41st Annual Meeting of the Society for Invertebrate Pathology
and 9th International Conference on Bacillus thuringiensis

Spodoptera litura is a major pest attacking important commercial crops like cotton in India. Commercial Bt cotton hybrids carrying Cry1Ac toxin (Bollgard I type) or Cry1Ac and Cry2Ab toxins (Bollgard II type) give adequate control of the target insect Helicoverpa armigera, however there is a common opinion among farmers that S. litura is an emerging problem in Bt cotton. There is an urgent need to screen for more toxic holotype and/or hybrid Cry proteins against S. litura to minimise the use of chemical insecticides in Bt cotton, the main objective of transgenic cotton technology. In laboratory screening experiments G27, the EEC hybrid toxin producing strain, was more toxic than other holotypes (1Ca and 1Fa) and hybrids (AbAbC and AcAcC) tested.

The use of transgenic Bt-maize is increasing yearly (last year accounting for about 19% of the total maize planted area in the world) because of the efficient control of the corn borers, in especial Ostrinia nubilalis . Resistance to Bacillus thuringiensis (Bt) insecticidal toxins has been linked to the 12-domain cadherin locus in 3 lepidopteran species. The O. nubilalis cadherin gene has been revealed as a complex gene of about 20 kbp in length, with 34 introns. In the present work, we have studied the size polymorphism of the gene in a Spanish population, by amplifying the genomic sequence of the gene in 16 overlapping regions. The variability observed was not uniformly distributed, with a maximum in region 14 and a minimum (no polymorphism) in region 4. All this size variability must be due to changes in the intronic regions because we found no detectable size differences in mRNA. This variability can be useful to select appropriate polymorphic regions to be used as markers of this gene in experiments such us to determine the genetic linkage of the cadherin to Bt resistance traits.

This work describes a novel strategy for cloning cry genes from Bacillus thuringiensis by constructing library with B. thuringiensis as host and shuttle vector pHT304 as cloning vector and then screening by checking the formation of crystals. B. thuringiensis strain YBT-1518 shows toxicity against root-knot nematode and produces 54kDa and 45kDa crystal proteins. Eight out of three hundred colonies were found to produce crystals and its crystal proteins showed toxicity to nematode, Meloidogyne hapla. Seven colonies formed the same rice-shaped crystal as strain YBT-1518 does, while the other one did typical bipyramidal crystal. The rice-shaped crystals consisted of either 54kDa protein or 45kDa protein, while the crystal protein of the bipyramidal crystal was estimated 140kDa. The 45kDa crystal protein is encoded by a novel gene, formly cry55Aa1, which has not any significant homology to any cry genes. The 54kDa protein was encoded by cry6Aa2. Surprisingly, the 140kDa protein was the product of gene cry5Ba2. There is neither this 140kDa protein in the crystal protein contents nor the bypyramidal crystal in the sporulation culture. The gene cloning strategy described in this work provides a novel way to isolate novel and/or silent crystal protein genes from B. thuringiensis.

Based on the known cry gene sequences of B. thuringiensis, three pairs of primers were designed from the 5 conserved blocks found in the delta-endotoxin coding region. Designed primer pairs amplify the regions between blocks 1 and 5, 2 and 5, and 1 and 4, respectively. In silico analyses indicated that up to 96% of the known sequences can be amplified by one or more of these pairs. Their ability to detect new cry genes was tested when DNA from B. thuringiensis strains showing atypical crystal morphology was used as template. Some 175 strains recorded as “atypical” in the CINVESTAV-IPN (LBIT-series) collection log were further selected by phase contras microscopy, SDS-PAGE, and SEM analyses. After a systematic amplification and sequencing of amplicons obtained from 27 strains, 5 putative cry genes showed highest identities between 25 and 43%; and 4 more between 63 and 69%, to known cry genes. Complete sequencing of new cry genes is in an advanced phase.

A new approach to the study of the diversity of natural microbial communities is to analyze PCR products generated with primers homologous to relatively conserved regions in the genome through denaturing gradient gel electrophoresis (DGGE). This methodology allows the separation of DNA molecules that differ by single bases and therefore has the potential to provide information about variations in target genes in bacterial populations in natural systems. In this study, we modify a two-step PCR-based approach. The strategy allowed us the amplification of unknown Bacillus thuringiensis cry-related sequences present in a single strain. In a first step we used a primer pair of the cry genes-specific PCR system, based on the degenerate primers (OL1 and OL5). The obtained amplicons were used in a second (semi-nested) PCR for DGGE, in which cry degenerate primers OL3GC and OL5 were used. The resulting products were separated after DGGE. Each stained band should correspond to a single cry gen. The DGGE assay developed here provides a rapid and reliable way to analyze the genetic diversity of cry genes present in a single strain of B. thuringiensis in pure cultures, as well as in environmental samples.

Hemipteran pests are not susceptible to the effects of known Bt toxins and have replaced the Lepidoptera as primary pests on Bt transgenic crops. Ideally, a strategy similar to the Bt transgenic plant technology could be applied for management of hemipteran pests. The objective of our current study is to delineate the physiological basis for the lack of insecticidal effect of the Bt toxins Cry1Ac and Cry3Aa on the pea aphid. We have shown that: (1) Both protoxins were stable in acidic buffers. (2) On treatment with cathepsin L, activated Cry1Ac was stable but Cry3Aa was digested to a single peptide of less than 20 kDa . (3) When incubated with membrane extracts from the pea aphid stomach, the Cry1Ac and Cry3Aa protoxins were hydrolyzed to a peptide with a molecular mass similar to that of the trypsin-activated toxins. This hydrolysis took 3 or 16 hr in the presence or absence of cysteine proteinase activators respectively. These results suggest that Cry protoxins are stable in the aphid foregut but could only be activated in the stomach of the pea aphid. The potential binding, oligomerization and insertion of Cry toxins into the aphid gut remain to be examined.

The main characteristic of Bacillus thuringiensis (Bt) is the formation of protein crystals during their sporulation phase. It is the most commonly used bacterium in the biological control of insect larvae of agricultural pests and disease vectors. Endophytic bacteria are important due to their potential to be used in the control of insect larvae that feed on plants and/or live in their interior. Approximately 800 endophytic bacteria were isolated from sugar cane and there are stocked at the Laboratory of Microbial Genetics (ESALQ/USP- Brazil). Among them, 43 isolates were classified as Bacillus spp. by their colony morphology. Observation of a parasporal crystal by optical microscopy revealed that one of the isolates was B. thuringiensis (CTH31RzB4). This strain has been characterized by means of optical and electron microscopy, protein SDS-PAGE profile, cry, cyt, and vip gene content, and toxicity assays against Lepidoptera larvae.

The general features of Bacillus thuringiensis Cry toxins mode of action in lepidopteran larvae have been clarified, but the molecular events that occur after ingestion and solubilization are not completely characterized. This characterization is pivotal to prevent and actively combat the development of resistance in insects exposed to pesticides based on B. thuringiensis products including GM crops. We have analyzed the effect of Cry1Ac toxin in terms of binding parameters and permeabilization capacity on brush border membrane vesicles (BBMV) prepared from the anterior and the posterior part of Manduca sexta and Helicoverpa armigera larval midgut. Cry1Ac bound specifically to BBMV from both larvae and no significant differences were detected in the binding parameters between the anterior and posterior regions within species. In contrast, in both species the permeabilization activity, measured by means of a voltage-sensitive dye, was significantly higher in the posterior region. We have also analyzed the inhibition of binding and pore formation by the sugar GalNac, a key residue in some membrane receptors. In the presence of GalNac, differences between anterior and posterior midgut regions and between species were detected.

A two-choice disc test was used to examine the effect of Cry3A expression on host-plant preference. Discrimination between a transgenic potato, Solanum tuberosum (Solanaceae) cv. Superior NewLeaf (Monsanto, USA) capable of synthesizing Bacillus thuringiensis toxin, and an isogenic cultivar was studied using the two-spotted spider mite, Tetranychus urticae (Acari: Tetranychidae) under laboratory conditions. Adult females of spider mites were individually placed on leaf discs (one half transgenic and one half control) and observed at regular intervals. In addition, the distribution of T. urticae eggs on the discs was recorded. T. urticae females were found more frequently on control leaves than on transgenic leaves. The distribution of spider mite eggs reflected the observed biased distribution of females. These results indicate that potatoes expressing Bt for resistance against Colorado potato beetle are less preferred by spider mites under a choice test condition using excised leaves.

Bacillus thuringiensis as bioinseticide has incited to significant knowledge on Cry proteins. However, the bacterium ecology remains poorly understood. Thus, a tractable B. thuringiensis developed for basic researches could help plant-microbe interactions studies, as well as, gene expression pattern in response to a particular environment. We constructed strain S76GFP+ by electrotransferring a green fluorescence (gfp) gene expression vector (pAD43-25) to strain S76, a Brazilian wild type B. thuringiensis kurstaki, containing ca. eleven plasmids¾two of then bearing five cry genes. Fluorescence microscopy showed green streptobacillus, as early as, two hours after inoculation in liquid medium containing the pAD43-25 selection marker. Interestingly, although the gfp gene expression is constitutively regulated in vector pAD43-25, the Green Fluorescence Protein (GFP) could be detected throughout the entire cell cycle and, even, green free spores were noticed. Besides GFP synthesis, S76GFP+ also maintained cry genes expression, as observed by SDS-PAGE. The present study revealed that after about 80 generations of S76GFP+ cells grown on rich and sporulation media, with no selective pressure, were still able to maintain Cry proteins and GFP production, as accessed by SDS-PAGE and fluorescence, directly scanned, respectively. These results indicate that our marker B. thuringisensis is a useful tool to study the biology of this bacterium.