Genetically improved potatoes: protection from damage by Colorado potato beetles

Russet Burbank potato plants have been genetically improved to resist insect attack and damage by Colorado potato beetles (Leptinotarsa decemlineata (Say)) by the insertion of a cryIIIA gene encoding the insect control protein of Bacillus thuringiensis var. tenebrionis. A modified gene that dramatically improved plant expression of this protein was utilized. Its expression in Russet Burbank potato plants resulted in protection from damage by all insect stages in the laboratory and in dramatic levels of protection at multiple field locations. Analysis of these genetically modified potatoes indicated that they conform to the standards for Russet Burbank potatoes in terms of agronomic and quality characteristics including taste.     

Elucidation of the mechanism of CryIIIA overproduction in a mutagenized strain of Bacillus thuringiensis var. tenebrionis

NB176 is a Bacillus thuringiensis mutant derived by λ-irradiation of NB125 Bacillus thuringiensis var. tenebrionis (Krieg). It exhibits two interesting phenotypes: (i) oligosporogeny and (ii) twofold to threefold overproduction of the CryIIIA protein. Southern profiles of the NB176 strain showed an additional copy(s) of the cryIIIA gene located on a 4 kb HindIII fragment, in addition to the expected cryIIIA gene on a 3 kb HindIII fragment. Each cryIIIA gene-bearing HindIII fragment was cloned from NB176. The restriction map of the 3 kb HindIII fragment was identical to that published by Donovan and coworkers. Sequencing of the 4 kb HindIII fragment showed no alterations in the promoter region of the cryIIIA gene but did show replacement of the region immediately following the cryIIIA open reading frame with a sequence encoding a transposase with 50% amino acid homology to that of Tn 1000. These findings suggest that the overproduction phenotype of NB176 results from extra copies of the cryIIIA gene produced from a transposition event(s) induced or stabilized by γ-irradiation. Integration of additional copies of the cryIIIA gene into the native 90MDa plasmid of the wild-type B. thuringiensis var. tenebrionis strain resulted in strains that made enormous crystals, many possessing greatly enhanced insecticidal activity.     

Characterization of a New Bacillus thuringiensis Strain That Is Toxic to Coleoptera

A new strain of Bacillus thuringiensis 2-7 was found to belong to the serotype H8. Cells of this strain contained irregular and flat crystalline inclusions and two large plasmids. The gene responsible for crystal formation is most likely located on the large plasmid greater than 105 MDa in size. Comparison of the cry gene of B. thuringiensis 2-7 and the cryIIIA gene of B. thuringiensis subsp. tenebrionis showed that their nucleotide sequences are identical.     

Characterization of Bacillus thuringiensis isolates from Argentina that are potentially useful in insect pest control

In order to find novel strains of Bacillus thuringiensis that are toxic to some of the major pests that impact economically important crops in Argentina, we initiated a search for B. thuringiensis isolates native to Argentina. We succeeded in assembling a collection of 41 isolates, some of which show a high potential to be used in biological control programs against lepidopteran and coleopteran pests. About 90% of the strains showed toxicity against Spodoptera frugiperda and Anticarsia gemmatalis, two important lepidopteran pests in Argentina. It is noteworthy that only one of these strains contained a cry1-type gene, while another isolate showed a dual toxicity against the lepidopteran and coleopteran insects assayed. Genetic characterization of the strains suggests that the collection likely harbors novel Cry proteins that may be of potential use in biological insect pest control.     

Expression of insecticidal activity in Rhizobium containing the δ-endotoxin gene cloned from Bacillus thuringiensis subsp. tenebrionis

Bacillus thuringiensis subsp. tenebrionis produces a 65 kilodalton polypeptide toxin which is lethal to various coleopteran insect larvae. The gene encoding this toxin was cloned in E. coli in the broad host range vector pKT230 and subsequently transferred to Rhizobium leguminosarum by conjugation. Western blot analysis showed that the toxin gene was expressed in the free living state of Rhizobium producing two major polypeptides of 73 and 68 kilodalton in size. The level of expression of the toxin gene in Rhizobium varied from strain to strain. Cell extracts from toxin-producing rhizobia were toxic to larvae of Gasterophysa viridula. Bioassays also showed that the -endotoxin was toxic to larvae of the clover weevil Sitona lepidus. Furthermore, pea (Pisum sativum) and white clover (Trifolium repens) plants suffered less root and nodule damage by Sitona larvae when they were inoculated with Rhizobium strains containing the toxin gene. This suggests that such rhizobia could be useful in the biological control of this important legume pest.Abbreviations B.t.t. Bacillus thuringiensis subsp. tenebrionis - IPTG isopropyl--D-thiogalactoside     

Effectiveness of beta-exotoxin ofBacillus thuringiensis var.thuringiensis on the ability ofMeloidogyne sp. from brinjal (Solanum melongena L.) to survive

Beta-exotoxin produced byBacillus thuringiensis var.thuringiensis grown in the acid hydrolysates of wheat and rice brans caused 95% and 85% mortality respectively ofMeloidogyne sp. as against 72% of β-exotoxin produced on farm yard manure within 7 days. Acid hydrolysate of wheat or rice bran and solid farm yard manure proved to be the best media for growth ofB. thuringiensis var.thuringiensis.     

Effects of genetic and environmental host plant variation on the susceptibility of two noctuids toBacillus thuringiensis

TwoApium graveolens var.rapaceum (L.) cultivars that differ in their suitability for the survival and growth ofSpodoptera exigua (Hübner) andTrichoplusia ni (Hübner) were used to examine the effect of genetic and seasonal environmental variation in host plant suitability on the efficacy ofBacillus thuringiensis subsp.kurstaki (Berliner). The effects of host plant genotype andB. thuringiensis were generally independent, so thatB. thuringiensis efficacy was greatest on the resistant host plant cultivar. Host plant suitability varied within growing season for both insect species but, while host plant suitability decreased with increasing plant age forT. ni, the response ofS. exigua to plant age was not as clear. Within season variation in host plant suitability affectedB. thuringiensis efficacy and the interaction betweenB. thuringiensis and host plant cultivar forS. exigua but not forT. ni. Soluble protein and Folin-Denis phenolic concentrations of host plant tissue were not correlated with changes in host plant suitability to either insect species.     

Response of the Carrot Weevil,Listronotus oregonensis(Coleoptera: Curculionidae), to Strains ofBacillus thuringiensis

Mortality and frass production bioassays were used to investigate the toxicity of seven strains ofBacillus thuringiensisagainst the adult carrot weevil,Listronotus oregonensis(Le Conte). A semi-artificial diet of carrot foliage with 4% agar was selected to maximize feeding by the insects.Bacillus thuringiensissubsp.tenebrionis(Krieg, Huger, Langenbruch, and Schnetter) (BTT) and two unidentifiedB. thuringiensisstrains, A30 and A429, gave the lowest LC₅₀values. The frass bioassay supported the conclusions of the mortality assay. Mortality of adults continued after their removal from the insecticidal medium, with the highest mortality being caused by strains A429 and BTT. Survivors from the frass bioassay, initially exposed to strains A30, A429, and BTT, did not resume normal levels of feeding after their removal from the insecticidal medium.     

Cloning and characterization of an insecticidal crystal protein gene from Bacillus thuringiensis subspecies kenyae

A sporulating culture ofBacillus thuringiensis subsp.kenyae strain HD549 is toxic to larvae of lepidopteran insect species such asSpodoptera litura, Helicoverpa armigera andPhthorimaea operculella, and a dipteran insect,Culex fatigans. A 1.9-kb DNA fragment, PCR-amplified from HD549 using cryII-gene-specific primers, was cloned and expressed inE. coli. The recombinant protein produced 92% mortality in first-instar larvae ofSpodoptera litura and 86% inhibition of adult emergence inPhthorimaea operculella, but showed very low toxicity againstHelicoverpa armigera, and lower mortality against third-instar larvae of dipteran insectsCulex fatigans, Anopheles stephensi andAedes aegypti. The sequence of the cloned crystal protein gene showed almost complete homology with a mosquitocidal toxin gene fromBacillus thuringiensis var.kurstaki, with only five mutations scattered in different regions. Amino acid alignment with different insecticidal crystal proteins using the MUTALIN program suggested presence of the conserved block 3 region in the sequence of this protein. A mutation in codon 409 of this gene that changes a highly conserved phenylalanine residue to serine lies in this block.     

Development of insect resistance in tomato plants expressing the δ-endotoxin gene ofBacillus thuringiensis subsp.tenebrionis

A crystal -endotoxin gene ofBacillus thuringiensis subsp.tenebrionis (B.t.t.) encoding a coleopteran insect-specific toxin was used to construct a chimeric gene which expressed the toxin in plant cells. Via anAgrobacterium tumefaciens binary vector system, the toxin gene was transferred into tomato cells. From leaf disks recombinant plants were regenerated. Hybridization experiments demonstrated that these plants synthesized toxin-specific mRNA of the expected size. Transgenic tomato plants with the chimericB.t.t. toxin gene contained a 74 kDa protein which cross-reacted with toxin antibodies. The expression caused a significant insecticidal activity of the transgenic tomato plants against Colorado potato beetle larvae.     

Insecticidal effects of some biological agents on Agelastica alni (Coleoptera: Chrysomelidae)

The alder leaf beetle (Agelastica alni L., Coleoptera: Chrysomelidae) causes approximately 10% of total economic damage to hazelnut product per year in Turkey. A. alni larvae are susceptible to several pathogens indigenous to the area in which these insects occur in Turkey. In the present study, in order to find a more effective and safer biological control agent against this common pest, we evaluated the various biological agents’ insecticidal activity during the four hazelnut seasons from 2002 to 2005 on the larvae of the alder leaf beetle collected from the vicinity of Trabzon, Turkey. The tested agents are 25 insect-originating bacteria, 2 bacterial toxins and 1 viral preparation. The results showed that the highest insecticidal activity was obtained by bacterial isolates at 1.8 × 10⁹ bacteria/mL dose, within ten days on the larvae of A. alni. These are 90% for Bacillus thuringiensis biovar tenebrionis (4AA1), Bacillus sphaericus (Ar4, isolated from Anoplus roboris L., Col.: Curculionidae), and Bacillus thuringiensis (Mm2, isolated from Melolontha melolontha L., Col.: Scarabaeidae). Our results indicate that these isolates may be valuable as biological control agent.     

Expression of parasporal crystal protein (δ—endotoxin) gene(s) ofBacillus thuringiensis var.israelensis in sporogenic and asporogenic mutant strains ofBacillus cereus

The expression of parasporal crystal protein (δ-endotoxin) coding gene(s) ofBacillus thuringlensis var.israelensis and its association, if any, with sporulation was studied in sporogenicBacillus cereus and its asporogenic mutant strains. Five asporogenous mutants ofBacillus cereus blocked at different stages of sporulation, were isolated from a streptomycin-resistant strain, The transconjugants isolated from the plasmid transfer experiments betweenBacillus thuringiensis var.israelensis and streptomycin resistantBacillus cereus and its asporogenous mutants, showed larvicidal activity. The crystal protein gene(s) are, therefore, expressed both in sporulating and in non-sporulating mutant strains ofBacillus cereus suggesting that the expression of crystal protein gene(s), is independent of sporulation specific functions inBacillus cereus. Part of the work was carried out at Biotechnology Programme, Jadavpur University, Calcutta 700 032, India.     

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).     

Use of the egg parasiteEdovum puttleri [Hym.: Eulophidae] in an IPM system developed for Colorado potato beetle [Col.: Chrysomelidae] control on potatoes,Beltsville, Maryland

Edovum puttleri Grissell in conjunction with the fungal pathogenBeauveria bassiana (Balsamo) Vuillimen, a β-exotoxin ofBacillus thuringiensis varthuringiensis Berliner, and the insect growth regulator triflumuron (Alsystin 4F) were evaluated in a pest management system to control Colorado potato beetle,Leptinotarsa decemlineata (Say). Soil treatments ofB. bassiana had no significant impact on emerging Colorado potato beetle adults. Foliar applications of the β-exotoxin and triflumuron significantly reduced 1 ^st larval generation. Releases ofE. puttleri resulted in 56 % egg mass parasitization, significant reduction of 2 ^nd generation larvae, and significantly higher potato yields.Leptinotarsa texana (Schaeffer) was successfully used as an alternate host for production ofE. puttleri.      

Survival of a strain of Bacillus megaterium carrying a lepidopteran-specific gene of Bacillus thuringiensis in the phyllospheres of various economically important plants

The colonizing ability of a transcipient strain of Bacillus megaterium carrying a lepidopteran-specific cryIA (a) gene of Bacillus thuringiensis in the phyllospheres of various economically important plants was studied. Similar experiments were also carried out using the parental B. thuringiensis var. kurstaki strain HD1 for a comparison. While the transcipient remained on the leaves of cotton and okra for more than 28 days, its survival in phyllospheres of mulberry, peanut, chickpea, tomato and rice was rather limited to about 3 – 5 days. The persistence of B. thuringiensis, on the other hand, was extremely short (i.e. less than 4 days) on all the crop plants tested.     

Expression and characterization of inhA gene from Bacillus thuringiensis 8010

InhA, a zinc metalloprotease secreted by Bacillus thuringiensis, specifically hydrolyzes antibacterial peptides produced by insect hosts. In this study, the inhA gene was cloned from B. thuringiensis 8010 using a pair of degenerate primers and the deduced 796 amino acid sequence showed a high degree of similarity with other InhA proteins in the Bacillus cereus group. The deduced amino acid sequence contained the zinc-binding motif (HEXXH), which is characteristic of the zinc-metalloprotease family. Additionally, the inhA gene was expressed in Escherichia coli BL21 (DE3). The expressed InhA protein was shown to be toxic to the third larvae of Plutella xylostella, contrary to preliminary study concerning the effect of InhA on Bombyx mori. This study provided insights into the potential of InhA for the biological control of certain lepidopteran insects.     

Transgenic rice plants with a synthetic cry1Ab gene from Bacillus thuringiensis were highly resistant to eight lepidopteran rice pest species

To fully explore the resistance potential of transgenic rice produced by Agrobacterium-mediated transformation, an elite line KMD1 was assessed for its resistance to eight lepidopteran rice pest species. KMD1 contained a synthetic cry1Ab gene from Bacillus thuringiensis under the control of a maize ubiquitin promoter. It was derived from a commercial japonica Chinese rice variety Xiushui 11, and bred true for both agronomic traits and a cry1Ab gene when the bioassays were done in 1998 in the R₅ generation. The eight lepidopteran pest species were: four Pyralidae species: Chilo suppressalis (striped stem borer, SSB), Scirpophaga incertulas (yellow stem borer, YSB), Cnaphalocrocis medinalis (leaf folder), Herpitogramma licarisalis; two Noctuidae: Sesamia inferens (pink stem borer, PSB) and Naranga anescens; one Stayridae: Mycalesis gotama; and one Hesperiidae, Parnara guttata. In laboratory bioassays, 100% mortality was observed in all insect species when their newly hatched or third-instar larvae were fed KMD1 leaf tissues, whereas only 9.65% of the neonates and none of the third-instar larvae died when fed the leaf tissues of non-transgenic control. Moreover, the leaf area of control tissues consumed in four days by stem borers was 20 to 40 times higher than that of KMD1 tissues, and the area of control tissues eaten by leaf-feeding species was 120 to 180 times greater than that of the transgenic tissues. Under natural infestation, no KMD1 plant was visibly damaged by the SSB, YSB and leaf folder in field evaluation. On the other hand, 80, 9.3 and 88.7% of control plants were injured by SSB, YSB, and leaf folder, respectively. These data disclosed that the transgenic line was highly resistant to a broad spectrum of lepidopteran insect species and could be useful in insect resistance breeding programs.     

Crystals of two mutants ofBacillus thuringiensis show increased toxicity against larvae ofSpodoptera littoralis

Crystals of two asporogenous mutants ofBacillus thuringiensis var.kurstaki strain HD-1 obtained following treatment with ozone and N-methyl-N′-nitro-N-nitrosoguanidine showed increased toxicity against larvae ofSpodoptera littoralis when compared to the wild-type crystal.