Impact of Bacillus thuringiensis strains on survival,reproduction and foraging behaviour in bumblebees (Bombus terrestris)

BACKGROUND: Bacillus thuringiensis (Bt) and its protein crystals are used worldwide, either as a spray or when expressed in transgenic crops, for the control of pest insects. However, owing to their intensive use, there exists a debate regarding the involvement of this microbial insecticide in bee colony losses. In this study, in a tiered approach using laboratory microcolonies, an evaluation was made of the potential lethal and sublethal hazards on colony reproduction and foraging behaviour of workers of the bumblebee Bombus terrestris (L.) of two commercial Bt strains: kurstaki (Dipel^®) and aizawai (Xentari^®). Bumblebees, like honey bees, are intensively used in modern agriculture for pollination and fulfil a crucial role in the natural ecosystem. RESULTS: Exposure of bumblebees dermally or via treated pollen to either of the two Bt formulations at their field recommended rates (0.1%) caused no reduction in survival. However, when applied in the feeding sugar water, aizawai killed all workers at a concentration of 0.1%, but this lethal effect was lost at 0.01%. With respect to reproductive effects, kurstaki was harmless, while aizawai at 0.1% delivered in the feeding sugar water and pollen reduced reproduction by 100 and 31% respectively. Lower doses of 0.01% aizawai in the sugar water showed no more effect. In addition, kurstaki at 0.1% and aizawai at 0.01% in the feeding sugar water did not impair the foraging behaviour, resulting in normal nest colony performance. CONCLUSION: The results with kurstaki and aizawai demonstrated that, in general, the Bt strains are safe to B. terrestris bumblebees, although in some cases there were detrimental effects that depended on strain and route of exposure. In addition, the authors believe that to draw firm conclusions regarding the hazards of Bt to bumblebees would require more information on relevant concentrations of Bt products in the environment. Hence, routine testing for lethal and sublethal effects is recommended to ascertain combined use of Bt products and bumblebees in modern agriculture. Copyright © 2009 Society of Chemical Industry     

Susceptibility pattern and development of resistance in the diamondback moth,Plutella xylostella L,to Bacillus thuringiensis Berl var kurstaki in India

Base‐line susceptibility for six‐day‐old larvae of the diamondback moth, Plutella xylostella, against Bacillus thuringiensis var kurstaki (Biobit^®) was studied by a cabbage leaf disc dip bioassay technique. Diamondback moth from 13 locations in seven different states spread over a distance of about 3000 km longitudinally was used for these studies. Forty‐eight‐hour LC₅₀ values varied from 1.0 to 10.97 mg AI litre⁻¹. Further investigations on the development of resistance under laboratory conditions showed an increase in LC₅₀ from 2.76 (for unselected F₁ generation) to 5.28 mg AI litre⁻¹ (for selected F₉ generation), using a selection concentration of 6.4 mg AI litre⁻¹. This suggested a possibility of the development of resistance under field conditions if there were to be extensive and indiscriminate use of B thuringiensis. These findings are discussed in relation to integrated pest management and the mechanisms of resistance in resistance management tactics. © 2000 Society of Chemical Industry     

Differential toxicity of Bacillus thuringiensis strains and their crystal toxins against high-altitude Himalayan populations of diamondback moth, Plutella xylostella L

BACKGROUND: Diamondback moth, Plutella xylostella (L.), is a major insect pest of crucifers in the biodiversity‐rich north‐western Indian Himalayan hills. The present investigation was aimed at determining the susceptibility pattern of P. xylostella populations collected from different locations of this region to autochthonous and standard Bacillus thuringiensis strains. RESULTS: Among the reference as well as indigenous B. thuringiensis strains tested, sub spp. kurstaki HD‐1, kurstaki HD‐73, galleriae HD‐8, local galleriae/colmeri strain BtOa1 and some of their Cry1 class toxins were found to be highly toxic. Surprisingly, the sub sp. tolworthi HD‐125, local tolworthi strain BtHa1 and Cry9 class toxins were found to be non‐toxic. Midgut homogenate from fourth‐instar larvae was found to activate 130 kDa protoxin from the local tolworthi strain BtHa1 into 68 kDa toxin, but failed to exert any larval mortality, probably owing to lack of receptor binding. CONCLUSION: The present study provides valuable baseline susceptibility data for the deployment of B. thuringiensis‐based control methods, as well as for future monitoring of development of resistance in P. xylostella to B. thuringiensis in this ecologically sensitive region. Copyright © 2008 Society of Chemical Industry     

Milling effect on the control efficacy of spray-dried Bacillus thuringiensis technical powder against diamondback moths

BACKGROUND: The relationship between the particle size of spray‐dried Bacillus thuringiensis NT0423 technical powder and its insecticidal activity against diamondback moth was investigated in laboratory and glasshouse conditions. A variety of sizes of Bt particles were made by homogenisation, followed by pilot‐scale milling to optimise further the current Bt formulation process. RESULTS: In laboratory conditions, homogenised Bt suspension with smaller particles had higher control efficacy against diamondback moth. However, in glasshouse conditions, the smallest particle treatment did not follow the size‐dependent insecticidal activity, rather showing decreased control efficacy. Secondly, air‐jet‐milled Bt powder (16.9 µm) had higher control efficacy than hammer‐milled Bt powder (35.1 µm) in glasshouse conditions. However, double air‐jet milling (5.3 µm) had lower control efficacy compared with one‐time milling. CONCLUSION: These results suggest that NT0423 technical powder with smaller particles is better in controlling diamondback moths, but excessively small particles (<10 µm) possibly reduce the insecticidal activity. Copyright © 2012 Society of Chemical Industry     

Control of the honeydew moth,Cryptoblabes gnidiella Mill. (Lepidoptera: Phycitidae), withBacillus thuringiensis berliner in avocado plantations

Field trials were conducted in 1971–1974 to control the honeydew moth,Cryptoblabes gnidiella Mill, withBacillus thuringiensis. Dipel, a commercial preparation containingBacillus thuringiensis var.kurstaki (HD-1 strain), was highly effective (at concentrations ranging from 0.25% to 1.5%) in killing caterpillars of all developmental stages. In both the field trials and additional laboratory trials, it had no adverse effect on the mealybugPseudococcus longispinus Targ.-Tozz., or on its parasites,Hungariella peregrina Compere andAnagyrus fusciventris Girault.     

Characterisation and toxicity of Bacillus thuringiensis strains from hazelnut pests and fields

BACKGROUND: In order to find and identify more toxic insecticidal Bacillus thuringiensis Berliner (Bt) strains, a survey was carried out of B. thuringiensis isolate pests belonging to Coleoptera, Lepidoptera and Diptera and from soils in hazelnut fields. Of 16 isolates having Bacillus cereus‐B. thuringiensis morphology, eight were classified as B. thuringiensis because of the production of parasporal δ‐endotoxin crystals. RESULTS: In this study, eight isolates of B. thuringiensis from hazelnut pests (isolates Bn1, Mm2, Mnd and Xd3) and from hazelnut soils (isolates 6, 27, 40 and 46) have been characterised in detail. These isolates were compared with reference strains by electron microscopy, SDS‐PAGE analysis, cry gene content, serological test and insecticidal activity. CONCLUSION: Results indicate that Bn1 and MnD are B. thuringiensis subsp. kurstaki, and Mm2 and Xd3 are B. thuringiensis subsp. tenebrionis. In addition, isolate 6 is B. thuringiensis subsp. israelensis, isolates 27 and 46 are B. thuringiensis subsp. kumamotoensis and isolate 40 is B. thuringiensis subsp. indiana. The four B. thuringiensis isolates from hazelnut pests may be valuable as biological control agents against coleopteran and lepidopteran insects. Copyright © 2009 Society of Chemical Industry     

Mode of inheritance and stability of resistance to Bacillus thuringiensis var kurstaki in a diamondback moth (Plutella xylostella) population from Malaysia

Genetic inheritance of resistance to Bacillus thuringiensis var kurstaki (BTK) was examined in a diamondback moth (Plutella xylostella) population collected from the Melaka region of Malaysia. A BTK‐selected sub‐population (BTK‐SEL) which was more than 100‐fold resistant to BTK compared with a susceptible (ROTH) population of P xylostella was used with standard reciprocal crosses and back‐crosses between ROTH and BTK‐SEL. Logit regression analysis of F ₁ reciprocal crosses indicated that BTK resistance was inherited as an incompletely recessive autosomal trait and controlled by a single locus. In contrast, other studies have shown that resistance to Cry1Ac is inherited as an incompletely dominant autosomal trait in a Cry1Ac‐selected sub‐population of the same Melaka population. The frequency of the allele responsible for resistance decreased without exposure to insecticide in the laboratory. © 2000 Society of Chemical Industry     

Evidence for Resistance to Bacillus thuringiensis (Bt) subsp. kurstaki HD-1, Bt subsp. aizawai and Abamectin in Field Populations of Plutella xylostella from Malaysia

The efficacy of Bacillus thuringiensis (Bt) subsp. kurstaki HD-1 (‘Dipel’™; Btk; CryIA & CryII) and Bt. subsp. aizawai (‘Florbac’™; Bta; CryIA & CryIC) was assessed against larvae from various field populations of Plutella xylostella (F2 generation) collected in the Cameron Highlands, Malaysia in April 1994 and a lowland population (SERD 2; F10 generation) collected in December 1993. Evidence of resistance to Btk and to a lesser extent Bta is reported in these populations (LC₅₀ Toxicity Ratios [TR]=3–14 and 2–8 respectively), most notably in SERD 2. The first recorded evidence of resistance to abamectin (TR=17–195-fold) in field populations of P. xylostella is also reported. In an unselected sub-population of SERD 2, the TR values for Btk, Bta and abamectin declined 2- to 3-fold (P<0·01) over six generations in the laboratory (F10–F16) while in sub-populations of SERD 2 selected with these products (F11–F15) there was a significant (P<0·01) increase in the TR (15-, 3- and 2·5-fold respectively) when compared with the F10 generation. This suggests the presence of marked resistance to Btk and some resistance to Bta and abamectin. There is also evidence of slight cross-resistance to Btk in the Bta-selected sub-population but no evidence for the reverse selection of resistance or for cross-resistance between Btk and abamectin. Concurrent selection studies (F11–F15) with another sub-population of SERD 2 demonstrated resistance to the acylurea insect growth regulator, teflubenzuron (‘Nomolt’™) (29-fold increase in TR). Based on the selection experiments with SERD 2, estimates of realised heritability (h²) of resistance gave very high values for teflubenzuron and Btk (c.0·7) and moderate values for abamectin and Bta (c.0·3). The results are discussed in relation to integrated pest management (IPM) and insecticide resistance management (IRM) strategies for P. xylostella.     

Factors in the Success and Failure of Microbial Insecticides in Vegetable Crops

A search for patterns in the success and failure of microbial insecticides in vegetable crops was conducted through review of four case studies: the use of Bacillus thuringiensis (B.t.) var. tenebrionis for control of the Colorado potato beetle, the use of B.t. var. kurstaki for control of the diamondback moth, the use of various B.t.s for control of lepidopterous pests in tomatoes and celery, and the use of a granulosis virus for control of potato tuber moth. With success defined in terms of achievement of technical goals (efficacy), commercial goals (end-user and insecticide manufacturer satisfaction) and social, or public goals (environmental and health safety), only certain of the case studies could be judged a success. These successes shared a variety of features including: (1) use of the microbial insecticide as a component, rather than as the sole agent, in an integrated crop management program; (2) unavailability of conventional insecticides, due to insecticide resistance, lack of registered products or mandatory IPM programs, provided incentive for the use of microbial insecticides; (3) modification of the expectation that microbial insecticides will perform within the chemical paradigm – fast, lethal and on contact; (4) exploitation of all possible benefits of the microbial insecticide, including safety to natural enemies, as well as efficacy against the target insect, and (5) support from large private and public institutions in the form of research, grower education, scouting programs, subsidized production, and economic and legal incentives to the use of microbial insecticides.     

Screening ofbacillus thuringiensis preparations for microbial control ofostrinia nubilalis in sweet corn

Bacillus thuringiensis Berliner (B.t.) preparations were bioassayed against larvae of the European corn borer,Ostrinia nubilalis Hübner, to determine their potency.B.t. serovar 3a3b,kurstaki (Dipel) was more potent thanB.t. serovar 1,thuringiensis (Bactospeine I);B.t. serovar 3a3b,kurstaki (Toarow CT) — in which the spores are inactive; or serovar 7,aizawai (ABG 6104). Another preparation of 3a3b,kurstaki (Bactospeine III), at concentrations of 0.5 and 1% sprayed on leaves of potted sweet corn seedlings and plants in the field, was active against larvae reared on an artificial diet in the laboratory. The pH of the corn leaf surface ranged from 6.9 to 7.5, before and after application.     

Entomopathogenic nematodes (Nematoda: Steinernematidae, heterorhabditidae) as control agents forParahypopta caestrum, a pest in the culture ofAsparagus officinalis

The entomopathogenic nematodes (EPNs)Steinernema feltiae (under the trade name Entonem) andHeterorhabditis bacteriophora (under the trade name Larvanem) were evaluated as potential control agents ofParahypopta caestrum, the major pest of the asparagus crop in Greece. In laboratory experiments the two nematodes provided equal insect suppression, but significant differences were found in the time it took them to kill the larvae.S. feltiae caused high levels of mortality within 24 h and the highest level at 48 h. In contrast,H. bacteriophora required 96 h to achieve the highest level of mortality. In field experiments, the nematodes provided equal insect suppression as compared with the insecticide cadusaphos and the mixture teflubenzuron +Bacillus thuringiensis var.kurstaki. No significant differences were found in the effectiveness of the insecticides used, but there were significant differences between the control and treatments. The findings showed thatS. feltiae andH. bacteriophora could be used to control the insectP. caestrum in asparagus culture. http://www.phytoparasitica.org posting Jan. 10, 2007.     

An effective strain ofBacillus thuringiensis against fourth-instar caterpillars ofBoarmia selenaria

A strain ofBacillus thuringiensis Berliner effective against fourth-instarBoarmk selenaria Schiffermüller (Lepidoptera: Geometridae) was found. Strain HD-251 of the varietykurstaki H-serotype 3a, 3b was more effective than the standard HD-1-S-1980 in laboratory bioassays. The potency of HD-251 is two to four times higher than that of the standard, and varies from 19,821 to 68,645 IU/mg. Two other strains of the varietyaizawai H-serotype 7,viz., HD-249 and T63L4, were inferior to HD-251, with potencies of 6,982 and 13,714 IU/mg, respectively, against first-instar larvae.     

Effect of Bacillus thuringiensis var. kurstaki on survival and mortality of immature and mature stages of Diadegma insulare parasitizing Plutella xylostella

Effects of Bacillus thuringiensis (Bt) on immature stages of Diadegma insulare (Cresson) (Hym., Ichneumonidae) within larvae of diamondback moth (DBM), Plutella xylostella (L.) (Lep., Plutellidae), and on the adult stage as a Bt-honey solution were evaluated at 25?±?1?°C, 65?±?10% r.h. and 16:8?h (L:D) photoperiod. P. xylostella and D. insulare pupae were collected from cabbage fields in Karaj, Iran. A commercial wettable powder formulation of Bt (var. kurstaki, serotype H-3a3b, strain Z-52) was used in these experiments. The value of LC₅₀ for third instar larvae of P. xylostella was 210?ppm. Parasitoid??s adult mortality at field rate of Bt was not significantly different from that of control. Formation of parasitoid??s pupae in Bt-parasitoid combined treatments was significantly lower than parasitoid alone. Formation of parasitoid??s pupae in larvae treated with LC₁₀, LC₃₀ and LC₅₀ of Bt and then exposed to parasitoids after 48?h was 5%, 3% and 2%, respectively, which was significantly lower than that of the parasitoid alone (41%). Mean percentage mortality of larvae of DBM in LC₃₀ and LC₅₀ concentrations of Bt combined with the parasitoid was approximately 100%. Our results showed that B. thuringiensis kills D. insulare larvae indirectly by killing susceptible hosts in which they are developing. Successful use of multiple biological control agents will depend on close monitoring of their compatibility in the field. Our results could be useful in this purpose.     

Bt maize and integrated pest management ‐ a European perspective

The European corn borer (Ostrinia nubilalis), the Mediterranean corn borer (Sesamia nonagrioides) and the western corn rootworm (Diabrotica virgifera virgifera) are the main arthropod pests in European maize production. Practised pest control includes chemical control, biological control and cultural control such as ploughing and crop rotation. A pest control option that is available since 1996 is maize varieties that are genetically engineered (GE) to produce insecticidal compounds. GE maize varieties available today express one or several genes from Bacillus thuringiensis (Bt) that target corn borers or corn rootworms. Incentives to growing Bt maize are simplified farm operations, high pest control efficiency, improved grain quality and ecological benefits. Limitations include the risk of resistance evolution in target pest populations, risk of secondary pest outbreaks and increased administration to comply with licence agreements. Growers willing to plant Bt maize in the European Union (EU) often face the problem that authorisation is denied. Only one Bt maize transformation event (MON810) is currently authorised for commercial cultivation, and some national authorities have banned cultivation. Spain is the only EU member state where Bt maize adoption levels are currently delivering farm income gains near full potential levels. In an integrated pest management (IPM) context, Bt maize can be regarded as a preventive (host plant resistance) or a responsive pest control measure. In any case, Bt maize is a highly specific tool that efficiently controls the main pests and allows combination with other preventive or responsive measures to solve other agricultural problems including those with secondary pests. Copyright © 2011 Society of Chemical Industry     

Testing insecticide resistance management strategies: mosaic versus rotations

BACKGROUND: Developing scientifically valid, economically acceptable insecticide resistance management (IRM) programs is critical for sustainable insect management. The diamondback moth, Plutella xylostella (L.), has demonstrated an ability to develop resistance to many different classes of insecticides, including proteins produced by the bacterium Bacillus thuringiensis Berliner (Bt). Recently it has developed resistance to the novel compounds spinosad and indoxacarb. In greenhouse cage experiments, a laboratory‐selected population of P. xylostella resistant to spinosad, indoxacarb and Bt was used to compare population growth and resistance evolution if these three insecticides were rotated or used in a mosaic fashion. RESULTS: The average population density through nine generations was lowest in the treatment in which the insecticide was rotated every generation (R‐1) (x? = 20.7 ± 3.20) compared with the treatment in which the insecticide was rotated every third generation (R‐3) (x? = 41.4 ± 17.6) or where the insecticides were applied as a mosaic (M) (x? = 41.8 ± 6.53). After nine generations, the survival of resistant individuals increased for each insecticide (7.2–73.5%) compared with the population without selection (CK) (0.73–3.1%). Survival on spinosad was significantly lower (23.7%) in the single‐generation rotation than for the other two treatments, both of which exceeded 72%. The calculated survival on all three insecticides treated simultaneously, according to the survival on each insecticide, was 0.26, 0.81 and 1.6% for R‐1, R‐3 and M treatments respectively. CONCLUSION: Results of both population density and resistance development indicated that insecticide rotation every generation was better for IRM than if the insecticide was rotated every third generation or if the three insecticides were applied as a mosaic. Copyright © 2010 Society of Chemical Industry     

Differing susceptibility to<Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> formulations of<Emphasis Type="Italic">Thaumetopoea wilkinsoni</Emphasis> populations between forests with different<Emphasis Type="Italic">Bt</Emphasis> management in Israel

The susceptibility ofThaumetopoea wilkinsoni larvae toBacillus thuringiensis (Bt) formulations was screened in 2003 and 2004. Eggs and larvae were collected from pine forests in 11 geographical locations in Israel. Larval mortality bioassays were conducted with commercial formulations (Delfin WG, Dipel DF and Foray 48B) at concentrations ranging from 0.001% to 0.1%. Significant differences in susceptibility toBt were recorded among populations that were treated withBt intensively, frequently, or never. The mortality recorded in a population that was never treated withBt was twice that in an intensivelyBt-treated population. The correlation between susceptibility toBt and the possible resistance to the microbe is discussed. http://www.phytoparasitica.org posting Jan. 31, 2007.     

The Provision and Subsequent Management of Plant Material for Herbicide Pot Experiments

Abstract

The marked resistance of Spodoptera spp. to endotoxins of Bacillus thuringiensis including the well recognized standards HD‐1–1971 and HD‐1‐S‐1980 has necessitated the search for a new active strain that can be used as a standard reference in the bioassays of these lepidopterous insects. The high activity of B. thuringiensis var. entomocidus HD‐635 vs. Spodoptera littoralis and S. exigua was found to fulfill these requirements. The sporeendotoxin complex of this strain, designated as HD‐635‐S‐1987, is proposed as a reference standard for bioassays against Spodoptera spp. and is assigned arbitrarily the value of 10,000 iu/mg. Potencies of an active strain, i.e. B. thuringiensis var. aizawai HD‐133 and of a low‐active preparation, i.e. HD‐1‐S‐1980 were carried out in reference to the newly proposed standard. Comparative studies revealed the possible reduction of bioassay period to 4 days as time saving and for a priori judgement on the activity of certain bioinsecticide preparations derived from B. thuringiensis.     

Greenhouse trials of newBacillus thuringiensis isolates for control ofChilo partellus larvae in sorghum

Eight newBacillus thuringiensis Berliner (Bt) isolates originally recovered from different source materials and geographic locations in Kenya were tested againstChilo partellus (Swinhoe) on sorghum grown in a greenhouse. All isolates had been demonstrated previously to be toxic to this pest.Bt strains were cultured in a liquid growth medium and preserved as powders for experimental application. Three-week-old sorghum plants (4-6 leaves) were artificially infested with 20 neonateC. partellus and sprayed 24 h later withBt suspensions. Non-infested sorghum and infested non-treated sorghum served as the comparative checks within each experimental trial. Plant development and dead-heart formation were monitored up to harvest; at harvest, grain yield was recorded. In each replicate trial, all of theBt strains tested reduced plant damage consistently, compared with the infested, non-treated checks. Dead-heart formation was significantly reduced and grain yields were significantly higher. Three novel isolates, A-3, A-C-2 and M44-2, provided superior levels of protection throughout. Statistically, there was no difference among these threeBt treatments and the non-infested check in terms of the number of dead-hearts (i.e., non-yielding plants) that formed during the course of the experiment. In the infested, non-treated check plots, dead-hearts developed in 74.5±10.0% of the plants, compared with only 3.1±1.2% of the non-infested plants, and 3.1 ±1.5%, 6.2±2.0% and 6.2±2.0% of the plants treated with M44-2, A-3 and A-C-2, respectively. At harvest, the weight of grain obtained from the non-infested control plots was considered to be 100% of the potential yield for each experimental replicate. In the infested, non-treated controls, only 14±5.6% of the potential yield was realized. When isolates M44-2, A-3 and A-C-2 were applied, proportionate yields of 83±5.4%, 93±2.3% and 98±1.2%, respectively, were obtained. Results showed that theBt strains selected may have a strategic role to play in the management ofC. partellus, providing applications are timed to target young larvae feeding in the leaf whorl.     

Development and characterisation of transgenic rice expressing two Bacillus thuringiensis genes

BACKGROUND: Transgenic crops that produce insecticidal toxins from the bacterium Bacillus thuringiensis Berliner (Bt) were first commercialised in 1996. The risk that pests have the potential to evolve resistance to Bt toxins is one of the most serious challenges to this technology. Gene stacking, pyramiding two Bt genes into one variety, is considered to be an effective insect resistance management (IRM) strategy. In this study, insect‐resistant rice expressing two Bt genes was developed by sexual crossing, and then characterised. RESULTS: Homozygous rice lines of two pyramided Bt genes were obtained in the F₃ generation. Quantification of Bt toxin showed that protein concentrations of Cry1Ab, Cry1Ac and Cry2A in the two‐gene lines were comparable with their single‐gene parents, while the expression of cry1C gene decreased after gene stacking. Four two‐gene lines showed higher activity to striped stem borer (Chilo suppressalis Walker) than parental lines in the laboratory bioassay. All pyramided lines and their hybrids exhibited excellent efficacy against stemborers and leaffolders in field evaluation, while most pyramided lines had no significant differences from original variety in yield under spraying of insecticide. CONCLUSION: These results demonstrate that the two‐gene lines have commercial potential and could serve as a valuable IRM strategy. Copyright © 2011 Society of Chemical Industry     

F2 screening for resistance to pyramided Bacillus thuringiensis maize in Louisiana and Mississippi populations of Diatraea saccharalis (Lepidoptera: Crambidae)

BACKGROUND: In the mid‐southern region of the United States, sugarcane borer, Diatraea saccharalis (F.), is a major target pest of transgenic maize expressing Bacillus thuringiensis (Bt) proteins. Novel transgenic maize technologies containing two or more pyramided Bt genes for controlling lepidopteran pests have recently become commercially available. Insect resistance management (IRM) is an important issue in the sustainable use of Bt crop technologies. The objective of this study was to determine the frequency of resistance alleles in field populations of D. saccharalis to the new pyramided Bt maize technologies. RESULTS: A total of 382 F₂ family lines derived from 735 feral larvae/pupae of D. saccharalis collected from six locations in Louisiana and Mississippi during 2008 and 2009 were screened for resistance to three new Bt maize technologies: MON 89034, Genuity? VT Triple Pro? and SmartStax?. None of the 382 F₂ isoline families survived on the Bt maize leaf tissue for ≥ 12 days in the F₂ screen. The joint frequency for two‐ or three‐gene resistance models with 95% probability in these populations was estimated to be < 0.0063 to MON 89034 and < 0.003 to VT Triple Pro? and SmartStax?. CONCLUSION: These results suggest that the resistance allele frequency in D. saccharalis to the three pyramided Bt maize technologies is low in the mid‐southern region of the United States, which should meet the rare resistance assumption of the currently used IRM strategy for Bt maize. Copyright © 2011 Society of Chemical Industry