Development of Bacillus thuringiensis in Galleria mellonella larvae exposed to gamma radiation

A suspension of Bacillus thuringiensis was inoculated at 24 and 72 hr into the oral cavity of Galleria mellonella larvae following exposure to 20, 50, and 70 Kr of gamma radiation, respectively. The cytopathology was conducted after B. thuringiensis had developed for 3, 5, and 7 hr and after radiation damage had developed for 27, 29, 31, 75, 77, and 79 hr in the larvae exposed to 20, 50, and 70 Kr, respectively.B. thuringiensis spores appeared in the midgut lumen from 3 to 7 hr after inoculation of 20 Kr irradiated larvae. At 7 hr after B. thuringiensis infection, and 79 hr after 20 Kr irradiation, the following changes were seen: B. thuringiensis rods appeared adsorbed onto the walls of epithelial cells, a few spores appeared in hemolymph, epithelial cells developed vacuoles, and villi appeared detached from the basement membrane.Within a period ranging from 3 to 5 hr after infection, B. thuringiensis rods attacked vacuolated epithelial cells of most of the 50 and 70 Kr irradiated larvae. At 7 hr after infection and at 31 hr after 70 Kr irradiation, the spores reached the interior of some epithelial cells and were also seen concentrated near the basement membrane.In general, the midgut epithelial cells of the 70 Kr-irradiated groups of larvae appeared highly vacuolated, badly disrupted, and in most cases undistinguishable as a result of attack of B. thuringiensis.In short, B. thuringiensis did not show a characteristic pattern of pathology on 20 and 50 Kr-irradiated midgut cells. The problem of permeability of B. thuringiensis toxin into the irradiated cells needs further investigation.     

Effect of exposure to soil on potency and spore viability of Bacillus thuringiensis

Ten-gram samples of a clay loam soil were inoculated with Bacillus thuringiensis var. galleriae (H-serotype V) and held at 25°C. Periodically the spores and δ endotoxin protein crystals of B. thuringiensis were extracted from soil samples. Numbers of viable spores were estimated by plate counts and pathogenicity determined by bioassay with larvae of Galleria mellonella. During 135 days, the number of viable spores fell slowly to 24% of the initial numbers, while pathogenicity fell rapidly to <1%, which suggests that the crystals were degraded far more rapidly than spores. Natural soil bacteria increased in numbers during the same period.     

Detection of Bacillus thuringiensis in soil by immunofluorescence

Persistence of viable and heat-killed vegetative cells, parasporal crystals, and spores of Bacillus thuringiensis in soil was monitored by immunofluorescence. The rates of disappearance of the different bacterial components decreased in the following order: viable cells, heat-killed cells, parasporal crystals, and spores. Vegetative cells disappeared at rapid, exponential rates; viable cells autolysed, whereas heat-killed cells were digested by an actinomycete-like, soil microorganism. Parasporal crystals disappeared at a slower, nonexponential rate. Numbers of spores remained unaltered throughout 91 days incubation at 25°C and no germination was detected in this period.     

Production of heat-stable exotoxin by Bacillus thuringiensis and related bacteria

Heat-stable exotoxin production by 740 strains of Bacillus thuringiensis and related bacteria was investigated using the housefly, Musca domestica, from the following viewpoints: (1) the relation-ship between B. thuringiensis flagellar (H) serotypes and exotoxin production and (2) the exotoxin production by Bacillus species other than B. thuringiensis. Of 437 isolates belonging to 11 serotypes of B. thuringiensis which had been confirmed to produce parasporal inclusions, 35 isolates belonging to serotypes 1, 3a:3b, 4a:4c, and 10 produced heat-stable exotoxin. Exotoxin was not detected in the isolates of serotypes 3a, 4a:4b, 5a:5b, 5a:5c, 6, 7, and 8a:8b. No heat-stable exotoxin was demonstrated in 28 acrystalliferous isolates which possessed H antigens of B. thuringiensis serotypes 1, 3a, 4a:4b, 4a:4c, 5a:5c, 6, 7, 10, 11a:11c, and 12. A total of 270 B. cereus isolates which did not possess B. thuringiensis H antigen were examined and three isolates were found to produce heat-stable exotoxin. No heat-stable exotoxin was produced by B. subtilis (two strains), B. natto (one strain), and B. megaterium (two strains). These results indicate that the heat-stable exotoxin production in B. thuringiensis is a strain-specific property rather than a serotype(subspecies)-specific property.     

Viable spore count as an index of effective dose of Bacillus thuringiensis

Leaf samples from Cercis occidentalis trees, collected 5–75 hr after application of varying concentrations of a Bacillus thuringiensis formulation, Dipel, were used as dose sources for third instar Schizura concinna larvae. Dose, defined as logarithmically transformed, estimated viable spore counts, and duration of time the formulation was on the leaf prior to sampling were considered in combination as predictors of insect mortality. By use of the multiple logistic model both factors were found to be significant predictors of insect mortality (P < 0.0001). Available methods for assessing goodness of fit indicate that the multiple logistic model adequately fits these data.     

Determination of heat-stable exotoxin of Bacillus thuringiensis by high-performance anion-exchange chromatography

A high-performance anion-exchange chromatography method for quantitative determination of heat-stable exotoxin in insecticide preparations and cultivation liquids is described. Heat-stable exotoxin-containing samples were chromatographed in a column packed with anion-exchange resin under gradient elution conditions, using HCl and NaCl solution, at 50°C. The calibration was linear in the exotoxin concentration range from 1 to 100 mg/ml. The sensitivity of the method allows one to determine 1 nmol and more of the heat-stable exotoxin in the applied sample, the relative standard deviation being not more than ±4%.     

Screen of Bacillus thuringiensis toxins for transgenic rice to control Sesamia inferens and Chilo suppressalis

Transgenic rice to control stem borer damage is under development in China. To assess the potential of Bacillus thuringiensis (Bt) transgenes in stem borer control, the toxicity of five Bt protoxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba and Cry1Ca) against two rice stem borers, Sesamia inferens (pink stem borer) and Chilo suppressalis (striped stem borer), was evaluated in the laboratory by feeding neonate larvae on artificial diets containing Bt protoxins. The results indicated that Cry1Ca exhibited the highest level of toxicity to both stem borers, with an LC₅₀ of 0.24 and 0.30 μg/g for C. suppressalis and S. inferens, respectively. However, S. inferens was 4-fold lower in susceptibility to Cry1Aa, and 6- and 47-fold less susceptible to Cry1Ab and Cry1Ba, respectively, compared to C. suppressalis. To evaluate interactions among Bt protoxins in stem borer larvae, toxicity assays were performed with mixtures of Cry1Aa/Cry1Ab, Cry1Aa/Cry1Ca, Cry1Ac/Cry1Ca, Cry1Ac/Cry1Ba, Cry1Ab/Cry1Ac, Cry1Ab/Cry1Ba, and Cry1Ab/Cry1Ca at 1:1 (w/w) ratios. All protoxin mixtures demonstrated significant synergistic toxicity activity against C. suppressalis, with values of 1.6- to 11-fold higher toxicity than the theoretical additive effect. Surprisingly, all but one of the Bt protoxin mixtures were antagonistic in toxicity to S. inferens. In mortality-time response experiments, S. inferens demonstrated increased tolerance to Cry1Ab and Cry1Ac compared to C. suppressalis when treated with low or high protoxin concentrations. The data indicate the utility of Cry1Ca protoxin and a Cry1Ac/Cry1Ca mixture to control both stem borer populations.     

Effect of some chemical insecticides on the germination and replication of commercial Bacillus thuringiensis

Experiments designed to determine the compatibility of commercial Bacillus thuringiensis and chemical insecticides showed that fenitrothion (2 ppm active ingredient), SBP 1382, and Gardona^® (1 ppm active ingredient) inhibited bacterial replication after 2 hr growth time in liquid broth culture. Spore germination and the size of the parasporal crystals were greatly reduced by high concentrations (1000 ppm) of these insecticide formulations most likely due to the presence of toxic emulsifiers and other additives in the emulsifiable concentrates. The commonly used emulsifiers, Atlox and Triton X-100, at 1000 ppm totally inhibited germination and reduced crystal size. Bacillus thuringiensis apparently metabolized fenitrothion and SBP 1382 during 2 hr of exposure in the cultures containing 10 and 100 ppm, respectively, of these insecticides.Orthene^® at 10,000 ppm for 2 hr had no significant inhibiting effect on the bacteria replication. Spore germination and crystal size were not affected by this concentration. Orthene is considered a potentially effective chemical insecticide in the integrated control of susceptible insect pests if used in concentrations low enough to spare natural control agents of the target species.     

Kinetics of pore formation by the Bacillus thuringiensis toxin Cry1Ac

After binding to specific receptors, Cry toxins form pores in the midgut apical membrane of susceptible insects. The receptors could form part of the pore structure or simply catalyze pore formation and consequently be recycled. To discriminate between these possibilities, the kinetics of pore formation in brush border membrane vesicles isolated from Manduca sexta was studied with an osmotic swelling assay. Pore formation, as deduced from changes in membrane permeability induced by Cry1Ac during a 60-min incubation period, was strongly dose-dependent, but rapidly reached a maximum as toxin concentration was increased. Following exposure of the vesicles to the toxin, the osmotic swelling rate reached a maximum shortly after a delay period. Under these conditions, at relatively high toxin concentrations, the maximal osmotic swelling rate increased linearly with toxin concentration. When vesicles were incubated for a short time with the toxin and then rapidly cooled to prevent the formation of new pores before and during the osmotic swelling experiment, a plateau in the rate of pore formation was observed as toxin concentration was increased. Taken together, these results suggest that the receptors do not act as simple catalysts of pore formation, but remain associated with the pores once they are formed.     

施氮方式对转基因棉花Bt蛋白含量及产量的影响

为研究氮肥运筹对棉花叶片、棉蕾和棉铃不同器官中Bt蛋白含量的影响,2009—2010年,以抗虫杂交棉中棉所72为试验材料,在大田条件下进行了不同基肥：花铃肥：盖顶肥施氮比例（分别为0:0.4:0.6、0.2:0.4:0.4、0.4:0.4:0.2、0.6:0.4:0）的试验。结果表明,施氮方式对棉花不同器官中Bt蛋白含量有明显影响。总体表现为随着氮肥前移,棉花幼嫩器官中Bt蛋白含量呈明显增加的趋势,而老熟器官中Bt蛋白含量呈明显降低的趋势。施氮方式对棉花幼嫩器官中Bt蛋白含量的影响比老熟器官明显,尤其是对幼嫩叶片Bt蛋白含量的影响大于幼小的棉蕾和棉铃器官。抗虫棉采用基肥：花铃肥：盖顶肥为0.4:0.4:0.2的施氮方式,总体能提高前中期棉花器官Bt蛋白的含量,有利于提高其抗虫性能;减少后期棉花器官Bt蛋白的含量,减轻对环境的压力;而且比其余3种施氮方式的籽棉产量和皮棉产量分别增加4.15%—11.24％、3.73%—12.01%。     

Characterization of resistance to Bacillus thuringiensis toxin Cry1Ac in Plutella xylostella from China

A field population (SZ) of Plutella xylostella, collected from the cabbage field in Shenzhen, Guangdong Province of China in 2002, showed 2.3-fold resistance to Cry1Aa, 110-fold to Cry1Ab, 30-fold to Cry1Ac, 2.1-fold to Cry1F, 5.3-fold to Cry2Aa and 6-fold resistance to Bacillus thuringiensis var. kurstaki (Btk) compared with a susceptible strain (ROTH). The SZBT strain was derived from the SZ population through 20 generations of selection with activated Cry1Ac in the laboratory. While the SZBT strain developed 1200-fold resistance to Cry1Ac after selection, resistance to Cry1Aa, Cry1Ab, Cry1F, and Btk increased to 31-, 1900-,>33- and 17-fold compared with the ROTH strain. However, little or no cross-resistance was detected to Cry1B, Cry1C and Cry2Aa in the SZBT strain. Genetic cross analyses between the SZBT and ROTH strains revealed that Cry1Ac-resistance in the SZBT strain was controlled by a single, autosomal, incompletely recessive gene. Binding studies with ¹²⁵I-labeled Cry1Ac showed that the brush border membrane vesicles (BBMVs) of midguts from the resistant SZBT insects had lost binding to Cry1Ac. Allelic complementation tests demonstrated that the major Bt resistance locus in the SZBT strain was same as that in the Cry1Ac-R strain which has “mode 1” resistance to Bt. An F₁ screen of 120 single-pair families between the SZBT strain and three field populations collected in 2008 was carried out. Based on this approach, the estimated frequencies of Cry1Ac-resistance alleles were 0.156 in the Yuxi population from Yunnan province, and 0.375 and 0.472 respectively in the Guangzhou and Huizhou populations from Guangdong province.     

Induced production of chitinase to enhance entomotoxicity of Bacillus thuringiensis employing starch industry wastewater as a substrate

Induced production of chitinase during bioconversion of starch industry wastewater (SIW) to Bacillus thuringiensis var. kurstaki HD-1 (Btk) based biopesticides was studied in shake flask as well as in computer-controlled fermentors. SIW was fortified with different concentrations (0%; 0.05%; 0.1%; 0.2%; 0.3% w/v) of colloidal chitin and its consequences were ascertained in terms of Btk growth (total cell count and viable spore count), chitinase, protease and amylase activities and entomotoxicity. At optimum concentration of 0.2% w/v colloidal chitin, the entomotoxicity of fermented broth and suspended pellet was enhanced from 12.4 × 10⁹ (without chitin) to 14.4 × 10⁹ SBU/L and from 18.2 × 10⁹ (without chitin) to 25.1 × 10⁹ SBU/L, respectively. Further, experiments were conducted for Btk growth in a computer-controlled 15 L bioreactor using SIW as a raw material with (0.2% w/v chitin, to induce chitinase) and without fortification of colloidal chitin. It was found that the total cell count, spore count, delta-endotoxin concentration (alkaline solubilised insecticidal crystal proteins), amylase and protease activities were reduced whereas the entomotoxicity and chitinase activity was increased with chitin fortification. The chitinase activity attained a maximum value at 24 h (15 mU/ml) and entomotoxicity of suspended pellet reached highest (26.7 × 10⁹ SBU/L) at 36 h of fermentation with chitin supplementation of SIW. In control (without chitin), the highest value of entomotoxicity of suspended pellet (20.5 × 10⁹ SBU/L) reached at 48 h of fermentation. A quantitative synergistic action of delta-endotoxin concentration, spore concentration and chitinase activity on the entomotoxicity against spruce budworm larvae was observed.     

Immunochemical analysis of parasporal crystal digests of Bacillus thuringiensis as an index of insecticidal activity

An immunoelectrophoretic method is described for analyzing alkali parasporal crystal digests of Bacillus thuringiensis preparations (Dipel^TM). There were two distinct immunoprecipitate peaks (A and B) in the direction of the anode. The higher A peak had no correlation with the bioactivity of B. thuringiensis samples, whereas, the height of the B peak was directly proportional to the bioactivity. There was excellent agreement between the immunoassay and the bioassay of insecticidal potency in terms of international units.     

Laboratory and field evaluations of industrial formulations of Bacillus thuringiensis serovar. israelensis against some mosquito species of central Italy

Two wettable powder formulations (Bactimos and Vectobac) and a flowable concentrate (Teknar) formulation of Bacillus thuringiensis serovar. israelensis were evaluated as larvicides of Culex pipiens, Aedes caspius, and Aedes detritus. In the laboratory, the levels of susceptibility of these species to the test formulations were essentially similar and corresponded to their relative potencies; the LC₉₀ values ranged from 0.042 to 0.37 ppm. C. pipiens, A. caspius, and A. detritus, in that order, were susceptible to the biocide. Under field conditions in central Italy. Bactimos at 0.5 kg/ha gave 98% control of C. pipiens in an irrigation canal. Teknar at 1.25 liters/ha gave 86–100%, and at 2.5 liters/ha gave 90–100% control of C. pipiens in two natural ponds. Against A. caspius in salt marsh habitats, Bactimos at 0.5 kg/ha and Teknar at 2.5 liters/ha yielded complete control of the larvae, while a lower rate (0.2 kg/ha) of Bactimos, and Vectobac at 0.5 kg/ha resulted in 82–94% and 67–91% control, respectively. Higher rates (0.75 and 1.0 kg/ha) of Vectobac gave 76–100% and 98–100% control of A. caspius. Bactimos at 0.15 kg/ha gave 93–98% control of A. detritus in two salt marsh ponds. B. thuringiensis serovar. israelensis is practically economical for the control of C. pipiens, A. caspius, and A. detritus in the various biotopes in central Italy.     

Reduction of Bacillus thuringiensis Cry1Ac toxicity against Helicoverpa armigera by a soluble toxin-binding cadherin fragment

A cadherin-like protein has been identified as a putative receptor for Bacillus thuringiensis (Bt) Cry1Ac toxin in Helicoverpa armigera and plays a key role in Bt insecticidal action. In this study, we produced a fragment from this H. armigera Cry1Ac toxin-binding cadherin that included the predicted toxin-binding region. Binding of Cry1Ac toxin to this cadherin fragment facilitated the formation of a 250-kDa toxin oligomer. The cadherin fragment was evaluated for its effect on Cry1Ac toxin-binding and toxicity by ligand blotting, binding assays, and bioassays. The results of ligand blotting and binding assays revealed that the binding of Cry1Ac to H. armigera midgut epithelial cells was reduced under denaturing or native conditions in vitro. Bioassay results indicated that toxicities from Cry1Ac protoxin or activated toxin were reduced in vivo by the H. armigera cadherin fragment. The addition of the cadherin fragment had no effect on Cry2Ab toxicity.     

A convenient procedure for the preparation of highly purified parasporal crystals of Bacillus thuringiensis

A method is described that provides, in a reproducible and very simple way, highly purified preparations of crystals of Bacillus thuringiensis. By switching from culture medium to distilled water, cells are induced to sporulate and complete autolysis ensues. The lysate can be layered directly on a 67% urografine solution and submitted to a 90 min centrifugation at 4660 × g_av. Spores are recovered as a pellet at the bottom of the tube, whereas crystals band just bellow the sample-solution interface. The purity of each fraction is higher than 99%.     

Persistence of Bacillus thuringiensis in foundation beeswax and beecomb in beehives for the control of Galleria mellonella

Beecomb was protected against wax moth attack by impregnating foundation beeswax with Bacillus thuringiensis. Bakthane, a serotype I product, gave good protection of broodcomb for 2 yr at 0.5% in wax and partial protection for 6 yr at 2%. The active component was exotoxin present at about 0.9% in Bakthane in a very insoluble state. No harm to the bees was detected, even when a partially purified preparation of exotoxin was used, but further tests on the leaching of pure exotoxin from comb into honey are required before the latter can be regarded as a practical method of wax moth control. The action of exotoxin on Galleria mellonella larvae was slower than that of the spore-crystal complex and less than that of exotoxin on some Diptera. Spores and crystals of serotype V were × 500 as potent in G. mellonella as those of serotype I and exotoxin on honey-rich artificial food in laboratory assays, but their activity deteriorated in the first hive trials with treated foundation wax. Prehive deterioration was due both to Teepol and soap in the liquid lubricating the wax mill rollers and to moist storage of the foundation. This deterioration was prevented by the use of Triton X-100 as lubricant, by drying the newly impregnated foundation wax and by storing it in dry conditions. This resulted in good protection of comb for one hive season by 1% of serotype V Thuricide in foundation wax. However, protection after two seasons varied, making the use of serotype V spores and crystals uneconomical for commercial practice, although safe for bee and man.     

The comparative potencies of the crystalline endotoxin of eight varieties of Bacillus thuringiensis to larvae of Pieris brassicae

A microdisc technique is described for the administration of a predetermined dose of the crystalline endotoxin of Bacillus thuringiensis to host insects allowed to feed freely. It combines a high degree of precision with ease of execution and allows the elimination of insects that fail to ingest the full dose.The technique was used to compare the potencies of eight varieties of B. thuringiensis in fifth-instar larvae of Pieris brassicae. The varieties thuringiensis, morrisoni, entomocidus, and galleriae all showed similar toxicities, variety tolworthi was one-third and aizawai one-twentieth as toxic, while the varieties alesti and sotto were too weakly toxic to allow their potencies to be accurately determined.Preliminary tests with the same varieties on larvae of Bombyx mori indicated an entirely different order of potencies, in which entomocidus, sotto, alesti, galleriae, and aizawai were the most toxic and tolworthi and thuringiensis the least.     

Scanning electron microscopy of the disruption of tobacco hornworm, Manduca sexta, midgut by Bacillus thuringiensis endotoxin

The ingestion of Bacillus thuringiensis crystal endotoxin by Manduca sexta causes the destruction of both goblet and columnar cells of the midgut. One hour after ingestion, the microvilli show pathological effects. Nearly complete destruction of the goblet and columnar cells has taken place after 4 hr exposure to the toxin.