K. van Frankenhuyzen

An in vitro system for testing Bacillus thuringiensis toxins: the lawn assay.

A cell assay system was developed that allows Bacillus thuringiensis delta-endotoxins activated at high pH (10.5) to be tested in vitro without causing alkaline injury to target cells. The assay is carried out on a lawn of gel-suspended cells, requires only 1 microliter of sample per dose, and is quantitative, rapid, and sensitive. The threshold dose for toxicity of B. thuringiensis subsp. kurstaki HD-73 with IPRI-CF-1 cells was 24 pg protein. The assay is also very useful for identifying antibodies which inhibit toxicity and for detecting beta-exotoxin.

Suppression of Bacillus thuringiensis δ-endotoxin activity by low alkaline pH

Abstract Reducing the alkaline pH (from 10.5 to 8) of enzyme-activated δ-endotoxin preparations from Bacillus thuringiensis subsp. kurstaki suppressed toxicity. The effect was reversible and readily observed in lawn assays against IPRICF-1 cells (a system in which alkaline injury to cells was prevented). Suppression of toxicity was also observed, though to a lesser extent, in force-feed assays against Bombyx mori. Doses in both types of bioassay were based on measurements of the total dissolved protein in activated toxin preparations. Inactivation caused by reducing the pH occurred in the presence of a chaotropic agent and was not affected by ultracentrifugation of toxin samples, indicating that molecular aggregation and precipitation of protein were probably not the immediate cause. A computer analysis of toxin proteins indicated that the effect might be related to the protonation of tyrosine residues when the pH is reduced or possibly to an overall change in molecular charge caused mostly by the protonation of these residues. The pH effect suggests that the highly alkaline environment of the lepidopteran midgut is important not only for solubilization and digestion of B. thuringiensis crystal protein, but for full expression of toxin activity as well.

Control of spruce budworm (Lepidoptera: Tortricidae) with Bacillus thuringiensis applications timed to conserve a larval parasitoid

Abstract Field trials conducted in 1990 and 1991 demonstrated that commercial formulations of Bacillus thuringiensis var. kurstaki Berliner applied late to fifth and sixth instar spruce budworm Choristoneura fumiferana (Clemens) expressly to avoid affecting the larval parasitoid Apanteles fumiferanae Viereck were as efficacious in controlling spruce budworm populations as a regularly timed spray that was targeted against third and fourth instars. In 1990, with high budworm populations (50 to 70 larvae per 45 cm branch), the corrected budworm population reduction after the early spray (34.6%) was less but not significantly different (p > 0.05) from that in the late treatment (55.3%). Defoliation of balsam fir Abies balsamea (L.) Miller in the early and late spray treatments was also not significantly different [87.5 ± 8.8% and 70.3 ± 12.8% ( x ± s.d. ), respectively]from that in the control plots (87.1 ± 9.2%), but was considered unacceptably high. In 1991, with moderately high budworm populations (16–45 larvae per branch) the corrected budworm population reductions resulting from the early and late treatments (76.7 and 92.8%, respectively) were not significantly different (p > 0.05). The percentage defoliation in the late treatment (28.6 ± 12.3%) was significantly higher statistically than that in the early treatment (18.7 ± 7.6%) but was biologically acceptable. Both levels were significantly less than the defoliation observed in the check plots (42.2 ± 14.8%), which was unacceptably high. These results suggest that commercial formulations of Bt applied undiluted to moderately high budworm populations at 30 billion (109) international units (BIU) ha−1 can be judiciously delayed to conserve the survival of A. fumiferanae and still be efficacious.

Use of Entomopathogens Against Forest Pests

Abstract Entomopathogens have been used extensively to control insect pests in forests with successful use of both augmentation and classical biological control strategies to apply or introduce bacteria, baculoviruses, fungi, and nematodes. In particular, Bacillus thuringiensis var. kurstaki has been used to effectively control numerous defoliators in forests. Baculoviruses provide excellent examples of successful classical biological control and augmentative introductions, also for the control of defoliators. The baculovirus infecting gypsy moth, Lymantria dispar, causes naturally occurring epizootics in defoliating host populations but in North America has largely been replaced since the introduction of the fungus Entomophaga maimaiga. Finally, the nematode Deladenus siricidicola has been used to control the invasive wood borer Sirex noctilio. With increasing numbers of invasive species, entomopathogens are also used as part of eradication programs or programs to slow invasive spread. All examples provided in this chapter outline specifically how achievements in the use of entomopathogens for pest control are contributing to the sustainable protection of forests around the world. Future challenges lie in store, but forest ecosystems, usually biodiverse areas that have relatively high economic or aesthetic injury levels (ie, some level of damage from pests can be tolerated), are excellent systems for effective control using entomopathogens.