Meir Broza

Cloning and expression of the lepidopteran toxin produced by Bacillus thuringiensis var. thuringiensis in Escherichia coli

The Bacillus thuringiensis var. thuringiensis strain 3A produces a proteinaceous parasporal crystal toxic to larvae of a variety of lepidopteran pests including Spodoptera littoralis (Egyptian cotton leaf worm), Heliothis zeae, H. virescens and Boarmia selenaria. By cloning of individual plasmids of B. thuringiensis in Escherichia coli, we localized a gene coding for the delta-endotoxin on the B. thuringiensis plasmid of about 17 kb designated pTN4. Following partial digestion of the B. thuringiensis plasmid pTN4 and cloning into the E. coli pACYC184 plasmid three clones were isolated in which toxin production was detected. One of these hybrid plasmids pTNG43 carried a 1.7-kb insert that hybridized to the 14-kb BamHI DNA fragments of B. thuringiensis var. thuringiensis strains 3A and berliner 1715. This BamHI DNA fragment of strain berliner 1715 has been shown to contain the gene that codes for the toxic protein of the crystal (Klier et al., 1982). No homologous sequences have been found between pTNG33 and the DNA of B. thuringiensis var. entomocidus strain 24, which exhibited insecticidal activity against S. littoralis similar to that of strain 3A.

A new sensitive method for determining the toxicity of a highly purified fraction from δ-endotoxin produced by Bacillus thuringiensis var. entomocidus on isolated larval midgut of Spodoptera littoralis (Lepidoptera, Noctuidae)

Abstract The purification procedure of the toxic fraction from the δ-endotoxin produced by Bacillus thuringiensis var. entomocidus was considerably shortened and simplified by combining the solubilization in a high p H (10.0) solution with glycine, opening SS bonds with dithiothreitol, and releasing hydrophobic connections with the detergents Triton N-101 and sodium cholate. The subsequent fractionation was immediately continued (without dialysis or concentration) on a Sepharose 6B column, yielding the active fraction designated C. Fraction C was then passed through an octyl Sepharose 4B column, yielding the active fraction designated I, followed by gel filtration on a Sepharose 6B column yielding the active fraction designated CI. All treatments were done with the same buffer solution at 4°C. Column elution was with the same buffer but with 0.075% detergents. The purity of the fraction CI was apparent by its appearance as a well-defined band on polyacrylamide gel isoelectric focusing at p H 6.1, accompanied by a small faint band. A sensitive method for evaluating the toxicity of endotoxin fractions on an isolated midgut system (from larvae of Spodoptera littoralis ) was developed. The system is based on measuring the activity of the enzyme, reduced glutathione S -transferase, released to the medium from epithelial cells ruptured by the toxin. Fraction CI, M r 64,000, was toxic to the epithelial cells of the isolated midgut in the absence of the peritrophic membrane. When the detergents were removed by dialysis, the active protein formed high molecular weight aggregates due to hydrophobic interactions. The passage of those aggregates to the sensitive epithelial cells of the midgut was prevented by the peritrophic membrane.

Evaluation of the effectiveness ofBacillus thuringiensis var.entomocidus as a pest control agent to replace chemical pesticides in alfalfa fields in Israel

The Egyptian cotton leafworm,Spodoptera littoralis Boisd., is the main important pest of alfalfa in Israel. Population levels of other pests usually do not reach the economic threshold. Nine chemical insecticide applications (7 spray applications and 2 granular fluorosilicate baits) were required to controlS. littoralis during the season of 1984 (in which infestation level was high) whereas only 5 applications of aBacillus thuringiensis var.entomocidus (Bte) preparation (3 sprays and 2 baits) produced the same effect. During 1983 and 1985 pest populations were moderate and only 6 chemical pesticide applications were required. A bait ofBte on wheat bran, Coax®, developed in the present study was effective in the control of large larvae (instars 4–6). Hay yields of fields treated withBte were similar to the yields of a conventionally treated field. The best control of large larvae was obtained by applying the bait preparation under the wind row during the alfalfa cutting.ZusammenfassungDie Baumwolleule,Spodoptera littoralis Boisd., ist der wichtigste Alfalfa-Schädling in Israel. Neun Anwendungen chemischer Insektizide (7 flüssig und 2 als Granulatköder) während der Saison 1984 (in welcher die Dichte des Schädlings sehr hoch war) zeigten dieselbe Wirkung wie nur 5 Anwendungen vonBac. thuringiensis var.entomocidus (3 flüssig und 2 Köder). In den Jahren 1983 und 1985 wechselte die Schädlingsdichte, und es waren nur 6 Anwendungen chemischer Präparate notwendig. EinBacillus-Köder auf Kleie-Basis, Coax®, der hierfür entwickelt wurde, erwies sich als wirksames Bekämpfungsmittel gegen ältere Raupen (L4–L6). Die Heu-Ernte von mitBacillus behandelten Feldern entsprach jener von konventionell behandelten. Der beste Bekämpfungseffekt gegen große Raupen ergab sich bei Anwendung derBacillus-Köder in der Lee-(unter dem Wind-) Reihe während des Alfalfa-Schnitts.

Seasonal changes in population of Heliothis armigera (Hb.) (Lepidoptera; Noctuidae) in cotton fields in Israel and its control with a Bacillus thuringiensis preparation

Heliothis armigera used to be an early‐season cotton pest in Israel, mainly in the Mediterranean Coastal Plain. Since 1984 this pest has expanded eastward to the Jordan Rift Valley and became a major pest throughout the season. In field trials Bacillus thuringiensis var. kurstaki (B. t.) was used as a pest control initially during the early part of the season in the Coastal Plain, larval mortality was somewhat lower, but very close to that compared to the chemically (Thionex) treated plots. It was concluded that in the early season B. t. may be a useful control agent of H. armigera with the advantage of preserving the populations of natural enemies. Field experiments were carried out to control an exceptionally high population (more than 32 larvae/2 m row) in the Jordan Rift Valley toward the end of the season. In both fields treated either with chemical or with B. t., survival of larvae was high (4,7–8 larvae/2 m). The feasibility of B. t. application was in doubt for the late part of the season, since during this period heavy chemical‐insecticides are applied against a number of important pests, which populate the cotton fields.