins also occurred in vivo, within the digestive system of L. hesperus. Excreted Cry1Ac and Cry2Ab retained activity toward lepidopteran larvae. Immunocytochemical in vivo localization studies showed negligible association of Cry1Ac with L. hesperus tissues. In contrast, strong extracellular association of Cry2Ab was observed with L. hesperus midgut brush border microvilli and basement membrane, as well as with cellular outlines within the hemolymph and fat body.     

Germinant Generation from δ-endotoxin of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strain 1.1

The novel finding of this study is that the δ-endotoxin present in the spore coat of Bacillus thuringiensis strain 1.1 (Bt1.1), plays a central role in spore germination by generation of germinant via its β-glucosidase activity and is based on the following: (i) the crystals of Bt1.1 consist of the 140 kDa δ-endotoxin which exhibits β-glucosidase enzymatic activity. Besides crystals, δ-endotoxin is also located in the spore coat and at this site displays β-glucosidase activity, resulting in glucose production; (ii) glucose is an efficient germinant of both Bt1.1 and acrystalliferous Bt4.1 strain; (iii) substrates of β-glucosidase can activate the germination of Bt1.1 spores, but not those of the acrystalliferous Bt4.1 sister strain that do not contain the 140 kDa δ-endotoxin; (iv) Reduction or enhancement of enzymatic activity of δ-endotoxin, results in retardation or acceleration of germination and outgrowth, respectively. Bt1.1 cells secrete a 60 kDa polypeptide which displays β-glucosidase activity as indicated by zymogram analysis and which is immunologically related to the 140 kDa δ-endotoxin.     

Biochemical and molecular characterization of δ-endotoxins in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

The δ-endotoxins (δ-ETX) of four native strains (RT7, RT19, RT25, and RT25), and one reference strain (4L1) of Bacillus thuringiensis were biochemically and molecularly characterized to determine their potential toxic activity against lepidopteran larvae. Crystals of δ-ETX were purified through a two-phase system to determine their morphology, molar mass, solubility, and resistance to proteinases. Toxic activity and cry gene content were also determined. Crystals from native strains exhibited polyhedral, irregular and cuboidal shapes, while those from 4L1 were bipyramidal. Seven proteins with estimated molar mass ≈30–134 kDa were detected as the main components of the native δ-ETX. Only crystals from 4L1, RT24, and RT25 underwend complete solubilization at pH >12.0. Crystals from all strains produced trypsinresistant peptides. None of the cry genes associated with toxicity in lepidopterans (cry1, cry2, cry9) was found in the native strains; however, 4L1 strain harbors cry1 and cry2 genes. Strains RT19 and RT25 caused significant mortality against Trichoplusia ni larvae with partial solubilization at pH 10, strain 4L1 caused 100 % mortality. Toxicity of native strains may come from a novel cry gene.     

Isolation and Characterization of a Strain of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Subsp. <Emphasis Type="Italic">morrisoni</Emphasis> PG-14 Encoding δ-Endotoxin Cry1Ac

Bacillus thuringiensis 656-3, isolated from a soil sample collected at mushroom houses, showed high toxicity to mushroom flies, Lycoriella mali and Coboldia fuscipes. B. thuringiensis 656-3 produced bipyramidal inclusions and reacted with the H antiserum of B. thuringiensis subsp. morrisoni (H8a8b). The plasmid and protein profiles of B. thuringiensis 656-3 were similar to those of its reference strain, subsp. morrisoni PG-14. However, PCR analysis using cry gene primers showed that B. thuringiensis 656-3, unlike its reference strain, had cry4A, cry4B, cry10A, cry11A, and cry1Ac genes, suggesting that B. thuringiensis 656-3 was a unique strain with respect to gene type. In addition, B. thuringiensis 656-3 showed a high level of toxicity against mushroom flies, L. mali and C. fuscipes.     

Activity of wild-type and hybrid<Emphasis Type="Italic"> Bacillus thuringiensis</Emphasis> δ-endotoxins against<Emphasis Type="Italic"> Agrotis ipsilon</Emphasis>

Twelve Cry1 and two Cry9 delta-endotoxins from Bacillus thuringiensis were tested for their activity against black cutworm ( Agrotis ipsilon). A. ipsilon was not susceptible to many toxins, but three toxins had significant activity. Cry9Ca was the most toxic, followed by Cry1Aa and Cry1Fb. Hybrids between these three active proteins were made by in vivo recombination and analyzed for activity against A. ipsilon. Analysis of hybrids between Cry1Aa and Cry1Fb indicated that domain I of Cry1Aa protein was involved in its higher activity.     

A new Tunisian strain of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis><Emphasis Type="Italic">kurstaki</Emphasis> having high insecticidal activity and δ-endotoxin yield

BLB1 is a new Bacillus thuringiensis kurstaki strain, isolated from a Tunisian soil sample. Assay of toxicity of BLB1 crystal proteins resulted in an LC50 of 70.32 ng of toxin per mg of flour against third instar Ephestia kuehniella with confidence limits of (31.6–109.04 ng). This LC50 is less than that of the commercial strains HD1 used as a reference. The characterization of this strain by scanning transmission electron microscopy, analysis of its cry genes content by PCR-sequencing, and analysis of its δ-endotoxin patterns demonstrate that it belongs to the same subgroup than HD1, but ruled out the involvement of cry gene content or protoxin activation in the hypertoxicity of this strain. Taking into account the δ-endotoxin/spore ratio for each strain, and by allowing the estimation of the production level per spore, it might be concluded that BLB1 production is the highest, when compared with that of HD1. On the basis of its toxicity, BLB1 could be considered as a strain of great interest and would allow the production of quantities of bioinsecticides at low cost.     

Morphological Characterization of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> (Hübner) (Lepidoptera: Noctuidae: Heliothinae)

The cotton bollworm Helicoverpa armigera (Hübner) is a widespread lepidopteran pest found in various crops worldwide. This highly polyphagous species, commonly found both in the Old and New World, has caused significant economic damage as an invasive agricultural pest in Brazil since 2013. The goal of the present study is to provide a detailed morphological assessment of adults and immature stages of H. armigera, as this species is often confused with H. zea (Boddie), a congeneric species that is native to the New World. The biology data were acquired during four full life cycles, and observations on general behavior, nocturnal habits of larvae and adults, and sensitivity of larvae to humidity were recorded. Larval chaetotaxy differs between the first and the remaining instars, which bear L2 on the meso- and metathorax and L3 on A3 through A6, along with conspicuous chalazae and longitudinal bands. Important morphological characters of this species include the following: eggs with four micropylar openings, lined with 12 cells arranged in the shape of a rosette; pupa adecticous and obtect, with prominent spiracles; adults with the distal antennomere striate. Adults exhibit sexual dimorphism in the number of setae on the frenulum and spines on the prothoracic leg. Illustrations of the critical morphological features of this species are provided.     

Brood cell size of <Emphasis Type="Italic">Apis</Emphasis> <Emphasis Type="Italic">mellifera</Emphasis> modifies the reproductive behavior of <Emphasis Type="Italic">Varroa</Emphasis> <Emphasis Type="Italic">destructor</Emphasis>

We undertook a field study to determine whether comb cell size affects the reproductive behavior of Varroa destructor under natural conditions. We examined the effect of brood cell width on the reproductive behavior of V. destructor in honey bee colonies, under natural conditions. Drone and worker brood combs were sampled from 11 colonies of Apis mellifera. A Pearson correlation test and a Tukey test were used to determine whether mite reproduction rate varied with brood cell width. Generalized additive model analysis showed that infestation rate increased positively and linearly with the width of worker and drone cells. The reproduction rate for viable mother mites was 0.96 viable female descendants per original invading female. No significant correlation was observed between brood cell width and number of offspring of V. destructor. Infertile mother mites were more frequent in narrower brood cells.     

Functional expression of the lipase gene Lip2 of <Emphasis Type="Italic">Pleurotus</Emphasis> <Emphasis Type="Italic">sapidus</Emphasis> in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically active lipase from a basidiomycete fungus.     

Co-expression of<Emphasis Type="Italic"> α</Emphasis>(1,3)galactosyltransferase and <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> PIPLC enhances hyperacute rejection of tumor cells

The use of α(1,3)galactosyltransferase (αGT) as a method of inducing hyperacute rejection of tumors has been gaining interest recently. However, the approach is based in part on the sensitivity of each tumor line to the effects of complement lysis. Tumors expressing complement resistance factors such as membrane cofactor (CD46), decay accelerating factor (CD55) and protectin (CD59) have been shown to be more resistant to complement mediated lysis. Anchored to the membrane by a glycosylphosphoinositol moiety (GPI-anchored), CD55 and CD59 can be cleaved by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PIPLC). Complement resistant A549 human lung carcinoma cells were engineered to express both the murine αGT gene and the B. thuringiensis PIPLC gene to alleviate complement resistance and enhance αgal-mediated cancer killing. The PIPLC native signal sequence was replaced with the human epidermal growth factor signal sequence, EGFssPIPLC, to induce secretion from A549. Expression of EGFssPIPLC resulted in complete removal of CD55 and CD59 while sparing the non-GPI-anchored CD46. Results demonstrated that A549 cells transduced with two recombinant retroviral vectors carrying the αGT and EGFssPIPLC genes expressed high levels of αgal epitope and exhibited a 5-fold increase in sensitivity to anti-αgal mediated complement lysis.     

Rapid induction of <Emphasis Type="Italic">Agrobacterium</Emphasis> <Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transgenic roots directly from adventitious roots in <Emphasis Type="Italic">Panax</Emphasis> <Emphasis Type="Italic">ginseng</Emphasis>

Root segments from seedlings of Panax ginseng produced adventitious roots directly when cultured on 1/2 MS solid medium lacking NH₄NO₃ and containing 3.0 mg l⁻¹ IBA. Using this adventitious root formation, we developed rapid and efficient transgenic root formation directly from adventitious root segments in P. ginseng. Root segments were co-cultivated with Agrobacterium tumefaciens (GV3101) caring β-glucuronidase (GUS) gene. Putative transgenic adventitious roots were formed directly from root segments on medium with 400 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin. Kanamycin resistant adventitious roots were selected and proliferated as individual lines by subculturing on medium with 300 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin at two weeks subculture interval. Frequency of transient and stable expression of GUS gene was enhanced by acetosyringon (50 mg l⁻¹) treatment. Integration of transgene into the plants was confirmed by the X-gluc reaction, PCR and Southern analysis. Production of transgenic plants was achieved via somatic embryogenesis from the embryogenic callus derived from independent lines of adventitious roots. The protocol for rapid induction of transgenic adventitious roots directly from adventitious roots can be applied for a new Agrobacterium tumefaciens-mediated genetic transformation protocol in P. ginseng.     

Insoluble but enzymatically active <Emphasis Type="Italic">α</Emphasis>-amylase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

The gene encoding thermostable α-amylase from Bacillus licheniformis consisting of 483 amino acid residues (mature protein) was cloned and expressed in Escherichia coli under the control of T7 promoter. The analysis of the soluble and insoluble fractions after lyzing the host cells revealed that recombinant α-amylase was produced in insoluble aggregates. Despite being produced in the insoluble aggregates the recombinant enzyme was highly active with a specific activity of 408 U/mg.     

Cry3Aa11: A New Cry3Aa δ-Endotoxin from a Local Isolate of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

A local isolate of Bacillus thuringiensis Mm2 had insecticidal activity against the larvae of Melolontha melolontha, Agelastica alni, Leptinotarsa decemlineata and Amphimallon solstitiale and produced a 65 kDa protein. SDS-PAGE profile of B. thuringiensis Mm2 was compared with those of 29 different Cry3Aa producers which verified Cry3Aa biosynthesis by the isolate. The cry3Aa gene of Mm2 was cloned, sequenced and the deduced amino acid sequence was compared with the cry3Aa sequences of ten different quaternary ranks. Its identity to these sequences ranged between 97.4% and 99.2%. The gene was next cloned into E. coli-Bacillus shuttle vector pNW33N and expressed at a low level in B. subtilis 168.     

<Emphasis Type="Italic">Carex</Emphasis> × <Emphasis Type="Italic">involuta</Emphasis> and <Emphasis Type="Italic">Carex juncella</Emphasis> in the flora of Slovakia

The paper brings information on an isolated occurrence and morphological characters of Carex × involuta and C. juncella populations in the Vel’ká Fatra Mts. Their presence has been known neither from the territory of Slovakia nor from the whole Western Carpathians till now.     

Cryopreservation of cell suspension cultures of <Emphasis Type="Italic">Taxus</Emphasis> × <Emphasis Type="Italic">media</Emphasis> and <Emphasis Type="Italic">Taxus floridana</Emphasis>

Different lines of cell suspension cultures of Taxus × media Rehd. and Taxus floridana Nutt. were cryopreserved with a two-step freezing method using a simple and inexpensive freezing container instead of a programmable freezer. Four to seven days old suspension cell cultures were precultured in growth medium supplemented with 0.5 M mannitol for 2 d. The medium was then replaced with cryoprotectant solution (1 M sucrose, 0.5 M glycerol and 0.5 M dimethylsulfoxide) and the cells incubated on ice for 1 h. Before being plunged into liquid nitrogen, cells were frozen with a cooling rate of approximately −1 °C per min to −80 °C. The highest post-thaw cell viability was 90 %. The recovery was line dependent. The cryopreservation procedure did not alter the nuclear DNA content of the cell lines. The results indicate that cryopreservation of Taxus cell suspension cultures using inexpensive freezing container is possible.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Codonopsis lanceolata</Emphasis> using the <Emphasis Type="Italic">γ-TMT</Emphasis> gene

Efficient transformation of leaf disc-derived callus of Codonopsis lanceolata was obtained using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector, pYBI121, that carries the neomycin phosphotransferase (npt II) gene as a selectable marker. The green shoots recovered from agroinfected explants on selection medium (containing 0.1 mg/l α-naphthaleneacetic acid (NAA), 1 mg/l 6-benzylaminopurine (BAP), 100 mg/l kanamycin, and 250 mg/l cefotaxime) were rooted on Murashige and Skoog (MS) medium supplemented with 2 mg/l IBA and 10 mg/l kanamycin. To optimize the transformation conditions, several factors were assessed, including the co-cultivation period, the duration of pre- and post-culture in darkness and light, the kanamycin concentration, and the Agrobacterium densities. We produced transgenic Codonopsis lanceolata overexpressing γ-tocopherol methyltransferase (γ-TMT) by this protocol. Moreover, the α-tocopherol content of the plants was enhanced by the overexpression of this gene. Bimal Kumar Ghimire and Eun Soo Seong contributed equally to this work.