Dosage-mortality studies with commercial Bacillus thuringiensis sprayed in a modified Potter's tower against some forest insects

Dosage-mortality tests were carried out with commercial Bacillus thuringiensis (B.T.) (Dipel)^® against various instars of the spruce budworm, Choristoneura fumiferana, the white-marked and Douglas fir tussock moths, Hemerocampa leucostigmata and Orgyia pseudotsugata, and the gypsy moth, Porthetria dispar. Dipel was applied as a dilute (10^?2) molasses suspension onto artificial diet surface in a spray tower designed to simulate aerial application. Probit analysis of the results showed that LD₅₀s expressed both in terms of gallons deposited per acre and as spores and crystals deposited per cm² increased with larval age for all species. The spruce budworm was the most sensitive to the bacteria, followed in decreasing order of sensitivity by the white-marked tussock moth, Douglas fir tussock moth, and the gypsy moth. The mean slopes for all instars of the four species were 1.6, 3.1, 2.6, and 2.2, respectively, indicating that precision-wise, the assay of B.T. on artificial medium was good. The relatively low slope for spruce budworm is explained by its peculiar feeding habit. Among all species tested, bacteria-treated larvae gained weight at a considerably reduced rate compared with untreated ones. Reduction in weight resulting from lowered feeding activity intensified as dosage rates increased. The implication of this in terms of mortality assessments in microbial control operations is discussed.It is suggested that 0.02 gallon (4 × 10⁶ International Units) of Dipel Molasses deposited per acre may achieve economic control of fourth- to sixth-instar budworm and first-to second-instar gypsy moths. A deposit rate for second- to fifth-instar white-marked or Douglas fir tussock moths appears to be in the vicinity of 0.01 gallon (2 × 10⁶ IU) per acre.     

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.     

Assessment ofTrichogramma species for biological control of forest lepidopteran defoliators

In a laboratory study, we determined the potential of threeTrichogramma (Hymenoptera: Trichogrammatidae) species,T. brassicae Bezdenko,T. minutum Riley andT. nr.sibiricum Sorokina, for biological control against six species of forest lepidopteran pests, black army cutworm, hemlock looper, eastern spruce budworm, western spruce budworm, white-marked tussock moth, and gypsy moth. Females of each parasitoid species were offered eggs from each of the six host species. Parasitization and the effect of the host species on the emerging progeny were examined and recorded.Trichogramma minutum had the broadest host range and successfully parasitized four host species out of the six offered.Trichogramma nr.sibiricum had the narrowest host range and parasitized only two species of hosts. Of the six host species, black army cutworm was the most preferred by all threeTrichogramma species; white-marked tussock moth and gypsy moth were not parasitized by any parasitoids. There was a positive correlation between the size of female offspring and their corresponding egg complement in all three parasitoid species. The developmental time of parasitoids from egg to adult was influenced by both the parasitoid and host species. Our results suggest thatT. minutum has the greatest potential for biological control against various forest lepidopteran pests and that the black army cutworm may be the best target candidate for further study.     

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.     

The delta-endotoxin of Bacillus thuringiensis. V. On the occurrence of endotoxin fragments in hemolymph

Leucine-³H labeled crystals of Bacillus thuringiensis δ-endotoxin were fed to last-instar larvae of spruce budworm, Choristoneura fumiferana, eastern forest tent caterpillar, Malacosoma disstria, and silkworm, Bombyx mori. Radioactivity was detected in hemolymph 1 min after feeding in the first two species, but not until 3–5 min after feeding in silkworm larvae. Most of the radioactivity from hemolymph of all three species eluted from gel filtration columns at the same elution volume indicating similar moleculear weights (<1800 daltons).     

Toxicity of Bacillus thuringiensis entomocidal protein toward cultured insect tissue

The entomocidal protein from crystalline inclusion bodies of Bacillus thuringiensis can be bioassayed in vitro using cultured insect tissue. Larval cells of the spruce budworm, Choristoneura fumiferana, are damaged by enzyme-digested (activated) protein isolated from B. thuringiensis crystals. Measurement of toxicity is accomplished by detection of adenosine triphosphate (ATP) in treated cultures using firefly bioluminescence. The ATP content of toxin-treated tissue is inversely proportional to the amount of toxin added. Tissue cells from the spruce budworm exhibited maximum susceptibility to activated δ-endotoxin after 120 hr incubation. Probit analysis of tissue ATP response to toxin dose indicated 50% of the cells were damaged by 14.6 μg or less of toxin protein per 2 × 10⁵ insect tissue cells. Activated δ-endotoxin was entomocidal to insects as well, as detemined by mortality studies with second-instar larvae of the European corn borer. Electron microscopic observations of insect tissue treated with activated δ-endotoxin protein for 60 min revealed massive outer membrane disruption and subsequent loss of cytoplasmic constituents, accompanied by swelling of the nuclear membrane.     

Interactions of Bacillus thuringiensis bioinsecticides and the predatory stink bug Podisus nigrispinus to control Plutella xylostella

The diamondback moth (DBM), Plutella xylostella (L.), is a major pest of brassica crops worldwide. Control of this pest is difficult because it rapidly develops resistance to synthetic and biological insecticides and because of the effects of insecticides on its natural enemies. Podisus nigrispinus (Dallas) is a predator that feeds on its prey, as well as on the host plants of its prey, and is an important biological control agent of DBMs. The aim of this study was to determine the susceptibility of P. xylostella larvae to two bioinsecticides: the HD1 strain of Bacillus thuringiensis (B. thuringiensis var. kurstaki) and the commercial product Agree^® (B. thuringiensis var. aizawai CG 91). In addition, the impact of these bioinsecticides on the P. nigrispinus consumption of DBM larvae and phytophagy was evaluated. Both the HD1 strain and Agree^® caused 100% mortality in P. xylostella larvae. P. nigrispinus nymphs fed only with kale leaves (Brassica oleracea var. acephala) sprayed with water, the HD1 strain, or Agree^® did not complete their nymphal development. When prey was also available, P. nigiripinus fed on kale leaves to obtain water. Both nymphs and adults of P. nigrispinus consumed greater numbers of DBM larvae, and fed less on kale leaves, when sprayed with the HD1 strain or Agree^®. These results suggest a positive interaction of B. thuringiensis‐based products and the predator P. nigrispinus in the control of P. xylostella larvae.     

Binding of individual Bacillus thuringiensis Cry proteins to the olive moth Prays oleae (Lepidoptera: Yponomeutidae)

The microlepidopteran Prays oleae is one of the main insect pests causing significant crop losses in the Mediterranean olive groves. Bacillus thuringiensis based insecticides are being successfully used to minimize the impact of the second and third generations of this pest. However, because of its very small size and difficulty of rearing, very few studies have been carried out to determine the potency and mode of action of B. thuringiensis Cry proteins in this insect. In this study, Cry1Ac, Cry1Ca, and Cry1Fa proteins were shown to be toxic to third instar larvae of P. oleae. Furthermore, binding assays with ¹²⁵I-Cry1Ac and brush border membrane vesicles from midguts of last-instar larvae showed specific binding sites for Cry1Ac that are shared, with low affinity, by Cry1Ca and Cry1Fa.     

Effects of bud phenology and foliage chemistry of balsam fir and white spruce trees on the efficacy of Bacillus thuringiensis against the spruce budworm, Choristoneura fumiferana

Abstract 1 Efficacy of commercial formulations of Bacillus thuringiensis ssp. kurstaki (Btk) against spruce budworm Choristoneura fumiferana was investigated in mixed balsam fir‐white spruce stands. Btk treatments were scheduled to coincide with early flaring of balsam fir shoots, and later with flaring of white spruce shoots. Btk efficacy on the two host trees was compared and examined according to the foliar content of nutrients and allelochemicals and the insect developmental stage at the time of spray. 2 Larvae fed white spruce foliage were less vulnerable to Btk ingestion than larvae fed balsam fir foliage. Higher larval survival on white spruce, observed 10 days after spray, was related to higher foliage content in tannins and a lower N/tannins ratio, which might have induced inactivation of Btk toxins. 3 Larval mortality due to Btk did not depend on spruce budworm larval age. 4 Foliage protection of both host trees was similar in plots treated with Btk: larval mortality due to Btk treatment reduced insect grazing pressure on balsam fir trees; meanwhile, suitability of white spruce foliage seemed to decrease very rapidly, which induced high larval mortality among spruce budworm fed on white spruce trees. Nevertheless, following Btk sprays, 50% more foliage remained on white spruce than on balsam fir trees, because of the higher white spruce foliage production. 5 Both spray timings achieved similar protection of white spruce trees, but Btk treatments had to be applied as early as possible (i.e. during the flaring of balsam fir shoots to optimally protect balsam fir trees in mixed balsam fir‐white spruce stands).     

Effects of tree age and stand thinning related variations in balsam fir secondary compounds on spruce budworm Choristoneura fumiferana development,growth and food utilization

• 1 The effect of tannins and monoterpenes on the development, mortality and food utilization of spruce budworm Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae) was investigated under laboratory conditions using an artificial diet. Tannins were extracted from balsam fir foliage of thinned and unthinned stands to reproduce stand thinning related variations in tannins. A mixture of synthetic monoterpenes was utilized to simulate the concentration found in young and old balsam fir trees.
• 2 Longer development time and lower pupal weight were observed for insects fed on diets with a lower nitrogen concentration and a higher tannin concentration (unthinned treatment). Tannins induced higher insect mortality at a low nitrogen concentration compared with the diet with a higher nitrogen concentration.
• 3 Approximate digestibility was higher for larvae fed on diets with high concentrations of nitrogen at both low and high concentrations of tannins. Efficiency of conversion of digested food (ECD) decreased with an increase in tannin concentration. Tannins reduced both the relative consumption and growth rate (RCR and RGR).
• 4 Monoterpenes increased spruce budworm mortality and this mortality reached almost 50% under concentrations of monoterpene typical of the young trees compared with 20% under monoterpene concentrations found in old trees.
• 5 A higher digestibility was observed for larvae fed on diet with a higher concentration of monoterpenes, whereas efficiency of conversion of ingested food (ECI), ECD, RCR, and RGR decreased with an increase in monoterpenes in the diet.
• 6 The results obtained in the present study are consistent with the defensive role of secondary compounds such as tannins and monoterpenes in the spruce budworm–balsam fir system.

     

A pathogenic strain of Bacillus cereus isolated from the cigarette beetle, Lasioderma serricorne

A noncrystalliferous, aerobic, spore-forming bacterium (accession number DD-1019) isolated from the cigarette beetle, Lasioderma serricorne, was identified as a strain of Bacillus cereus based on morphological, biochemical, and cultural similarities. Pathogenicity was established by exposing hatching larvae of the cigarette beetle to doses of inocula ranging from 1.17 to 600 × 10⁶ spores per gram of rearing medium. The LD₅₀ and the LD₉₀ were calculated to be 4.29 × 10⁶ and 371 × 10⁶ spores per gram of medium, respectively. The cigarette beetle was effectively controlled by both the DD-1019 strain of B. cereus and the CM1-1 strain (originally isolated from and pathogenic to codling moth, Laspeyresia pomonella) but proved quite refractory to Bacillus thuringiensis var. thuringiensis.     

Some Effects of Douglas Fir Terpenes on Certain Microorganisms

The Douglas fir terpene α-pinene was shown to inhibit the growth of a variety of bacteria and a yeast. Other terpenes of the Douglas fir, including limonene, camphene, and isobornyl acetate, were also inhibitory to Bacillus thuringiensis. All terpenes were inhibitory at concentrations normally present in the fir needle diet of Douglas fir tussock moth larvae. The presence of such terpenes in the diet of these insects was found to strongly influence the infectivity of B. thuringiensis spores for the Douglas fir tussock moth larvae. The terpene α-pinene destroyed the cellular integrity and modified mitochondrial activity in certain microorganisms.     

The susceptibility of the pea moth, Cydia nigricana, to infection by the granulosis virus of the codling moth, Cydia pomonella

Laboratory studies demonstrated that neonate larvae of the pea moth, Cydia nigricana, are susceptible to infection with a granulosis virus (CpGV) isolated from the codling moth, Cydia pomonella. Comparative LC₅₀ values for C. nigricana and C. pomonella are 1.90 × 10⁵ and 1.54 × 10⁴ capsules/ml of diet, respectively. The virus extracted from CpGV-infected pea moth larvae is serologically related, and probably identical, to CpGV.     

Insecticidal activity of spore-free mutants of Bacillus thuringiensis against the Indian meal moth and almond moth

Three oligosporogenic mutants of Bacillus thuringiensis were assayed for toxicity against larvae of the Indian meal moth, Plodia interpunctella, and the almond moth, Ephestia cautella. The results were compared with insecticidal activity obtained from the parent strain (HD-1) and two standard B. thuringiensis formulations (HD-1-S-1971 and HD-1-S-1980) against the same insect species. The toxicity of the sporeless mutant preparations was significantly diminished against the Indian meal moth (10- to 26-fold increase in LC₅₀) but exceeded the toxicity of the standards against the almond moth. The toxicities of the B. thuringiensis preparations toward the Indian meal moth were consistent with the number of spores in the test samples, but spores did not contribute to toxicity to E. cautella larvae. A rationale for basing dosage on soluble protein was demonstrated for use in situations where spores are not a contributing factor in toxicity.     

Histopathological effects of Bacillus thuringiensis on the midgut of tobacco hornworm larvae (Manduca sexta): Low doses compared with fasting

The cytology and ultrastructure of the midgut cells of Manduca sexta larvae are described for untreated controls, larvae which fed on a spore preparation of Bacillus thuringiensis, and larvae which were fasted for either 24 or 48 hr. New observations on the ultrastructure of midgut cells in Manduca larvae included the finding of specialized Golgi vesicles in anteriormost columnar cells and of regular arrays of expanded rough endoplasmic reticulum in goblet cells of the posterior midgut region. The present observations reveal that the columnar cells of the midgut responded cytologically in the same way to fasting as they did to exposure to the toxic spores of B. thuringiensis. The goblet cells, however, appeared unaffected by fasting but became swollen in response to feeding of B. thuringiensis spore preparation.     

Cellular immune responses of spruce budworm larvae to Entomophthora egressa protoplasts and other test particles

The protoplast stage of two isolates of Entomophthora egressa developed normally and eventually produced conidiophores when injected into larvae of the spruce budworm, Choristoneura fumiferana. The spruce budworm hemocytes never made long-term contact with the protoplasts either in vivo or in vitro. The protoplasts made active, short-term contact with spruce budworm granulocytes both in vivo and in vitro. Total larval hemocyte counts (THC) initially declined when larvae were injected with protoplasts, growth medium (MGM), or Escherichia coli. The recovery rate to THC control levels was similar for MGM and protoplasts and supports the concept of nonrecognition of protoplasts by the hemocytes. The granulocytes were important in both nodulation and phagocytosis of E. coli and Bacillus cereus, whereas the plasmatocytes were important in phagocytosis. In in vitro studies, spruce budworm granulocytes did not adhere to rod-shaped hyphal bodies, spherical hyphal bodies, or germinating spherical hyphal bodies of E. egressa, whereas the granulocytes readily encapsulated the hyphae. There was no evidence for the production by the protoplasts of metabolites which might interfere with hemocyte adhesion. When protoplasts contacted Tenebrio molitor granulocytes, the protoplasts reacted by increasing the number of protoplasmic extensions and by granule discharge. The process of granule discharge may be an active protoplast defense mechanism. The sporangiospores of Absidia repens and Rhizopus nigricans adhered to spruce budworm granulocytes; however, the number of A. repens spores per granulocyte and the level of granulocytes with spores decreased in the presence of phenylthiourea. The adhesion of A. repens spores to granulocytes was enhanced by N-acetylglucosamine, whereas glucosamine, sucrose, fucose, fructose, arabinose, and galactose either had no effect on or reduced spore adhesion. Thus, the chitin (or its subunits) in the hyphal wall may initiate the granulocyte response.     

Spruce budworm growth,development and food utilization on young and old balsam fir trees

Laboratory rearing of spruce budworm, Choristoneura fumiferana, in conjunction with field rearing, gravimetric analyses, a transfer experiment, and foliage chemical analyses at six dates during the period of budworm feeding activity indicated that the age of balsam fir, Abies balsamea, trees (70-year-old mature trees or 30-year-old juvenile trees) affected tree suitability for the spruce budworm via the chemical profile of the foliage. Insects reared on old trees had greater survival and pupal weight, shorter development times, and caused more defoliation than those reared on young trees. Young trees were more suitable for the development of young larvae (instars 2–5), while old trees were more suitable for the development of older, sixth-instar larvae. These results were confirmed by the laboratory transfer experiment. Young larvae fed foliage from young trees had higher relative growth rates (RGR), digestibility (AD), and efficiency of conversion of ingested foliage (ECI) than those fed foliage from old trees. These differences appeared to be related to the high N:tannins ratio, and the high contents of P present in young trees during the development of the young larvae. Old larvae fed foliage from old trees had higher relative growth rates, relative consumption rates (RCR), and digestibility of the foliage than those fed foliage from young trees. The high digestibility of the foliage of old trees was compensated for by a lower efficiency of conversion of digested food (ECD), which in turn resulted in no significant effect of tree age on the efficiency of conversion of ingested foliage by old larvae. The low relative consumption rate of old larvae fed foliage from young trees appeared to be related to the low N:tannins ratio, and the high contents of bornyl acetate, terpinolene, and °-3-carene present in young trees during the budworm sixth instar. Variations in these compounds in relation to tree age may serve as mechanisms of balsam fir resistance to spruce budworm by reducing the feeding rate of sixth instar larvae.     

A bioassay technique for Entomophthora sphaerosperma on the spruce budworm,Choristoneura fumiferana

A technique for conducting bioassays of Entomophthora sphaerosperma on sixth-instar larvae of the spruce budworm, Choristoneura fumiferana, was developed. Four assays were conducted by showering conidia on 10 larvae for each of 10 to 20 doses per assay. Dose was estimated by averaging estimates of the concentration of spores falling on water agar dishes before and after insect exposure. Maximum-likelihood probit analysis indicated significant regressions between log dose and probit mortality for all four assays. LC₅₀ values ranged from 11.21 to 18.77 spores/mm² with a weighted mean of 16.13 spores/mm². Slope estimates ranged from 0.92 to 1.87 with a weighted mean of 1.13. These low slope values may have been indicative of a highly variable test insect population, but also suggested a nontoxic infection process by the pathogen.     

Comparison of energy budgets for spruce budworm Choristoneura fumiferana (Clemens) on balsam fir and white spruce

Summary A determination was made of the differences in the utilization of energy by laboratory reared larval Choristoneura fumiferana fed either balsam fir or white spruce foliage. This enabled us to quantitatively measure the quality of these foliages vis a vis the spruce budworm.The larval strategy was to feed rapidly and develop quickly. Development time was longer on spruce than on fir. Total consumption was virtually identical on both foliage types though production was ca 20% greater on a white spruce diet. Calories/gram increased with insect development and with the development of balsam fir foliage but declined over time in white spruce. Assimilation efficiencies (A/C) were 33.9% to 40.2% while gross production efficiencies (P/C) were 9.5% to 13.3% and net production efficiencies (P/A) were 26.1% to 38.8%. The spruce fed insects gave higher values than those on fir, but all values were at the low end of the range for lepidopterous defoliators (possibly due to characteristics of conifer foliage).The enhanced growth found in spruce fed larvae contrasts the relationship between balsam fir and high insect densities. This is discussed together with the influence of spruce budworm on forest composition and the possible competitive benefits of spruce budworm induced tree mortality.Parts of this paper appeared in a Master's thesis (Koller 1978) submitted to the University of Maine     

Potential of Margosan-O, an azadirachtin-containing formulation from neem seed extract, as a control agent for spruce budworm, Choristoneura fumiferana

The effects on spruce budworm larvae, Choristoneura fumiferana (Clem.), produced by ingestion of Margosan-O, a commercially available neem seed extract formulation containing 0.3% azadirachtin, were investigated. Bioassays with the test material were conducted using various instars of spruce budworm larvae, with either artificial diet, cut branches of balsam fir, Abies balsamea (L.) Mill., or small growing balsam fir trees as substrates. The dose-response data on feeding reduction, developmental retardation, and mortality (LC50, LC95, and LD50) suggest that Margosan-O has promise as a control agent for spruce budworm in an integrated pest management program.