Host range and gene contents of Bacillus thuringiensis strains toxic towards Spodoptera exigua

Thirty-five strains of the entomopathogenic bacterium Bacillus thuringiensisactive on Spodoptera exigua, were characterized by means of serological identification and determination of crygene contents by PCR. The insecticidal activity of these 35 strains was further confirmed against S. exiguaand tested against two other species of the same genus: S. littoralisand S. frugiperda. The results indicate that serovars aizawai, thuringiensis, and kurstakiwere the most frequent within S. exigua-active strains and that serovar aizawaihad the highest number of strains exhibiting toxicity against the three species bioassayed. The presence in crygenes as determined by PCR suggests a non random distribution of some crygenes among serovars. Genes cry1C, cry1D, and cry1E, which are known to code for proteins toxic against Spodopteraspecies, were very common within S. exigua-active strains, specially in those belonging to serovar aizawai. However, some strains harbouring one or more of these genes were not toxic to S. littoralisor S. frugiperda; and some strains lacking all of the Spodoptera-active genes were found to be toxic to all three species. This suggests differences in the expression levels among strains bearing toxic genes and the involvement of other genes toxic to Spodopteraspecies. Since strains sharing the same crygenes exhibited different host ranges, the results indicate the need to perform toxicity bioassays in addition to other tests (serological identification and PCR) in order to determine the insecticidal activity of B. thuringiensisstrains.     

The circadian rhythm of flight activity of Spodoptera exigua males in response to sex pheromone

In many moths, male attraction to the blend of synthetic sex pheromone releasing continuously in the field shows an apparent circadian rhythm similar to that of locomotion activity. In this study, the daily rhythms of electroantennography (EAG) and behavioral responses to sex pheromone, and the daily rhythms of locomotion activity were measured in male beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae). The peaks of males trapped by light and sex pheromone were all during the latter part of the night in the field. However, there was no significant variation among time intervals in the EAG responses of male antennae to sex pheromone stimuli. The principal period of locomotion activity under L15:D9 (LD) continued to occur during the scotophase and the subjective scotophase in the day of constant darkness (DD1) and the second of two consecutive days of constant darkness (DD2). The majority of males contacted the sex pheromone source in a wind tunnel during the latter part of the scotophase under LD and the subjective scotophase for DD1 and DD2. There were significant associations between the rhythm of the behavioral response to sex pheromone and locomotion activity. These results suggested that the male's behavioral response to sex pheromone in the beet armyworm could be observed only until locomotion activity of the male occurred at the end of the dark period, despite sex pheromone being released continuously from synthetic pheromone‐baited traps in the field.     

对甜菜夜蛾高毒苏云金芽孢杆菌菌株的选育*

采用物理诱变——虫体传代模式,选育获得一株对甜菜夜蛾高毒菌株BtCZE 99985。通过摇瓶和40t发酵罐3年10批发酵试验,表明该菌株具有良好的发酵性能。摇瓶试验表明,与出发菌株93005、对照菌株HD-1-580、GC-91相比较,该菌株对甜菜夜蛾的毒效分别提高429％、655％、114％。40t发酵罐发酵试验表明,该菌株对甜菜夜蛾测定的LC50平均值为0．076μL/mL,比GC-91菌株(平均0．213μL/mL)的毒效提高180％。     

Vegetative insecticidal protein enhancing the toxicity of Bacillus thuringiensis subsp kurstaki against Spodoptera exigua

AIMS: The objective of this work was to enhance the insecticidal activity or widen the pesticidal spectrum of a commercial Bacillus thuringiensis strain YBT1520. METHODS AND RESULTS: A vegetative insecticidal protein gene vip3Aa7, under the control of its native promoter and cry3A promoter, was subcloned into B. thuringiensis acrystalliferous BMB171 to generate BMB8901 and BMBvip respectively. It was found that the amount of Vip3Aa7 protein produced by BMBvip was 3.2-fold more than that produced by BMB8901. Therefore, the vip3Aa7 gene under the control of cry3A promoter was transformed into strain YBT1520. The toxicity of the resulting strain BMB218V against Spodoptera exigua was 10-fold more than that of YBT1520, and that the toxicity of BMB218V against Helicoverpa armigera retained the same level as that of strain YBT1520. CONCLUSIONS: Strain YBT1520 obtained high toxicity against S. exigua after it was transformed and expressed the foreign vip3Aa7 gene. SIGNIFICANCE AND IMPACT OF THE STUDY: Commercial B. thuringiensis strain YBT1520 has high toxicity against H. armigera and Plutella xylostella, but almost no activity against S. exigua, which is a major crop pest in China. This work provides a new strategy for widening the activity spectrum of B. thuringiensis against agriculture pests.     

Sublethal effects of Bt toxin and chlorpyrifos on various Spodoptera exigua populations

Bt cotton (Cry1Ac) has been commercially grown in China since 1997, saving China's cotton production from attack by Bt‐target pests and also tremendously reducing pesticide usage. In recent years, however, Bt cotton, with 4.2 million ha of cultivation, has suffered from a secondary target pest, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae). In China, growers have even had to re‐adopt conventional pesticides to control the pest, and this practice has already caused serious pesticide residue. In order to clarify the sublethal effects of chemical pesticide, the responses of a Bt‐susceptible and a Bt‐tolerant (Bt10) S. exigua strain to three treatment combinations were examined, including Bt toxin, sublethal chlorpyrifos, and Bt + sublethal chlorpyrifos. The susceptible and the Bt10 strain responded differently to dual pressure. Bt toxin + sublethal chlorpyrifos treatment lowered larval mortality and stimulated population increase of the susceptible S. exigua, whereas it delayed growth and development of the Bt10 strain. Under dual pressure, although larvae of the Bt10 strain developed faster than larvae of the susceptible strain, the Bt10 population experienced higher larval mortality, prolonged pupal duration, decreased pupal weight, decreased emergence rate, and shortened adult longevity. Compared with the susceptible strain, the Bt10 strain was deleteriously affected by sublethal chlorpyrifos. The Bt‐tolerant/resistant S. exigua population was more vulnerable to chemical pesticides like chlorpyrifos regardless of whether it was exposed to Bt toxin or not. Our study provides a reference for increasing the efficacy of control of S. exigua in Bt‐cotton planting areas.     

The synergistic activity between Cry1Aa and Cry1c from Bacillus thuringiensis against Spodoptera exigua and Helicoverpa armigera

AIMS: To investigate the interaction between two crystal proteins, Cry1Aa and Cry1C, for future development of biopesticides based on Bacillus thuringiensis, toxicities of the two individual proteins and in combinations have been determined against Spodoptera exigua and Helicoverpa armigera larvae, and synergism between the proteins has been evaluated using synergistic factor. METHODS AND RESULTS: SDS-PAGE showed that Cry1Aa and Cry1C proteins could be expressed in acrystalliferous B. thuringiensis 4Q7 strain, with molecular weights of 135 and 130 kDa respectively. The bioassay results indicated a synergistic activity between Cry1Aa and Cry1C against S. exigua and H. armigera, and the highest toxicities could be observed in the combination of Cry1Aa and Cry1C at a ratio of 1 : 1. CONCLUSION: The two toxins, Cry1Aa and Cry1C, interact synergistically to exhibit higher toxicity against S. exigua and H. armigera. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the investigation on the synergistic activity between two B. thuringiensis Cry1 toxins. It can be applied to the rational design of new generations of B. thuringiensis biopesticides and to strategies for management of resistant insects.     

Nuclear polyhedrosis virus for control of Spodoptera exigua larvae on glasshouse crops

the efficacy of a nuclear polyhedrosis virus, SeNPV, for controlling larval populations of the beet armyworm, Spodoptera exigua (Noctuidae) was studied in chrysanthemum, gerbera, kalanchoë and tomato crops in glasshouses. Applications of 1×10⁸ PIBs/m² on the various crops resulted in 95–100% larval mortality. Virus applications caused comparable levels of mortality in populations of early and late instar larvae. Reduction in feeding damage to the crops, however, was more pronounced when the larvae were early instars at the time of virus application. Single applications with the virus resulted in higher levels of larval mortality as double applications with 0.1% methomyl and diflubenzuron.The virus appears to be a potential control agent for the beet armyworm in Dutch glasshouses.

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Résumé Les résultats d'applications d'un virus de la polyédrose nucléaire contre des populations de chenilles de Spodoptera exigua sont examinés dans des chrysanthèmes, gerberas, kalanchoës et tomates. Une dose de 1×10⁸ PIBs/m² résulte à 95–100% mortalité larvale dans tous les végétaux. L'effet sur chemilles de première et seconde stade larvale et sur chenilles de troisième et quatrième stade larvale était pareille. Malgré le niveau élevé du mortalité larvale les végéteaux étaient damagés sérieusement, particulièrement quand le virus était appliqué contre des chenilles de troisième et quatrième stade larvale.Comparée avec des pesticides chimiques, méthomyl et diflubenzuron, applications de virus résultent à un mortalité larvale plus haute. Le conclusion était que le virus est un candidat sérieus pour l'utilisation dans le lutte biologique contre les chenilles de S. exigua dans des serres en Pays Bas.

     

Diversity of Colombian strains of Bacillus thuringiensis with insecticidal activity against dipteran and lepidopteran insects

AIM: To evaluate the genetic and molecular diversity and insecticidal activity of Bacillus thuringiensis isolates from all the natural regions of Colombia. METHODS AND RESULTS: A total of 445 isolates from a collection of B. thuringiensis were characterized. The parasporal crystal morphology that was most abundant was bipyramidal (60%). Almost 10% of the isolates were toxic to Spodoptera frugiperda and 5.6% against Culex quinquefasciatus larvae. cry gene content determined by PCR indicated that 10.6% of the isolates contained cry1 genes and 1.1% contained cry2, cry4 or cry11 genes. Protein content of the parasporal crystal was determined by SDS-PAGE; 25 and 18 different protein profiles were found in isolates active against S. frugiperda and C. quinquefasciatus, respectively. CONCLUSIONS: Bacillus thuringiensis presents great genetic and molecular diversity even in isolates from the same soil sample. Moreover, the diversity and activity of the isolates might have a relationship with the geographical origin of the samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained here indicate that some of the B. thuringiensis isolates characterized in this study are potential control agents that could be used in programmes against mosquitoes and S. frugiperda.     

Influence of tannic acid and Cry1Ac toxin of Bacillus thuringiensis on larval survival, growth, and development of Helicoverpa armigera

Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) causes huge economic losses in cotton production around the world. Tannin, one of the important secondary substances in cotton plants, can increase the δ‐endotoxin activity of Bacillus thuringiensis ssp. kurstaki. The mechanism of interaction between tannin and Bt toxin on H. armigera is unclear. We investigated the interaction between tannic acid and Cry1Ac toxin in H. armigera, and monitored survival, growth, and development during the larval period after treating the larvae with four concentrations of Cry1Ac toxin (0, 2, 8, and 14 μg^?1) alone or in combination with four concentrations of tannic acid (0, 0.5, 1, and 2 mg g^?1). Mortality of larvae treated with both tannic acid and Cry1Ac was higher than the mortality of larvae treated with tannic acid or Cry1Ac alone. Mortality was 47.5 and 51.5% in larvae treated with 14 μg g^?1 Cry1Ac alone or 2 mg g^?1 tannic acid alone, respectively. In contrast, larval mortality was 75% when treated with the mixture of 14 μg g^?1 Cry1Ac and 2 mg g^?1 tannic acid, suggesting that a mixture of the two enhanced the effectiveness of each one alone. The developmental time of larvae treated with the combination of tannic acid and Cry1Ac was significantly longer than when they were treated with Cry1Ac or tannic acid alone. Larval weight, pupal weight, and pupation rate were also significantly reduced in larvae treated with both toxins, compared with the larvae treated with either toxin alone. These results showed that the interactive effect of tannic acid and Cry1Ac on larval growth inhibition is additive, and that tannic acid improves Cry1Ac toxicity to insects. Tannic acid used in combination with B. thuringiensis might potentially reduce overall insecticide use, thus delaying development of insecticide resistance.     

Insecticidal activity of mixtures of Bacillus thuringiensis crystals with plant oils of Sinapis alba and Azadirachta indica

The main purpose of this study was to determine the insecticidal activity of mixtures of Bacillus thuringiensis crystalline toxins and plant substances that could contribute to create a new bioinsecticide of high efficiency. We decided to determine the toxicity of crystals of B. thuringiensis MPU B9 isolate, azadirachtin (NeemAzal), mustard oil and their mixtures against lepidopteran pests: Cydia pomonella, Spodoptera exigua and Dendrolimus pini. The interaction of the components in mixtures against insects was evaluated and their utility in plant protection was assessed. The crystals and plant substances showed synergistic effect against all insect species. The occurrence of synergism between two components depended on the ratio of their concentration in mixtures. Slight amount of one of the components added to the mixture led to enhanced toxicity of the preparation. The supplementation of mustard oil with B. thuringiensis toxins from up to 0.3% of the mixture resulted in more than twofold higher observed mortality of L1 larvae of D. pini in comparison with expected mortality. The beneficial insecticidal effect of the mustard oil and crystals mixture was also noted when crystals comprised up to 1.3% and from 7 to 20% against L1 caterpillars of C. pomonella and L3 larvae of S. exigua, respectively. The insecticidal efficient effect was achieved when mixture of B. thuringiensis crystals and azadirachtin was used against the three species of lepidopteran pests. The combinations in which components interacted synergistically against caterpillars are potentially useful for making a new bioinsecticide for controlling lepidopteran pests in plant protection.     

Feeding and dispersion of Spodoptera exigua larvae and its relevance for control with a nuclear polyhedrosis virus

On chrysanthemum crops the larvae of the beet armyworm Spodoptera exigua (Hübner) (Lep.: Noctuidae) moved upwards to the top of plants after hatching and predominantly fed on the upper foliage layers. On tomato, however, the larvae did not move upwards and mainly fed on the lower leaves. On chrysanthemums up to the fourth instar most feeding occurred at the underside of foliage while the upper-epidermis remained intact. Larvae dispersing from a single egg batch of 35 eggs damaged about 90 small and 50 tall chrysanthemum plants during their development. The successive larval stages contributed respectively 0.1, 0.4, 4, 20 and 75 percent to the total foliage consumption.The results indicated that the virus preferably should be applied to the lower leaves of tomatoes and chrysanthemums when young instars are present, but to the upper-middle and top leaves of chrysanthemums when the larvae are older than second instars.

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Résumé La consommation et la dispersion des chenilles de Spodoptera exigua sont examinées sur chrysanthèmes et tomates de serre. Les adults déposent les oeufs généralement au feuilles près du sol. Apres l'éclosion les chenilles graduellement se mouvent en haute dans les chrysanthèmes et consomment principalement les feuilles les plus hautes. Dans tomate, cependant, les chenilles s'alimentent principalement au feuilles près du sol.Pendant leur dévelopment les chenilles originaires d'une seule pond de 35 oeufs peuvent endommager environ go petits ou so hautes crysanthè mes. Jusqu'à la quatrième stade larvale les chenilles s'alimentent principalement avec le surface inférieure des feuilles, sans consommer l'épiderme supérieure.Les cinq stades larvales contribuent respectivement 0,1, 0,4, 4, 20 et 75% a la consommation totale des feuilles de chrysanthème.Les resultats des expériments sur la conduite larvale suggestent que les viruses de la polyédrose nucléaire doivent être appliqués préférablement sur la face inférieure des feuilles bas de chrysanthème et de tomate avant que les chenilles se développent a la troisième stade larvale. Quand les larves sur chrysanthèmes sont déja dans le troisième stade larvale, application du virus sur les feuilles plus haute probablement donnait des resultats optimal.

     

Effect of host plants on the infectivity of nucleopolyhedrovirus to Spodoptera exigua larvae

Previous studies have shown that the infectivity of baculovirus to herbivores is affected by phytochemicals ingested during the acquisition of viral inoculum on the foliage of host plants. Here, we measured the effects of 14 host plant species on the infectivity of Spodoptera exigua nucleopolyhedrovirus (SeNPV) to its larvae. The order of the LD₅₀ values of SeNPV among the host plants was Ipomoea aquatica > Brassica oleracea > Raphanus sativus > Amaranthus tricolor > Spinacia oleracea > Vigna unguiculata > Solanum melongena > Capsicum annuum > Apium graveolens > Allium fistulosum > Lactuca sativa > Brassica chinensis > Zea mays > Glycine max, with 940.1 ± 2.26, 424.0 ± 0.60, 295.2 ± 1.13, 147.3 ± 0.63, 138.6 ± 0.22, 119.9 ± 0.07, 119.8 ± 0.02, 109.2 ± 0.18, 104.8 ± 0.62, 102.1 ± 0.66, 97.9 ± 0.22, 89.9 ± 0.32, 79.0 ± 0.13 and 64.0 ± 0.38 OBs per larva, respectively, and the values of mean time to death of virus‐infected larvae were 6.21 ± 0.11, 7.12 ± 0.10, 7.33 ± 0.21, 6.97 ± 0.02, 7.06 ± 0.01, 7.29 ± 0.03, 7.32 ± 0.05, 7.07 ± 0.08, 7.24 ± 0.11, 7.09 ± 0.13, 7.50 ± 0.06, 7.23 ± 0.01, 7.30 ± 0.02 and 7.19 ± 0.07 days, respectively. The mean time to death of larvae decreased with increasing viral dose, and corrected mortality decreased as the larval mean time to death increased. These findings have significance for understanding the effects of host plants on the infectivity of baculovirus to noctuids.     

Toxicity of a <Emphasis Type="Italic">Bacillus thuringiensis israelensis</Emphasis>-like strain against <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis>

Bacillus thuringiensis (Bt) Berliner is a promising agent for microbial control of agriculturally and medically important insects. This study aimed at searching for Bt strains encoding Cry proteins that act more efficiently against fall armyworm. Thirty Bt strains were isolated from soil samples in Pernambuco State and evaluated through bioassays. Among these, strain I4A7 was the most efficient against the fall armyworm, Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera: Noctuidae), and thus it was characterized by biochemical sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and molecular (polymerase chain reaction (PCR) and sequencing reaction) methods. The protein pattern of this strain on a SDS–PAGE was similar to that of B. thuringiensis israelensis (Bti). Moreover, I4A7 cry DNA sequence showed high identity (99–100%) to genes cry4Aa, 4Ba, 10Aa, 11Aa, cyt1Aa and cyt2B from Bti. The toxicity of the newly isolated Bti-like strain upon S. frugiperda should be considered as this strain might be used in combination with other Bt strains, such as B. thuringiensis var. kurstaki (Btk). Handling Editor: Helen Roy.     

Activity and Persistence of Steinernema carpocapsae and Spodoptera exigua Nuclear Polyhedrosis Virus against S. exigua Larvae on Soybean

Experiments were conducted to assess the effects of the entomopathogenic nematode Steinernema carpocapsae and Spodoptera exigua multinucleocapsid nuclear polyhedrosis virus (SeMNPV), alone and in combinations, on mortality of the beet armyworm, S. exigua, larvae on soybean. In 1991 tests, field-grown soybean plants were treated with S. carpocapsae at 0.3 and 0.6 nematodes/cm² of leaflet, SeMNPV at 20 and 40 polyhedral inclusion bodies (PIB)/cm², and all possible combinations. Treated leaflets were collected from plants and bioassayed with 5-day-old larvae. The combination of S. carpocapsae at 0.6 nematodes/cm² + SeMNPV at 40 PIB/cm² produced significantly higher larval mortality (61.7%) compared with either S. carpocapsae (24.8-35.1%) or SeMNPV (26.5-33.7%) alone. In 1992, similar tests were repeated using S. carpocapsae at 0.2 and 0.5 nematodes/cm², and SeMNPV at 14 and 35 PIB/cm². The combination of 0.5 nematodes/cm² + 35 PIB/cm² resulted in significantly higher larval mortality (64.0%) than either pathogen alone (41.5-49.0%). Steinernema carpocapsae and SeMNPV produced additive effects on beet arlnyworm mortality. Persistence of S. carpocapsae was 12-24 hours and SeMNPV was 96-120 hours on soybean.     

Insecticidal activities of Ginkgo biloba seed coat extracts against Spodoptera exigua (Lepidoptera: Noctuidae)

The present study relates to a methanol extract of the seed coat of Ginkgo biloba, and tested particularly on the third instar larvae of Spodoptera exigua. The extract was found to have an inhibitory effect on the growth of the larvae besides bringing a change in the nutrient reserves in the body of the insect. Topical application of five different doses of the methanol extract resulted in a mortal effect to third instar larvae of S. exigua that is very much dependent on the dose as well as duration of exposure. Lower doses revealed lower mortality after 24 h of application. At doses of 1.00, 2.00, 4.00, 8.00 and 16.00 ng/larva, mortalities were 9.25, 26.07, 50.32, 56.28 and 92.44%, respectively. The dose for 50% mortality (LD₅₀) of methanol extracts by applied by a topical method with 1 µL of acetone solution was 1.92 ng/larva. Nutrient reserves like protein, glycogen and lipid are known to regulate pupation and adult emergence. These reserves have been found to be lower in treated larvae, indicating the insecticidal role of methanol extracts from G. biloba against third instar larvae of S. exigua.     

A proteomic analysis approach to study insecticidal crystal proteins from different strains of Bacillus thuringiensis

We obtained and compared a new cry2Ac6 gene from Bacillus wuhanensis 140, and Bacillus thuringiensis (Bt) subsp. kurstaki 4.0718 and B.t. kurstaki XL004 that share a similar genetic background but occupy different ecological niches. Using a proteomic approach and function-based activity profiling, we systemically identified the insecticidal crystal proteins (ICPs) from the three Bt species, which were found to be mainly distributed at pH 4–7 on two-dimensional electrophoresis (2DE) gels by PDQuest software. The proteins that exhibited a significant difference in expression were excised, digested in-gel and identified by MALDI-TOF-MS. Thirty-three differently expressed proteins were identified from the three Bt strains. The Cry2Ac6, Cry1Ab16, CryIG, CryH2, CryI, CryINA67-1 and CryI⁺ crystal protein mixture from B.t. wuhanensis 140, Cry1Ac, Cry2Aa and endotoxin delta1 from B.t. kurstaki 4.0718 were further analyzed by bioinformatics analysis. Two common proteins were founded in three strains, the heat shock proteins (HSP60) and the translation elongation factor Tu, which help with protein refolding and prevent protein degradation. The different enzymes of metabolism, including glutamate racemase, chemotaxis protein histidine kinase and related kinases pyruvate dehydrogenase complex E1orE3 were identified. Some protein spots could not be identified. The results indicate that each Bt strain has unique ICPs as well as some common proteins related to ICPs formation, and that the virulence of Bt strains is closely related to the expression of specific ICPs.     

Cloning and characterisation of a novel holotype crystal protein gene,cry59Ba1, from Bacillus thuringiensis strain Bm59-2, toxic to Spodoptera exigua (Lepidoptera)

Abstract

A novel cry59-type gene, cry59Ba1, was obtained from isolate Bm59-2 and identified from an assembled plasmid genome sequence. This gene was found to encode a polypeptide of 674 amino acid residues with a predicted molecular mass of 75.2 kDa. This polypeptide was 62.1% identical to cry59Aa1. The Cry59Ba1 protein was expressed in the acrystalliferous mutant strain HD73^? and tested against Culex quinquefasciatus (Diptera), Spodoptera exigua (Lepidoptera) and Helicoverpa armigera (Lepidoptera). The bioassay showed Cry59Ba1 protein to be highly toxic to S. exigua (Lepidoptera) (LC50 =26.2 µg/ml, 95% confidence limit, 16.2-75.3 µg/ml). The cloning of cry59Ba1 gene may provide a novel type insecticidal resource for resolving the problem of lepidopteran insects developing resistance to the Cry1 proteins.     

Compatibility and interaction between Bacillus thuringiensis and certain insecticides: perspective in management of Tuta absoluta (Lepidoptera: Gelechiidae)

Bacillus thuringiensis var. kurstaki (Berliner) (Bt) has been suggested as a biological control agent for Tuta absoluta (Meyrick). The objective of this study was to determine the interaction between abamectin, azadirachtin, indoxacarb, chlorantraniliprole, dichlorvos and metaflumizone with Bt. Effect of recommended doses of the chemical insecticides on colonisation of Bt was also investigated in culture medium. Except for metaflumizone, none of the chemicals tested reduced the colonisation of Bt compared with control. Interaction between Bt and the chemical insecticides on 2nd-instar larvae was also assessed. In interaction tests, Bt was applied at LC₅₀ level, 0, 12, 24 or 36 h after treating the larvae with LC₁₀ or LC₂₅ of the chemical insecticides. An antagonistic effect was observed in all treatments where Bt was applied immediately after the chemical insecticide. Also, antagonism was observed when treatment with Bt was done 12 h after azadirachtin and metaflumizone applications. Applying Bt 12 and 24 h after treatment with LC₂₅ of chlorantraniliprole, dichlorvos and abamectin resulted in synergism. But, synergism with LC₁₀ of dichlorvos and abamectin was observed only after 12 h. Additive effect was observed in the rest of the time and concentration combinations. Based on the results obtained, simultaneous use of the chemical insecticides tested and Bt is not recommended for T. absoluta control; and an appropriate time interval should be taken into consideration accordingly.     

Improved production of insecticidal proteins in Bacillus thuringiensis strains carrying an additional cry1C gene in its chromosome

A cryIC gene, whose product is active against Spodoptera exigua, was introduced into wildtype Bacillus thuringiensis kurstaki strain YBT1520 using an integrative and thermosensitive vector, pBMB-FLCE, which was developed based on B. thuringiensis transposon Tn4430 harboring a tnpI-tnpA gene. With the mediation of TnpI-TnpA, the cry1C gene was integrated into the chromosome of the host strain. To prevent secondary integration, the integrative vector was eliminated by moving recombinant cultures to 46 degrees C for generations. Two integrative recombinant B. thuringiensis strains BMB1520-E and BMB1520-F were obtained. In recombinant BMB1520-F, the cry1C gene was expressed stably at a significant level and did not reduce the expression of endogenous crystal protein genes. Bioassay results indicated that BMB1520-E and BMB1520-F showed a higher level of activity against S. exigua third-instar larvae than did their parent strains, in addition to the high toxicity to Plutella xylostella third-instar later larvae.