Susceptibility of the spruce budworm, Choristoneura fumiferana, and the white-marked tussock moth, Orgyia leucostigmata, to Bacillus thuringiensis: Chemical insecticide combinations

Bacillus thuringiensis mixed with the organophosphate insecticides, fenitrothion (Sumithion), Gardona^®, and Orthene^®, or the synthetic pyrethroid, SBP 1382, was incorporated into synthetic diet and fed larvae of the spruce budworm, Choristoneura fumiferana, and the white-marked tussock moth, Orgyia leucostigma. Mortality was highest when larvae were fed combinations of low concentrations of the insecticides and low to moderate concentrations of the pathogen. The data indicated that applications of a B. thuringiensis dosage expected to produce about 45% mortality of third and fourth instar larvae of the spruce budworm combined with a dosage of fenitrothion causing about 40% mortality or a dosage of Orthene causing from 5 to 25% mortality should result in low budworm survival. With a B. thuringiensis dosage causing 20–60% mortality combined with a fenitrothion dosage causing 15–50% mortality or a sublethal dosage of Gardona, a low survival rate of young white-marked tussock moth larvae may be expected.     

Selectivity of different biological products to the egg parasitoid Telenomus remus (Hymenoptera: Platygastridae)

The selectivity of five entomopathogens and a chemical insecticide (positive control) to pupae and adults of the egg parasitoid Telenomus remus (Nixon) was evaluated in the laboratory under controlled environmental conditions according to protocols established by the International Organization for Biological Control. Baculovirus anticarsia (Baculovirus AEE^®), Bacillus thuringiensis var. kurstaki (Thuricide^®), Bacillus thuringiensis var. aizawai (Agree^®), Beauveria bassiana (Boveril^®), Metarhizium anisopliae (Metarril^®) and Trichoderma harzianum (Trichodermil^®) are harmless to T. remus pupae, and adults. Thus, our results suggest that the insect control strategies applied here are compatible since entomopathogens were classified as harmless to T. remus in most examined cases and therefore facilitate a joint application to control different pests. Bacillus thuringiensis var. kurstaki (Dipel^®), despite being classified as slightly harmful in some of the evaluations, can still be considered compatible for use together with T. remus, especially when compared with chemical insecticides such as chlorpyrifos that might be considered harmful to the parasitoid survival.     

MALDI-TOF-MS and 16S rRNA characterization of lead tolerant metallophile bacteria isolated from saffron soils of Kashmir for their sequestration potential

Toxic metal contamination in soils due industrialization is nowadays a concern to the scientists worldwide. The current study deals with the evaluation of response and tolerance by isolated metallophilic bacteria in different lead concentrations (100 ppm to 1000 ppm). By taking optical densities of the isolates, the minimum inhibitory concentration (MIC) of Pb²⁺ were determined.16S rRNA and MALDI-TOF MS were used for the identification of the bacteria. Total of 37 isolates were observed, among them 04 (Staphylococcus equorum, Staphylococcus warneri, Bacillus safensis and Bacillus thuringiensis), isolated were detected having efficacy of Pb²⁺tolerance and sequestration at varying MIC. Furthermore, B. thuringiensis was observed to have highest (900 ppm) tolerance for lead and lowest (500 ppm) for Staphylococcus warneri. Moreover, the highest (65.3%) sequestration potential has been observed for B. thuringiensis and least (52.8%) for S. warneri. The tolerance and sequestration potential properties of these isolated species can be utilised to exterminate heavy metals and reduce their toxicity from the contaminated environment.     

Rumen Bacterial and Protozoal Responses to Insecticide Substrates

Insecticides containing organophosphate, chlorinated hydrocarbon, and carbamate were tested with bovine ruminal ingesta fractions. Rumen bacteria exposed to insecticide levels of 0 to 500 ppm in rumen fluid for 4 hr were inoculated into rumen fluid-starch feed extract medium. No apparent significant bacterial count inhibitions were noted. Also, when insecticides were used as carbon sources at concentrations of 500 ppm in carbohydrate-limited media, no increases in bacterial counts were indicated. Warburg manometric data showed that paraffin oil-Triton X-155 preparations of dimethoate, Diazinon, lindane, Thiodan and Sevin stimulated gas production in holotrich protozoa. Entodinium simplex, an oligotrich, produced less gas with insecticide substrates per unit of dry weight than did an Isotricha sp. Rumen bacteria and plant debris fractions from ruminal ingesta provided with insecticides did not give increased manometric responses over the endogenous control vessels. Washed suspensions of I. intestinalis produced volatile fatty acids in excess of the endogenous suspensions when provided insecticide substrates. Thiodan dissimilation by I. intestinalis was followed colorimetrically with 15% loss in substrate in 1 hr of incubation at 39 C. Diazinon-C¹⁴ substrate uptake was demonstrated with suspensions of E. simplex and I. intestinalis. Rumen ciliates are suggested as a possible means for screening out useful insecticides susceptible to microbial dissimilation for use on forage and other cattle-feed crops.     

Cyt1Ab1 and Cyt2Ba1 from Bacillus thuringiensis subsp. medellin and B. thuringiensis subsp. israelensis Synergize Bacillus sphaericus against Aedes aegypti and Resistant Culex quinquefasciatus (Diptera: Culicidae)

The interaction of two cytolytic toxins, Cyt1Ab from Bacillus thuringiensis subsp. medellin and Cyt2Ba from Bacillus thuringiensis subsp. israelensis, with Bacillus sphaericus was evaluated against susceptible and resistant Culex quinquefasciatus and the nonsensitive species Aedes aegypti. Mixtures of B. sphaericus with either cytolytic toxin were synergistic, and B. sphaericus resistance in C. quinquefasciatus was suppressed from >17,000- to 2-fold with a 3:1 mixture of B. sphaericus and Cyt1Ab. This trait may prove useful for combating insecticide resistance and for improving the activity of microbial insecticides.     

Survival of Bacillus thuringiensis Spores in Soil

Bacillus thuringiensis spores and parasporal crystals were incubated in natural soil, both in the laboratory and in nature. During the first 2 weeks, the spore count decreased by approximately 1 log. Thereafter, the number of spore CFU remained constant for at least 8 months. B. thuringiensis did not lose its ability to make the parasporal crystals during its residence in soil. Spore survival was similar for a commercial spore-crystal preparation (the insecticide) and for laboratory-grown spores. In contrast to these results, spores that were produced in situ in soil through multiplication of added vegetative cells survived for only a short time. For spore additions to soil, variations in soil pH had little effect on survival for those spores that survived the first 2 weeks of incubation. Also without effect were various pretreatments of the spores before incubation in soil or nutritional amendment or desiccation of the soil. Remoistening of a desiccated soil, however, caused a decrease in spore numbers. Spores incubated in soil in the field did not show this, but the degree of soil desiccation in nature probably never reached that for the laboratory samples. The good survival of B. thuringiensis spores after the first 2 weeks in soil seemed to be a result of their inability to germinate in soil. We found no evidence for the hypothesis that rapid germination ability for spores in soil conferred a survival advantage.     

The effectiveness ofBacillus thuringiensis formulations for the control of larvae ofSchizura concinna onCercis occidentalis trees in California

Five commercial formulations ofBacillus thuringiensis (Berliner) were found to be effective for control of larvae of the red-humped caterpillar,Schizura concinna (Smith & Abbot), an important defoliator of broad-leaved trees in California. The formulations tested were Biotrol® BTB 183 25 W, Biotrol® XK, Dipel®, Thuricide® HPC, and Thuricide® 90TS 950T. As a result of these trials,B. thuringiensis replaced chemical insecticides for the control of this pest in 1970, and has provided effective control as part of an operational pest management programme for four years.     

Infection of Tribolium castaneum with Bacillus thuringiensis: Quantification of Bacterial Replication within Cadavers,Transmission via Cannibalism,and Inhibition of Spore Germination

Reproduction within a host and transmission to the next host are crucial for the virulence and fitness of pathogens. Nevertheless, basic knowledge about such parameters is often missing from the literature, even for well-studied bacteria, such as Bacillus thuringiensis, an endospore-forming insect pathogen, which infects its hosts via the oral route. To characterize bacterial replication success, we made use of an experimental oral infection system for the red flour beetle Tribolium castaneum and developed a flow cytometric assay for the quantification of both spore ingestion by the individual beetle larvae and the resulting spore load after bacterial replication and resporulation within cadavers. On average, spore numbers increased 460-fold, showing that Bacillus thuringiensis grows and replicates successfully in insect cadavers. By inoculating cadaver-derived spores and spores from bacterial stock cultures into nutrient medium, we next investigated outgrowth characteristics of vegetative cells and found that cadaver-derived bacteria showed reduced growth compared to bacteria from the stock cultures. Interestingly, this reduced growth was a consequence of inhibited spore germination, probably originating from the host and resulting in reduced host mortality in subsequent infections by cadaver-derived spores. Nevertheless, we further showed that Bacillus thuringiensis transmission was possible via larval cannibalism when no other food was offered. These results contribute to our understanding of the ecology of Bacillus thuringiensis as an insect pathogen.     

Inhibitory Effects of Turf Pesticides on Bacillus popilliae and the Prevalence of Milky Disease

Fourteen pesticides (fungicides, herbicides, and insecticides) were tested to determine whether they had deleterious effects on the bioinsecticide Bacillus popilliae, the causal agent of milky disease. All of these pesticides reduced levels of spore viability, spore germination, and/or vegetative cell growth when they were tested over a range of concentrations from 0 to 1,000 ppm of active ingredient, and the fungicides had the greatest detrimental effects. As determined by tests in water, the level of spore viability was significantly reduced by chlorothalonil, iprodione, (2,4-dichlorophenoxy)acetic acid plus 2-(2,4-dichlorophenoxy)propionic acid, and 2-[(4-chloro-o-tolyl)oxy]propionic acid plus (2,4-dichlorophenoxy)acetic acid. In tests performed with iprodione, loss of viability was evident at concentrations less than the concentration calculated to result from recommended use. Tests performed in soil demonstrated that triadimefon, chlorothalonil, (2,4-dichlorophenoxy)acetic acid plus 2-(2,4-dichlorophenoxy)propionic acid, and pendimethalin at concentrations resulting from recommended rates of application reduced spore titers. Spore germination did not occur in the continued presence of 2-[(4-chloro-otolyl)oxy]propionic acid plus (2,4-dichlorophenoxy)acetic acid, isofenphos, and chlordane, whereas exposure of spores to triadimefon or pendimethalin for 2 days stimulated germination. The tests to determine effects on spore germination were inconclusive for all other pesticides. Triadimefon, chlorothalonil, iprodione, pendimethalin, and chlorpyrifos at concentrations less than the concentrations recommended for use inhibited vegetative cell growth of B. popilliae, and chlordane at a concentration that was twice the concentration expected to result from the recommended rate of application repressed cell growth. My data support the hypothesis that use of synthetic pesticides can contribute to a low incidence of milky disease in white grubs.     

Evaluation of Bacillus thuringiensis as a biocide of blackfly larvae (Diptera: Simuliidae)

Thirteen strains of Bacillus thuringiensis were bioassayed against late-instar larvae of field-collected Simulium vittatum. All 13 strains caused significant blackfly mortality. The mortalities ranged from 64% for the HD 225 strain to 88% for HD 39 at 10 ppm for a 24-hr exposure period. A minimum 24 hr of exposure to a minimum concentration of 10 ppm was required to produce mortalities approximating 90%. The LC₅₀ values for the HD 39 and HD 225 strains were 1.1 and 1.0 ppm, respectively.     

Effect of weather on a microbial insecticide

A commercial formulation of Bacillus thuringiensis was exposed to the weather in the field at Fargo, North Dakota, during the summers of 1969 and 1970. A serial dilution technique was utilized in counting viable bacterial spores after exposure to the weather. Weather suppresses populations of B. thuringiensis in the field. The greatest effect occurred during the first 24 hr of exposure. Rain and solar radiation caused a significant reduction in the number of viable spores. Solar radiation had the greatest effect. The information has practical value when determining the time of application, the number of applications, and the amount of a microbial insecticide to use to control a pest.     

Sporulation and toxin production by Bacillus thuringiensis var. israelensis in cadavers of mosquito larvae (Diptera: Culicidae)

Laboratory experiments with 4th-instar larvae of Aedes aegypti and Anopheles albimanus (Diptera: Culicidae) demonstrated that the entomocidal bacterium, Bacillus thuringiensis var. israelensis, can grow vegetatively, sporulate, and produce toxin in cadavers of mosquito larvae. In A. aegypti, spore counts rose from 2 × 10²/cadaver 4 hr after treatment to 1.4 × 10⁵/cadaver approximately 72 hr later, whereas in A. albimanus spore counts per cadaver increased from 2.2 × 10³ between 4 and 24 hr to 3.2 × 10⁵ at 72 hr post-treatment. Bioassays of larval cadavers indicated that toxicity associated with sporulation of B. thuringiensis var. israelensis reached a maximum level approximately 72 hr after treatment. These results demonstrate that under appropriate conditions B. thuringiensis var. israelensis can use the substrates available in larval cadavers for growth and sporulation.     

Toxicity of Bacillus thuringiensis toward Aedes aegypti larvae.

Among six strains of Bacillus thuringiensis and five other species of Bacillus, only two strains of B. thuringiensis, strains HD-1 and BA-068, were toxic to Aedes aegypti larvae within 24 hr. The LC₅₀s were 5.6 × 10⁴ and 2.4 × 10⁵ spores/ml for strains HD-1 and BA-068, respectively. The toxic factor(s) was heat sensitive and γ ray resistant and preliminary evidences indicated that it was associated with the crystalline body of B. thuringiensis.     

Production and utilization of Bacillus thuringiensis

Biological insecticide formulations based on the entomogenous bacterium. Bacillus thuringiensis, have begun to be used widely in agriculture. This spore-forming bacillus can be grown in submerged culture and formulated to provide stable agricultural formulations compatible with aerial and ground application systems. The safety of these products to nontarget organisms and man is responsible for the growing interest in their use on a variety of crops including pastures and forests. No longer a laboratory curiosity, B. thuringiensis is an economic alternative to chemical insecticides.     

Recovery of Bacillus thuringiensis and other bacteria from larvae of Spodoptera littoralis previously fed B. thuringiensis-treated leaves

Exposure of a spore-crystal suspension of Bacillus thuringiensis to UV irradiation for (200 lx) 8.5 min killed most of the spores ($\text{P}\text{P}{}_0\text{= 2.6 × 10}{}^{\mathrm{?4}}$), while the insecticidal activity of the suspension to larvae of Spodoptera littoralis was only slightly affected. Numbers of colony-forming units (CFU) of B. thuringiensis recovered from larvae after ingestion of spores decreased with time as long as the larvae lived and several hours after larval death. Only 3–6 hr after larval death, the spores germinated and multiplied, reaching up to 100-fold after 24 hr. When UV-irradiated suspensions were used, numbers of CFU per larva were too scarce to be recovered from living larvae. However, 1.5 × 10⁶ CFU/larva were recovered 24 hr after death. It seems that the disruption of the gut epithelium by the endotoxin caused a change in the unfavorable conditions for endospore germination, thus providing the suitable ambient for germination and multiplication of B. thuringiensis. Numbers of other bacteria present per milligram of healthy larva increased with larval weight, predominantly Streptococcus sp. and Erwinia sp. In dead larvae, the increase of Erwinia sp. was higher than that of Streptococcus sp. Other bacterial species isolated were: Corynebacterium sp., Micrococcus sp., Serratia marcescens, and Bacillus sp.     

Growth ofDiacrisia obliqua [Lep.: Arctiidae] with low doses ofBacillus thuringiensis var.Kurstaki

Early 3rd instarDiacrisia obliqua Walk. larvae were treated with concentrations ofBacillus thuringiensis var.kurstaki (Dipel^®) and the growth of treated larvae was assessed. All the doses reduced significantly the weight and survival of the insects (p<0.001).     

Response of adult parasitoids ofBemisia tabaci (Hom.: Aleyrodidae) to leaf residues of selected cotton insecticides

The contact toxicity of eight insecticides to adults of four parasitoids of the sweetpotato whiteflyBemisia tabaci was evaluated in the laboratory. Two common Texas species,Eretmocerus sp. andEncarsia pergandiella Howard, and two exotic species,Eretmocerus mundus Mercet from Spain andEncarsia formosa Gahan from Greece were tested. Insecticides, applied as sprays to greenhouse-grown cotton plants at recommended rates were amitraz (Ovasyn^®), azinphosmethyl (Guthion^®), bifenthrin (Capture^®), buprofezin (Applaud^®), cypermethrin (Ammo^®), methyl parathion and thiodicarb (Larvin^®), with a water control. Parasitoid adults were confined on discs cut from leaves (1) sprayed the same day and (2) sprayed two days previously. Survival in both treatments was measured two and four days following exposure. Significant differences in toxicity were detected among the insecticides. Buprofezin was not toxic to any of the four parasitoids. When caged on leaves sprayed two days previously, only amitraz of the remaining compounds allowed significant general parasitoid survival after two days.E. mundus exhibited the greatest overall tolerance to insecticides, with 40% or more surviving 48 hr after confinement on leaves sprayed with amitraz, thiodicarb and cypermethrin. Survival was generally much reduced after 96 hr. In a separate test, fresh residues of endosulfan (Thiodan^®) were highly toxic at the two rates tested, but two day old residues at the lower rate allowed 76.7% survival ofE. mundus and 35% survival ofE. pergandiella after 48 hrs.     

Effect of insecticides on the mortalities of three whitefly parasitoid species, Eretmocerusmundus, Eretmoceruseremicus and Encarsiaformosa (Hymenoptera: Aphelinidae)

The toxicities of 24 insecticides for the biological control of whiteflies were evaluated for Eretmocerus mundus (Mercet), Eretmocerus eremicus Rose and Zolnerowich and Encarsia formosa Gahan using the residual film method (for adults) and the dipping method (for pupae). Mortalities from insect growth regulators (IGRs) (flufenoxuron and lufenuron), Bacillus thuringiensis (Bt), pymetrozine and sulfur were <30% for both pupae and adults of all three species, indicating that the parasitoids were not seriously affected by these insecticides. Neonicotinoids (acetamiprid, clothianidin, dinotefuran, imidacloprid and nitenpyram), synthetic pyrethroids (etofenprox and permethrin), organophosphates (acephate and fenitrothion), chlorphenapyr, emamectin benzoate, spinosad and tolfenpyrad were seriously harmful (100% mortality) and acaricides (chinomethionat, milbemectin and pyridaben) were moderately harmful or seriously harmful to adult parasitoids (leading to mortalities of >92%). For each insecticide, the mortality of pupae was generally lower than that of adults, even though the toxicity classification for the two groups was similar. The results indicate that IGRs, Bt, pymetrozine and sulfur are relatively harmless, and are compatible with the use of parasitoids to help control whiteflies for integrated pest management in greenhouses.     

Characterization of Bacillus thuringiensis isolated from soils in the Jazan region of Saudi Arabia,and their efficacy against Spodoptera littoralis and Aedes aegypti larvae

Pest control in Saudi Arabia depends on applying chemical insecticides, which have many undesirable considerations and impacts on the environment. Therefore, the aim of this study was to isolate Bacillus thuringiensis from different rhizosphere soil samples in the Jazan region for the biological control of Spodoptera littoralis and Aedes aegypti larvae. The samples were collected from the rhizosphere of different plants located in eight agricultural areas in Jazan, Saudi Arabia. Out of 100 bacterial isolates, four bacterial isolates belonging to Bacillus species were selected namely JZ1, JZ2, JZ3, and JZ4, and identified using classical bacteriological and molecular identification using 16S rRNA. JZ1 and JZ2 isolates were identified as Bacillus thuringiensis. SDS-PAGE analysis and the detection of the Cry1 gene were used to describe the two isolates JZ1 and JZ2 in comparison to Bacillus thuringiensis reference strain Kurstaki HD1 (BTSK) were revealed that slightly different from each other due to the place of their isolation and namely Khlab JZ1 and Ayash JZ2. The EC₅₀ of JZ1 and JZ2 isolates, BTSK, and the commercial biopesticide DiPEL 6.4 DF against the second-instar larvae of Aedes aegypti were 207, 932, 400, and 500 ppm respectively, while EC₅₀ against first-instar larvae of Spodoptera littoralis were 193.93, 589.7, 265.108, and 342.9, ppm respectively. Isolate JZ1 recorded the highest mortality while JZ2 isolate gave the lowest mortality. It can be concluded that the local isolate of JZ1 and JZ2 can be developed for bio formulations to be used in Spodoptera littoralis and Aedes aegypti biological control programs.     

Distribution of 2-kb miniplasmid pBMB2062 from Bacillus thuringiensis kurstaki YBT-1520 strain in Bacillus species

A multi-copy and small plasmid pBMB2062 from Bacillus thuringiensis kurstaki YBT-1520 strain was cloned and characterized and its distribution was analyzed using dot-blot analysis with the ORF1 fragment as a probe. Bacillus species of 84 serotypes were evaluated. The pBMB2062 plasmid was found to be present in commercial B. thuringiensis kurstaki (H3abc) and aizawai (H7) insecticides of various serotypes, and one Bacillus cereus UW85 strain (produced Zwittermicin fungicide and Cry toxin synergist). The sequences of 7 pBMB2062-like plasmids from randomly selected Bacillus species (positive signal in the dot-blot analysis) were highly conserved. Two open reading frames (ORFs), ORF1 and ORF2, were present in this plasmid. ORF1 was found to be necessary for plasmid replication, whereas ORF2 did not play a role in replication or stability. Based on its sequence homology, ORF2 was a putative solitary antitoxin. Furthermore, the copy number of the replicon of pBMB2062 was higher than those of ori1030 and ori44 based on the thermogenic data, and ori2062 could drive the stable replication of a recombinant plasmid (11 kb total size) in B. thuringiensis.