H.D. Burges

Survival of Bacillus thuringiensis and Bacillus cereus spore inocula in soil: effects of pH, moisture, nutrient availability and indigenous microorganisms

Abstract The influence of soil pH, moisture, nutrient availability and indigenous microorganisms on the survival and growth of Bacillus thuringiensis and B. cereus spore inocula in soil was investigated. The factor of greatest importance was nutrient availability. B. thuringiensis could not grow under most natural soil conditions, whilst B. cereus grew only slowly. Supplementing soil with additional nutrients, or autoclaving, stimulated growth of the Bacillus populations. Growth was reduced by a low soil pH (5.2 compared to 7.3), whilst both Bacillus species grew faster and survived better in wetter (at 0, −0.01 MPa) than drier (at −0.10, −1.00 MPa) soils. The death of B. thuringiensis in natural soil probably accounts for its rarity in the outdoor environment. It is suggested that this demise is attributable to a failure of B. thuringiensis to germinate in soil.

Ecology of Bacillus thuringiensis in storage moths

Abstract A disease-free stock of Plodia interpunctella was produced by a continuous rearing technique. In dense populations of this stock, 10 4 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 10 7 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 × 10 8 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 × 10 9 spores/200 g of food is required for effective insect control.

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.

Importance of spores and δ-endotoxin protein crystals of Bacillus thuringiensis in Galleria mellonella

Abstract Larvae of Galleria mellonella were fed on a honey-rich artificial food containing live spores or toxic crystals of Bacillus thuringiensis serotype V or various combinations of both. In this food; 1:1 combinations were 10 times more potent than live spores alone and about 104 times more potent than crystals alone. Reduction in the proportion of spores, but not in that of crystals, decreased the slope of probit lines from 3.4 to 0.6. One or more factors in the spore are at least partly responsible for the potency of serotype V in G. mellonella. The results suggest than an observed gross loss of potency of this serotype in beehives is more likely to be due to death of spores than to deterioration of crystals. The reaction of G. mellonella to serotype V is nearest to that of a type 3 host species. Spores of serotype I are almost inactive in this host.

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

Abstract 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

Effect of ultraviolet and gamma rays on the activity of δ-endotoxin protein crystals of Bacillus thuringiensis

Abstract Sensitive bioassays with larvae of Pieris brassicae revealed no reduction of insecticidal activity as a result of severe gamma or ultraviolet irradiation of crystals of Bacillus thuringiensis (serotype V). The measured response was the inhibition of larval feeding by the crystals over exposure periods short enough for the presence of live spores not to influence feeding. The results were analyzed using a logistic model.

Effect of incubation in natural and autoclaved soil upon potency and viability of Bacillus thuringiensis

Abstract Spores and parasporal crystals of a Bacillus thuringiensis var. aizawai H-serotype 7, strain HD137, streptomycin-resistant mutant, were added to normal and autoclaved aliquots of p H 5 soil incubated at 25°C and −0.10 MPa water availability. Viable B. thuringiensis in soil samples were estimated by dilution-plating on a streptomycin-based medium, and combined spore and crystal insecticidal activity was bioassayed with larvae of Galleria mellonella . Populations of B. thuringiensis in both soil treatments suffered exponential rates of mortality, which were represented by segmented linear regression. Mortality was far greater in natural than autoclaved soil. Potency also fell in both soil treatments. This loss of potency was greater in natural soil, although the rates of potency loss in either soil treatment correlated poorly with the respective mortality rates of the B. thuringiensis populations, as potency losses were not exponential functions. The results suggest that the presence of indigenous microorganisms in natural soil accelerated the rate of mortality and loss of potency of B. thuringiensis .

Detection of Bacillus thuringiensis in soil by immunofluorescence

Abstract 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.

Effect of incubation in non-sterilised and autoclaved arable soil on survival of Bacillus thuringiensis and Bacillus cereus spore inocula

Abstract Inocula of 103, 104, and 105 Bacillus thuringiensis spores/g non-sterilised soil apparently failed to germinate and viability declined by up to 55% during 90 days incubation at 25°C and -0.10 MPa. In autoclaved (121°C, 20 min) soil, no net mortality was detected in any of the 3 levels of inocula and detection of germination was erratic. No germination was detected yet sporadic growth occurred with inocula of 103 spores/g soil, and both (sporadic) germination and growth were detected at 104 spores/g soil. Final levels of these inocula were 500–2000% greater than their initial levels. Germination and growth were not detected in the inoculum of 105 spores/g soil. By contrast, inocula of 104 Bacillus cereus spores/g germinated in both non-sterilised and autoclaved soil, but then exhibited bacteriostasis. No net changes in inoculum size were detected in either soil treatment. We suggest that the mortality of B. thuringiensis spores in non-sterilised soil resulted from an inherent inability of the spor...

Occurrence of pathogens of the flour beetle, Tribolium castaneum

All but one of the specific pathogens of Tribolium castaneum were found in samples of insects from British food stores. The commonest pathogen, Nosema whitei, occurred in an average of 10.1% of the insects in 27% of the samples. At the time of sampling, diseases were probably not limiting appreciably the numbers of insects in stored food. Disease is not likely to curb infestations before the level of economic importance is reached. However, diseases may be of some economic importance by reducing or eliminating sources of infestation in undisturbed residues of food.