Enterotoxin-producing strains of Bacillus thuringiensis isolated from food

P.H. DAMGAARD, H.D. LARSEN, B.M. HANSEN, J. BRESCIANI AND K. JØRGENSEN. 1996. Strains of Bacillus thuringiensis were isolated from various food items (pasta, pitta bread and milk) and were found to belong to either H-serotype kurstaki or neoleonensis. The strains were bioassayed against Pieris brassicae and insecticidal activity of strains was found to correspond to the presence of the cry1.A -gene. All strains, except one, were found to express cytotoxic effects on Vero cells as an indicator of enterotoxin activity. Further, the B. thuringiensis strains HD-1 (serotype kurstuki ), NB-125 (serotype tenebrionis ) and HD-567 (serotype isruelensis ) which are used commercially for insect pest management, were also found to have cytotoxic effects on Vero cells.     

Diarrhoeal enterotoxin production by strains of Bacillus thuringiensis isolated from commercial Bacillus thuringiensis-based insecticides

Abstract Strains of Bacillus cereus and B. thuringiensis were tested by the Tecra VIA kit for the ability to produce a diarrhoeal enterotoxin. The strains of B. thuringiensis were isolated from commercial B. thuringiensis -based insecticides (Bactimos^TM, DiPel^TM, Florbac^TM FC, Foray^TM 48B, Novodor^TM FC, Turex^TM, VecTobac^TM, XenTari^TM). The production of diarrhoeal enterotoxin varied by a factor of more than 100 among the different strains tested. B. cereus (F4433/73) produced the highest amount of enterotoxin and the B. thuringiensis strain isolated from DiPel^TM the lowest. The products were tested for their content of diarrhoeal enterotoxin and all products, except MVP^TM which does not contain viable B. thuringiensis spores, contained diarrhoeal enterotoxins. The results indicates an potential risk for gastroenteritis outbreak caused by B. thuringiensis .     

Characterization of two Bacillus thuringiensis ssp. morrisoni strains isolated from Thaumetopoea pityocampa (Lep., Thaumetopoeidae)

The pine processionary moth Thaumetopoea pityocampa Den. and Schiff. (Lep., Thaumetopoeidae) is one of the most harmful insect pests for pine species in Mediterranean countries including Turkey. Two Bacillus thuringiensis isolates obtained from T. pityocampa were identified and characterized in terms of crystal shape using electron microscopy, SDS–PAGE analysis, cry gene contents, H-serotype and insecticidal activity. Examination by a scanning electron microscope showed that Tp6 and Tp14 isolates have flat square and bipyramidal crystal shapes, respectively. PCR analysis showed that Tp6 contains cry3 gene and Tp14 isolate contains cry1 and cry2 genes. On the other hand, the presence of Cry3 and Cry1 proteins were confirmed by observation of approximately 65- and 130-kDa proteins by SDS–PAGE in Tp6 and Tp14 isolates, respectively. According to H-serotype results, these isolates were identified as Bacillus thuringiensis ssp. morrisoni (H8a8b). Toxicity tests were performed against six insect species belonging to Lepidoptera and Coleoptera. The highest insecticidal activity was 100% for Tp6 isolate on larvae of Agelastica alni and Leptinotarsa decemlineata and 100% for Tp14 isolate on larvae of Malacosoma neustria. Our results indicate that isolates Tp6 and Tp14 may be valuable biological control agents for various coleopteran and lepidopteran pests.     

Characterization of filament forming Bacillus strains isolated from bulking sludge

Summary Filament-forming bacilli were isolated from bulking sludge. They were physiologically very similar. However, they developed into rhizoid or non-rhizoid colonies. According to their morphological, physiological, and genetical properties the isolates were identified as Bacillus mycoides and Bacillus cereus.     

Molecular characterization of Bacillus thuringiensis strains from Argentina

Bacillus thuringiensis INTA 7-3, INTA 51-3, INTA Mo9-5 and INTA Mo14-4 strains were obtained from Argentina and characterized by determination of serotype, toxicity, plasmid composition, insecticidal gene content ( cry and vip ) and the cloning of the single- vip3A gene of the INTA Mo9-5 strain. The serotype analysis identified the serovars tohokuensis and darmstadiensis for the INTA 51-3 and INTA Mo14-4 strains, respectively, whereas the INTA Mo9-5 strain was classified as "autoagglutinated". In contrast to the plasmid patterns of INTA 7-3, INTA 51-3 and INTA Mo9-5 (which were similar to B. thuringiensis HD-1 strain), strain INTA Mo14-4 showed a unique plasmid array. PCR analysis of the four strains revealed the presence of cry genes and vip3A genes. Interestingly, it was found that B. thuringiensis 4Q7 strain, which is a plasmid cured strain, contained vip3A genes indicating the presence of these insecticidal genes in the chromosome. Bioassays towards various lepidopteran species revealed that B. thuringiensis INTA Mo9-5 and INTA 7-3 strains were highly active. In particular, the mean LC(50) obtained against A. gemmatalis larvae with the INTA Mo9-5 and INTA 7-3 strains were 7 (5.7-8.6) and 6.7 (5.6-8.0) ppm, respectively. The INTA Mo14-4 strain was non-toxic and strain INTA 51-3 showed only a weak larvicidal activity.     

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.     

Characterization of a mosquitocidal Bacillus thuringiensis serovar sotto strain isolated from Okinawa, Japan

AIMS: To characterize the mosquitocidal activity of parasporal inclusions of the Bacillus thuringiensis serovar sotto strain 96-OK-85-24, for comparison with two well-characterized mosquitocidal strains. METHODS AND RESULTS: The strain 96-OK-85-24 significantly differed from the existing mosquitocidal B. thuringiensis strains in: (1) lacking the larvicidal activity against Culex pipiens molestus and haemolytic activity, and (2) SDS-PAGE profiles, immunological properties and N-terminal amino acid sequences of parasporal inclusion proteins. CONCLUSIONS: It is clear from the results that the strain 96-OK-85-24 synthesizes a novel mosquitocidal Cry protein with a unique toxicity spectrum. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of the occurrence of a mosquitocidal B. thuringiensis strain with an unusual toxicity spectrum, lacking the activity against the culicine mosquito.     

Characterization of a Bacillus thuringiensis strain collection isolated from diverse Costa Rican natural ecosystems

Costa Rican natural ecosystems are among the most diverse in the world. For this reason, we isolated strains of the entomopathogenic bacteria Bacillus thuringiensis (Bt) to determine their diversity, distribution and abundance. A total of 146 Bt strains were obtained from environmental samples collected from diverse natural ecosystems and life zones of Costa Rica. We recovered Bt strains from 71%, 63%, 61% and 54% of soil samples, fresh leaves, other substrates and leaf litter respectively. Bt was isolated in 65% of the samples collected in the humid tropical forest in national parks (Braulio Carrillo, Gandoca Manzanillo, Sierpe, Hitoy Cerere, and Cahuita), and in 59% of the samples collected in the dry tropical forest (Parque Nacional Marino las Baulas, Palo Verde and Santa Rosa). In the very humid tropical forest (Tortuguero) Bt was isolated in 75% of the samples and in the very humid tropical forest transition perhumid (Carara) it was found in 69% of the samples. The strains exhibit a diverse number, size and morphology of parasporal inclusion bodies: irregular (47%), oval (20%), bipyramidal (3%), bipyramidal and cubic (1%), bipyramidal, oval and irregular (5%) and bipyramidal, oval and cubic crystals (2%). Strains isolated from Braulio Carrillo, Tortuguero and Cahuita, presented predominantly irregular crystals. On the other hand, more than 60% of the isolates from Térraba-Sierpe and Hitoy-Cerere had medium oval crystals. Strains from Gandoca-Manzanillo, Palo Verde and Carara presented mainly combinations of oval and irregular crystals. Nevertheless, the greatest diversity in crystal morphology was observed in those from Santa Rosa, Llanos del Rio Medio Queso and Parque Marino las Baulas. Protein analyses of the crystal-spore preparations showed delta-endotoxin with diverse electrophoretic patterns, with molecular weights in the range of 20 to 160 kDa. Fifty six percent of the strains amplified with the cry2 primer, 54% with vip3, 20% with cry1, 9% with cry3-cry7 and 8% with cry8. The cry11 and cyt genes were found in 8% and 7% of the strains, respectively. When analyzed with specific primers for the cryl subfamily, 13 different genetic profiles were obtained. In addition, twenty-four strains did not amplify with any of the primers used, suggesting they contain novel cry genes. The diversity of Bt genes found in this collection indicates it could have great potential for the control of different species of insect pests. The toxicological characterization of the strains by bioassays against important insect pests will provide useful information about their potential use for the formulation of biological insecticides and their respective cry and vip genes for the transformation of crops to confer resistance to insects.     

Diversity of Bacillus thuringiensis strains isolated from coffee plantations infested with the coffee berry borer Hypothenemus hampei

The coffee berry borer Hypothenemus hampei Ferrari (Coleoptera: Scolytidae) was first reported infecting Costa Rican coffee plantations in the year 2000. Due to the impact that this plague has in the economy of the country, we were interested in seeking new alternatives for the biological control of H. hampei, based on the entomopathogenic bacteria Bacillus thuringiensis. A total of 202 B. thuringiensis isolates obtained from Costa Rican coffee plantations infested with H. hampei were analyzed through crystal morphology of the crystal inclusions and SDS-PAGE of 6-endotoxins, while 105 strains were further evaluated by PCR for the presence cry, cyt and vip genes. Most of the Bt strains showed diverse crystal morphologies: pleomorphic (35%), oval (37%), bipyramidal (3%), bipyramidal and oval (12%), bipyramidal, oval and pleomorphic (10%) and bipyramidal, oval and cubic (3%). The SDS-PAGE analyses of the crystal preparations showed five strains with delta-endotoxin from 20 to 40 kDa, six from 40 to 50 kDa, seven from 50 to 60 kDa, 19 from 60 to 70 kDa, 29 from 70 to 100 kDa and 39 from 100-145 kDa. PCR analyses demonstrated that the collection showed diverse cry genes profiles having several genes per strain: 78 strains contained the vip3 gene, 82 the cry2 gene, 45 the cry1 and 29 strains harbored cry3-cry7 genes. A total of 13 strains did not amplified with any of the cry primers used: cry1, cry2, cry3-7, cry5, cry11, cry12 and cry14. Forty-three different genetic profiles were found, mainly due to the combination of cry1A genes with other cry and vip genes. The genetic characterization of the collection provides opportunities for the selection of strains to be tested in bioassays against H. hampei and other insect pests of agricultural importance.     

Characterization of Mexican Bacillus thuringiensis strains toxic for lepidopteran and coleopteran larvae

Bacillus thuringiensis strains C-4, C-9, GM-7, and GM-10, isolated from northeast Mexico and selected for their high toxicity against lepidopteran and coleopteran pests, were characterized following United States Environmental Protection Agency (EPA)'s guidelines. Flagellar serotyping revealed that GM-7 and GM-10 belonged to serotype aizawai, whereas C-4, C-9 corresponded to the kumamotoensis serotype. GM-10 and C-9 were also shown to be the most effective against lepidoptera and coleoptera larvae, respectively. None of the tested strains produced beta-exotoxin or showed activity against mosquitoes. GM-7 and GM-10 were sensitive to R-41 and CP-51 phages. All strains synthesized crystal proteins of 130-140 kDa. PCR analysis showed that C-4, GM-7, and GM-10 strains expressed cry1 genes, and C-9 expressed cry3 and cry7/8 genes, but not cry1. However, the C-9 strain had no cross-reaction with antisera raised against Cry3A and Cry7A proteins. GM-7 and GM-10 were sensitive to R-41 and CP-51 phages. When the delta-endotoxin (crystal) from the four strains was subcutaneously injected to Balb/c mice, alone or in combination with spores, only C-4 and C-9 provoked tissue necrosis similar to that caused by the beta-exotoxin producer HD-41. Tissue necrosis was prevented with the injection of pentoxifylline, an inhibitor of tumor necrosis factor alpha (TNF-alpha) production, suggesting a role of this cytokine in the observed effect. Our results demonstrated that GM-7 and GM-10 strains are effective and suitable for control of lepidopteran pests and safe for mammals under EPA regulations. The potential of the C-9 strain for the control of several coleopteran pests, and the induction of tissue necrosis in mice by C-4 and C-9 strains, are discussed.     

Characterization of Bacillus cereus strains isolated from drugs and evaluation of their toxins

The microbial contamination of 68 samples of topical and 324 samples of oral medicaments has been studied. The most common group of contaminants was members of the genus Bacillus (34.4%). Because of the pathogenic significance of B. cereus, 39 strains were characterized by morphology and biochemical properties. All except three showed most of the characteristics of the type strain. They were highly resistant to lincomycin, polymyxin B and penicillin G-cephalosporin and were susceptible to streptomycin, erythromycin and chloramphenicol. Enterotoxin, phospholipase C and haemolysin production were also studied: 33 strains gave a positive vascular permeability reaction, four of them causing necrosis, and 24 showed positive mouse lethal tests. All the strains had phospholipase activity. The majority also exhibited differing degrees of haemolysis. Permeability factor was related to mouse lethality and haemolytic activity. Phospholipase C was not related to any of the above activities.     

Characterization of Bacillus cereus strains isolated from drugs and evaluation of their toxins

The microbial contamination of 68 samples of topical and 324 samples of oral medicaments has been studied. The most common group of contaminants was members of the genus Bacillus (34.4%). Because of the pathogenic significance of B. cereus , 39 strains were characterized by morphology and biochemical properties. All except three showed most of the characteristics of the type strain. They were highly resistant to lincomycin, polymyxin B and penicillin G-cephalosporin and were susceptible to streptomycin, erythromycin and chloramphenicol. Enterotoxin, phospholipase C and haemolysin production were also studied: 33 strains gave a positive vascular permeability reaction, four of them causing necrosis, and 24 showed positive mouse lethal tests. All the strains had phospholipase activity. The majority also exhibited differing degrees of haemolysis. Permeability factor was related to mouse lethality and haemolytic activity. Phospholipase C was not related to any of the above activities.     

Characterization of chitinase‐producing Serratia and Bacillus strains isolated from insects

Chitinases (EC 3.2.1.14) are enzymes that hydrolyze chitin by cleaving β‐1,4 N‐glycosidic bonds. These enzymes have been used for multiple applications in biotechnology, especially for controlling insect pests and phytopathogenic fungi. In the present study, we isolated two chitinase‐producing bacteria strains from insects (strain SCH‐1 from Moechotypa diphysis and strain SCH‐2 from Sphedanolestes impressicollis). Serratia sp. SCH‐1 was a short, rod‐shaped facultative anaerobe, while Bacillus strain SCH‐2 was a rod‐shaped endospore‐forming anaerobe. Strains SCH‐1 and SCH‐2 were identified as Serratia sp. and Bacillus sp., respectively based on 16S rRNA gene sequencing. Strain SCH‐1 shared maximum homology (99.44%) with Serratia nematodiphila DZ0503SBS1 and Serratia marcescens subsp. sakuensis KRED. Strain SCH‐2 had a maximum homology of 99.24% with Bacillus thuringiensis ATCC 10792 and Bacillus toyonensis BCT‐7112. Serratia sp. SCH‐1 contained greater levels of saturated fatty acids, but the concentration of branched acids, especially iso‐C_15:0, was highest in Bacillus sp. SCH‐2. Serratia sp. SCH‐1 possessed chitinase activity of 1.59 unit/mg protein after 5 days of incubation in culture medium. In contrast, Bacillus sp. SCH‐2 had a maximum activity of 0.84 unit/mg protein after 4 days of incubation. Chitinase isozymes produced by Serratia sp. SCH‐1 appeared as five bands with sizes of 20, 26, 36, 45 and 54 kDa. Bacillus sp. SCH‐2 showed a chitinase isozyme profile with three bands having sizes of 36, 45 and 50 kDa on SDS‐PAGE gels.     

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.     

Characterisation of endophytic Bacillus thuringiensis strains isolated from wheat plants as biocontrol agents against wheat flag smut

To explore effective and ecofriendly means of controlling wheat flag smut (WFS) using biocontrol agents, three endophytic strains (58-2-1, 37-1 and YC-1) of Bacillus sp. were isolated from winter wheat plants in China and identified as Bacillus thuringiensis based on their 16S rDNA sequences as well as phenotypic characteristics. Four morphological (leaf length, root length, dry weight and tiller numbers) and one physiological [root vigour (RV)] parameters of wheat plants treated by strains 58-2-1 and 37-1 were significantly enhanced compared to the control. The soluble sugar contents in the roots of wheat samples treated by the two strains were significantly lower than the control. The resistance of wheat varieties to WFS was investigated by inoculation tests. Of the 12 wheat varieties tested, 6 (Yunhan-618, Bainongaikang-58, Kaimai-20, Zhengmai-9023, 04-zhong-36 and Yanzhan-4110) were identified as WFS-highly resistant (HR), 3 (Pumai-9, Jinboshi-1 and Yunong-202) WFS-moderately resistant (MR), 1 (Yubao-1) WFS-susceptible (S) and 2 (Yumai-012 and Yunong-416) WFS-highly susceptible (HS) varieties. The Urocystis tritici-induced yield loss on the S/HS varieties was significantly higher than that on the HR/MR ones. The strains 58-2-1 and 37-1 had control efficacies of 6.7–100% (av.54.8%) and 33.3–100% (av. 66.5%) on 9 and 7 out of 12 varieties, respectively. The strains 58-2-1 and 37-1 had enhanced yields of 10.2–54.9% (av. 32.9%) and 2.8–43.4% (av. 24.8%) on 10 and 8 out of 12 varieties, respectively. This is the first report on endophytic B. thuringiensis strains isolated from wheat plants with the abilities to suppress WFS and to enhance yields on multiple wheat varieties.