Classification ofBacillus thuringiensis strains

An updated classification ofB. thuringiensis strains according to flagellar antigens is presented. Twenty-seven antigenic groups and seven subgroups have enabled us to distiguish 34 serovars. Biochemical tests are unable to separate these serovars. The significance of serovars is discussed in comparison with pathogenicity and with reference to the Bacteriological Code.      

Transfer of plasmids and chromosomal genes amongst subspecies ofBacillus thuringiensis

Summary The plasmids pBC16 and pC194 fromBacilus thuringiensis subsp.israelensis strains A084-16-194 were transferred to 25 subspecies ofB. thuringiensis by a conjugation-like process using broth mating technique. The frequencies of transfer varied considerably between different mating pairs, ranging from 1.1×10^–9 to 9.8×10^–5. Additionally, chromosomal transfer could also be demonstrated in tenB. thuringiensis subspecies with very low frequencies (4.3×10^–9 to 3.7×10^–7). The intersubspecies matings within a group of eight subspecies strains gave higher frequencies of transfer than the matings between the subspecies. Furthermore, the results indicated that the capability to transfer plasmids among these various subspecies did not depend on the presence of large plasmid.     

Screening of the insecticidal activity ofBacillus thuringiensis strains against the egyptian cotton leaf wormSpodoptera littoralis

A screening of the larvicidal activity of the more than 900 strains ofBacillus thuringiensis strains, combining the Institut Pasteur collection was realized. A quick bioassay using 1st instar larvae and semi-synthetic medium was developed. Many strains were toxic toSpodoptera littoralis, but only a few belonging mainly to serovarsaizawai, kenyae andentomocidus showed high level of toxicity. The profiles of strain activities differed from serovar to serovar, but within the same serovar toxicity can vary with different strains. Oneaizawai strain tested in the field gave satisfactory results, better than a commercially used strain, tested in the same experiment.

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Résumé Le criblage de notre collection deB. thuringiensis (plus de 900 souches) a été effectué contreSpodoptera littoralis. Une technique rapide de bioessai utilisant des chenilles néonates et un milieu semisynthétique a été mise au point. Beaucoup de souches se sont montrées actives à forte dose, mais seulement quelques-unes appartenant principalement aux sérovaraizawai etkenyae à doses plus faibles. Les différents sérotypes ont un profil d'activité différent mais un même sérotype peut comprendre des souches de toxicité variée. Une souche du sérovaraizawai a été testée en champs et a donné de bons résultats, son activité ayant été évaluée à environ 5 fois celle d'une souche couramment commercialisée.

     

Persistence ofBacillus thuringiensis andBacillus cereus in soil supplemented with grass or manure

Summary Persistance of inocula ofBacillus thuringiensis spores, parasporal crystals, andBacillus cereus spores in soil supplemented with dried-grass or partly composted, dried-chicken manure (100 mg supplement per 900 mg soil,^–0.01 MPa water availability, 25°C) were monitored over a period of up to 64 days by dilution plating and bioassay with larvae ofPieris brassicae. The inoculantB. thuringiensis population increased 22 x in level in grass-supplemented soil, but declined in manure-supplemented soil to 0.22 x the original level. TheB. cereus inocula declined in both soil treatments to approximately 0.1 x the original level. Insecticidal activity of theB. thuringiensis parasporal crystal decreased exponentially in grass and manuresupplemented soils, with half-lives of approximately 9.5 and 8.5 days respectively.     

Transfer of an insecticidal protein gene ofBacillus thuringiensis into plant-colonizingAzospirillum

A fusion plasmid, pRKC, was constructed, using pACYC184, RSF1010 and a kanamycin-resistance cartridge from pUC4K, to convey thecryIA(a) gene intoAzospirillum spp. With the pRKC plasmid, the number of putative transconjugants obtained inA. lipoferum was about 300-fold higher than inA. brasilense. Conjugation frequency and plasmid stability inA. lipoferum were less for pBTF8, which carries thecryIA(a) gene in the correct orientation for a constitutive promoter, than for pBTF9, which carries the gene in the opposite orientation. Expression of thecryIA(a) gene was not apparent in SDS-PAGE analysis ofA. lipoferum transconjugants harbouring pBTF8. However,Escherichia coli transformants with the pBTF8 rescued fromA. lipoferum transconjugants produced an approximately 135 kDa Cry protein, indicating that thecry gene is intact in the transconjugants.V. Udayasuriyan was and A. Nakamura, H. Masaki and T. Uozumi are with the Department of Biotechnology, Faculty of Agriculture, The University of Tokyo, Yayoi 1-1-1, Bunkyo-Ku, Tokyo 113, Japan; V. Udayasuriyan is now with the Department of Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultral University, Coimbatore-641 003, India.     

Effectiveness of beta-exotoxin ofBacillus thuringiensis var.thuringiensis on the ability ofMeloidogyne sp. from brinjal (Solanum melongena L.) to survive

Beta-exotoxin produced byBacillus thuringiensis var.thuringiensis grown in the acid hydrolysates of wheat and rice brans caused 95% and 85% mortality respectively ofMeloidogyne sp. as against 72% of β-exotoxin produced on farm yard manure within 7 days. Acid hydrolysate of wheat or rice bran and solid farm yard manure proved to be the best media for growth ofB. thuringiensis var.thuringiensis.     

Larvicidal efficacy ofBacillus thuringiensis var. israelensis andBacillus sphaericus onAnopheles arabiensis in Ethiopia

The second instar larvae of the malaria vector mosquito,Anopheles arabiensis,were more susceptible to Bacillus thuringiensis var. israelensis (IPS-82) and B. sphaericus (SPH-88) than the third instar larvae. The LC ₅₀ values were 1.0 Μg^I-1 and 1.8 Μg_I-1 for IPS-82 against second and third instar larvae respectively, after 48 h of exposure. The LC₅₀ values for SPH-88 were 3.6 Μg {siI-1} against the second instar larvae and 7.6 Μg_I-1 against the third instar larvae of An. arabiensis. The larvicidal efficacy of SPH-88 was significantly less than IPS-82. The potential of IPS-82 for the control of An. arabiensis in malaria endemic areas is promising.     

Arabidopsis thaliana small subunit leader and transit peptide enhance the expression ofBacillus thuringiensis proteins in transgenic plants

The expression of the modified gene for a truncated form of thecryIA(c) gene, encoding the insecticidal portion of the lepidopteran-active CryIA(c) protein fromBacillus thuringiensis var.kurstaki (B.t.k.) HD73, under control of theArabidopsis thaliana ribulose-1,5-bisphosphate carboxylase (Rubisco) small subunitats1A promoter with and without its associated transit peptide was analyzed in transgenic tobacco plants. Examination of leaf tissue revealed that theats1A promoter with its transit peptide sequence fused to the truncated CryIA(c) protein provided a 10-fold to 20-fold increase incryIA(c) mRNA and protein levels compared to gene constructs in which the cauliflower mosaic virus 35S promoter with a duplication of the enhancer region (CaMV-En35S) was used to express the samecryIA(c) gene. Transient expression assays in tobacco protoplasts and the whole plant results support the conclusion that the transit peptide plus untranslated sequences upstream of that region are both required for the increase in expression of the CryIA(c) protein. Furthermore, the CaMV-En35S promoter can be used with theArabidopsis ats1A untranslated leader and transit peptide to increase expression of this protein. While subcellular fractionation revealed that the truncated CryIA(c) protein fused to theats1A transit peptide is located in the chloroplast, the increase in gene expression is independent of targeting of the CryIA(c) protein to the chloroplast. The results reported here provide new insight into the role of 5 untranslated leader sequences and translational fusions to increase heterologous gene expression, and they demonstrate the utility of this approach in the development of insect-resistant crops.     

Cytolytic differences among lepidopteran cell lines exposed to toxins ofBacillus thuringiensis subst.Kurstaki. (HD-263) andAizawai (HD-112): Effect of aminosugars and N-glycosylation

Summary Comparison of lytic-dose response behavior of seven lepidopteran cell lines to the activated delta-endotoxin polypeptides ofBacillus thuringiensis subspecieskurstaki (HD-263) andaizawai (HD-112) indicated distinct differences among the lines. The lines derived fromSpodoptera speciesS. exigua (URC-SE-1A) andS. littoralis (UIV-SL-575) were more susceptible to lysis byaizawai towin (Bta) thankurstaki toxin (Btk) as were cells from theLymantria dispar line (IPLB-LD652Y). However, the concentrations of Bta required for lysis of 50% of URC-SE-1A and IPLB-LD652Y cells (LC₅₀) were 0.2 to 0.8 μg/ml compared to 5 to 9 μg/ml for UIV-SL-575 cells. In comparison, Btk LC₅₀ concentrations for the three lines were similar (14 to 19 μg/ml). Cells fromS. frugiperda (IPLB-SF-21AE) andTrichoplusia ni (TN368) were similar in their response to Bta (LC₅₀=2.5 to 3.7 μg/ml) and Btk (LC₅₀=1.0 to 2.8 μg/ml) whereas the lines derived fromHeliothis spp. were the least susceptible to both toxins. The LC₅₀ concentrations for Bta with theH. zea line (IPLB-HA-1075) andH. virescens line (BCIRL-HV-AM1) were >50 μg/ml and for Btk were >50 μg/ml and 42 to 50 μg/ml, respectively, yet for both lines Btk was the more cytolytic. Cytolysis of TN368 cells could be inhibited to varying extents by preincubation of the toxins with the aminosugars of galactose, mannose, and glucose and theirN-acetyl derivatives. The unsubstituted hexoses were not inhibitory. The order of decreasing inhibitory effectiveness was the same for both toxins regardless of the derivative species and followed the order galactose, mannose, and glucose. Also, inhibition of cytolysis could be achieved to varying extents by assaying cells grown in medium with tunicamycin. Lysis with Btk was inhibited 68 and 37% using treated cells of TN368 and IPLB-LD652Y, respectively; however, no inhibition was observed with URC-SE-1A cells. Further, no inhibition of Bta-mediated lysis was obtained with tunicamycin-grown cells of the three lines.     

Growth ofDiacrisia obliqua [Lep.: Arctiidae] with low doses ofBacillus thuringiensis var.Kurstaki

Early 3rd instarDiacrisia obliqua Walk. larvae were treated with concentrations ofBacillus thuringiensis var.kurstaki (Dipel^®) and the growth of treated larvae was assessed. All the doses reduced significantly the weight and survival of the insects (p<0.001).     

Crystals of two mutants ofBacillus thuringiensis show increased toxicity against larvae ofSpodoptera littoralis

Crystals of two asporogenous mutants ofBacillus thuringiensis var.kurstaki strain HD-1 obtained following treatment with ozone and N-methyl-N′-nitro-N-nitrosoguanidine showed increased toxicity against larvae ofSpodoptera littoralis when compared to the wild-type crystal.      

Optimised expression in Escherichia coli and purification of the functional form of the Bacillus thuringiensis Cry4Aa delta-endotoxin

Achieving high-level expression of the Bacillus thuringiensis Cry4Aa mosquito-larvicidal protein was demonstrated. The 130-kDa Cry4Aa protoxin was overexpressed as an inclusion body in Escherichia coli under the control of the tac promoter together with the cry4Ba promoter. The solubility of the toxin inclusions in carbonate buffer, pH 10.0, was markedly enhanced at a cultivation temperature of 30 degrees C. Elimination of the tryptic cleavage site at Arg-235 in the loop between helices 5 and 6 still retained the high-level toxicity of E. coli cells expressing the Cry4Aa mutant against Aedes aegypti larvae. Trypsin digestion of the R235Q mutant protoxin produced a protease-resistant fragment of ca. 65kDa. A homogeneous product of the 65-kDa trypsin-treated R203Q protein was obtained after size-exclusion chromatography that would pave the way for the further crystallisation and X-ray crystallographic studies.     

Behavioral and physiological responses of susceptible and resistant diamondback moth larvae to Bacillus thuringiensis

To determine whether field-selected resistance of diamondback moth (Plutella xylostella L.) (Lepidoptera: Plutellidae) to Bacillus thuringiensis is based on behavioral or physiological adaptation, we measured mortality, consumption, and movement of larvae from a susceptible and a resistant colony when placed on untreated and B. thuringiensis treated cabbage. Colonies did not differ in mortality, consumption, or movement on untreated cabbage. However, for a given amount of consumption of treated cabbage, resistant larvae had lower mortality than susceptible larvae, demonstrating that resistance had a physiological basis. The movement patterns could not account for the differences between colonies in survival. Resistant larvae did not avoid B. thuringiensis more than did susceptible larvae. Thus, we found no evidence for behavioral resistance.     

Effects ofBacillus thuringiensis var.San diego on predation effectiveness,development and mortality ofColeomegilla maculata lengi (Col.: Coccinellidae) larvae

Laboratory experiments were conducted to determine the effects of M-One^™ (Bacillus thuringiensis var.san diego) on larval instars ofColeomegilla maculata lengi Timberlake. Coccinellid larval development (from egg hatch to adult), completed on pollen treated with suspensions of M-One^™ at 20 ml/litre (5.6×10⁸ CPBIU/litre) and 200 ml/litre, took respectively 29.3 and 38.5 days compared with 21.9 days for the control (water). M-One^™ did not cause larval mortality.C. maculata third instars did not show any preference between eggs ofLeptinotarsa decemlineata (Say) treated with water or with M-One^™ at 20 ml/litre. However, at 200 ml M-One^™/litre, the number of eggs attacked was 34.7% lower than the eggs treated with water only, 48 h after the beginning of the test. These results indicate that the use of M-One^™, at the manufacturer's recommended field rate of 20 ml/litre, does not cause a major threat to larvalC. maculata populations.

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Résumé Des bioessais en laboratoire ont été effectués afin de déterminer si les larves de la coccinelle maculée,Coleomegilla maculata lengi Timberlake (Col.: Coccinellidae), sont affectées par M-One^™, un insecticide biologique préparé à partir de la bactérieBacillus thuringiensis var.san diego Berliner et utilisé dans la lutte contre le doryphore de la pomme de terre,Leptinotarsa decemlineata (Say) (Col.: Chrysomelidae). Le développement larvaire, effectué sur du pollen traité avec des concentrations de 20 ml M-One^™/litre (5,6×10⁸ unités internationales de doryphore/litre) et 200 ml M-One^™/litre, a nécessité 29,3 et 38,5 jours respectivement, comparativement à 21,9 jours pour le témoin (eau). M-One^™ n'a pas causé de mortalité chez les larves. Au cours d'un test de 48 h, les larves de stade III n'ont montré aucune préférence entre des œufs traités avec 20 ml M-One^™/litre et des œufs traités avec de l'eau. Par contre avec 200 ml M-One^™/litre, le nombre d'œufs attaqués a diminué significativement de 34,7% par rapport au témoin, 48 h après le début du test. Ces résultats indiquent que l'utilisation de M-One^™ à la concentration recommandée de 20 ml/litre ne représente pas une menace pour les populations larvaires de la coccinelle maculée.

     

Lethality of chlorine, chlorine dioxide, and a commercial fruit and vegetable sanitizer to vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis

Chlorine, chlorine dioxide (ClO₂), and a commercial raw fruit and vegetable sanitizer (Fit powder) were evaluated for their effectiveness in killing vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis. The ultimate goal was to use one or both species as a potential surrogate(s) for Bacillus anthracis in studies that focus on determining the efficacy of sanitizers in killing the pathogen on food contact surfaces and foods. Treatment with alkaline (pH 10.5–11.0) ClO₂ (200 mg/mL) produced by electrochemical technologies reduced populations of a five-strain mixture of vegetative cells and a five-strain mixture of spores of B. cereus by more than 5.4 and more than 6.4 log cfu/mL, respectively, within 5 min. This finding compares with respective reductions of 4.5 and 1.8 log cfu/mL resulting from treatment with 200 mg/mL chlorine. Treatment with a 1.5% acidified (pH 3.0) solution of Fit powder product was less effective, causing 2.5-log and 0.4-log cfu/mL reductions in the number of B. cereus cells and spores, respectively. Treatment with alkaline ClO₂ (85 mg/mL), acidified (pH 3.4) ClO₂ (85 mg/mL), and a mixture of ClO₂ (85 mg/mL) and Fit powder product (0.5%) (pH 3.5) caused reductions in vegetative cell/spore populations of more than 5.3/5.6, 5.3/5.7, and 5.3/6.0 log cfu/mL, respectively. Treatment of B. cereus and B. thuringiensis spores in a medium (3.4 mg/mL organic and inorganic solids) in which cells had grown and produced spores with an equal volume of alkaline (pH 12.1) ClO₂ (400 mg/mL) for 30 min reduced populations by 4.6 and 5.2 log cfu/mL, respectively, indicating high lethality in the presence of materials other than spores that would potentially react with and neutralize the sporicidal activity of ClO₂.Published by permission of the International Association for Food Protection: Journal of Food Protection (2004) 60:1702–1708This revised version was published online in April 2005 with corrections to the text and the section heading.In section Preparation of treatment solutions the phrase 22-28°C was replaced by 22±2°C.     

Selection of chitinolytic strains of Bacillus thuringiensis

Three selected strains of Bacillus thuringiensis native to Mexico produced endochitinases, chitobiosidases, and N-acetyl--glucosaminidases in a medium containing colloidal chitin as a main carbon source. Two types of chitinases were clearly identified: endochitinases and chitobiosidases. Chromosomal location of a chitinase gene in B. thuringiensis LBIT-82 was resolved.     

Application of RAPD technique to study polymorphism among Bacillus thuringiensis isolates from Jordan

The soil-borne bacterium Bacillus thuringiensis (Bt) is an important biological agent used against human and plant pests and diseases. Seven Jordanian Bt isolates, which have been analysed for toxicity against important pests, were also differentiated through serotyping. In this study, they were analysed at the molecular level using random amplified polymorphic DNA markers. Five more international strains were incorporated in the analysis. The DNA markers used showed high polymorphism among the isolates tested. However, the data did not align completely with earlier serotyping for most isolates. Therefore, it is recommended to engage several analyses (e.g. biochemical and molecular) when classifying newly surveyed Bt isolates in the world.     

Effect of L-cystine on macromolecular changes during spore and parasporal crystal formation inBacillus thuringiensis var.thuringiensis

High concentration of L-cystine (0.25%) when present in a glucose-mineral salt medium inhibited sporulation-specific events like protease production, calcium uptake and dipicolinic acid synthesis inBacillus thuringiensis var.thuringiensis. In addition, the enzymes of the Krebs cycle from aconitase onwards were completely inhibited by a high concentration of cystine. At a low concentration of cystine (0.05%), none of the above mentioned macromolecular changes were affected. Lipid synthesis monitored by [1,2¹⁴ C]-acetate incorporation into lipid as well as into whole cells was completely inhibited.     

Molecular typing of Bacillus thuringiensis serovars by RAPD-PCR

One hundred and twenty-six strains of Bacillus thuringiensis representing 57 serovars were allocated to 58 genomic types using random amplified polymorphic DNA (RAPD)-PCR patterns. Serovars darmstadiensis, israelensis, kenyae, kumamotoensis, kurstaki, morrisoni, pakistani, sotto, thuringiensis and tolworthi each encompassed identical or closely related strains. Despite this genomic homogeneity, most of these serovars also included at least one variant strain. Serovars aizawai, canadensis, entomocidus and sotto biotype dendrolimus, on the other hand, were genomically heterogeneous. Of the 57 serovars examined, 31 contained at least one strain with a closely related or identical RAPD pattern to a strain from a different serovar. We conclude that while the species is genomically diverse, the homogeneous serovars represent clonal lineages of successful insect pathogens.