Differentiation of <Emphasis Type="Italic">Aspergillus niger</Emphasis> by random amplification of polymorphic DNA

The randomly amplified polymorphic DNA (RAPD) patterns of whole-cell lysates from five Aspergillus niger isolates, including one reference strain, two isolated from deep freeze, and two environmental strains from soil and plant infections, were investigated. PCR-RAPD analysis of genomic DNA was performed using eight primers (Tube-A1, Tube-A6, Tube-A17, Tube-B8, Tube-B11, Tube-B15, Tube-C5, Tube-C6). The RAPD assay discriminated between all strains. Comparison of deep freeze isolates showed identical RAPD patterns in some of the reference and environmental isolates. The data indicates that the RAPD technique is useful for fingerprinting A. niger.     

Phenotypic and genotypic features of new autoagglutinating Bacillus thuringiensis strains

A total of 28 autoagglutinating strains of Bacillus thuringiensis were isolated from different ecologic niches and distinct sites. Twenty-six strains demonstrated toxicity to mosquito larvae of Aedes aegypti and Culex quinquefasciatus. The electrophoretic protein profiles of the crystal components were studied. Twenty-three out of the 28 strains showed the same larvicidal activity and the same protein profiles as B. thuringiensis serovar israelensis. Using isoenzyme analysis (MLEE), it was observed the presence of three electrophoretic types (ETs). The mosquitocidal strains grouped into one ET. The random amplified polymorphic DNA analysis (RAPD) was evaluated using six primers, which demonstrated three different patterns for the 28 autoagglutinating strains, allowing correlation of the profiles obtained with the toxicity observed in the bioassays. The RAPD patterns for mosquitocidal strains were identical to the one of serovar israelensis. However, to strains of low toxicity, each primer generated distinctive RAPD patterns, which demonstrated that these strains belong to different serovars. Although the antigenic classification the 26 autoagglutinating strains of B. thuringiensis could not be determined by classical flagellar serotyping, MLEE and RAPD profiles proved these strains to be compatible with B. thuringiensis serovar israelensis.     

Competition and reproduction in mixed infections of pathogenic and non-pathogenic Bacillus spp

Diamondback moth, Plutella xylostella, larvae were infected with a primary pathogen, Bacillus thuringiensis kurstaki (Btk) in single strain and mixed infections. Mixed infections comprised Btk and a non-pathogenic isolate, either Bacillus thuringiensis tenebrionis (Btt) or Bacillus cereus (Bc). All strains reproduced in larval cadavers, but there was evidence of competition between different isolates within hosts. Non-pathogenic isolates (Btt, Bc) had growth rates that were faster than Btk in vivo, whereas Btk outcompeted Btt in vitro. Passage through insects increased the in vitro competitive ability of Btk against Btt.     

Properties of Bacillus thuringiensis isolated from bank voles

AIMS: To assess the properties of B. thuringiensis naturally occurring in the intestines of bank voles. METHODS AND RESULTS: Seventeen Bacillus thuringiensis strains, exhibiting typical growth on selective medium for the B. cereus group and characterized by the ability to produce parasporal crystals, were isolated from bank voles trapped in the ?omza Landscape Park of the Narew River Valley (north-east Poland). All isolates were characterized by pulsed field gel electrophoresis (PFGE) of chromosomal DNA and SDS polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins. Six pulsotypes were found with PFGE typing, using SmaI or NotI as restriction enzymes. Significant differences in chromosome size, ranging from 2.4 to 4.2 Mb for the B. thuringiensis strains studied, were noted. Strain heterogeneity in pulsotypes was also reflected by the similarity of whole-cell protein profiles of the strains. Environmental isolates and reference strains grouped at 71% similarity according to SDS-PAGE data and at 84% on the basis of biochemical tests. CONCLUSIONS: B. thuringiensis from intestines of bank voles demonstrated an important level of heterogeneity. The comparison of PFGE profiles and SDS-PAGE of whole-cell protein patterns may be useful to evaluate the relationship between B. thuringiensis isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The results presented in this paper may help to explain the diversity of B. thuringiensis.     

Comparative genotyping of Clostridium thermocellum strains isolated from biogas plants: Genetic markers and characterization of cellulolytic potential

Clostridium thermocellum is among the most prevalent of known anaerobic cellulolytic bacteria. In this study, genetic and phenotypic variations among C. thermocellum strains isolated from different biogas plants were determined and different genotyping methods were evaluated on these isolates. At least two C. thermocellum strains were isolated independently from each of nine different biogas plants via enrichment on cellulose. Various DNA-based genotyping methods such as ribotyping, RAPD (Random Amplified Polymorphic DNA) and VNTR (Variable Number of Tandem Repeats) were applied to these isolates. One novel approach – the amplification of unknown target sequences between copies of a previously discovered Random Inserted Mobile Element (RIME) – was also tested. The genotyping method with the highest discriminatory power was found to be the amplification of the sequences between the insertion elements, where isolates from each biogas plant yielded a different band pattern. Cellulolytic potentials, optimal growth conditions and substrate spectra of all isolates were characterized to help identify phenotypic variations. Irrespective of the genotyping method used, the isolates from each individual biogas plant always exhibited identical patterns. This is suggestive of a single C. thermocellum strain exhibiting dominance in each biogas plant. The genotypic groups reflect the results of the physiological characterization of the isolates like substrate diversity and cellulase activity. Conversely, strains isolated across a range of biogas plants differed in their genotyping results and physiological properties. Both strains isolated from one biogas plant had the best specific cellulose-degrading properties and might therefore achieve superior substrate utilization yields in biogas fermenters.     

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.     

The symbiont Capsaspora owczarzaki,nov. gen. nov. sp., isolated from three strains of the pulmonate snail Biomphalaria glabrata is related to members of the Mesomycetozoea

While investigating the resistance of some strains of Biomphalaria glabrata to infection with Schistosoma mansoni, a unicellular eukaryotic symbiont was noted in the snail haemolymph. It was similar in appearance to Nuclearia sp. reported from B. glabrata. Sequences comprising the 18S, ITS1, 5.8S, ITS2 and the beginning of the 28S rDNA gene regions were obtained from symbionts isolated from three strains of B. glabrata, and compared with the same sequences obtained from a culture of Nuclearia sp. 18S rDNA sequences were identical for all four isolates. 18S rDNA sequences were used in a phylogenetic analysis to produce minimum evolution, maximum parsimony, maximum likelihood and Bayesian trees. All four analyses indicated that the B. glabrata symbiont is not closely related to Nuclearia but instead to the Mesomycetozoea, a recently recognised clade of symbiotic eukaryotes. Based on phylogenetic analysis, life history and morphological differences, the symbiont is described as a new genus and species, Capsaspora owczarzaki. Distinguishing characters are the presence of life cycle stage(s) that occur within snail haemolymph; ability to kill and ingest digenetic trematode larvae; ability to undergo asexual fission to produce daughter cells; absence of flagella, a mucous sheath and membranes containing chitin, elastin, or collagen; and presence of long unbranching pseudopodia and a penetration process. Using both polymerase chain reaction (PCR) and culturing techniques, the S. mansoni-resistant Salvador and 13-16-R1 strains were found to be significantly more likely to harbour the symbiont than the susceptible M line strain. Small but consistent sequence differences were noted among symbiont isolates from different snail strains, raising the possibility that the symbiont has diverged in different snail lineages. This suggests further that the symbiont is not restricted to albino lab-reared snails. A role, if any, of the symbiont in resistance awaits further study.     

Isolation of a non-insecticidal Bacillus thuringiensis strain belonging to serotype H8a8b

Bacillus thuringiensis strains non-toxic to Lepidoptera, Bombyx mori and Diptera, Culex pipiens pallens larvae were isolated from Korean soil samples during an investigation of B. thuringiensis isolates highly toxic to insect pests. One of these isolates, NTB-88, produces parasporal inclusions about 138 kDa in size and is non-toxic to 19 insect species of three orders, Lepidoptera, Diptera and Coleoptera, even though it is highly susceptible to tryptic cleavage. Study of flagellar (H) antibodies of 33 B. thuringiensis strains revealed that NTB-88 has an H antigen identical with that of subsp. morrisoni (serotype 8a8b). Comparison of parasporal inclusion proteins and plasmid DNA patterns of strain NTB-88 with B. thuringiensis subsp. morrisoni HD-12 and B. thuringiensis subsp. morrisoni PG-14 showed that the isolate is a novel non-insecticidal B. thuringiensis strain belonging to serotype 8a8b.     

Genomic fingerprinting of Bradyrhizobium japonicum isolates by RAPD and rep-PCR

Genetic diversity of indigenous Bradyrhizobium japonicum population in Croatia was studied by using different PCR-based fingerprinting methods. Characteristic DNA profiles for 20 B. japonicum field isolates and two reference strains were obtained using random primers (RAPD) and two sets of repetitive primers (REP- and ERIC-PCR). In comparison with the REP, the ERIC primer set generates fingerprints of lower complexity, but still several strain-specific bands were detected. Different B. japonicum isolates could be more efficiently distinguished by using combined results from REP- and ERIC-PCR. The most polymorphic bands were observed after amplification with four different RAPD primers. Both methods, RAPD and rep-PCR, resulted in identical grouping of the strains. Cluster analysis, irrespective of the fingerprinting method used, revealed that all the isolates could be divided into three major groups. Within the major groups, the degree of relative similarity between B. japonicum isolates was dependent upon the method used. Our results indicate that both RAPD and rep-PCR fingerprinting can effectively distinguish different B. japonicum strains. RAPD fingerprinting proved to be slightly more discriminatory than rep-PCR.     

Temporal association of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) and bacteria

Galleria mellonella L. larvae were infected with three species (seven strains) of Steinernema spp. or three species (three strains) of Heterorhabditis spp. Infected larvae were incubated at 22, 27, and 32 degrees C. Larvae were dorsally dissected every 6h over a 48-h period. Hemolymph was collected and streaked on tryptic soy agar plates. Several non-symbiotic bacterial species were identified from infected insect cadavers: Enterobacter gergoviae, Vibrio spp., Pseudomonas fluorescens type C, Serratia marcescens, Citrobacter freundii, and Serratia proteomaculans. At 18-24 h incubation, the nematode-associated symbiont occurred almost exclusively. Bacterial associates generally appeared outside the 18-24 h window. Infective juveniles of Steinernema feltiae (Filipjev) (27), Steinernema riobrave Cabanillas, Poinar, and Raulston (Oscar), or Steinernema carpocapsae (Weiser) (Kapow) were left untreated, or surface sterilized using thimerosal, then pipetted under sterile conditions onto tryptic soy agar plates. Several additional species of associated bacteria were identified using this method compared with the less extensive range of species isolated from infected G. mellonella. There was no difference in bacterial species identified from non-sterile or surface sterilized nematodes, suggesting that the bacteria identified originated from either inside the nematode or between second and third stage juvenile cuticles. Infective juveniles of S. feltiae (Cowles), S. carpocapsae (Cowles), and H. bacteriophora Poinar (Cowles) were isolated from field samples. Nematodes were surface-sterilized using sodium hypochlorite, mixed with G. mellonella hemolymph, and pipetted onto Biolog BUG (with blood) agar. Only the relevant symbionts were isolated from the limited number of samples available. The nematodes were then cultured in the laboratory for 14 months (sub-cultured in G. mellonella 7-times). Other Enterobacteriaceae could then be isolated from the steinernematid nematodes including S. marcescens, Salmonella sp., and E. gergoviae, indicating the ability of the nematodes to associate with other bacteria in laboratory culture.     

Genotypic analyses of lactobacilli with a range of tannase activities isolated from human feces and fermented foods

A total of 77 tannase producing lactobacilli strains isolated from human feces or fermented foods were examined for their genotypic profiles and intensities of tannase production. With a PCR-based assay targeting recA gene, all strains except one isolate were assigned to either Lactobacillus plantarum, L. paraplantarum, or L. pentosus whereas a 16/23S rDNA targeted PCR-based assay identified all except 6 isolates (inclusive of the above one isolate) as one of the closely related species. Subsequent DNA/DNA hybridization assays revealed that these 6 exceptional isolates showed low homology (between 1.2% and 55.8% relative DNA binding) against type strains of the three species. Supplemental carbohydrate fermentation profiles on the 6 isolates indicated that two of them were identified as L. acidophilus, one as Pediococcus acidilactici, one as P. pentosaceus, and two remained unidentifiable. The evidence suggests that the 16/23S rDNA targeted PCR assay can be used as a reliable identification tool for the closely related lactobacilli, and that the tannase gene is widely distributed within members of the Lactobacillaceae family. Meanwhile, a randomly amplified polymorphism DNA (RAPD) analysis revealed that all except 8 isolates were well allocated in 4 major RAPD clusters, though not species specific, consisting of two L. plantarum predominant clusters, one L. paraplantarum predominant, and one L. pentosus predominant. The RAPD patterns of the 8 non-clustered isolates, which consisted of the 6 unidentifiable isolates and 2 isolates identified as L. pentosus, were <40% similarity to those belonging to the 4 clusters. A quantitative assay of the tannase activities showed that there was a marked variation in the activities among the strains, which did not correlate with either species identification or clustering by RAPD.     

Novel Endophytes of Rice form a Taxonomically Distinct Subgroup of Serratia marcescens

Six endophytic strains isolated from surface-sterilized rice roots and stems of different rice varieties grown in the Philippines were characterized. They were analyzed by physiological and biochemical tests, SDS-PAGE of whole-cell protein patterns, DNA-DNA hybridization and 16S rDNA sequencing. SDS-PAGE of whole-cell patterns showed that the six isolates fell into two subgroups which were similar but not identical in protein patterns to S. marcescens. The phylogenetic analysis of 16S rDNA sequences of two representative strains IRBG 500 and IRBG 501 indicated that they were closely related to S. marcescens(more than 99% identity). Physiological and biochemical tests corroborated that the isolates were highly related to each other and to S. marcescens. In cluster analysis, all six isolates were clustered together at 93% similarity level and grouped closely with Serratia marcescens at 86% similarity level. DNA-DNA hybridization studies revealed that the isolates shared high similarity levels with S. marcescens(≥86% DNA-DNA binding), indicating they belong to the same species. However, the isolates differed in several biochemical characteristics from the type strain. They produce urease and utilize urea and L(+) sorbose as a substrate, which is different from all known Serratia reference strains. These results suggest that the six endophytic isolates represent a novel, non-pigmented subgroup of S. marcescens.     

Isolation and toxicity of Bacillus thuringiensis from potato-growing areas in Bolivia

Bacillus thuringiensis was isolated from 116 samples collected in high altitude potato-growing areas in Bolivia. In these regions, main potato pests are the potato tuberworm Phthorimaea operculella, and the Andean weevils Premnotrypes latithorax and Rhigopsidius tucumanus. B. thuringiensis was found in 60% of the samples. The main percentage of samples with B. thuringiensis was found in larvae of R. tucumanus (78%). Bioassays were performed with 112 isolates. None resulted toxic to either larvae or adults of the two Andean weevils. However, 18 isolates from this study showed more toxicity against the beet armyworm Spodoptera exigua than the standard strain var. kurstaki isolated from DELFIN. Among these isolates, three were also effective against P. operculella, conferring better or equal protection to the tubers than the reference strain HD-1 isolated from DIPEL. The most toxic strains against S. exigua and P. operculella were characterized in terms of serotyping, crystal morphology, protein profile, and cry gene content. PCR was performed with primers amplifying genes from the cry1, cry2, cry3, cry4, cry7, 8, and cry9Aa families. The toxic strains presented bipyramidal crystals, at least a band of 130kDa in SDS-PAGE, and showed an amplification product with cry1 family primers. One of the isolates did not amplify with any specific primer belonging to known cry1 genes. Restriction Fragment Length Polymorphism (RFLP) confirmed the presence of a novel gene and sequence comparison showed that this gene had homology to cry1G.     

The occurrence and properties of Bacillus thuringiensis isolated from free-living animals

AIMS: To assess the prevalence and properties of Bacillus thuringiensis isolated from the intestines of small mammals. METHODS AND RESULTS: Bacillus thuringiensis was found in 11% of rodents and 17% of insectivores. Using PFGE of chromosomal DNA, SDS-PAGE of whole-cell proteins and biochemical tests (API system), 12 isolates and three reference strains were classified. Numerical analysis revealed 61% and 89% similarity of protein profiles and biochemical properties of the bacilli, respectively. The results of PFGE were consistent with the outcomes of the analysis of protein profiles. CONCLUSIONS: Although B. thuringiensis is not common in the intestines of small mammals, it is heterogeneous at the genotypic and phenotypic level. SIGNIFICANCE AND IMPACT OF THE STUDY: The results presented here help to explain the diversity and the ecological significance of B. thuringiensis. Future study should focus on the toxic activity of the isolated strains.     

Identification and differentiation of Lactobacillus, Streptococcus and Bifidobacterium species in fermented milk products with bifidobacteria

The aim of this study was to identify and discriminate bacteria contained in commercial fermented milks with bifidobacteria by the use of amplified ribosomal DNA restriction analysis (ARDRA) and randomly amplified polymorphic DNA (RAPD) techniques. ARDRA of the 16S rDNA gene and RAPD were performed on 13 Lactobacillus strains, 13 Streptococcus and 13 Bifidobacterium strains isolated from commercial fermented milk. Lactobacillus delbrueckii, Streptococcus thermophilus and Bifidobacterium animalis isolates were identified by genus- and species-PCR and also, they were differentiated at genus and species level by ARDRA using MwoI restriction enzyme. The ARDRA technique allowed for the discrimination among these three related genus with the use of only one restriction enzyme, since distinctive profiles were obtained for each genus. Therefore it can be a simple, rapid and useful method for routine identification. Also, RAPD technique allowed the discrimination of all bacteria contained in dairy products, at genus- and strain-level by the performance of one PCR reaction.     

16S rDNA-based phylogeny of non-symbiotic bacteria of Entorno-pathogenic nematodes from infected insect cadavers

Using 16S rDNA gene sequencing technique, three different species of non-symbiotic bacteria of entomopatho-genic nematodes (EPNs) (Steinernema sp.and Heterorhabditis sp.) were isolated and identified from infected insect cadavers(Galleria mellonella larvae) after 48-hour post infections.Sequence similarity analysis revealed that the strains SRK3, SRK4 and SRK5 belong to Ochrobactrum cytisi,Schineria larvae and Ochrobactrum anthropi,respectively.The isolates O.anthropi and S.larvae were found to be associated with Heterorhabditis indica strains BDU-17 and Yer-136,respectively,whereas O.cytisi was associated with Steinernema siamkayai strain BDU-87. Phenotypically, temporal EPN bacteria were fairly related to symbiotic EPN bacteria (Photorhabdus and Xenorhabdus genera). The strains SRK3 and SRK5 were phylogeographically similar to several non-symbionts and contaminated EPN bacteria isolated in Germany(LMG3311T) and China (X-14),while the strain SRK4 was identical to the isolates of S.larvae (L1/57,L1/58, L1/68 and L2/11) from Wohlfahrtia magnifica in Hungary.The result was further confirmed by RNA secondary structure and minimum energy calculations of aligned sequences.This study suggested that the non-symbionts of these nematodes are phylogeographically diverged in some extent due to phase variation.Therefore,these strains are not host-dependent, but environment-specific isolates.     

Plasmid transfer between strains of Bacillus thuringiensis infecting Galleria mellonella and Spodoptera littoralis.

To determine the possibility of plasmid transfer occurring between strains of Bacillus thuringiensis in infected lepidopterous larvae, Galleria mellonella and Spodoptera littoralis were infected with two or more strains of B. thuringiensis and the resulting bacteria from the dead insects were examined for plasmid transfer. Transfer rates of plasmids coding for crystal production and tetracycline resistance were high, reaching levels similar to those obtained in laboratory broth cultures. Transfer was higher in G. mellonella than S. littoralis, probably due to the greater ability of B. thuringiensis to colonize the larvae. In broth cultures, B. thuringiensis was also able to transfer plasmids into sporeforming bacteria present in soil samples. The results suggest that plasmid transfer between strains of B. thuringiensis occurs in nature, resulting in the production of new combinations of delta-endotoxins within populations of the bacteria.     

Inhibition of the growth of Paenibacillus larvae, the causal agent of American foulbrood of honeybees, by selected strains of aerobic spore-forming bacteria isolated from apiarian sources

The bacterium Paenibacillus larvae, the causative agent of American foulbrood disease of honeybee larvae, occurs throughout the world and is found in many beekeeping areas of Argentina. The potential as biocontrol agents of antagonic aerobic spore-forming bacteria isolated from honey samples and other apiarian sources were evaluated. Each isolate was screened against one strain of Paenibacillus larvae (ATCC 9545) by using a perpendicular streak technique. Ten randomly selected bacterial strains from the group that showed the best antagonistic effect to P. larvae ATCC 9545 were selected for further study. These were identified as Bacillus subtilis (m351), B. pumilus (m350), B. licheniformis (m347), B. cereus (mv33), B. cereus (m387), B. cereus (m6c), B. megaterium (m404), Brevibacillus laterosporus (BLAT169), B. laterosporus (BLAT170), and B. laterosporus (BLAT171). The antagonistic strains were tested against 17 P. larvae strains from different geographical origins by means of a spot test in wells. The analysis of variance and posterior comparison of means by Tukey method (P < 0.01) showed that the best antagonists were B. megaterium (m404), B. licheniformis (m347), B. cereus (m6c), B. cereus (mv33), and B. cereus (m387).     

Investigations on bacteria as a potential biological control agent of summer chafer, Amphimallon solstitiale L. (Coleoptera: Scarabaeidae)

Studying the bacteria of hazardous insects allows the opportunity to find potentially better biological control agents. Therefore, in this study, bacteria from summer chafer (Amphimallon solstitiale L., Coleoptera: Scarabaeidae) we isolated and identified the insecticidal effects of bacteria isolated from A. solstitiale and Melolontha melolontha L. (common cockchafer, Coleoptera: Scarabaeidae) and the mixtures of these bacterial isolates were investigated on A. solstitiale larvae. Crystals from Bacillus sp. isolated from M. melolontha were also purified, and tested against the second and third-stage larvae of A. solstitiale. The bacterial isolates of A. solstitiale were identified as Pseudomonas sp., Pseudomonas sp., Bacillus cereus and Micrococcus luteus, based on their morphology, spore formation, nutritional features, and physiological and biochemical characteristics. The insecticidal effects of the bacterial isolates determined on the larvae of A. solstitiale were 90% with B. cereus isolated from A. solstitiale, and 75% with B. cereus, B. sphaericus and B. thuringiensis isolated from M. melolontha within ten days. The highest insecticidal effects of the mixed infections on the larvae of A. solstitiale were 100% both with B. cereus+B. sphaericus and with B. cereus+B. thuringiensis. In the crystal protein bioassays, the highest insecticidal effect was 65% with crystals of B. thuringiensis and B. sphaericus isolated from M. melolontha within seven days. Finally, our results showed that the mixed infections could be utilized as microbial control agents, as they have a 100% insecticidal effect on the larvae of A. solstitiale.     

Plasmid transfer between strains of Bacillus thuringiensis infecting Galleria mellonella and Spodoptera littoralis

To determine the possibility of plasmid transfer occurring between strains of Bacillus thuringiensis in infected lepidopterous larvae, Galleria mellonella and Spodoptera littoralis were infected with two or more strains of B. thuringiensis and the resulting bacteria from the dead insects were examined for plasmid transfer. Transfer rates of plasmids coding for crystal production and tetracycline resistance were high, reaching levels similar to those obtained in laboratory broth cultures. Transfer was higher in G. mellonella than S. littoralis, probably due to the greater ability of B. thuringiensis to colonize the larvae. In broth cultures, B. thuringiensis was also able to transfer plasmids into sporeforming bacteria present in soil samples. The results suggest that plasmid transfer between strains of B. thuringiensis occurs in nature, resulting in the production of new combinations of delta-endotoxins within populations of the bacteria.