Two new subspecies of Bacillus thuringiensis isolated in Japan: Bacillus thuringiensis subsp. kumamotoensis (serotype 18) and Bacillus thuringiensis subsp. tochigiensis (serotype 19)

Bacteriological and serological characteristics of three Bacillus thuringiensis isolates obtained in Japan were investigated. They formed typical rhomboidal parasporal inclusions but flagellar (H) antigens of these isolates were different from those of the known 17 H serotypes of B. thuringiensis. The three isolates were divided into two new serotypes (serotypes 18 and 19). The serotype 18 isolate (3–71) produced thermostable exotoxin and the inclusions of this isolate were toxic to larvae of the silkworm, Bombyx mori, but nontoxic to larvae of the mosquito, Aedes aegypti. The other isolate (119-72) belonging to serotype 18 produced inclusions nontoxic to larvae of B. mori and A. aegypti and did not produce thermostable exotoxin. However, other bacteriological properties of the isolate 119-72 were similar to those of the isolate 3–71. The serotype 19 isolate (117-72) produced inclusions nontoxic to larvae of B. mori and A. aegypti and did not produce thermostable exotoxin. Acid production from saccharose and the production of brownish purple pigment were observed in the two serotype 18 isolates, while neither of them was observed in the serotype 19 isolate. In other 29 biochemical properties tested, there was no difference among the three isolates. Based on these characteristics, the following two subspecies names are proposed: Bacillus thuringiensis subsp. kumamotoensis (serotype 18) for the type strain 3–71 and Bacillus thuringiensis subsp. tochigiensis (serotype 19) for the type strain 117-72.     

Larvicidal activity of Bacillus thuringiensis subsp. israelensis, serovar H14 in Aedes aegypti: Histopathological studies

Bacillus thuringiensis subsp. israelensis, serovar H14, when applied as a primary commercial powder, caused the rapid death of Aedes aegypti larvae. Mortality started 6 min after application of 4 μg/ml of the pathogen and reached a maximum 27 min later. When the LC₅₀ (10 ng/ml) was applied, mortality began after 37 min and reached a maximum 120 min later. Histopathological changes in B. thuringiensis israelensis-treated larvae could be observed only in the midgut and caeca. In B. thuringiensis israelensis-treated “dead larvae”, the epithelial layer is disorganized, most of the cells have disappeared and the peritrophic membrane is broken. The epithelium in the B. thuringiensis israelensis-treated “living larvae” still maintains its monolayer structure, but with marked cellular hypertrophy and vacuolized cytoplasm. Also, the “brush border” is thinner and disrupted. Based on the fact that mortality of A. aegypti is a quick process, and because the histopathological changes caused by B. thuringiensis israelensis are similar to those found in lepidopterous larvae treated with pure δ-endotoxin of other B. thuringiensis variants, it is suggested that larvicidal activity of B. thuringiensis israelensis in A. aegypti is due to its δ-endotoxin.     

Insecticidal activity of Bacillus thuringiensis crystal proteins

Published data on insecticidal activity of crystal proteins from Bacillus thuringiensis are incorporated into the Bt toxin specificity relational database. To date, 125 of the 174 holotype known toxins have been tested in ∼1700 bioassays against 163 test species; 49 toxins have not been tested at all; 59 were tested against 71 Lepidoptera species in 1182 bioassays; 53 toxins were tested against 23 Diptera species in 233 bioassays; and 47 were tested against 39 Coleoptera species in 190 bioassays. Activity spectra of the tested toxins were summarized for each order. Comparisons of LC₅₀ values are confounded by high variability of the estimates, mostly due to within-species variation in susceptibility, and errors associated with estimation of toxin protein content. Limited analyses suggest that crystal protein toxicity is not affected by quarternary toxin rank or host used for gene expression, but that pre-ingestion treatment by solubilization or enzymatic processing has a large effect. There is an increasing number of toxin families with cross-order activity, as 15 of the 87 families (secondary rank) that are pesticidal are active against more than one order. Cross-order activity does not threaten environmental safety of B. thuringiensis-based pest control because toxins tend to be much less toxic to taxa outside the family’s primary specificity range.     

Persistence of Bacillus thuringiensis parasporal crystal insecticidal activity in soil

Spores and parasporal crystals of a Bacillus thuringiensis var. aizawai (H-serotype 7), strain HD137, streptomycin-resistant mutant were added to acidic (pH 5.0) natural and autoclaved soil and incubated at ?0.10 MPa, 25°C. Populations of B. thuringiensis in both soil treatments showed exponential rates of mortality which were represented by linear regression, the loss of viability being greater in natural than autoclaved soil. In natural soil, parasporal crystal insecticidal activity was lost at a complex, nonexponential rate. The initial, rapid decrease of activity gradually slowed, and the level of activity stabilized at 10% of the original inoculum level after 250 days incubation, until the cessation of sampling at >2 years. In autoclaved soil no significant (P > 0.2) loss of parasporal crystal insecticidal activity was detected over the same period, which suggested that soil microorganisms were responsible for the loss of crystal insecticidal activity in the natural, nonsterilized soil. The rate of loss of crystal activity in natural soil correlated well with assay data reported in the literature using Galleria mellonella, which measures the combined activity of spore and crystal. In autoclaved soil correlation was poor, probably due to variability in the bioassay data.     

Sporeless mutants of Bacillus thuringiensis. III. The process of crystal formation

In order to investigate the formation of the parasporal crystal of B. thuringiensis with special reference to the spore, sequential ultrastructural analysis of sporulation was performed using a sporeless mutant strain (sp^?) as well as its parent wild strain (sp⁺). From the logarithmic growth to the end of forespore formation, the same sequential process of sporulation proceeded in both strains and a forespore with double membranes appeared. Thereafter, subsequent sporulation in the sp strain was either partly or completely arrested and finally spore (mainly the forespore) became deformed. On the other hand, crystal formation took place throughout by the same processes both in sp⁺ and sp^? strains. During the forespore formation, a primordial crystal and an ovoid inclusion appeared and after this stage, the crystal displayed a characteristic diamond-shaped body with lattice fringes increasing its size. No regularity was found in the position of the crystal with respect to the spore. As far as the present ultrastructural observations were concerned, the crystal developed without any special association with the membranes of the spore. However, without the formation of the forespore (including the incipient forespore), no crystal formation was observed.     

Sporeless mutants of Bacillus thuringiensis

Five sporeless mutant strains of Bacillus thuringiensis were selected after treatment with the mutagen N-methyl-N′-nitro-N-nitrosoguanidine. Two mutant strains were derived from alesti and three from aizawai. All mutants were completely lacking in ability to form spores. Bipyramidal crystalline bodies of the mutants were very regular in shape, as seen with the parent strains, and lay free in the culture broth with autolysis. The insecticidal activity of mutants was, in principle, the same as that of original strains.Cells of the mutants tended to autolyze easily at the end of cultivation. However, 1–10% of cells still remain living. They are completely killed by heat treatment, e.g., 60°C for 30 min, which, however, causes a slight but nonsignificant reduction of toxicity.Thus, use of these sporeless mutants of Bacillus thuringiensis as microbial insecticide having no viable cells is suggested. They may serve as a biochemical starting material for β-endotoxin.     

Characterization of crystal prototoxin and an activated toxin from Bacillus thuringiensis var. entomocidus

Toxin crystals from Bacillus thuringiensis var. entomocidus were lysed by proteases present in gut juice from larval Philosamia ricini (Lepidoptera) with the release of a prototoxin and an activated toxin. Some of the toxic activity of the lysate was complexed with a pheophytinlike pigment and this complex was retarded on filtration through Sephadex gels. A method is described for the removal of the pheophytin from larval protease preparations. The prototoxin has a molecular weight greater than 200,000, determined by its exclusion from Sephadex G-200 and on activation produces a toxin of molecular weight about 50,000. Isoelectric focusing of crystal lysates gave pI values of 4.5 and 6.4 for the prototoxin and toxin, respectively. The antigenic composition of the prototoxin and of the toxin are compared and the significance of antigen h as an indicator of activation is discussed.     

Binding of Bacillus thuringiensis subsp. israelensis Cry4Ba to Cyt1Aa has an important role in synergism

Bacillus thuringiensis subsp. israelensis (Bti) produces at least four different crystal proteins that are specifically toxic to different mosquito species and that belong to two non-related family of toxins, Cry and Cyt named Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa. Cyt1Aa enhances the activity of Cry4Aa, Cry4Ba or Cry11Aa and overcomes resistance of Culex quinquefasciatus populations resistant to Cry11Aa, Cry4Aa or Cry4Ba. Cyt1Aa synergized Cry11Aa by their specific interaction since single point mutants on both Cyt1Aa and Cry11Aa that affected their binding interaction affected their synergistic insecticidal activity. In this work we show that Cyt1Aa loop β6-αE K198A, E204A and β7 K225A mutants affected binding and synergism with Cry4Ba. In addition, site directed mutagenesis showed that Cry4Ba domain II loop α-8 is involved in binding and in synergism with Cyt1Aa since Cry4Ba SI303-304AA double mutant showed decreased binding and synergism with Cyt1Aa. These data suggest that similarly to the synergism between Cry11Aa and Cyt1Aa toxins, the Cyt1Aa also functions as a receptor for Cry4Ba explaining the mechanism of synergism between these two Bti toxins.     

Interactions between Bacillus thuringiensis subsp. kurstaki HD-1 and midgut bacteria in larvae of gypsy moth and spruce budworm

We examined interaction between Bacillus thuringiensis subsp. kurstaki HD-1 (Foray 48B) and larval midgut bacteria in two lepidopteran hosts, Lymantria dispar and Choristoneura fumiferana. The pathogen multiplied in either moribund (C. fumiferana) or dead (L. dispar) larvae, regardless of the presence of midgut bacteria. Inoculation of L. dispar resulted in a pronounced proliferation of enteric bacteria, which did not contribute to larval death because B. thuringiensis was able to kill larvae in absence of midgut bacteria. Sterile, aureomycin- or ampicillin-treated larvae were killed in a dose-dependent manner but there was no mortality among larvae treated with the antibiotic cocktail used by [Broderick et al., 2006] and [Broderick et al., 2009]. These results do not support an obligate role of midgut bacteria in insecticidal activity of HD-1. The outcome of experiments on the role of midgut bacteria may be more dependent on which bacterial species are dominant at the time of experimentation than on host species per se. The L. dispar cohorts used in our study had a microflora, that was dominated by Enterococcus and Staphylococcus and lacked Enterobacter. Another factor that can confound experimental results is the disk-feeding method for inoculation, which biases mortality estimates towards the least susceptible portion of the test population.     

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.     

Bacillus thuringiensis serovar mogi (flagellar serotype 3a3b3d), a novel serogroup with a mosquitocidal activity

The flagellated vegetative cells of the Bacillus thuringiensis strain K4 were agglutinated with the H3 reference antiserum and further, agglutinated with 3b and 3d monospecific factor sera but non-reactive for 3c and 3e factor sera. This creates a new serogroup with flagellar antigenic structure of 3a3b3d: B. thuringiensis serovar mogi. The strain K4 showed high activity against dipteran larvae, Anopheles sinensis and Culex pipienspallens while no lepidopteran toxicity. It produced ovoidal parasporal inclusions (crystals) whose SDS-PAGE protein profile consisted of several bands ranging from 75 to 30 kDa. Through the protein identification by nano-LC-ESI-IT MS analysis, the putative peptides of Cry19Ba, Cry40ORF2, Cry27Aa and Cry20Aa were detected.     

Ecology of Bacillus thuringiensis in storage moths

A disease-free stock of Plodia interpunctella was produced by a continuous rearing technique. In dense populations of this stock, 10⁴ or more spores of H serotype V Bacillus thuringiensis applied at one point on the surface of 200 g of food were required to cause epizootics, compared with 10⁷ or more when spread evenly over the surface. In infected populations, spores contaminated the surfaces of all stages of the insect. In diseased larval cadavers there were 5.6–42.2 × 10⁸ spores/g of dry insect (P. interpunctella, Ephestia cautella, Anagasta kuehniella, Ephestia elutella, and Galleria mellonella). Larvae did not cannibalize live larvae while food was present though they sometimes ate cadavers. This is the most potent means of natural spread of the disease. Occurring mainly in protected situations such as food stores, natural infections are usually light, but occasionally spectacular surface accumulations of dead larvae occur, possibly associated with stress, physiological condition of the larvae, serotype of the bacterium, or behavior pattern such as migration. Natural disease may curb infestations in debris, but it attacks too late to prevent excessive damage to stored food. A prophylactic, even admixture of 2 × 10⁹ spores/200 g of food is required for effective insect control.     

The control of Monomorium pharaonis (Hymenoptera: Formicidae) with Bacillus thuringiensis

In this study, the effect of different preparations made from Bacillus thuringiensis var. thuringiensis (strains: CCEB 555 and CCEB 058) on ants, Monomorium pharaonis, under laboratory conditions is reported. The different preparations tested consisted of (1) a liquid culture of the strain B. thuringiensis CCEB 555 (containing spores and exotoxin), (2) the supernatant of the culture broth of strain CCEB 555 (containing exotoxin), and (3) the biological preparation “Bathurin” prepared from the strain B. thuringiensis CCEB 058 (containing spores and inclusions, without exotoxin). The preparations were used either pure or in alternation with borax, i.e., 1 wk borax, 3 wk the respective preparation for several months. All preparations were found to be toxic to M. pharaonis and their effect was characterized by a slow extinction of the ant colony. Administration of “Bathurin” (1.3%) yielded a 100% mortality after 20 wk. Using a liquid culture of B. thuringiensis var. thuringiensis, 100% mortality was recorded after 21 wk, a period of time which did not differ from that obtained with the supernatant of the culture containing exotoxin. The alternation with borax was found to accelerate ant mortality by 9–10 wk after administration. In all experiments, the worker ants died first, the queen ants surviving them by 1–3 wk.In experiments employing worker ants only, a 100 and 98% mortality, respectively, occurred within 3 wk after administration of a liquid culture of B. thuringiensis and “Bathurin” supplemented with borax.     

Distribution of phenotypes among Bacillus thuringiensis strains

An extensive collection of Bacillus thuringiensis isolates from around the world were phenotypically profiled using standard biochemical tests. Six phenotypic traits occurred in 20–86% of the isolates and were useful in distinguishing isolates: production of urease (U; 20.5% of isolates), hydrolysis of esculin (E; 32.3% of isolates), acid production from salicin (A; 37.4% of isolates), acid production from sucrose (S; 34.0% of isolates), production of phospholipase C or lecithinase (L; 79.7% of isolates), and hydrolysis of starch (T; 85.8% of isolates). With the exception of acid production from salicin and hydrolysis of esculin, which were associated, the traits assorted independently. Of the 64 possible combinations of these six phenotypic characteristics, 15 combinations accounted for ca. 80% of all isolates, with the most common phenotype being TL (23.6% of isolates). Surprisingly, while the biochemical traits generally assorted independently, certain phenotypic traits associated with the parasporal crystal were correlated with certain combinations of biochemical traits. Crystals that remained attached to spores (which tended to be non-toxic to insects) were highly correlated with the phenotypes that included both L and S. Among the 15 most abundant phenotypes characterizing B. thuringiensis strains, amorphous crystals were associated with TLE, TL, T, and Ø (the absence of positive tested biochemical traits). Amorphous crystal types displayed a distinct bias toward toxicity to dipteran insects. Although all common phenotypes included B. thuringiensis isolates producing bipyramidal crystals toxic to lepidopteran insects, those with the highest abundance of these toxic crystals displayed phenotypes TLU, TLUA, TLUAE, and TLAE.     

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.     

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.     

Single concentration tests show synergism among Bacillus thuringiensis subsp. israelensis toxins against the malaria vector mosquito Anophelesalbimanus

Bioassays of insecticidal proteins from Bacillus thuringiensis subsp. israelensis with larvae of the malaria vector mosquito Anophelesalbimanus showed that the cytolytic protein Cyt1Aa was not toxic alone, but it increased the toxicity of the crystalline proteins Cry4Ba and Cry11Aa. Synergism also occurred between Cry4Ba and Cry11Aa toxins. Whereas many previous analyses of synergism have been based on a series of toxin concentrations leading to comparisons between expected and observed values for the concentration killing 50% of insects tested (LC₅₀), we describe and apply a method here that enables testing for synergism based on single concentrations of toxins.     

Pathogenesis and midgut histopathology of Bacillus thuringiensis in Simulium vittatum (Diptera: Simuliidae)

The pathogenesis and midgut histopathology which resulted when larvae of the blackfly, Simulium vittatum, were exposed to Bacillus thuringiensis at various temperatures and periods of exposure were investigated. The onset of mortality was studied at 10°, 15°, 19°, and 24°C. For each 4–5°C increase in temperature above 15°C, the onset of mortality was shortened by 24 hr. Exposures as brief as 15 min to 10 ppm of a whole spore preparation resulted in an average mortality of 29% in late-instar larvae. Mortality increased sharply for exposures up to 3 hr, approaching a maximum of 80%.The gross signs of disease included cessation of feeding and tetany with brachytosis. The tissue most affected was the midgut epithelium in the regions of the gastric caeca and posterior stomach. The formation of cytoplasmic vacuoles followed by cell lysis and/or sloughing were very apparent in moribund larvae. Death resulted without bacteremia.     

Bacillus thuringiensis parasporal crystal toxin: Dissociation into toxic low molecular weight peptides

Parasporal crystals of Bacillus thuringiensis can be dissociated into low molecular weight peptides (< 5000 daltons) by dissolving them in 0.1 M N-morpholinopropane sulfonic acid buffer pH 7.8 containing 0.05 M dithiothreitol and 2M–4M KSCN, or by performic acid oxidation. The peptides obtained by dissolving in KSCN were still toxic to silkworm larvae.