Studies on Bacillus thuringiensis strains isolated from Swedish soils: insect toxicity and production of B. cereus-diarrhoeal-type enterotoxin

At moderate concentration, 23 of 40 strains of Bacillus thuringiensis isolated from Sweden were toxic to Trichoplusia ni and five were toxic to Aedes aegypti. Five of the strains were toxic to Diabrotica undecimpunctata at high concentration, two were toxic to Heliothis virescens at low concentration and five produced thuringiensin (formerly called -exotoxin). No strain was toxic towards the beet armyworm Spodoptera exigua at low concentration. Twenty-three of the strains produced a B. cereus-diarrhoeal-type enterotoxin.A. Abdel-Hameed is with the Department of Microbiology, Faculty of Pharmacy, Cairo University, Kasr-El-Aini Street, Cairo, Egypt. R. Landén is with the Department of Microbiology, Stockholm University, S-10691 Stockholm, Sweden. A. Abdel-Hameed's present address is the Department of Applied Chemistry and Microbiology, P. O. Box 27, Viikki, Building B, FIN-00014 University of Helsinki;     

Toxicity of the blue-green alga Oscillatoria agardhii to the mosquito Aedes aegypti and the shrimp Artemia salina

The cyanobacterium Oscillatoria agardhii 27, which does not produce mammalian neuro- or hepatotoxins, was highly toxic to the larval stages of the yellow fever mosquito Aedes aegypti: its 24-h LC₅₀ values against fourth-and second-instar larvae of A. aegypti were 8.7 and 6.1 g live cells/ml, respectively. The toxin was water-soluble and was partially purified but the chemical nature of the toxic compound(s) is still unknown. Aqueous solutions were also toxic to the newborn larvae of the brine shrimp, Artemia salina, used for the bioassay. The toxic activity of these solutions decreased markedly on heating to 90°C for 15 min.J. Kiviranta is with the Department of Pharmacy, P.O. Box 15, FIN-00014 University of Helsinki, Helsinki, Finland; A. Abdel-Hameed is usually with the Department of Microbiology, Faculty of Pharmacy, Cairo University, Kasr-El-Aini Street, Cairo, Egypt, but is presently with the Department of Applied Chemistry and Microbiology, P.O. Box 27, Viikki, Building B, FIN-00014 University of Helsinki, Helsinki, Finland.     

Detection of Vibrio cholerae O1 in the aquatic environment in Brazil employing direct immunofluorescence microscopy

Culturing and immunofluorescence (FA) methods for detection of Vibrio cholerae O1 in samples collected from the aquatic environment at selected sites in Brazil were compared. Of the samples examined, 90% were positive for V. cholerae O1 by FA but none was positive by culture, although strains of V. cholerae other than O1 strains were readily isolated. Evidence for V. cholerae O1 being autochthonous to the aquatic environment of Brazil is presented. Furthermore, FA methods are recommended for cholera surveillance programmes directed at the natural environment.M.T. Martins is with the Department of Microbiology, I.C.B. II, University of Sao Paulo, Sao Paulo, S.P. CEP 05508, Brazil. P.S. Sanchez and M.I.Z. Sato are with the State Agency for Environmental Control-CETESB, Sao Paulo, S.P. CEP 05459, Brazil. P.R. Brayton and R.R. Colwell are with the Department of Microbiology, University of Maryland, College Park, MD 20742, USA; R.R. Colwell is also with the Center of Marine Biotechnology, Maryland Biotechnology Institute, 600 East Lombard St, Baltimore, MD 21202, USA.     

Isolation and characterization of Bacillus thuringiensis strains from olive-related habitats in Turkey

Aims: To isolate Bacillus thuringiensis strains from different olive‐related habitats (olive groves and olive oil factories) in Turkey and to characterize these strains by molecular methods. Methods and Results: A total of 150 samples, consisting of olive grove soil, green olive leaves, olive leaf residues, animal faeces, olive pomace and dust, were examined for the presence of B. thuringiensis. One hundred B. thuringiensis strains were isolated from 54 environmental samples (36%) and characterized in terms of crystal morphology, cry and cyt gene content by polymerase chain reaction, plasmid profiles and 16S‐internal transcribed spacer ribosomal DNA restriction fragment length polymorphism (16S‐ITS rDNA RFLP). The highest percentage of samples containing B. thuringiensis was found in 38 out of 54 total soil samples (70%). Of the 100 B. thuringiensis isolates, the most frequent crystal shapes were irregularly shaped (24%), spherical‐irregular pointed (19%), cuboidal (17%) and spherical (16%). The cry1 plus cry4 genotype was the most abundant genotype in our collection (21%). RFLP analysis of the amplified 16S‐ITS rDNA revealed 11 distinct patterns for the isolates and 10 reference strains. Conclusions: Bacillus thuringiensis isolates showed a great genetic diversity and crystal shape heterogeneity. Significance and Impact of the Study: This is the first study on the isolation and characterization of B. thuringiensis from olive‐related habitats in Turkey. No correlation was observed between the cry genotypes and insecticidal crystal shapes of the isolates. Restriction profiles of 23% of the isolates were found to be different from those of the 10 reference strains used.     

Characterization of Bacillus thuringiensis ser. balearica (Serotype H48) and ser. navarrensis (Serotype H50): Two Novel Serovars Isolated in Spain

The novel strains of Bacillus thuringiensis PM9 and NA69, isolated from soil samples in Spain, were classified and characterized in terms of their crystal proteins, plasmid profile, cry genes content, and their toxicological properties against several species of Lepidoptera, Coleoptera, and Diptera. Both strains share morphological and biochemical characteristics with previously described B. thuringiensis strains, although their unique H antigens identify them as two new serotypes. Two new serovar names, B. thuringiensis serovar balearica (H serotype 48) and B. thuringiensis serovar navarrensis (H serotype 50) are proposed for the type strains PM9 and NA69, respectively. Received: 22 June 1999 / Accepted: 2 August 1999     

Genetic Diversity of Bacillus cereus/B. thuringiensis Isolates from Natural Sources

The genetic diversity and relationships among 154 Bacillus cereus/B. thuringiensis isolates recovered from soil samples from five geographic areas in Norway were investigated with multilocus enzyme electrophoresis (MEE). Cluster analysis revealed two major groups (designated cluster I and cluster II) separated at genetic distance greater than 0.55. Cluster I included 62 electrophoretic types (ETs) originating from all five locations, whereas, in cluster II, all but one isolate were from the same location. The isolates were also serotyped with B. thuringiensis flagellar antisera, and 28 distinct serotypes were identified. In general, serotyping did not show correlation to the genetic diversity of the isolates. The presence of IS231- and IS240-like transposable elements was detected in 14% of the strains of cluster II only. Parasporal crystals were observed in three strains; ten other strains were toxic to Trichoplusia ni. We conclude that B. cereus/B. thuringiensis from soil exhibit a high degree of recombination. Received: 15 December 1997 / Accepted: 26 January 1998     

Growth,sporulation and toxin production byBacillus thuringiensis subsp.israelensis andB. sphaericus in media based on mustard-seed meal

Bacillus thuringiensis subsp.israelensis andB. sphaericus strains 2362 and 1593 were grown in media based on defatted mustard-seed meal (MSM). The meal contains 40% (w/w) protein, with glutamic acid and arginine as the major amino acids. The toxic potencies of the final bacterial powders towardsCulex pipens quinquefasciatus Say, compared with those of the respective international reference standards, were 46% forB. thuringiensis subsp.israelensis, 62% forB. sphaericus 2362 and 88% forB. sphaericus 1593 when 2% (w/v) MSM was used for growth. With 4% (w/v) MSM,B. thuringiensis subsp.israelensis grew better but had undetectable larvicidal activity, whereas theB. sphaericus strains not only grew better but gave a higher degree of sporulation and toxicity. The potencies ofB. sphaericus in medium with 4% MSM were comparable with those of international reference standards.The authors are with the Department of Life Sciences, University of Bombay, Bombay 400 098, India.     

Polysaccharide production by Aureobasidium pullulans: factors affecting polysaccharide formation

Aureobasidium pullulans NRRL 6220 synthesized polysaccharide most actively in media containing sucrose, fructose or maltose with (NH₄)₂SO₄ (0.6 g/l) or ammonium acetate giving greatest yields of the polysaccharide. With (NH₄)₂SO₄ at 1.2 g/l, production of polysaccharide was decreased considerably. Polysaccharide production was highest with an initial pH of 6.5 while biomass formation was better below an initial pH of 5.5. Optimum phosphate concentration for polysaccharide production was 0.03 m.S.M. Badr-Eldin, H.G. El-Masry and O.A. Abd El-Rahman are with the Microbial Chemistry Department, National Research Center, Dokki, Cairo, Egypt; F.H.A. Mohamad is with the Chemical Engineering and Pilot Plant Department, National Research Center, Dokki, Cairo, Egypt. O.M. El-Tayeb is with the Microbiology Department, Faculty of Pharmacy, Cairo University, Egypt.     

Role of membrane potential in ammonium inhibition of nitrogenase activity in the cultured cyanobiont Nostoc ANTH

Ammonium at 5mM completely inhibited nitrogenase activity of Nostoc ANTH but only slightly inhibited the membrane potential, indicating that these two events are independent and that nitrogenase activity is not regulated by the latter.B.B. Singh and P.S. Bisen are with the Department of Microbiology, Barkatullah University, Bhopal-462026, India; S. Singh was with the Department of Microbiology, Barkatullah University, Bhopal, India; he is now with the Department of Microbiology, North Maharastra University, Jalgaom-425001, India.     

A new species of Gigaspora from desert soils of Rajasthan,India

A new species of the endogonaceous fungus Gigaspora, isolated from the Indian semi-arid region, is described. The fungus, named G. tuberculata, produces rusty-brown azygospores with septate subtending hypha. The azygospores bear warts all over the outer wall. The shape, size and general appearance of these spores resemble those of Scutellospora persica.Neeraj and A.K. Varma are with the Microbiology Unit, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, India; K.G. Mukerji is with the Applied Mycology Laboratory, Botany Department, University of Delhi, Delhi 110 006, India. B.C. Sharma is with the Department of Textile Engineering, Indian Institute of Technology, New Delhi 110 016, India.     

Toxicity of a <Emphasis Type="Italic">Bacillus thuringiensis israelensis</Emphasis>-like strain against <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis>

Bacillus thuringiensis (Bt) Berliner is a promising agent for microbial control of agriculturally and medically important insects. This study aimed at searching for Bt strains encoding Cry proteins that act more efficiently against fall armyworm. Thirty Bt strains were isolated from soil samples in Pernambuco State and evaluated through bioassays. Among these, strain I4A7 was the most efficient against the fall armyworm, Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera: Noctuidae), and thus it was characterized by biochemical sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and molecular (polymerase chain reaction (PCR) and sequencing reaction) methods. The protein pattern of this strain on a SDS–PAGE was similar to that of B. thuringiensis israelensis (Bti). Moreover, I4A7 cry DNA sequence showed high identity (99–100%) to genes cry4Aa, 4Ba, 10Aa, 11Aa, cyt1Aa and cyt2B from Bti. The toxicity of the newly isolated Bti-like strain upon S. frugiperda should be considered as this strain might be used in combination with other Bt strains, such as B. thuringiensis var. kurstaki (Btk). Handling Editor: Helen Roy.     

Flagellin (FliC) protein sequence diversity among <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> does not correlate with H serotype diversity

In Escherichia coli, the fliC gene encodes flagellin, the protein responsible for eliciting the immunological reaction in H serotyping. Here, the presence of the flagellin fliC gene was studied in 86 Bacillus thuringiensis strains encompassing 67 H serotypes. Nineteen strains from four additional species in the B. cereus sensu lato group, B. cereus, B. anthracis, B. mycoides, and B. weihenstephanensis, were added for comparison purposes. The fliC genes were amplified, cloned and their nucleotide sequences determined and translated into amino acid sequences. A bootstrapped neighbor-joining tree was generated from the alignment of the translated amino acid sequences of the amplicons. Although most B. thuringiensis H serotypes had different flagellin amino acid sequences, some different B. thuringiensis serovars shared identical flagellin amino acid sequences. In addition, although serovars from the same H serotype were sometimes found clustered together, several serovars from the same H serotype carried flagellins with sufficiently different amino acid sequences as to be located on distant clusters. No correlations could be established between flagellin (FliC) protein sequence diversity among B. thuringiensis H serotypes and H serotype diversity. These suggest that the B. thuringiensis fliC gene does not code for the flagellin copy responsible for eliciting the immunological reaction in H serotyping. In a previous study, the authors have shown that the B. thuringiensis hag gene codes for the flagellin copy responsible for eliciting the immunological reaction in H serotyping. It is proposed that the B. thuringiensis fliC gene studied here be renamed and that the so-called hag gene studied before be renamed fliC, both in accordance with the E. coli nomenclature. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Physiological and molecular detection of crystalliferous Bacillus thuringiensis strains from habitats in the South Central United States

Gram-positive, endospore-forming Bacillus thuringiensis-like strains were isolated from 95 of 413 samples collected at the 0–5 cm depth of noncultivated soils and stagnant or dried-up ponds as well as from dust from stored grain products in South Central United States. Based on the production of parasporal crystals, 25 isolates were identified as B. thuringiensis after examining 227 B. thuringiensis-like colonies. The greatest proportion of samples yielding B. thuringiensis were from the dust from grain storage. The sodium acetate selective medium, heat processing, and crystal staining used in the initial screening revealed diverse populations of B. thuringiensis, which were categorized into distinct crystal morphological groups. Sugar fermentation, antibiotic sensitivity, growth characteristics and PCR studies showed diversity among the isolates that were distributed among 25 of the 58 known strains. The most frequently isolated strains were kurstaki, aizawai, morrisoni, thuringiensis, sotto and kenyae that together represented more than 90% of the characterized isolates. PCR analysis using 30 family primer pairs for cry and cyt genes showed that the frequency of the cry1 gene (62%) was predominant followed by the cry2 genes (30%), and the rest (8%) were other cry gene types, such as cry3, cry4, cry10, cry11, cry14, cry15, cry20, cry24 and cry26. Both cyt1 and -2 genes were also detected. Several isolates showed PCR products on the gel that were not consistent with the expected sizes of nucleotides targeted by the primers. These were suggestive of nonspecific amplifications and were not used in the characterization process. Journal of Industrial Microbiology & Biotechnology (2002) 28, 284–290 DOI: 10.1038/sj/jim/7000244 Received 30 May 2001/ Accepted in revised form 10 January 2002     

Alteration of lipopolysaccharide and protein profiles in SDS-PAGE of rhizobia by osmotic and heat stress

The effects of osmotic and heat stress on lipopolysaccharides and proteins of rhizobia isolated from the root nodules of leguminous trees grown in semi-arid soils of the Sudan, and of agricultural legumes grown in salt-affected soils of Egypt, were determined by SDS-PAGE. The rhizobia were of three types: (1) sensitive strains, unable to grow in 3% (w/v) NaCl in yeast mannitol medium; (2) tolerant strains which could grow in 3% (w/v) NaCl; and (3) halophytic strains which grew with 3 to 10% (w/v) NaCl. The sensitive strains changed their gel pattern or the amount of lipopolysaccharide they synthesized when grown in 1% (w/v) NaCl. The tolerant and halophytic strains often modified their lipopolysaccharides in 3% NaCl, which was evident by a shift in the banding patterns towards longer chain length. Similar effects were observed in cells incubated with sucrose and, to a lesser extent, in cells incubated at growth temperatures near the recorded maximum temperature for growth. The stress-induced changes in lipopolysaccharides were not associated with specific banding patterns of the lipopolysaccharides. During incubation in medium containing elevated concentrations of NaCl or sucrose, the protein patterns of the rhizobia were also changed. A protein with relative mobility of 65 kDa appeared during temperature stress. The maximum growth temperature of the Sudanese rhizobia were up to 44.2°C.H.H. Zahran and M. Karsisto were and L.A. Räsänen and K. Lindström are with the Department of Applied Chemistry and Microbiology, University of Helsinki, POB 27, SF-00014 University of Helsinki, Finland. H.H. Zahran is now with the Department of Botany, Faculty of Science, Beni-Suef, Egypt. M. Karsisto is now with the Finnish Forest Research Institute, PL 18, SF-01301 Vantaa, Finland.     

Discrimination among <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> H serotypes,serovars and strains based on 16S rRNA, <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">aroE</Emphasis> gene sequence analyses

Our aim was to investigate the capability of each of three genes, 16S rRNA, gyrB and aroE, to discriminate, first, among Bacillus thuringiensis H serotypes; second, among B. thuringiensis serovars from the same H serotype; and third, among B. thuringiensis strains from the same serovar. The 16S rRNA, gyrB and aroE genes were amplified from 21 B. thuringiensis H serotypes and their nucleotide sequences determined. Additional strains from four B. cereus sensu lato species were included for comparison purposes. These sequences were pair-wise compared and phylogenetic relationships were revealed. Each of the three genes under study could discriminate among B. thuringiensis H serotypes. The gyrB and aroE genes showed a discriminatory power among B. thuringiensis H serotypes up to nine fold greater than that of the 16S rRNA gene. The gyrB gene was retained for subsequent analyses to discriminate B. thuringiensis serovars from the same H serotype and to discriminate strains from same serovar. A total of 42 B. thuringiensis strains, which encompassed 25 serovars from 12 H serotypes, were analyzed. The gyrB gene nucleotide sequences were different enough as to be sufficient to discriminate among B. thuringiensis serovars from the same H serotype and among B. thuringiensis strains from the same serovar. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Virulence of some native Bacillus thuringiensis isolates against Ephestia kuehniella (Zeller) (Lep., Pyralidae) and Pieris brassicae (Lep., Pieridae) larvae isolated from stored products of Urmia city

Bacillus thuringiensis isolates were recovered from numerous sources including soil, grain dust, plant leaves, diseased insect larvae from insectariums and sericulture environments. B. thuringiensis strains were isolated using acetate selection method with 0.025?M. concentration. The morphology of crystals was studied using light microscopy. Bioassay tests were conducted on Ephestia kuehniella (Zeller) (L.) as well as Pieris brassicae (L.). Based on the results, 35 B. thuringiensis strains were isolated from 140 samples. Majority of strains (%31.42) had bipyramidal crystals. There was a significant difference in toxicity to insects among B. thuringiensis isolates; 28.57 and 14.28% of the isolates were toxic to the larvae of P. brassicae and E. kuehniella, respectively, causing more than 50% mortality. Results indicated that B. thuringiensis isolates with insecticidal activity could be used in integrated pest management to control farm and stored product pests.     

Investigation of the cyt gene in Bacillus thuringiensis and the biological activities of Bt isolates from the soil of China

The presence of cyt genes was investigated in 80 type strains of Bacillus thuringiensis and 143 isolates obtained from soil samples of China by PCR amplification using two pairs of primers for the cyt1 and cyt2 genes. Three type strains of serotypes H11ac, H14 and H36, eight isolates belonging to H3, H14, H18 and H21, and one isolate of unknown serotype harbored cyt genes. We also tested the cytolytic activity for mammal cells, the hemolytic activity for sheep erythrocytes and insecticidal activity against mosquitoes of five isolates that contained cyt genes but did not belong to B. thuringiensis serovar israelensis. The protein profiles of the five isolates were different from those of the type strains of B. thuringiensis serovar israelensis, and among the five isolates, only Y-5 showed mosquitocidal activity against larvae of Culex quinquefasciatus. All five of the isolates exhibited hemolytic activity, but only three could cause the cell death of A549 cells. The cytopathological changes induced by NX-4 in some A549 cells were characterized with cell-ballooning.     

Survival of lux-marked bacteria introduced into soil and the rhizosphere of bean (Phaseolus vulgaris L.)

The survival of lux-marked recombinants of Escherichia coli and Bacillus subtilis was studied in the rhizosphere of bean (Phaseolus vulgaris L.) and in bulk soil. The number of E. coli (pSB343) containing a complete lux operon did not differ significantly according to whether they were introduced into soil separately or together with a non-luminescent mutant Pseudomonas fluorescens R2fN. When genetically altered strains of E. coli and B. subtilis bearing a complete or an incomplete lux-reporter system were introduced into soil, the numbers of surviving cells were the same both in the rhizosphere and bulk soil. The insertion of lux genes into bacterial strains therefore does not affect their competitiveness and survival in the rhizosphere and bulk soil.The author is with the Department of Microbiology, University of Silesia, Jagielloska 28, 40-032 Katowice, Poland     

Lactic acid bacteria in fermented foods in Thailand

Traditional fermented foods (fish, meat and vegetable products), produced by many different processes, are eaten in many parts of Thailand. Lactic acid bacteria are responsible for the souring and ripening of these foods. Homofermentative strains of Lactobacillus pentosus, L. plantarum and Pediococcus pentosaceus are dominant in foods with low salt concentrations whereas P. halophilus strains are present in foods containing high salt. Strains of Lactobacillus sake, other Lactobacillus spp., P. acidilactici and P. urinaeequi are frequently found. Heterofermentative strains of L. brevis, L. confusus, L. fermentum, L. vaccinostercus, other Lactobacillus spp., and of Leuconostoc spp. are distributed as minor bacteria and strains of Staphylococcus, Enterococcus and Halobacterium are occasionally isolated.S. Tanasupawat is with the Department of Microbiology, Faculty of Pharmaceutical Sciences. Chulalongkorn University, Bangkok 10330, Thailand; K. Komagata is with the Department of Agricultural Chemistry, Tokyo University of Agriculture, Sakuragaoka 1-1-1, Setagaya-ku, Tokyo 156, Japan.     

Inhibition of different strains of enteropathogens in a lactic-fermenting cereal gruel

Twenty-eight strains of enteropathogens, including Campylobacter, Salmonella, Shigella, enterotoxigenic Escherichica coli (ETEC), Staphylococcus and Bacillus were added to cereal gruels prepared from low-tannin sorghum and inoculated with a lactic acid starter culture. Campylobacter strains were not detectable after 6 h, and Salmonella, Shigella and Staphylococcus strains not after 12 h. No viable cells of Bacillus strains were detected after 16 h of fermentation and the ETEC strains were completely inhibited after 24 h. No strain variability was observed within the different genera. In control gruels (no starter culture added), all the enteropathogens increased in number during incubation at 32°C except for the Campylobacter strains which decreased after 12 h of incubation.R. Kingamkono is with the Tanzania Food and Nutrition Centre, Dar-es-Salaam, Tanzania. E. Sjögren and B. Kaijser are with the Department of Clinical Bacteriology, Göteborg University, Gothenburg, Sweden. U. Svanberg is with the Chalmers University of Technology, c/o SIK, Box 5401, 5-402 29 Gothenburg, Sweden