Bacillus thuringiensis var. israelensis production involving re-use of the supernatant

The supernatant arising after biomass separation of Bacillus thuringiensis var. israelensis by flocculation/sedimentation was re-used after being supplemented with 25, 50 and 75% (w/v) of the original culture medium, based on corn steep liquor, glucose and mineral salts. Supplementation at 75% gave a spore concentration (1 x 10(10) c.f.u. ml(-1)) five times greater than that obtained with the other supplements.     

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.     

Soil respiration of Alaskan tundra at elevated atmospheric carbon dioxide concentrations

Summary CO₂ efflux from tussock tundra in Alaska that had been exposed to elevated CO₂ for 2.5 growing seasons was measured to assess the effect of long- and short-term CO₂ enrichment on soil respiration. Long-term treatments were: 348, 514, and 683 μll⁻¹ CO₂ and 680 μll⁻¹ CO₂+4°C above ambient. Measurements were made at 5 CO₂ concentrations between 87 and 680 μll⁻¹ CO₂. Neither long- or short-term CO₂ enrichment significantly affected soil CO₂ efflux. Tundra developed at elevated temperature and 680 μll⁻¹ CO₂ had slightly higher, but not statistically different, mean respiration rates compared to untreated tundra and to tundra under CO₂ control alone.     

Distribution of Bacillus thuringiensis strains in Southern Sweden

Bacillus thuringiensis strains were found to be naturally present in the soils of southern Sweden, being isolated from nine out of 12 sites examined. Forest soil samples were more rich in B. thuringiensis strains than soil samples collected from cultivated areas. A wide diversity of B. thuringiensis strains, representing different biochemical groups, was isolated; samples from Aspö and Fogdö regions showed the highest degree of diversity.R. Landén and M. Bryne are with the Department of Microbiology, Stockholm University, S-10691 Stockholm, Sweden. A. Abdel-Hameed is with the Department of Microbiology, Faculty of Pharmacy, Cairo University, Kasr-El-Aini Street, Cairo, Egypt. A. Abdel-Hameed's present address is the Department of Applied Chemistry and Microbiology, PO Box 27, Viikki, Building B, SF-00014 University of Helsinki, Helsinki, Finland     

Responses of soil biota to elevated atmospheric carbon dioxide

Increasing concentrations of atmospheric CO₂ could have dramatic effects upon terrestrial ecosystems including changes in ecosystem structure, nutrient cycling rates, net primary production, C source-sink relationships and successional patterns. All of these potential changes will be constrained to some degree by below ground processes and mediated by responses of soil biota to indirect effects of CO₂ enrichment. A review of our current state of knowledge regarding responses of soil biota is presented, covering responses of mycorrhizae, N-fixing bacteria and actinomycetes, soil microbiota, plant pathogens, and soil fauna. Emphasis will be placed on consequences to biota of increasing C input through the rhizosphere and resulting feedbacks to above ground systems. Rising CO₂ may also result in altered nutrient concentrations of plant litter, potentially changing decomposition rates through indirect effects upon decomposer communities. Thus, this review will also cover current information on decomposition of litter produced at elevated CO₂. Summary Predictably, the responses of soil biota to CO₂ enrichment and the degree of experimental emphasis on them increase with proximity to, and intimacy with, roots. Symbiotic associations are all stimulated to some degree. Total plant mycorrhization increases with elevated CO₂. VAM fungi increase proportionately with fine root length/mass increase. ECM fungi, however, exhibit greater colonization per unit root length/mass at elevated CO₂ than at current atmospheric levels. Total N-fixation per plant increases in all species examined, although the mechanisms of increase, as well as the eventual benefit to the host relative to N uptake may vary. Microbial responses are unclear. The assumption that changes in root exudation will drive increased mineralization and facilitate nutrient uptake should be examined experimentally, in light of recent models. Microbial results to date suggest that metabolic activity (measured as changes in process rates) is stimulated by root C input, rather than population size (measured by cell or colony counts). Insufficient evidence exists to predict responses of either soil-borne plant pathogens or soil fauna (i.e., food web responses). These are areas requiring attention, the first for its potential to limit ecosystem production through disease and the second because of its importance to nutrient cycling processes. Preliminary data on foliar litter decomposition suggests that neither nutrient ratios nor decomposition rates will be affected by rising CO₂. This is another important area that may be better understood as the number of longer term studies with more realistic CO₂ exposures increase. Evidence continues to mount that C fixation increases with CO₂ enrichment and that the bulk of this C enters the belowground component of ecosystems. The global fate and effects of this additional C may affect all hierarchical levels, from organisms to ecosystems, and will be largely determined by responses of soil biota.     

Cloning and characterization of an insecticidal crystal protein gene from Bacillus thuringiensis subspecies kenyae

A sporulating culture ofBacillus thuringiensis subsp.kenyae strain HD549 is toxic to larvae of lepidopteran insect species such asSpodoptera litura, Helicoverpa armigera andPhthorimaea operculella, and a dipteran insect,Culex fatigans. A 1.9-kb DNA fragment, PCR-amplified from HD549 using cryII-gene-specific primers, was cloned and expressed inE. coli. The recombinant protein produced 92% mortality in first-instar larvae ofSpodoptera litura and 86% inhibition of adult emergence inPhthorimaea operculella, but showed very low toxicity againstHelicoverpa armigera, and lower mortality against third-instar larvae of dipteran insectsCulex fatigans, Anopheles stephensi andAedes aegypti. The sequence of the cloned crystal protein gene showed almost complete homology with a mosquitocidal toxin gene fromBacillus thuringiensis var.kurstaki, with only five mutations scattered in different regions. Amino acid alignment with different insecticidal crystal proteins using the MUTALIN program suggested presence of the conserved block 3 region in the sequence of this protein. A mutation in codon 409 of this gene that changes a highly conserved phenylalanine residue to serine lies in this block.     

Photosynthetic inhibition after long-term exposure to elevated levels of atmospheric carbon dioxide

The effect of long-term exposure to elevated levels of CO₂ on biomass partitioning, net photosynthesis and starch metabolism was examined in cotton. Plants were grown under controlled conditions at 350, 675 and 1000 l l^-1 CO₂. Plants grown at 675 and 1000 l l^-1 had 72% and 115% more dry weight respectively than plants grown at 350 l l^-1. Increases in weight were partially due to corresponding increases in leaf starch. CO₂ enrichment also caused a decrease in chlorophyll concentration and a change in the chlorophyll a/b ratio. High CO₂ grown plants had lower photosynthetic capacity than 350 l l^-1 grown plants when measured at each CO₂ concentration. Reduced photosynthetic rates were correlated with high internal (non-stomatal) resistances and higher starch levels. It is suggested that carbohydrate accumulation causes a decline in photosynthesis by feedback inhibition and/or physical damage at the chloroplast level.Abbreviations Ci internal CO₂ concentration - Chl chlorophyll - DMSO dimethylsulfoxide - HSD honestly significant difference (procedure) - MCW methanolchloroform-water - Pi inorganic phosphate - S.E.M. standard error of mean     

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.     

Hemolysin II is more characteristic of Bacillus thuringiensis than Bacillus cereus

To investigate the distribution of the hemolysin II determinant among strains of Bacillus cereus and Bacillus thuringiensis, thirteen strains of B. cereus and fourteen strains of B. thuringiensis strains were tested for hybridization of their chromosomal DNAs with a DNA probe containing the B. cereus hemolysin II gene. In addition, the production of hemolysin II, whose activity is not inhibited by cholesterol, was tested. The presence (absence) of the hybridization response in the microorganism's genome correlated with the presence (absence) of cholesterol-unaffected hemolysin production. Only four out of thirteen B. cereus strains were found to give a positive response in hybridization experiments, whereas thirteen out of fourteen B. thuringiensis strains responded positively. DNAs from ten B. thuringiensis strains contained a 3.5 kb EcoRV fragment, which hybridized with the B. cereus hemolysin II gene probe. The 3.5 kb EcoRV DNA fragment from one of these strains (B. thuringiensis VKM-B1555) was cloned and expressed in Escherichia coli cells. The hemolysin encoded by the cloned DNA fragment was not inhibited by cholesterol and possessed all other properties of B. cereus hemolysin II. The obtained data clearly show limited distribution of hemolysin II among B. cereus strains and demonstrate that hemolysin II is more characteristic of B. thuringiensis than B. cereus.     

Rising atmospheric carbon dioxide concentration and the future of C4 crops for food and fuel

Crops with the C₄ photosynthetic pathway are vital to global food supply, particularly in the tropical regions where human well-being and agricultural productivity are most closely linked. While rising atmospheric [CO₂] is the driving force behind the greater temperatures and water stress, which threaten to reduce future crop yields, it also has the potential to directly benefit crop physiology. The nature of C₄ plant responses to elevated [CO₂] has been controversial. Recent evidence from free-air CO₂ enrichment (FACE) experiments suggests that elevated [CO₂] does not directly stimulate C₄ photosynthesis. Nonetheless, drought stress can be ameliorated at elevated [CO₂] as a result of lower stomatal conductance and greater intercellular [CO₂]. Therefore, unlike C₃ crops for which there is a direct enhancement of photosynthesis by elevated [CO₂], C₄ crops will only benefit from elevated [CO₂] in times and places of drought stress. Current projections of future crop yields have assumed that rising [CO₂] will directly enhance photosynthesis in all situations and, therefore, are likely to be overly optimistic. Additional experiments are needed to evaluate the extent to which amelioration of drought stress by elevated [CO₂] will improve C₄ crop yields for food and fuel over the range of C₄ crop growing conditions and genotypes.     

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.     

Effect of light and atmospheric carbon dioxide concentration on nitrogen fixation by herbage legumes

Summary Lucerne, red clover and white clover were grown at two atmospheric concentrations of CO₂ (300 and 1000 μl l⁻¹) and the effects on N₂ fixation, nodule mass/number and root/shoot dry matter production determined. Pea plants were similarly evaluated as a comparison with grain legumes. CO₂ enrichment increased N₂ fixation activity in all cases but activity/unit nodule mass was significantly increased only in the pea. The enhancement of N₂ fixation in herbage legumes by CO₂ enrichment reflected an increase in nodule mass which in turn was attributed to increased nodule number, and results show that under the experimental conditions obtaining here photosynthate supply did not limit nodule N₂ fixation in these plants though it was limiting in the case of peas. White clover growing in a 6 and 14 hour photoperiod was studied for response of the N₂ fixing system to light. Long photoperiod (14 hour) plants assayed at constant temperature (20°C) did not show a significant response to light at the end of the dark period either in terms of fixation per plant or per unit nodule mass, in contrast with short photoperiod (6 hour) plants which showed significant responses. Short photoperiod plants compensated for reduced photosynthates by maintaining only half the root nodule mass and fixation activity of 14 hour photoperiod plants though plants in both systems supported similar rates of N₂ fixation per unit mass of nodule during the photoperiod. Comparison of N₂ fixation activities in whole and decapitated plant systems indicates the importance of shoot reserves for sustaining nitrogenase activity in white clover during short-term interruption of photosynthesis. These results support the conclusion of the CO₂ enrichment studies, that herbage legumes have the potential for supplying their nodule photosynthate requirements for sustaining optimum rates of N₂ fixation and excess carbon supply is used solely to promote further nodulation. Nodules of short photoperiod white clover plants were less efficient in N₂ fixation in that they evolved more H₂ relative to N₂ (C₂H₂) reduced than did long photoperiod plants.     

On the Adaptive Nature of the Dissociation Process in Bacillus thuringiensis

The process of dissociation into variants that differ in colony morphology occurring in batch cloned cultures of two Bacillus thuringiensis strains belonging to different subspecies was studied at optimal and elevated temperatures. An increase in the cultivation temperature to 40°C resulted in an increase in the fraction of R variants to 100% after 72 h of cultivation of either of the strains. This increase was not due to the selection of forms with greater resistance to elevated temperature. The level of resistance to elevated temperature was determined by the strain genotype and did not correlate with morphological characteristics of the colonies.     

Survival of a strain of Bacillus megaterium carrying a lepidopteran-specific gene of Bacillus thuringiensis in the phyllospheres of various economically important plants

The colonizing ability of a transcipient strain of Bacillus megaterium carrying a lepidopteran-specific cryIA (a) gene of Bacillus thuringiensis in the phyllospheres of various economically important plants was studied. Similar experiments were also carried out using the parental B. thuringiensis var. kurstaki strain HD1 for a comparison. While the transcipient remained on the leaves of cotton and okra for more than 28 days, its survival in phyllospheres of mulberry, peanut, chickpea, tomato and rice was rather limited to about 3 – 5 days. The persistence of B. thuringiensis, on the other hand, was extremely short (i.e. less than 4 days) on all the crop plants tested.     

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.     

Bacteriocin-like inhibitor substances produced by Mexican strains of Bacillus thuringiensis

Bacteriocins are antimicrobial peptides synthesized and secreted by bacteria and could potentially be used as natural food preservatives. Here, we report the production of bacteriocin-like inhibitor substances (Bt-BLIS) by five Mexican strains of Bacillus thuringiensis. Bacillus thuringiensis subsp. morrisoni (LBIT 269), B. thuringiensis subsp. kurstaki (LBIT 287), B. thuringiensis subsp kenyae (LBIT 404), B. thuringiensis subsp. entomocidus (LBIT 420) and B. thuringiensis subsp. tolworthi (LBIT 524) produced proteinaceous Bt-BLIS with high levels of activity against Bacillus cereus and other gram-positive bacteria. Although none was active against the gram-negative bacteria, Escherichia coli, Shigella species and Pseudomonas aeruginosa, the five Bt-BLIS demonstrated antimicrobial activity against Vibrio cholerae, the etiologic agent of cholera. Biochemical and biophysical studies demonstrated that the five Bt-BLIS could be categorized into two groups, those produced by LBIT 269 and 287 (Group A) and LBIT 404, 420, 524 (Group B), based on relative time of peptide synthesis, distinctive bacterial target specificity and stability in a wide range of temperatures and pH. Because of their stability and bactericidal activities against B. cereus and V. cholerae agents of emetic, diarrheal and lethal syndromes in humans, these Bt-BLIS could potentially be used as biodegradable preservatives in the food industry.     

Effect of CryIC toxin from Bacillus thuringiensis on larval feeding behavior of Spodoptera exigua

The lack of data on the effect of Bacillus thuringiensis (B.t.) toxins on larval feeding behavior of the pest Spodoptera exigua (Hübner) (Noctuidae: Amphypyrini) prompted us to investigate the effect of three delivery systems of CryIC, a commercial formulation, inclusion bodies, and the activated CryIC toxin. The commercial formulation was the least and CryIC toxin the most lethal form to neonates of susceptible colonies. All but two of the treatments in choice tests with neonates and third instars showed significant avoidance of B.t. treated diet, with greater proportion of larvae from susceptible (UCR-S and AUBURN-S) and resistant (AUBURN-R) colonies on untreated diet than on diet treated with any of the CryIC forms and concentrations tested. Furthermore, third instars consumed significantly more control than treated diet for all CryIC forms, colonies and concentrations. The avoidance of CryIC toxin by neonates and third instars strongly suggests that CryIC, which also is present in the commercial formulation and in the inclusion bodies, is responsible for eliciting avoidance behavior by S. exigua larvae. Behavioral observations of third instars in a no-choice test on either treated or control diet indicated that questing behavior in susceptible larvae appears to be positively related with presence of CryIC toxin in the diet. Furthermore, resistant third instars were on the whole more active than susceptible thirds on both treated and control diet. Resistant thirds raised on CryIC treated diet (AUBURN-RC) spent more time eating treated diet than resistant larvae raised on control diet (AUBURN-R), suggesting that diet conditioning plays an important role on feeding behavior of S. exigua. The implications of these results are discussed.     

The impact of elevated carbon dioxide on the phosphorus nutrition of plants: a review

Background Increasing attention is being focused on the influence of rapid increases in atmospheric CO₂ concentration on nutrient cycling in ecosystems. An understanding of how elevated CO₂ affects plant utilization and acquisition of phosphorus (P) will be critical for P management to maintain ecosystem sustainability in P-deficient regions.Scope This review focuses on the impact of elevated CO₂ on plant P demand, utilization in plants and P acquisition from soil. Several knowledge gaps on elevated CO₂-P associations are highlighted.Conclusions Significant increases in P demand by plants are likely to happen under elevated CO₂ due to the stimulation of photosynthesis, and subsequent growth responses. Elevated CO₂ alters P acquisition through changes in root morphology and increases in rooting depth. Moreover, the quantity and composition of root exudates are likely to change under elevated CO₂, due to the changes in carbon fluxes along the glycolytic pathway and the tricarboxylic acid cycle. As a consequence, these root exudates may lead to P mobilization by the chelation of P from sparingly soluble P complexes, by the alteration of the biochemical environment and by changes to microbial activity in the rhizosphere. Future research on chemical, molecular, microbiological and physiological aspects is needed to improve understanding of how elevated CO₂ might affect the use and acquisition of P by plants.     

An index for determining the culture time of inoculum in cultivation of Bacillus thuringiensis

The intensity of motility of Bacillus thuringiensis was used to determine the optimal culture time of the inoculum being used for thuringiensin production. Thuringiensin production with an inoculum taken at its maximum motility was about 3 times higher than that with an inoculum taken in the late exponential phase.     

Secondary structure of the entomocidal toxin from Bacillus thuringiensis subsp. kurstaki HD-73

The secondary structure of the toxin fromBacillus thuringiensis subsp.kurstaki (Btk) HD-73 was estimated by Raman, infrared, and circular dichroism spectroscopy, and by predictive methods. Circular dichroism and infrared spectroscopy gave an estimate of 33–40% -helix, whereas Raman and predictive methods gave approximately 20%. Raman and circular dichroism spectra, as well as predictive methods, indicated that the toxin contains 32–40% -sheet structure, whereas infrared spectroscopy gave a slightly lower estimate. Thus, all of these approaches are in agreement that the native conformation of Btk HD-73 toxin is highly folded and contains considerable amounts of both -helical and -sheet structures. No significant differences were detected in the secondary structure of the toxin either in solution or as a hydrated pellet.