Transgenic plants expressing two Bacillus thuringiensis toxins delay insect resistance evolution

Preventing insect pests from developing resistance to Bacillus thuringiensis (Bt) toxins produced by transgenic crops is a major challenge for agriculture. Theoretical models suggest that plants containing two dissimilar Bt toxin genes ('pyramided' plants) have the potential to delay resistance more effectively than single-toxin plants used sequentially or in mosaics. To test these predictions, we developed a unique model system consisting of Bt transgenic broccoli plants and the diamondback moth, Plutella xylostella. We conducted a greenhouse study using an artificial population of diamondback moths carrying genes for resistance to the Bt toxins Cry1Ac and Cry1C at frequencies of about 0.10 and 0.20, respectively. After 24 generations of selection, resistance to pyramided two-gene plants was significantly delayed as compared with resistance to single-gene plants deployed in mosaics, and to Cry1Ac toxin when it was the first used in a sequence. These results have important implications for the development and regulation of transgenic insecticidal plants.     

Larvicidal activities against agricultural pests of transgenic Escherichia coli expressing combinations of four genes from Bacillus thuringiensis

The genes cry1Ac and cry1Ca from Bacillus thuringiensis subsps. kurstaki HD-73 and aizawai 4J4, respectively, encoding δ-endotoxins against lepidopteran larvae were isolated, cloned and expressed in Escherichia coli, with and without cyt1Aa (encoding cytolytic protein) and p20 (accessory protein) from subsp. israelensis. Nine combinations of the genes under control of an early T7, P _A1 inducible promoter, produced the encoding proteins. Toxicities were examined against larvae of three major agricultural pests: Pectinophora gossypiella, Helicoverpa armigera and Spodoptera littoralis. The clones expressing cyt1Aa, with or without p20, were not toxic. The clone expressing cry1Ac (pBt-1A) was the most toxic to P. gossypiella (LC₅₀ of 0.27 × 10⁸ cells g⁻¹). Clone pBt-1CA expressing cry1Ca and cry1Ac displayed the highest toxicity (LC₅₀ of 0.12 × 10⁸ cells ml⁻¹) against S. littoralis. Clone pBt-1CARCy expressing all four genes (cry1Ca, cry1Ac, p20, cyt1Aa) in tandem exhibited the highest toxicity to H. armigera (LC₅₀ of 0.16 × 10⁸ cells ml⁻¹). Cyt1Aa failed to raise the toxicity of these Cry toxins against P. gossypiella and S. littoralis but significantly enhanced toxicity against H. armigera. Two additional clones expressing either cry1Ac or cry1Ca under tandem promoters, P _A1 and P _psbA (constitutive), displayed significantly higher toxicities (7.5- to 140-fold) than their counterparts with P _A1 alone, reducing the LC₅₀ values to below 10⁷ cells ml⁻¹. Vadim Khasdan and Maria Sapojnik are contributed equally to this work.     

Transgenic crops expressing Bacillus thuringiensis toxins and biological control

The area devoted to growing transgenic plants expressing insecticidal Cry proteins derived from Bacillus thuringiensis (Bt) is increasing worldwide. A major concern with the adoption of Bt crops is their potential impact on nontarget organisms including biological control organisms. Regulatory frameworks should advocate a step-wise (tiered) approach to assess possible nontarget effects of Bt crops. Laboratory and glasshouse studies have revealed effects on natural enemies only when Bt-susceptible, sublethally damaged herbivores were used as prey or host, with no indication of direct toxic effects. Field studies have confirmed that the abundance and activity of parasitoids and predators are similar in Bt and non-Bt crops. In contrast, applications of conventional insecticides have usually resulted in negative impacts on biological control organisms. Because Bt-transgenic varieties can lead to substantial reductions in insecticide use in some crops, they can contribute to integrated pest management systems with a strong biological control component.     

Transgenic rice expressing Allium sativum leaf lectin with enhanced resistance against sap-sucking insect pests

Mannose binding Allium sativum leaf agglutinin (ASAL) has been shown to be antifeedant and insecticidal against sap-sucking insects. In the present investigation, ASAL coding sequence was expressed under the control of CaMV35S promoter in a chimeric gene cassette containing plant selection marker, hpt and gusA reporter gene of pCAMBIA1301 binary vector in an elite indica rice cv. IR64. Many fertile transgenic plants were generated using scutellar calli as initial explants through Agrobacterium-mediated transformation technology. GUS activity was observed in selected calli and in mature plants. Transformation frequency was calculated to be ~12.1%±0.351 (mean ± SE). Southern blot analyses revealed the integration of ASAL gene into rice genome with a predominant single copy insertion. Transgene localization was detected on chromosomes of transformed plants using PRINS and C-PRINS techniques. Northern and western blot analyses determined the expression of transgene in transformed lines. ELISA analyses estimated ASAL expression up to 0.72 and 0.67% of total soluble protein in T₀ and T₁ plants, respectively. Survival and fecundity of brown planthopper and green leafhopper were reduced to 36% (P<0.01), 32% (P<0.05) and 40.5, 29.5% (P<0.001), respectively, when tested on selected plants in comparison to control plants. Specific binding of expressed ASAL to receptor proteins of insect gut was analysed. Analysis of T₁ progenies confirmed the inheritance of the transgenes. Thus, ASAL promises to be a potential component in insect resistance rice breeding programme.     

Transgenic avidin maize is resistant to storage insect pests

Avidin is a glycoprotein found in chicken egg white, that sequesters the vitamin biotin. Here we show that when present in maize at levels of > or =100 p.p.m., avidin is toxic to and prevents development of insects that damage grains during storage. Insect toxicity is caused by a biotin deficiency, as shown by prevention of toxicity with biotin supplementation. The avidin maize is not, however, toxic to mice when administered as the sole component of their diet for 21 days. These dates suggest that avidin expression in food or feed grain crops can be used as a biopesticide against a spectrum of stored-produce insect pests.     

Bacillus thuringiensis and control of plant pests

Summary The feasibility ofB. thuringiensis as an economic insect control agent is dependent upon various factors. Consideration of three situations. whereB. thuringiensis could be used illustrates the interaction of these factors, and their contribution to determining the success or failure of the organism as an insecticide. Apple crops suffer from attack by a complex of insect pests, many of which are very susceptible to theB. thuringiensis toxin. However chemical control methods are preferred by growers in order to satisfy the consumers' demand for top quality blemish-free fruit. By contrast cotton growers are beset with problems of pest resistance to chemical insecticides. This problem is tackled by using pest management strategies which orchestrate all possible methods of control. Such a situation is ideally suited toB. thuringiensis products, but as yet they have proven to be of inadequate efficacy. The softwood forest industry is an example whereB. thuringiensis is both needed and effective. The pest complex is relatively simple, and there is public concern about the health risks of chemical sprays. Recent developments of high performance formulations means thatB. thuringiensis is an economic, cost-effective, biological alternative to chemical control for forestry pests.

---

Resumen La posibilidad de utilizarB. thuringiensis para el control de insectos de una forma económica depende de varios factores. La consideración de tres situaciones distintas en las queB. thuringiensis puede ser utilizado ilustra la interacción de estos factores y su contribución al éxito o fracaso del organismo como insecticida. El cultivo de manzanas sufre el ataque de una serie de plagas de insectos, muchas de las cuales son susceptibles a la toxina deB. thuringiensis. Sin embargo, los agricultores prefieren utilizar métodos de control químico a fin de satisfacer la demanda del consumidor por un fruto de primera calidad sin marcas ni señales. Por el contrario, los cultivadores de algodon estan sensibilizados a los problemas de la aparición de plagas resistentes a los insecticidas químicos. Este problema se ha abordado utilizando sistemas de control integrado que aglutinan todos los posibles métodos de control. Esta situación parece la ideal para el uso de productos a base deB. thuringiensis, pero hasta ahora estos han resultado ser poco eficaces. La industria de la madera blanda constituye en ejemplo en el cualB. thuringiensis es a la vez necesario y efectivo. Las plagas en cuestión forman un sistema relativamente simple y los efectos de los sprays químicos sobre la salud pública son causa de preocupación. Los desarrollos recientes en formulaciones de elevada eficacia convierten aB. thuringiensis en una alternativa biológica económicamente factible frente al control químico de plagas forestales.

---

Résumé La possibilité d'utiliser économiquementB. thuringiensis dans la lutte contre les insectes nuisibles dépend de facteurs divers. L'examen de trois situations dans lesquellesB. thuringiensis peut être utilisé illustre l'interaction de ces facteurs et leur contribution dans le succès ou l'échec de cet organisme comme insecticide. Les récoltes de pommes sont menacées par les attaques d'un mélange complexe d'insectes, dont beaucoup sont sensibles à la toxine deB. thuringiensis. Cependant, les horticulteurs préfèrent les méthodes chimiques à cause de l'exigence des consommateurs pour des fruits de haute qualité et sans aucune tache. Par contre, les cultivateurs de coton sont obsédés par les problèmes de résistance aux insecticides chimiques. Ce problème est affronté par des stratégies faisant appel à tous les moyens de lutte possibles. C'est une situation convenant bien aux produits qui contiennentB. thuringiensis, mais jusqu'ici ces produits se sont montrés inefficaces. L'industrie du bois tendre forestier est toutefois un exemple de cas oùB. thuringiensis est à la fois nécessaire et efficace. En effet, le complexe des insectes agressifs est relativement simple et il existe en outre une prévention du public contre les risques sanitaires des pulvérisations de produits chimiques. La mise au point récente de formulations à haute performance démontre queB. thuringiensis est, pour la lutte contre les insectes nuisibles des forêts, une alternative économique et bon-marché.

---

---

Invited paper presented at the VII International Conference on the Global Impacts of Applied Microbiology, Helsiki, 12–16 August 1985. Session 8     

Characterization of novel Brazilian Bacillus thuringiensis strains active against Spodoptera frugiperda and other insect pests

Brazilian strains of Bacillus thuringiensis, namely S701, S764 and S1265 were analysed regarding their cry gene and protein contents, crystal type, and activity against larvae of the lepidopteran fall armyworm (Spodoptera frugiperda Smith), the velvet caterpillar (Anticarsia gemmatalis), the dipterans (Culex quinquefasciatus and Aedes aegypti) and the coleopteran (Tenebrio molitor). The LC₅₀ of the strains against second instar larvae of S. frugiperda or A. gemmatalis revealed a high potency against those insect species. The spore–crystal mixtures of the isolates were analysed by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and showed similar protein pattern as the B. thuringiensis subsp. kurstaki strain HD‐1 (proteins approximately 130 and 65 kDa) for isolates S701 and S764, respectively, and only one major protein of approximately 130 kDa for isolate S1265. The polymerase chain reaction (PCR) using total DNA of the isolates and general and specific primers showed the presence of cry1Aa, cry1Ac, cry1Ia and cry2Ab genes in the two isolates serotyped as B. thuringiensis kurstaki (S701 and S764) and the presence of cry1D and cry2Ad in B. thuringiensis morrisoni S1265 strain. Scanning electron microscopy of strains S701 and S764, showed the presence of bipyramidal, cuboidal and round crystals, like in strain HD‐1 and bipyramidal and round crystals like in strain S1265.     

Toxicity and synergism in transgenic Escherichia coli expressing four genes from Bacillus thuringiensis subsp. israelensis

The genes cyt1Aa and p20 , encoding, respectively, cytolytic and accessory proteins of Bacillus thuringiensis subsp. israelensis , were introduced into previously constructed clones expressing cry4Aa and cry11Aa in Escherichia coli ( Ben-Dov et al ., 1995 ). Fifteen clones with all possible combinations of the four genes were obtained and found to express the genes included. Two new combinations, pVE4-ADRC and pVE4-ARC, expressing cyt1Aa , p20 and cry4Aa , with or without cry11Aa , respectively, were more toxic than their counterparts without cyt1Aa . They displayed the highest toxicity against Aedes aegypti larvae ever reached in transgenic bacteria. Five out of the six clones (except pVE4-DC) containing cry4Aa or cry11Aa (with or without p20 ) displayed varying levels of synergism with cyt1Aa : they are 1.5-to 34-fold more toxic than the respective clones without cyt1Aa against exposed larvae. Their lethal times also decreased (they kill larvae quicker), more so at higher cell concentrations. These clones are anticipated to dramatically reduce the likelihood of resistant development in the target organisms ( Wirth et al ., 1997 ).     

Mosquito larvicidal activity of transgenic Anabaena PCC 7120 expressing toxin genes from Bacillus thuringiensis subsp. israelensis

Genes encoding the mosquito larvicidal toxins Cry4Aa, Cry11Aa, Cyt1Aa and the regulatory P20 from Bacillus thuringiensis subsp. israelensis were introduced into the nitrogen-fixing, filamentous cyanobacterium Anabaena PCC 7120 for expression under control of two strong promoters P(psbA) and P(A1). The clone pRVE4-ADRC displayed toxicity against fourth-instar larvae of Aedes aegypti, the highest ever achieved in cyanobacteria. It was about 2.5-fold more toxic than the respective clone without cyt1Aa [Wu et al., Appl. Environ. Microbiol. 63 (1997) 4971-4975]. Cyt1Aa synergized the combination of Crys by about five-fold. Consistently, the lethal times exerted by pRVE4-ADRC were also reduced (it killed exposed larvae more quickly). This clone may become a useful biological control agent which reduces the probability of resistance development in the target organisms [Wirth et al., Proc. Natl. Acad. Sci. USA 94 (1997) 10536-10540].     

Experimental evidence that refuges delay insect adaptation to Bacillus thuringiensis

Theoretical projections suggest that refuges from exposure can delay insect adaptation to environmentally benign insecticides derived from Bacillus thuringiensis, but experimental tests of this approach have been limited. We tested the refuge tactic by selecting two sets of two colonies of diamondback moth (Plutella xylostella) for resistance to B. thuringiensis subsp. aizawai in the laboratory. In each set, one colony was selected with no refuge and the other with a 10 per cent refuge from exposure to B. thuringiensis subsp. aizawai. Bioassays conducted after nine selections were completed show that mortality caused by B. thuringiensis subsp. aizawai was significantly greater in the refuge colonies than in the no-refuge colonies. These results demonstrate that the refuges delayed the evolution of resistance. Relative to a susceptible colony, final resistance ratios were 19 and 8 for the two no-refuge colonies compared to 6 and 5 for the refuge colonies. The mean realized heritability of resistance to B. thuringiensis subsp. aizawai was 0.046 for colonies without refuges, and -0.002 for colonies with refuges. Selection with B. thuringiensis subsp. aizawai decreased susceptibility to B. thuringiensis toxin Cry1Ab, but not to Cry1C or B. thuringiensis subsp. kurstaki. Although the ultimate test of refuges will occur in the field, the experimental evidence reported here confirms modelling results indicating that refuges can slow the evolution of insect resistance to B. thuringiensis.     

Enduring toxicity of transgenic Anabaena PCC 7120 expressing mosquito larvicidal genes from Bacillus thuringiensis ssp. israelensis

Persistence of biological control agents against mosquito larvae was tested under simulated field conditions. Mosquito larvicidal activity of transgenic Anabaena PCC 7120 expressing cry4Aa, cry11Aa and p20 from Bacillus thuringiensis ssp. israelensis was greater than B. thuringiensis ssp. israelensis primary powder (fun 89C06D) or wettable powder (WP) (Bactimos products) when either mixed with silt or exposed to sunlight outdoors. Reduction of Bactimos primary powder toxicity was at least 10-fold higher than Anabaena's after mixing with silt. In outdoors experiments, Bactimos WP remained toxic (over 30% mortality of 3rd instar Aedes aegypti larvae) for 2-4 days only, while transgenic Anabaena's toxicity endured 8-21 days.     

A naturally occurring plant cysteine protease possesses remarkable toxicity against insect pests and synergizes Bacillus thuringiensis toxin

When caterpillars feed on maize (Zea maize L.) lines with native resistance to several Lepidopteran pests, a defensive cysteine protease, Mir1-CP, rapidly accumulates at the wound site. Mir1-CP has been shown to inhibit caterpillar growth in vivo by attacking and permeabilizing the insect's peritrophic matrix (PM), a structure that surrounds the food bolus, assists in digestion and protects the midgut from microbes and toxins. PM permeabilization weakens the caterpillar defenses by facilitating the movement of other insecticidal proteins in the diet to the midgut microvilli and thereby enhancing their toxicity. To directly determine the toxicity of Mir1-CP, the purified recombinant enzyme was directly tested against four economically significant Lepidopteran pests in bioassays. Mir1-CP LC(50) values were 1.8, 3.6, 0.6, and 8.0 ppm for corn earworm, tobacco budworm, fall armyworm and southwestern corn borer, respectively. These values were the same order of magnitude as those determined for the Bacillus thuringiensis toxin Bt-CryIIA. In addition to being directly toxic to the larvae, 60 ppb Mir1-CP synergized sublethal concentrations of Bt-CryIIA in all four species. Permeabilization of the PM by Mir1-CP probably provides ready access to Bt-binding sites on the midgut microvilli and increases its activity. Consequently, Mir1-CP could be used for controlling caterpillar pests in maize using non-transgenic approaches and potentially could be used in other crops either singly or in combination with Bt-toxins.     

Genetically engineered vegetables expressing proteins from Bacillus thuringiensis for insect resistance: successes, disappointments, challenges and ways to move forward

Genetically engineered (GE) insect-resistant crops that express proteins from Bacillus thuringiensis (Bt) have been widely adopted in the two field crops currently commercially available, Bt cotton and Bt corn. However, the development and commercialization of Bt vegetables has lagged in comparison, which is unfortunate since vegetables tend to be insecticide-intensive crops due to high pest pressure and cosmetic standards required for the market. While it is often stated that consumer choice has played a major role in companies avoiding development of Bt vegetables, this concept requires re-evaluation. In market studies in North America when consumers have been provided basic information about Bt genetic engineering, then given a choice between Bt and conventional sweet corn, they have often preferred the former. Likewise, 77% of consumers in a US survey said they would likely purchase foods produced through biotechnology for their ability to reduce pesticide use. Presently, however, the only commercialized Bt vegetable is sweet corn. Perhaps more critical obstacles to Bt vegetables are their relatively smaller acreages and the cost of government biosafety regulations that inadvertently favor large acreage of field crops because companies can obtain a better return on investment. In developing countries, private-public partnerships may provide the vehicle to bring Bt vegetables to market. However, these can be subverted by misinformation from anti-biotech campaigns, as is the case with Bt eggplant in India. Without the use of Bt vegetables as a tool for integrated pest management, farmers and the general public will not be able to realize the substantial environmental and economic benefits that have been well documented with Bt cotton and Bt corn.     

Ingested particles reduce susceptibility of insect larvae to Bacillus thuringiensis

Susceptibility to Bacillus thuringiensis of mosquito and lepidopteran larvae is affected by feeding behaviour and nutritional value of the available food. Reduced mortality is attributed to feeding inhibition and dilution of the pathogen in the presence of nutritional and inert particles, which limit the amount of ingested toxin. These reasons are, however, not sufficient to explain the data presented here. Values of LC₅₀ (the concentration that kills 50% of exposed population) of B. thuringiensis subsp. israelensis (Berliner) against Aedes aegypti (L.) larvae and of B. thuringiensis subsp. kenyae (Berliner) against Spodoptera littoralis (Boisduval) larvae were about 20–217 and 2.3–44‐fold higher, respectively, in the presence of nutritional or biologically inert (non‐nutritional) particles than without. The number of B. thuringiensis spores in carcasses of B. thuringiensis ‐killed A. aegypti and S. littoralis larvae were between 1.9 and 5.6‐fold and between 8.5 and 12‐fold higher, respectively, in the presence of particles than without. In all cases, non‐nutritional particles better protected the exposed larvae than nutritious particles. We propose that another basic mechanism exists, that ingested particles protect midgut epithelial cells by covering their surface and thus preventing availability of the toxin to the gut receptors. Understanding the defence mechanisms of insects against B. thuringiensis toxicity may lead to improved pest management methods.     

Cross-resistance spectra of Culex quinquefasciatus resistant to mosquitocidal toxins of Bacillus thuringiensis towards recombinant Escherichia coli expressing genes from B. thuringiensis ssp. israelensis

Sixteen Escherichia coli clones were assayed against susceptible and Bacillus thuringiensis-resistant Culex quinquefasciatus larvae. The clones expressed different combinations of four genes from Bacillus thuringiensis ssp. israelensis; three genes encoded mosquitocidal toxins (Cry11Aa, Cry4Aa and Cyt1Aa) and the fourth encoded an accessory protein (P20). The cross-resistance spectra of the mosquitoes were similar to the profiles for recombinant B. thuringiensis strains expressing B. thuringiensis toxin genes, but with varied toxicity levels. The toxicity of the recombinants towards resistant mosquito larvae was improved when p20 and cyt1Aa were expressed in combination with cry4Aa and/or cry11Aa. Recombinant pVE4-ADRC, expressing cry4Aa, cry11Aa, p20 and cyt1Aa, was the most active against the resistant Culex, and resistance levels did not exceed fourfold. These results indicate that B. thuringiensis ssp. israelensis genes expressed in a heterologous host such as E. coli can be effective against susceptible and B. thuringiensis-resistant larvae and suppress resistance.     

Distribution of cryV-type insecticidal protein genes in Bacillus thuringiensis and cloning of cryV-type genes from Bacillus thuringiensis subsp. kurstaki and Bacillus thuringiensis subsp. entomocidus.

DNA dot blot hybridizations with a cryV-specific probe and a cryI-specific probe were performed to screen 24 Bacillus thuringiensis strains for their cryV-type (lepidopteran- and coleopteran-specific) and cryI-type (lepidopteran-specific) insecticidal crystal protein gene contents, respectively. The cryV-specific probe hybridized to 12 of the B. thuringiensis strains examined. Most of the cryV-positive strains also hybridized to the cryI-specific probe, indicating that the cryV genes are closely related to cryI genes. Two cryV-type genes, cryV1 and cryV465, were cloned from B. thuringiensis subsp. kurstaki HD-1 and B. thuringiensis subsp. entomocidus BP465, respectively, and their nucleotide sequences were determined. The CryV1 protein was toxic to Plutella xylostella and Bombyx mori, whereas the CryV465 protein was toxic only to Plutella xylostella.