Spore-crystal Mixture Research Articles

Proteome Response of Tribolium castaneum Larvae to Bacillus thuringiensis Toxin Producing Strains

Susceptibility of Tribolium castaneum (Tc) larvae was determined against spore-crystal mixtures of five coleopteran specific and one lepidopteran specific Bacillus thuringiensis Cry toxin producing strains and those containing the structurally unrelated Cry3Ba and Cry23Aa/Cry37Aa proteins were found toxic (LC50 values 13.53 and 6.30 µg spore-crystal mixture/µL flour disc, respectively). Using iTRAQ combined with LC-MS/MS allowed the discovery of seven novel differentially expressed proteins in early response of Tc larvae to the two active spore-crystal mixtures. Proteins showing a statistically significant change in treated larvae compared to non-intoxicated larvae fell into two major categories; up-regulated proteins were involved in host defense (odorant binding protein C12, apolipophorin-III and chemosensory protein 18) and down-regulated proteins were linked to metabolic pathways affecting larval metabolism and development (pyruvate dehydrogenase Eα subunit, cuticular protein, ribosomal protein L13a and apolipoprotein LI-II). Among increased proteins, Odorant binding protein C12 showed the highest change, 4-fold increase in both toxin treatments. The protein displayed amino acid sequence and structural homology to Tenebrio molitor 12 kDa hemolymph protein b precursor, a non-olfactory odorant binding protein. Analysis of mRNA expression and mortality assays in Odorant binding protein C12 silenced larvae were consistent with a general immune defense function of non-olfactory odorant binding proteins. Regarding down-regulated proteins, at the transcriptional level, pyruvate dehydrogenase and cuticular genes were decreased in Tc larvae exposed to the Cry3Ba producing strain compared to the Cry23Aa/Cry37Aa producing strain, which may contribute to the developmental arrest that we observed with larvae fed the Cry3Ba producing strain. Results demonstrated a distinct host transcriptional regulation depending upon the Cry toxin treatment. Knowledge on how insects respond to Bt intoxication will allow designing more effective management strategies for pest control.

Toxicity of native Bacillus thuringiensis isolates on the larval stages of pine processionary moth Thaumetopoea wilkinsoni at different temperatures

Pine processionary moth, Thaumetopoea wilkinsoni Tams, is an important defoliating lepidopteran pest of pine trees. The aim of this study was to determine the required spore-crystal concentration of local Bacillus thuringiensis (Bt) isolates, optimal ambient temperature, and larval stage to control this troublesome forest pest. The susceptibility of T. wilkinsoni larvae decreased with older stage and lower temperatures. The optimum temperature was found to be 15 °C or higher for the control of early larval stages. At the highest spore-crystal concentration (500 µg g^{-1}), the most effective isolate (SY49.1) caused 83% mortality for the second-stage larvae at 5 °C. However, an approximately 4-fold decrease in mortalities was observed in late-stage larvae for all isolates examined at this temperature. Nevertheless, other Bt isolates, excluding SY27.3, caused nearly complete mortality at 25 °C for early-stage larvae. Considering the distribution of seasonal temperature, Bt products should be applied at the highest ambient temperature to earlier stages for efficient control. We propose that local Bt isolates SY27.1, SY49.1, and SY62.1 could be used to develop environmentally safe bioinsecticides to control this important pest species. These results indicate that larval stage and environmental temperature should be taken into consideration for efficient control of T. wilkinsoni using the spore-crystal mixture of Bacillus thuringiensis isolates.

A novel Bacillus thuringiensis strain and its pathogenicity against three important pest insects

A highly pathogenic Bacillus thuringiensis (Bt) strain was isolated from a soil sample in Turkey and characterized in terms of both its 16S-ITS rDNA region and cry gene content. This strain (SY49.1) harboured several cry genes producing crystalline inclusions known to have toxicity on lepidopteran, dipteran and coleopteran pests. The 16S-ITS sequence analysis of Bt SY49.1 showed 98% similarity to Bt serovar andalousiensis BGSC 4AW1. The larvae of Ephestia kuehniella and Plodia interpunctella were treated with a spore-crystal mixture of this strain in the dose range of 50–1000 μg g−1. Mortality rates were higher than 90% at the highest concentration for these pests. For Tribolium castaneum much higher concentrations were needed than with lepidopteran pests larvae. At the highest concentration tested of 10 mg g−1, about 62% mortality was obtained. It was concluded that this native strain of Bt could be used as an effective biocontrol agent against various lepidopteran pests.

Isolation and characterization of native Bacillus thuringiensis isolates from Syrian soil and testing of their insecticidal activities against some insect pests

Bacillus thuringiensis was detected in 12.5% of soil samples collected from different regions in Syria and 25 B. thuringiensis isolates were found to be highly toxic to larvae of Ephestia kuehniella Zeller, Phthorimaea operculella Zeller, and Cydia pomonella L. (Lepidoptera), but not to the larvae of Culex quinquefasciatus (Diptera). Light microscopy investigation showed the presence of bipyramidal and cuboidal parasporal bodies produced by these isolates. The existence of different cry genes in the tested isolates was studied using a PCR strategy with a set of general primers recognizing some of the cry genes reported in the relevant literature. Primers corresponding to 2 types of cry genes (cry1 and cry2) successfully amplified DNA in all of the tested isolates. Moreover, the proteins encoded by these genes were detected in the SDS-PAGE of the purified parasporal bodies. The 50% lethal concentration of the spore-crystal mixture of the 25 isolates against E. kuehniella larvae varied from 8.4 to 97.6 µg g^{-1}. A comparison of the LC_{50} values of the tested isolates with those of the reference strains B. thuringiensis kurstaki HD-1 and HD-73 (20.8 and 46 µg g^{-1}, respectively) showed that some of these isolates have a higher toxicity potential. Moreover, flagellar serotyping revealed that 4 isolates, which were among the most toxic, belonged to serotype kurstaki. This study constitutes the first isolation and characterization of local B. thuringiensis isolates in Syria. Some of these isolates exhibit toxic potential and, therefore, could be adopted for future applications to control some important insect pests.

Cry Genes profiling and the toxicity of isolates of Bacillus thuringiensis from soil samples against American bollworm, Helicoverpa armigera

The aim of this study was to search for Bacillus thuringiensis (Bt) harbouring cry1A gene which could effectively control cotton pest, American bollworm, Helicoverpa armigera. cry gene profiling of 50 Bt isolates showed the presence of cry1, cry2, cry3, cry4, cry7, cry8 and cry9 genes. None of the isolates harboured cry1 gene alone. It was always found in combination with cry3. There was no isolate positive for cry10 gene. Considering isolates with single cry genes, the frequency of cry4 was predominant (22%) followed cry2 (6%), cry3 (4%) and cry8 (2%). Isolates having two cry genes in combination had 14% incidence for cry2 + cry4, 12% for cry3 + cry4 and 10% for cry1 + cry3. The most dominant three gene linkage was cry1 + cry3 + cry4. Further profiling of cry1 gene showed that cry1K gene was abundantly present in all combinations such as cry1A, cry1D, cry1F and cry1I. However, cry1C existed independent of other subtypes. Finally, the Bt isolates with cry1A were analyzed for 16S rRNA gene, which showed two distinct groups of isolates on the basis of sequence homology. Bioassays of spore-crystal mixtures of SBS-Bt4, 8, 17, 21 and 26 harbouring cry1 against neonate larvae of H. armigera showed LC(50) 1288, 1202, 467·7, 524·8 and 108·5 μg ml(-1) . The SBS-Bt26 showed fourfold higher toxicity than the cry 1Ac harbouring positive control, HD-73. None of the isolates harboured single cry 1 gene. They were always in combination of two or three genes. A Bt isolate (Bt26) had fourfold higher toxicity against H. armigera larvae compared with the positive control HD 73 and hence can be commercially exploited to control insect pest. The inter relationship between the cry genes content and the toxicity may allow better understanding of Bt ecology.

Cloning and characterization of truncated cry1Ab gene from a new indigenous isolate of Bacillus thuringiensis

The insecticidal crystal protein(s) encoded by cry gene(s) of Bacillus thuringiensis (Bt) have been used for insect control both as biopesticides and in transgenic plants. A new 3'-truncated cry1Ab gene was cloned from an indigenous isolate of Bt, A19-31. Nucleotide sequencing and homology search revealed that the deduced amino acid sequence of Cry1Ab toxin of Bt strain A19-31 had a variation of two amino acid residues with the holotype sequence, Cry1Ab1. Expression of the 3'-truncated cry1Ab gene was studied in an acrystalliferous strain of Bt (4Q7). SDS-PAGE and immunostrip analysis of spore-crystal mixture revealed a low level expression of the 3'-truncated cry1Ab gene. Insecticidal activity assay showed that the recombinant 3'-truncated cry1Ab gene product was toxic to larvae of both Helicoverpa armigera and Spodoptera litura.

Evidence of the Involvement of E358, A498 and C571 of a New Cry1Ac δ-endotoxin of Bacillus thuringiensis in its High Insecticidal Activity Against Ephestia kuehniella

A new cry1Ac-type gene was cloned from Bacillus thuringiensis strain BLB1, sequenced and expressed. The deduced amino acid sequence of the polypeptide has a predicted molecular mass of 132.186 kDa. The amino acid sequence alignment of BLB1 Cry1Ac with those of the published ones showed that this is a new delta-endotoxin. When compared with Cry1Ac of Bacillus thuringiensis strain HD1, it was found that BLB1 Cry1Ac harbours three mutations: V358E localized in domain II and V498A and Y571C localized in domain III. When the BLB1 Cry1Ac toxin was expressed in an acrystalliferous strain of B. thuringiensis (HD1CryB), bipyramidal crystals were produced. The spore-crystal mixture of this recombinant strain was at least two-fold more active against larvae of the lepidopteran Ephestia kuehniella than that of the recombinant strain expressing Cry1Ac of HD1. The study of the structural effect of these mutations suggested that they may stabilize key regions involved in the binding of the domains II and III to insect receptors.

Bioinsecticide production by the bacterium Bacillus thuringiensis. 1. Pattern of cell growth, toxin production and by-product synthesis

To develop a cost-effective process for the production of Bacillus thuringiensis-based insecticide, it is important to cultivate the bacterial strain in rich medium to obtain the highest yields of spore-crystal complexes. It was found that cultivation of the bacterium in medium with high concentrations of glucose (50–90 g l−1) resulted in much lower bacterial spores, crystal protein and lower toxicity, when tested against Spodoptera littoralis and Anagasta kuehniella larvae. The best results were obtained with glucose concentration of 20.0 g 1−1 as 7.1 × 1011 spores ml−1 and 3.4 g l−1 of crystal protein were achieved with LC50 of 40.1 and 50.2 mg kg−1 meal against S. littoralis and A. kuehniella, respectively. However, >21% of the consumed glucose was diverted into by-product synthesis at the expense of spore-crystal protein mixture. Only 78.3% of consumed glucose was converted into spores and crystal protein. Among by-products formed, acetic acid and β-hydroxybutyric acid (PHB) were produced during the phase of active growth and glutamic acid and succinic acid during the phase of active toxin production.

Removal of nickel from aqueous solution by the bacterium Bacillus thuringiensis

The biosorption of the toxic metal (nickel) from aqueous solutions on dried vegetative cell and spore–crystal mixture of Bacillus thuringiensis var. thuringiensis was tested under laboratory conditions as a function of pH, initial metal ion concentration and temperature. The characteristics of the adsorption process were investigated using Scatchard analysis. Scatchard analysis of the equilibrium binding data for metal ions on vegetative cell and spore–crystal mixture of B. thuringiensis gave rise to a linear plot, indicating that the Langmuir model could be applied successfully. Adsorption behaviour of nickel(II) ion on B. thuringiensis is expressed by both Langmuir and Freundlich isotherms. The adsorption data with respect to the metal provided an excellent fit to both isotherms. Ni(II) ion uptake of B. thuringiensis's spore–crystal mixture at 250 mg l −1 was 15.7%, whereas its vegetative cell metal uptake was 10%. The best temperature for ion uptake was found to be at 35 °C.

Cloning, Characterization, and Expression of a New cry2Ab Gene From Bacillus thuringiensis Strain 14-1

Bacillus thuringiensis is the major source for transfer of genes to impart insect resistance in transgenic plants. Cry2A proteins of B. thuringiensis are promising candidates for management of resistance development in insects owing to their difference from the currently used Cry1A proteins, in structure and insecticidal mechanism. The cry2Ab gene was found to lack a functional promoter and, hence, is cryptic in nature. The cry2Ab7 gene was cloned from a new indigenous B. thuringiensis strain, 14-1. Nucleotide sequencing of the cry2Ab gene cloned from B. thuringiensis strain 14-1 revealed an open reading frame of 1902 bp. The deduced amino acid sequence of Cry2Ab of B. thuringiensis strain 14-1 showed a variation in three amino acid residues in comparison to the holotype sequence, Cry2Ab1. Expression of the newly cloned cry2Ab gene was studied in an acrystalliferous strain of B. thuringiensis (4Q7) by fusing the cry2Ab gene downstream of cry2Aa promoter and orf1 + orf2 sequences. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of a spore-crystal mixture obtained from transformants of B. thuringiensis strain 4Q7 showed production of Cry2Ab protein of about 65 kDa. Alkali solubilized Cry2Ab7 protein showed toxicity against Helicoverpa armigera neonates.

Biosorption of chromium(VI) ions from aqueous solution by the bacterium Bacillus thuringiensis

The biosorption of chromium(VI) ions from aqueous solutions on dried vegetative cell and spore–crystal mixture of Bacillus thuringiensis var. thuringiensis was tested using the batch method as a function of pH, initial metal ion concentration and temperature. The optimum adsorption pH value observed for chromium(VI) ions was 2.0. At the optimal conditions, metal ion uptake has rised with increasing initial metal ion concentration. Chromium(VI) ions uptake of B. thuringiensis’ spore–crystal mixture at 250 mg l −1 was 24.1%, whereas its vegetative cell metal uptake was 18.0%. Chromium(VI) biosorption experiments were carried out at three different temperatures, 15, 25 and 35 °C. The best temperature for biosorption was 25 °C. Scatchard plot analysis were used to obtain more compact information about the interaction between chromium(VI) ions and biosorbents. The plot results are further studied to determine if they fit Langmuir and Freundlich models. Scatchard analysis of the equilibrium binding data for chromium(VI) ions on vegetative cell and spore–crystal mixture of B. thuringiensis gave rise to a linear plot, indicating that the Langmuir model could be applied. The adsorption data with respect to the metal provided an excellent fit to both Langmuir and Freundlich isotherms.

Isolation, geographical diversity and insecticidal activity of Bacillus thuringiensis from soils in Spain

Bacillus thuringiensis is a spore-forming bacterium showing the unusual ability to produce endogenous crystals during sporulation that are toxic for some pest insects. This work was performed to study the composition, ecological distribution and insecticidal activity of isolates of this entomopathogenic bacterium from the Spanish territory. Using a standard isolation method, B. thuringiensis was isolated from 115 out of 493 soil samples collected in the Iberian Peninsula and the Canary and Balearic Archipelagos. The percentages of samples with B. thuringiensis were 31.7, 27.6 and 18.5 and the B. thuringiensis index 0.065, 0.067 and 0.11 for the Iberian Peninsula, Canary and Balearic Archipelagos, respectively. The prairies were shown to be the worst source of B. thuringiensis while forests, urban and agricultural habitats showed similar percentages. Strain classification based on H-antigen agglutination showed a great diversity among the Spanish isolates, which were distributed among 24 subspecies, including three new ones andaluciensis, asturiensis and palmanyolensis. We differentiated 65 different protein profiles of spore–crystal mixtures by sulfate-polyacrylamide gel electrophoresis and we selected 109 isolates representative of these profiles to evaluate their insecticidal activity against insects from the Orders Orthoptera, Dictyoptera, Coleoptera, Lepidoptera and Diptera. We found variable percentages of isolates active against Coleoptera and Lepidoptera, one isolate highly active against mosquito larvae and for the first time, three isolates active against cockroaches and locusts.

Characterization of novel Brazilian Bacillus thuringiensis strains active against Spodoptera frugiperda and other insect pests

Abstract:Brazilian strains of Bacillus thuringiensis, namely S701, S764 and S1265 were analysed regarding their cry gene and protein contents, crystal type, and activity against larvae of the lepidopteran fall armyworm (Spodoptera frugiperda Smith), the velvet caterpillar (Anticarsia gemmatalis), the dipterans (Culex quinquefasciatus and Aedes aegypti) and the coleopteran (Tenebrio molitor). The LC50 of the strains against second instar larvae of S. frugiperda or A. gemmatalis revealed a high potency against those insect species. The spore–crystal mixtures of the isolates were analysed by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and showed similar protein pattern as the B. thuringiensis subsp. kurstaki strain HD‐1 (proteins approximately 130 and 65 kDa) for isolates S701 and S764, respectively, and only one major protein of approximately 130 kDa for isolate S1265. The polymerase chain reaction (PCR) using total DNA of the isolates and general and specific primers showed the presence of cry1Aa, cry1Ac, cry1Ia and cry2Ab genes in the two isolates serotyped as B. thuringiensis kurstaki (S701 and S764) and the presence of cry1D and cry2Ad in B. thuringiensis morrisoni S1265 strain. Scanning electron microscopy of strains S701 and S764, showed the presence of bipyramidal, cuboidal and round crystals, like in strain HD‐1 and bipyramidal and round crystals like in strain S1265.

Geographical variation in larval susceptibility of the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae) to Bacillus thuringiensis spore–crystal mixtures and purified crystal proteins and associated resistance development in India

The susceptibility of larvae of the diamondback moth, Plutella xylostella Linnaeus to purified crystal proteins and spore-crystal preparations of Bacillus thuringiensis was investigated for 13 populations from seven states in India. The LC50 (microg ml(-1), 48 h) values of Cry proteins for different populations of P. xylostella ranged from 0.14-3.74 (Cry1Aa), 0.007-1.25 (Cry1Ab), 0.18-2.47 (Cry1Ac) and 0.12-3.0 (Cry1C). The LC50 (mg (ai) l(-1), 48 h) of spore-crystal preparations ranged from 0.02-0.98 (HD-1) and 0.06-2.14 (HD-73). Significantly higher LC50 values for all tested toxins and strains were obtained with populations collected from Iruttupallam and Ottanchathiram in the southern state of Tamil Nadu, whereas some of the populations collected from the northern part of India were more susceptible than the susceptible IARI 17-65 population. The high levels of resistance in the Iruttupallam and Ottanchathiram populations to Cry1Ab suggested selection pressure by Cry1Ab, which is the predominant toxin in B. thuringiensis formulations used in India. Cry1Ab was found to be more toxic than the other toxins. The population from Iruttupallam showed increased resistance following selection with Cry1Ab in the laboratory (LC50 from 1.25 to 4.31 microg ml(-1) over two generations) and also showed cross resistance to CrylAa and CrylAc. The resistance to Biobit in the field population from Iruttupallam declined slowly; requiring c. 33 generations for an overall 10-fold decline in LC50 when the insects were reared in the laboratory without exposure to B. thuringensis.

Expression of a cry2Aa gene in an acrystalliferous Bacillus thuringiensis strain and toxicity of Cry2Aa against Helicoverpa armigera

In the recent past research has been mainly focused on the expression of cry1 genes of Bacillus thuringiensis (Bt) to engineer lepidopteran insect resistance in plants. Search for structurally different toxins is necessary for the management of resistance development in insects. The intact cry2Aa operon (3.95 kb) of a new isolate of Bt, 47-8, was subcloned into a Bt shuttle vector, pHT3101 (6.7 kb). Recombinant pHT3101 containing the cry2Aa operon of Bt strain 47-8 was named as pTN2Aa and used to transform acrystalliferous Bt strain 4Q7 by electroporation. Phase contrast microscopic observation revealed the presence of crystalline inclusions in the transformants of Bt strain 4Q7 harbouring pTN2Aa. SDS–PAGE of a spore–crystal mixture prepared from transformants of acrystalliferous Bt strain 4Q7 harbouring pTN2Aa showed a single band of about 65 kDa alone confirming the expression of the cloned cry2Aa. Bioassay with Helicoverpa armigera showed 71.4% mortality caused by the proteins encoded by the newly cloned cry2Aa gene (at the concentration of 2.3 μg/μl) on the seventh day and all the survivors that escaped from Cry2Aa toxicity showed severe (81–99%) inhibition in larval growth.

Characterization ofBacillus thuringiensissubsp.kurstakistrain S93 effective against the fall armywormSpodoptera frugiperda)

A Brazilian strain of Bacillus thuringiensis subsp. kurstaki, designated S93, was analyzed regarding its cry gene and protein contents and activity against the fall armyworm (Spodoptera frugiperda, Smith 1797). Bioassays using lyophilized powders of S93 or HD-1 and third instar larvae of S. frugiperda showed a 12.3-fold lower LC50for the S93 strain when compared with the standard HD-1 strain. The spore-crystal mixture, analyzed by SDS-PAGE, showed two major polypeptides of 130 and 65 kDa, corresponding to Cry1 and Cry2 toxins, respectively. Western blot analysis showed that these proteins were immunologically related to the Cry1A protein from B. thuringiensis subsp. kurstaki HD-73. The polymerase chain reaction technique (PCR) using total DNA from the S93 strain and specific primers showed the presence of cry1Aa, cry1Ab, and cry1Ac genes, and a cry1A-type gene was localized in a plasmid of about 44 MDa. A cry1Ab gene was isolated from a S93 plasmid DNA library and completely sequenced. Computer analysis showed that the gene sequence (GenBank acession number AF059670) is identical to cry1Ab1 and has 91.6 and 85.9% identity with cry1Aa1 and cry1Ac1 genes, respectively. The deduced amino-acid sequence showed a high degree of similarity with the amino-acid sequences of the Cry1Ab1 (100%), Cry1Aa1 (93.8%), and Cry1Ac1 (90.6%) proteins.Key words: Bacillus thuringiensis, Spodoptera frugiperda, biological control, crystal protein, cry genes.

Spore coat protein synergizes bacillus thuringiensis crystal toxicity for the indianmeal moth

Spores from Bacillus thuringiensis serovars kurstaki and entomocidus synergized crystal protein toxicity for larvae of the Indianmeal moth (Plodia interpunctella). Preparations of spore-crystal mixtures of either serovar were more toxic for the larvae than either purified spores or crystals alone (based on dry weight). Spores lost 53% of their toxicity for the Indianmeal moth after 2 h of UV-irradiation, but remained partially toxic (28%) even after 4 h of irradiation. Spore coat protein was toxic for the Indianmeal moth and was synergistic with B. thuringiensis serovar kurstaki HD-1 crystal protein. Enhanced toxicity of the combined spore-crystal preparation was attributed to a combination of crystal and spore coat protein, and included the effects of spore germination and resulting septicemia in the larval hemolymph. Ultraviolet irradiation of spores reduced the toxicity from septicemia but not the synergism caused by spore coat protein. The potencies of spore-crystal preparations must be carefully evaluated on the basis of contributions from all three factors.

Synergism between Bacillus thuringiensis Spores and Toxins against Resistant and Susceptible Diamondback Moths (Plutella xylostella).

We studied the effects of combinations of Bacillus thuringiensis spores and toxins on the mortality of diamondback moth (Plutella xylostella) larvae in leaf residue bioassays. Spores of B. thuringiensis subsp. kurstaki increased the toxicity of crystals of B. thuringiensis subsp. kurstaki to both resistant and susceptible larvae. For B. thuringiensis subsp. kurstaki, resistance ratios were 1,200 for a spore-crystal mixture and 56,000 for crystals without spores. Treatment of a spore-crystal formulation of B. thuringiensis subsp. kurstaki with the antibiotic streptomycin to inhibit spore germination reduced toxicity to resistant larvae but not to susceptible larvae. In contrast, analogous experiments with B. thuringiensis subsp. aizawai revealed no significant effects of adding spores to crystals or of treating a spore-crystal formulation with streptomycin. Synergism occurred between Cry2A and B. thuringiensis subsp. kurstaki spores against susceptible larvae and between Cry1C and B. thuringiensis subsp. aizawai spores against resistant and susceptible larvae. The results show that B. thuringiensis toxins combined with spores can be toxic even though the toxins and spores have little or no independent toxicity. Results reported here and previously suggest that, for diamondback moth larvae, the extent of synergism between spores and toxins of B. thuringiensis depends on the strain of insect, the type of spore, the set of toxins, the presence of other materials such as formulation ingredients, and the concentrations of spores and toxins.

Environmental Distribution and Diversity of Bacillus thuringiensis in Spain

Bacillus thuringiensis was isolated from 301 out of 1,005 samples collected in Spain from agricultural and non-cultivated soils, dust from stored products, and dead insects. Based on the production of parasporal crystals, 1,401 isolates were identified as B. thuringiensis after examining 11,982 B. thuringiensis-like colonies. We found a greater presence of B. thuringiensis in dust from grain storages than in other habitats. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the spore-crystal mixtures revealed diverse populations of B. thuringiensis which were differentiated in at least 92 distinct protein profiles. Serological identification also showed great diversity among the Spanish isolates which were distributed among 38 of the 58 known serovars. The most frequently found serovars were aizawai, kurstaki, konkukian, morrisoni, and thuringiensis, which together represented more than 50% of the serotyped isolates. In preliminary toxicity assays, a number of isolates were found to show significant insecticidal activity against the lepidopterans Heliothis armigera (76.1% of the assayed isolates), Spodoptera exigua (50.5%), and Plutella xylostella (19.7%). Thirty five isolates were toxic to both H. armigera and S. exigua, and eight were toxic to S. exigua and P. xylostella. Four and one isolates were toxic to the coleopterans Leptinotarsa decemlineata and Colaspidema atrum, respectively, and three to the dipteran Tipula oleracea. The electrophoretic pattern and serovar of most of the isolates with toxic activity were consistent with those reported in the literature, although other isolates revealed unusual protein profiles, were assigned to new H serovars, or were included in H serovars not previously reported within such pathotypes.

Dominion.Niles Eldredge

Previous articleNext article No AccessNew Biological BooksDominion. Niles Eldredge Carole L. CrumleyCarole L. Crumley Search for more articles by this author PDFPDF PLUS Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinkedInRedditEmail SectionsMoreDetailsFiguresReferencesCited by The Quarterly Review of Biology Volume 71, Number 4Dec., 1996 Published in association with Stony Brook University Article DOIhttps://doi.org/10.1086/419564 Citations: 2Citations are reported from Crossref Copyright 1996 The University of ChicagoPDF download Crossref reports the following articles citing this article:M. Mohan, G.T. Gujar Geographical variation in larval susceptibility of the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae) to Bacillus thuringiensis spore–crystal mixtures and purified crystal proteins and associated resistance development in India, Bulletin of Entomological Research 92, no.0606 (Mar 2007).https://doi.org/10.1079/BER2002195M. D. Ferrandis, R. Andrew, M. Porcar, J. Iriarte, V. Cosmao-Dumanoir, M. -M. Lecadet, P. Caballero, J. Ferre Characterization of Bacillus thuringiensis serovar bolivia (serotype H63), a novel serovar isolated from the Bolivian high valleys, Letters in Applied Microbiology 28, no.66 (Jun 1999): 440–444.https://doi.org/10.1046/j.1365-2672.1999.00558.x