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.     

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.     

Efficacy of Aldicarb to Rotylenchulus reniformis and Biodegradation in Cotton Field Soils

The microbial degradation of aldicarb was examined in the greenhouse using soil from four cotton fields with a history of aldicarb use. The addition of aldicarb at 0.59 kg a.i./ha to natural soil increased Rotylenchulus reniformis numbers 6.6% in one soil and decreased R. reniformis numbers only 25.8% in another soil as compared to the corresponding natural soil without aldicarb. The use of increasing rates of aldicarb did not increase the efficacy of aldicarb in these soils. Rotylenchulus reniformis numbers were reduced 39.8, 22.6, and 6.8%, and increased 5.7% for aldicarb applied at 0.29, 0.59, 0.85, and 1.19 kg a.i./ha, respectively, in one natural soil. In another natural soil, R. reniformis numbers were reduced 42.5 and 21.9% for aldicarb applied at 0.29 and 1.19 kg a.i./ha, respectively, but increased 19.1 and 10.6% for aldicarb applied at 0.59 and 0.85 kg a.i./ha, respectively. Autoclaving the soils restored aldicarb toxicity in both soils, and R. reniformis numbers were reduced 96 and 99%, respectively, as compared to autoclaved soil without aldicarb. Bacterial populations were greater in the natural soils where aldicarb did not reduce R. reniformis numbers relative to the same soils that were autoclaved. However, no bacterial species was consistently associated with aldicarb degradation.     

Efficacy of Bacillus thuringiensis,Paecilomyces marquandii,and Streptomyces costaricanus with and without Organic Amendments against Meloidogyne hapla Infecting Lettuce

Chitin, wheat mash, or brewery compost were incorporated into unfumigated and methyl bromide-fumigated organic soils placed in microplots formed from cylindrical drainage tiles (0.25 m-diam. clay tile). After 3 weeks, Meloidogyne hapla and cell or spore suspensions of Bacillus thuringiensis, Paecilomyces marquandii, and Streptomyces costaricanus were individually added to the soils of designated microplots. A B. thuringiensis + S. costaricanus combination was also tested. Lettuce seedlings, cv. Montello, were transplanted into the soils 3 to 4 days later. All the bacterial and fungal antagonists applied without a soil amendment, except the B. thuringiensis + S. costaricanus treatment, reduced root galling and increased lettuce head weight in the unfumigated organic soil, but not in the fumigated soil. All three amendments were also effective against M. hapla and reduced root galling in fumigated and unfumigated soils. Wheat mash amendment increased lettuce head weight in the unfumigated soil. In general, no antagonist × amendment interaction was detected. Soil populations of B. thuringiensis were maintained at ≥4.0 log10 colony-forming units/g organic soil during the first 14 days after planting. However, viable cells of B. thuringiensis were not detected after 49 days.     

Distribution of Bacillus thuringiensis in the complex of spore-forming bacteria of the genus Bacillus Cohn in the soils of the nut-fruit forests of southern Kyrgyzstan

Three-year-long investigation of the microflora in the soil of nut-fruit forests in the south of Kyrgyzstan showed a wide distribution of spore-forming bacteria Bacillus subtilis, B. cereus, B. idosus, and B. megaterium. In the complex of these bacteria, crystal-forming bacteria B. thuringiensis are abundant. The occurrence of B. thuringiensis in mountain-forest black-brown soils reaches 80% of soil samples. Dominating subspecies are tohokuensis (H17), israelensis (H14), and toguchini (H31). Abundance of B. thuringiensis was insignificant and accounted for only 1% of spore-forming bacteria. The abundance B. thuringiensis in oozy biocenoses on the shores of water bodies within the Sary-Chelek Biosphere Reserve reaches 12.6–18.0% of soil-forming bacteria. Thus, it is possible to assume that B. thuringiensis not only are conserved in soils but also can reproduce.     

Development and Field Performance of a Broad-Spectrum Nonviable Asporogenic Recombinant Strain of Bacillus thuringiensis with Greater Potency and UV Resistance

The main problems with Bacillus thuringiensis products for pest control are their often narrow activity spectrum, high sensitivity to UV degradation, and low cost effectiveness (high potency required). We constructed a sporulation-deficient SigK⁻ B. thuringiensis strain that expressed a chimeric cry1C/Ab gene, the product of which had high activity against various lepidopteran pests, including Spodoptera littoralis (Egyptian cotton leaf worm) and Spodoptera exigua (lesser [beet] armyworm), which are not readily controlled by other Cry δ-endotoxins. The SigK⁻ host strain carried the cry1Ac gene, the product of which is highly active against the larvae of the major pests Ostrinia nubilalis (European corn borer) and Heliothis virescens (tobacco budworm). This new strain had greater potency and a broader activity spectrum than the parent strain. The crystals produced by the asporogenic strain remained encapsulated within the cells, which protected them from UV degradation. The cry1C/Ab gene was introduced into the B. thuringiensis host via a site-specific recombination vector so that unwanted DNA was eliminated. Therefore, the final construct contained no sequences of non-B. thuringiensis origin. As the recombinant strain is a mutant blocked at late sporulation, it does not produce viable spores and therefore cannot compete with wild-type B. thuringiensis strains in the environment. It is thus a very safe biopesticide. In field trials, this new recombinant strain protected cabbage and broccoli against a pest complex under natural infestation conditions.     

Activity of a Brazilian Strain of Bacillus thuringiensis israelensis Against the Cotton Boll Weevil Anthonomus grandis Boheman (Coleoptera: Tenebrionidae)

A Brazilian Bacillus thuringiensis subspecies israelensis, toxic to Diptera, including mosquitoes, was found also to show toxicity to the coleopteran boll weevil Anthonomus grandis Boheman at an equivalent level to that of the standard coleopteran-active B. thuringiensis subspecies tenebrionis T08017. Recombinant B. thuringiensis strains expressing the individual Cyt1Aa, Cry4Aa, Cry4Ba and Cry11Aa toxins from this strain were assessed to evaluate their potential contribution to the activity against A. grandis, either alone or in combination. Whilst individual toxins produced mortality, none was sufficiently potent to allow calculation of LC₅₀ values. Combinations of toxins were unable to attain the same potency as the parental B. thuringiensis subsp. israelensis, suggesting a major role for other factors produced by this strain.     

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.     

Interaction of Rotylenchulus reniformis with Seedling Disease Pathogens of Cotton

The impact of 10 Fusarium species in concomitant association with Rotylenchulus reniformis on cotton seedling disease was examined under greenhouse conditions. In experiment 1, fungal treatments consisted of Fusarium chlamydosporum, F. equiseti, F. lateritium, F. moniliforme, F. oxysporum, F. oxysporum f.sp. vasinfectum, F. proliferatum, F. semitectum, F. solani, and F. sporotrichioides; Rhizoctonia solani; and Thielaviopsis basicola. The experimental design was a 2 × 14 factorial consisting of the presence or absence of R. reniformis and the 12 fungal treatments plus two controls in autoclaved field soil. In experiment 2, the same fungal and nematode treatments were examined in autoclaved or non-autoclaved soil. This experimental design was a 2 × 2 × 14 factorial consisting of field or autoclaved soil, presence or absence of R. reniformis, and the 12 fungal treatments plus two controls. In both tests, Fusarium oxysporum f. sp. vasinfectum, F. solani, R. solani, and T. basicola consistently displayed extensive root and hypocotyl necrosis that was more severe (P ≤ 0.05) in the presence of R. reniformis. Soil treatment (autoclaved vs. non-autoclaved) influenced the impact of the Fusarium species on cotton seedling disease, with disease being more severe in the autoclaved soil. Rotylenchulus reniformis reproduction on cotton seedlings was greater in field soil compared to autoclaved soil (P ≤ 0.05). This study suggests the importance of Fusarium species and R. reniformis in cotton seedling disease.     

Specificity of Bacillus thuringiensis Delta-Endotoxin

The insecticidal activity of the delta-endotoxins of 14 Bacillus thuringiensis strains belonging to 12 subspecies was determined against Pieris brassicae, Heliothis virescens, and Spodoptera littoralis. Larvae of P. brassicae were highly susceptible to purified crystals of strains of B. thuringiensis subsp. thuringiensis and B. thuringiensis subsp. morrisoni, whereas H. virescens responded best to B. thuringiensis subsp. kenyae and B. thuringiensis subsp. kurstaki. The crystals of the B. thuringiensis subsp. entomocidus strain were the most potent against S. littoralis. It was shown that the solubility of the crystals within the gut of the three insect species is a first important step in the mode of action. Predissolution of the crystals especially enhanced the insecticidal activity against H. virescens. When in vitro-activated toxins were applied, the relative potency range varied greatly from one insect species to another. It can be concluded that at least three factors influence the potency of B. thuringiensis delta-endotoxins: the strain-related origin of the toxin, the degree of solubility of the crystals in the gut juice, and the intrinsic susceptibility of the insect to the toxin.     

Novel Isolate of Bacillus thuringiensis subsp. thuringiensis That Produces a Quasicuboidal Crystal of Cry1Ab21 Toxic to Larvae of Trichoplusia ni

A new isolate (IS5056) of Bacillus thuringiensis subsp. thuringiensis that produces a novel variant of Cry1Ab, Cry1Ab21, was isolated from soil collected in northeastern Poland. Cry1Ab21 was composed of 1,155 amino acids and had a molecular mass of 130.5 kDa, and a single copy of the gene coding for this endotoxin was located on a ~75-kbp plasmid. When synthesized by the wild-type strain, Cry1Ab21 produced a unique, irregular, bipyramidal crystal whose long and short axes were both approximately 1 μm long, which gave it a cuboidal appearance in wet mount preparations. In diet incorporation bioassays, the 50% lethal concentrations of the crystal-spore complex were 16.9 and 29.7 μg ml⁻¹ for second- and fourth-instar larvae of the cabbage looper, Trichoplusia ni, respectively, but the isolate was essentially nontoxic to larvae of the beet armyworm, Spodoptera exigua. A bioassay of autoclaved spore-crystal preparations showed no evidence of β-exotoxin activity, indicating that toxicity was due primarily to Cry1Ab21. Studies of the pathogenesis of isolate IS5056 in second-instar larvae of T. ni showed that after larval death the bacterium colonized and subsequently sporulated extensively throughout the cadaver, suggesting that other bacteria inhabiting the midgut lumen played little if any role in mortality. As T. ni is among the most destructive pests of vegetable crops in North America and has developed resistance to B. thuringiensis, this new isolate may have applied value.     

Genetic Differentiation between Sympatric Populations of Bacillus cereus and Bacillus thuringiensis

Little is known about genetic exchanges in natural populations of bacteria of the spore-forming Bacillus cereus group, because no population genetics studies have been performed with local sympatric populations. We isolated strains of Bacillus thuringiensis and B. cereus from small samples of soil collected at the same time from two separate geographical sites, one within the forest and the other at the edge of the forest. A total of 100 B. cereus and 98 B. thuringiensis strains were isolated and characterized by electrophoresis to determine allelic composition at nine enzymatic loci. We observed genetic differentiation between populations of B. cereus and B. thuringiensis. Populations of a given Bacillus species—B. thuringiensis or B. cereus—were genetically more similar to each other than to populations of the other Bacillus species. Hemolytic activity provided further evidence of this genetic divergence, which remained evident even if putative clones were removed from the data set. Our results suggest that the rate of gene flow was higher between strains of the same species, but that exchanges between B. cereus and B. thuringiensis were nonetheless possible. Linkage disequilibrium analysis revealed sufficient recombination for B. cereus populations to be considered panmictic units. In B. thuringiensis, the balance between clonal proliferation and recombination seemed to depend on location. Overall, our data indicate that it is not important for risk assessment purposes to determine whether B. cereus and B. thuringiensis belong to a single or two species. Assessment of the biosafety of pest control based on B. thuringiensis requires evaluation of the extent of genetic exchange between strains in realistic natural conditions.     

Plasmid Transfer between the Bacillus thuringiensis Subspecies kurstaki and tenebrionis in Laboratory Culture and Soil and in Lepidopteran and Coleopteran Larvae

Plasmid transfer between Bacillus thuringiensis subsp. kurstaki HD1 and B. thuringiensis subsp. tenebrionis donor strains and a streptomycin-resistant B. thuringiensis subsp. kurstaki recipient was studied under environmentally relevant laboratory conditions in vitro, in soil, and in insects. Plasmid transfer was detected in vitro at temperatures of 5 to 37°C, at pH 5.9 to 9.0, and at water activities of 0.965 to 0.995, and the highest transfer ratios (up to 10⁻¹ transconjugant/donor) were detected within 4 h. In contrast, no plasmid transfer was detected in nonsterile soil, and rapid formation of spores by the introduced strains probably contributed most to the lack of plasmid transfer observed. When a B. thuringiensis subsp. kurstaki strain was used as the donor strain, plasmid transfer was detected in killed susceptible lepidopteran insect (Lacanobia oleracea) larvae but not in the nonsusceptible coleopteran insect Phaedon chocleriae. When a B. thuringiensis subsp. tenerbrionis strain was used as the donor strain, no plasmid transfer was detected in either of these insects even when they were killed. These results show that in larger susceptible lepidopteran insects there is a greater opportunity for growth of B. thuringiensis strains, and this finding, combined with decreased competition due to a low initial background bacterial population, can provide suitable conditions for efficient plasmid transfer in the environment.     

Three years of aerial field experiments with Bacillus thuringiensis plus chitinase formulation against the spruce budworm

From 1971 to 1973 several Bacillus thuringiensis formulations were tested in the field against larvae of the spruce budworm under various conditions of population and tree defoliation. The results showed B. thuringiensis treatments can be a weapon in the control of spruce budworm outbreaks and the beneficial effect of B. thuringiensis treatments appear to be prolonged over 1 or 2 years. A new compact formulation was developed making B. thuringiensis treatments more economical and competitive with chemical insecticides.     

Bacillus thuringiensis isolates from soil and diseased insects in Egyptian cotton fields and their activity against lepidopterous insects

The distribution of Bacillus thuringiensis in Egyptian soils of cotton cultivations represented by 11 governorates was studied. This study aimed to isolate indigenous strains that may be potent against some lepidopterous insect pests. Out of 45 isolates, only 10 were effective and they showed high levels of toxicity against the target insects at 500 μg/ml (80% larval mortality or higher). Among these 10 isolates, two isolates were potent against Spodoptera littoralis, two isolates were potent against Helicoverpa (= Heliothis) armigera, seven isolates were potent against Pectinophora gossypiella, but none were potent against Agrotis ypsilon. LC₅₀ and LC₉₀ values and the potency of the B. thuringiensis isolates have been determined. Trials were conducted to isolate B. thuringiensis from diseased lepidopterous insects collected from the same locations, but all isolates showed low potential activity against the target insects. These findings showed promise for the possible use of some indigenous B. thuringiensis strains in the control of lepidopterous pests in Egypt.     

Increasing Soil Water Retention with Native‐Sourced Mulch Improves Seedling Establishment in Postmine Mediterranean Sandy Soils

Seasonal drought and heavily impeded soils reduce restoration success in Mediterranean‐type postmine soils, where up to 90% seedling mortality has been observed after 2 years. To alleviate these barriers, amendments were incorporated into the soil profile of a freshly mined sand quarry. Within the quarry, three 223 m² replicate sites contained two substrate amendments: 12% v/v native‐sourced mulch or gravel incorporated within the top 50 cm of the soil profile. Three remnant sites provided a “natural” reference system. Seeds of two autochthonous trees, Banksia attenuata (R.Br.) and B. menziesii (R.Br.) were sown across all treatments. Soil impedance, moisture, and seedling stomatal conductance were monitored for 2 years, at which point seedlings were excavated, and nutrient concentration, root morphology, and soil chemical properties were measured. Roots in all restoration treatments were restricted to the top 40 cm of the profile due to increases in soil impedance, regardless of amendment, compared to >70 cm in the natural system. Seedling mortality was greatest after the second summer in the control and inorganic amendment treatments, with stomatal conductance indicating severe drought stress. Survival in the organic treatment was 24–42% greater than the control, with higher soil moisture and stomatal conductance rates 2.5 times that of the control. The increased soil water retention by a native‐sourced mulch was shown to improve postmine restoration success for these native trees.     

生草栽培对山核桃林地土壤养分及微生物多样性的影响

高强度经营导致山核桃林地土壤性质改变,设置了白三叶、黑麦草、油菜、紫云英、自然杂草和清耕(对照)6个处理,以3次重复,随机排列的2年田间试验,研究了不同生草栽培对山核桃林地土壤养分和微生物多样性的影响。结果表明:与清耕相比,生草栽培均能改善土壤养分状况,除全钾外,白三叶和紫云英处理较其他处理能显著提高土壤养分含量,其余几个处理间差异不明显;生草栽培显著提高了土壤微生物生物量碳(MBC)含量,白三叶、黑麦草、紫云英、油菜和自然杂草处理分别较清耕提高了169.6%、159.7%、144.1%、138.6%和58.6%,差异达显著水平(P0.05)。6种不同处理的土壤微生物活性(AWCD)、微生物Shannon指数和均匀度指数均存在明显差异。不同处理的土壤AWCD为白三叶紫云英油菜自然杂草黑麦草清耕;白三叶处理的土壤微生物Shannon指数高于其他处理;白三叶、紫云英和油菜处理的土壤微生物均匀度指数显著高于其他处理。相关性分析表明,土壤微生物活性、微生物Shannon指数和均匀度指数两两之间均达到极显著差异(P0.01),三者与土壤各养分指标之间未达显著差异,但表现为正相关关系。白三叶、紫云英和油菜对改善土壤微生物特性效果较好。     

Experimental evidence that refuges delay insect adaptation to Bacillus thuringiensis

Theoretical projections suggest that refuges from exposure can delay insect adaptation to environmentally benign insecticides derived from Bacillus thuringiensis, but experimental tests of this approach have been limited. We tested the refuge tactic by selecting two sets of two colonies of diamondback moth (Plutella xylostella) for resistance to B. thuringiensis subsp. aizawai in the laboratory. In each set, one colony was selected with no refuge and the other with a 10 per cent refuge from exposure to B. thuringiensis subsp. aizawai. Bioassays conducted after nine selections were completed show that mortality caused by B. thuringiensis subsp. aizawai was significantly greater in the refuge colonies than in the no-refuge colonies. These results demonstrate that the refuges delayed the evolution of resistance. Relative to a susceptible colony, final resistance ratios were 19 and 8 for the two no-refuge colonies compared to 6 and 5 for the refuge colonies. The mean realized heritability of resistance to B. thuringiensis subsp. aizawai was 0.046 for colonies without refuges, and -0.002 for colonies with refuges. Selection with B. thuringiensis subsp. aizawai decreased susceptibility to B. thuringiensis toxin Cry1Ab, but not to Cry1C or B. thuringiensis subsp. kurstaki. Although the ultimate test of refuges will occur in the field, the experimental evidence reported here confirms modelling results indicating that refuges can slow the evolution of insect resistance to B. thuringiensis.     

Bioassay of a nuclear-polyhedrosis virus and Bacillus thuringiensis against the American bollworm, Heliothis armigera, in Botswana

Heliothis armigera is a serious pest in Botswana. Preparations of a local nuclear-polyhedrosis virus and Bacillus thuringiensis (Biotrol BTB-183) are being considered as treatments for control of this pest. Laboratory bioassays of these two preparations were carried out to provide a firm basis for comparison with past field trials and a standard for future field testing. The LC₅₀ of the virus and LD₅₀ of B. thuringiensis were found to compare favorably with those for similar materials against closely and distantly related lepidopterous pests. The majority of larvae were killed very rapidly by the B. thuringiensis, although some survived up to 14 days before death. The minimum time to mortality for the virus was 4 days. B. thuringiensis lowered the weight of both male and female surviving pupae, whereas the virus had no significant effect on its survivors. Concentrations of the two materials calculated from the LC₉₀ and LD₉₀ were considerably lower than those already found to give some or good control of H. armigera on sorghum. Improved spray coverage and protection of the materials against environmental degradation are required if spraying concentrations are to be reduced and yet remain effective.     

Persistence and Recycling of Bioinsecticidal Bacillus thuringiensis subsp. israelensis Spores in Contrasting Environments: Evidence from Field Monitoring and Laboratory Experiments

Sprays of commercial preparations of the bacterium Bacillus thuringiensis subsp. israelensis are widely used for the control of mosquito larvae. Despite an abundant literature on B. thuringiensis subsp. israelensis field efficiency on mosquito control, few studies have evaluated the fate of spores in the environment after treatments. In the present article, two complementary experiments were conducted to study the effect of different parameters on B. thuringiensis subsp. israelensis persistence and recycling, in field conditions and in the laboratory. First, we monitored B. thuringiensis subsp. israelensis persistence in the field in two contrasting regions in France: the Rhône-Alpes region, where mosquito breeding sites are temporary ponds under forest cover with large amounts of decaying leaf matter on the ground and the Mediterranean region characterized by open breeding sites such as brackish marshes. Viable B. thuringiensis subsp. israelensis spores can persist for months after a treatment, and their quantity is explained both by the vegetation type and by the number of local treatments. We found no evidence of B. thuringiensis subsp. israelensis recycling in the field. Then, we tested the effect of water level, substrate type, salinity and presence of mosquito larvae on the persistence/recycling of B. thuringiensis subsp. israelensis spores in controlled laboratory conditions (microcosms). We found no effect of change in water level or salinity on B. thuringiensis subsp. israelensis persistence over time (75 days). B. thuringiensis subsp. israelensis spores tended to persist longer in substrates containing organic matter compared to sand-only substrates. B. thuringiensis subsp. israelensis recycling only occurred in presence of mosquito larvae but was unrelated to the presence of organic matter.