Development and field performance of a broad-spectrum nonviable asporogenic recombinant strain of Bacillus thuringiensis with greater potency and UV resistance

Abstract

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 delta-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.

FIG. 1

Construction of plasmids pHTBS-F3-1C/Ab, pHTF3-1C/Ab-IRS-K, and pHTF3-1C/Ab-IRS-T. pHTBS-F3-1C/Ab was constructed as follows: the 2.3-kbp BglII-EcoRI DNA fragment of pHT81, which contains the 3′ end and downstream adjacent regions of the cry1C/Ab chimeric gene, was purified and ligated with the 9-kbp BglII-EcoRI fragment of pHTBS-F3-1C. pHTF3-1C/Ab-IRS-K was obtained by ligating the SphI-AlwNI and XhoI-AlwNI fragments of pHT-IRS-BSK (each carrying an IRS of Tn4430) to the 6.82-kbp DNA fragment of pHTBS-F3-1C/Ab that contains the origin of the replication of pHT1030 (ori B. thuringiensis) and the chimeric cry1C/Ab gene under the control of the promoter of the cry3A gene (Pcry3A). pHTF3-1C/Ab-IRS-T was constructed by a three-way ligation as follows: the 1.6-kbp Ecl136II-EcoRI DNA fragment of pHTBS2 (28) harboring a tetracycline resistance marker was purified and ligated with the SmaI-EcoRI and EcoRI-EcoRI fragments of pHTF3-1C/Ab-IRS-K. Restriction sites destroyed during the manipulation are indicated by square brackets, and only restriction sites relevant to the experimental design are shown. The various boxes representing genes or IRSs are not drawn to scale. Bt, B. thuringiensis.

FIG. 2

Agarose gel electrophoresis of BamHI-XhoI-EcoRI-digested plasmid DNA from native and transformed B. thuringiensis Kto and Kto SigK⁻ strains. Lanes: 1 and 8, 1-kb DNA ladder; 2, Kto recipient; 3, Kto (pHTF3-1C/Ab-IRS-T-Δ); 4 and 7, pHTF3-1C/Ab-IRS-T isolated from E. coli; 5, Kto SigK⁻ (pHTF3-1C/Ab-IRS-T-Δ); 6, Kto SigK⁻ recipient. The 2.9-kbp BamHI DNA fragment corresponding to the pBluescript II KS(−) and the 1.6-kbp BamHI-EcoRI DNA fragment corresponding to the tet gene of B. cereus are indicated by black arrows (lane 7). These DNA fragments are absent from the Kto and Kto SigK⁻ transformants harboring pHTF3-1C/Ab-IRS-T-Δ. Only the 2.6-kbp EcoRI-XhoI and 4.3-kbp BamHI-EcoRI DNA fragments corresponding to the origin of replication of pHT1030 and the cry1C/Ab gene, respectively, remain (indicated by open triangles in lanes 3 and 5) after the site-specific recombination that removes the tet gene and pBluescript II KS(−). The sizes (in kilobase pairs) of the 1-kb ladder are shown on the right.