Bt Strains Research Articles

Engineering of <i>Bacillus thuringiensis</i> insecticidal proteins

Bacillus thuringiensis (Bt) has been used as sprayable pesticides for many decades. Bt strains utilized in these products produce multiple insecticidal proteins to complement a narrow insect specificity of each protein. In the late 1990s, genes encoding Bt insecticidal proteins were expressed in crop plants such as cotton and corn to protect these crops from insect damage. The first Bt protein used in transgenic cotton was Cry1Ac to control Heliothis virescens (tobacco budworm). Cry1Ab was applied to corn to control Ostrinia nubilalis (European corn borer). Since these insects have developed resistance to Cry1Ac and Cry1Ab, new Bt proteins are required to overcome the resistance. In order to protect corn furthermore, it is desired to control Diabrotica virgifera (Western corn rootworm), Helicoverpa zea (corn earworm) and Spodoptera frugiperda (fall armyworm). Recently, many new Bt insecticidal proteins have been discovered, but most of them require protein engineering to meet the high activity standard for commercialization. The engineering process for higher activity necessary for Bt crops is called optimization. The seed industry has been optimizing Bt insecticidal proteins to improve their insecticidal activity. In this review, several optimization projects, which have led to substantial activity increases of Bt insecticidal proteins, are described.

Isolation and molecular characterization of Bacillus thuringiensis subsp. kurstaki toxic to lepidopteran pests Spodoptera spp. and Plutella xylostella.

Bacillus thuringiensis (Bt) has been widely used as a biological control agent for lepidopteran pests. However, resistance to Bt is a major concern associated with Spodoptera spp. (Noctuidae) and Plutella xylostella (Plutellidae). For efficient control of Noctuidae and Plutellidae, novel Bt strains which have high toxicity and a broad host range are needed. To develop novel Bt strains as used for bio-insecticides, the Bt IMBL-B9 with high toxicity against Spodoptera exigua, Spodoptera frugiperda and P. xylostella was isolated and characterized. The Bt kurstaki IMBL-B9 strain produced bipyramidal and cuboidal crystals consisting of cry toxins with molecular weights of 130 and 65 kDa, respectively. This strain harbors eight crystal protein genes in total, including cry1Ea and one vegetative insecticidal protein gene. The median lethal concentration (LC50 ) values of IMBL-B9 against S. exigua and S. frugiperda were 21.8- and 19.3-fold lower than those of the Bt kusrstaki strain, and 5.6- and 4.9-fold lower than those of Bt aizawai strain, respectively. To evaluate the insecticidal activity of Cry proteins from IMBL-B9, cry gene-sourced recombinant Bt strains were constructed. These strains have insecticidal activity and synergic action against lepidopteran pests. In this study, a novel Bt kurstaki IMBL-B9 strain was isolated and this could be useful for the development of new bio-insecticide or cry gene-based recombinant products as an alternative solution against lepidopterans, including Noctuidae and Plutellidae. © 2022 Society of Chemical Industry.

Oral Immune Priming Treatment Alters Microbiome Composition in the Red Flour Beetle Tribolium castaneum.

It is now well-established that the microbiome is relevant for many of an organism’s properties and that its composition reacts dynamically to various conditions. The microbiome interacts with host immunity and can play important roles in the defenses against pathogens. In invertebrates, immune priming, that is, improved survival upon secondary exposure to a previously encountered pathogen, can be dependent upon the presence of the gut microbiome. However, it is currently unknown whether the microbiome changes upon priming treatment. We here addressed this question in a well-established model for immune priming, the red flour beetle Tribolium castaneum exposed to the entomopathogenic bacterium Bacillus thuringiensis (Bt). After priming treatments, the microbiota composition of beetle larvae was assessed by deep sequencing of the V1-V2 region of the bacterial 16S rRNA gene. We compared the effect of two established routes of priming treatments in this system: injection priming with heat-killed Bt and oral priming via ingestion of filtered sterilized bacterial spore culture supernatants. For oral priming, we used several strains of Bt known to vary in their ability to induce priming. Our study revealed changes in microbiome composition following the oral priming treatment with two different strains of Bt, only one of which (Bt tenebrionis, Btt) is known to lead to improved survival. In contrast, injection priming treatment with the same bacterial strain did not result in microbiome changes. Combined with the previous results indicating that oral priming with Btt depends on the larval microbiome, this suggests that certain members of the microbiome could be involved in forming an oral priming response in the red flour beetle.

PHB2 affects the virulence of Vip3Aa to Sf9 cells through internalization and mitochondrial stability

ABSTRACT The vegetative insecticidal proteins (Vip3A) secreted by some Bacillus thuringiensis (Bt) strains during vegetative growth are regarded as a new generation of insecticidal toxins. Like insecticidal crystal proteins, they are also used in transgenic crops to control pests. However, their insecticidal mechanisms are far less defined than those of insecticidal crystal protein. Prohibitin 2 (PHB2) is a potential Vip3Aa binding receptor identified from the membrane of Sf9 cells in our previous work. In this paper, we demonstrated the interaction between Vip3Aa and PHB2 using pull-down, dot blotting, microscale thermophoresis, and co-immunoprecipitation assays. PHB2 is distributed on the cell membrane and in the cytoplasm, and the co-localization of PHB2 and Vip3Aa was observed in Sf9 cells using a confocal laser scanning microscope. Moreover, PHB2 could interact with scavenger receptor-C via its SPFH (stomatin, prohibitin, flotillin, and HflK/C) domain. Downregulation of phb2 expression reduced the degree of internalization of Vip3Aa, exacerbated Vip3Aa-mediated mitochondrial damage, and increased Vip3Aa toxicity to Sf9 cells. This suggested that PHB2 performs two different functions: Acting as an interacting partner to facilitate the internalization of Vip3Aa into Sf9 cells and maintaining the stability of mitochondria. The latter has a more important influence on the virulence of Vip3Aa.

Characterization of insecticidal Cry1Cb2 protein from Bacillus thuringiensis toxic to Myzus persicae (Sulzer)

The toxins produced by Bacillus thuringiensis (Bt) are well known for their insecticidal activity against Lepidoptera, Diptera and Coleoptera; however, the sap-sucking insects (Hemiptera) are not particularly susceptible to Bt toxins. We describe the aphicidal effect of Cry toxin from Bt strain GP919 against one of the most pernicious hemipterans in the agricultural environment, Myzus persicae. The mortality bioassay shows that the strain cause mortality rates above 80% at concentration of 10 ng/µl with a LC50 of 9.01 ng/µl; whereas it showed no lethal toxicity against the lepidopteran Spodoptera frugiperda. The mayor protein (∼130 kDa) expressed by this strain was subjected to purification, solubilization and trypsin digestion, the band of ∼ 65 kDa which was obtained from trypsin digestion was purified by ion-exchange chromatography and was used to feed the aphid. The bioassay shows mortality rates above 85% at concentration of 10 ng/µl and the LC50 was 6.58 ng/µl. The resulting fragment from the digestion was identified by mass spectrometry and the candidate protein showed an overall 100% amino acid sequence identity to the reported Cry1Cb2 (WP 033698561.1) protein from Bt. Koch’s postulated also was carried out with the GP919 strain and also, we document the signs of infection caused by this strain. This is the first report of a Cry1Cb2 protein that is toxic to a sucking insect and this protein may become a promising environmentally friendly tool for the control of M. persicae and possible also for other sap sucking insect pests.

Characterization of native Bacillus thuringiensis strains against Spodoptera litura and Spodoptera exigua

In the present study 12 native Bt strains isolated from insect cadavers were screened for their bioefficacy against neonates of economically important polyphagous pests Spodoptera litura and Spodoptera exigua by feeding assays at single concentrations of 10 μg/g of diet. Toxicity of Bt strains against neonates of S. litura and S. exigua varies from 39.04% (VKK-EV and VKK-PX2) to 70.97% (VKK-AC1) and 20.0% (VKK-AC1 and VKK-MPS) to 88.00% (VKK-AC2) on 7th day after treatment respectively. The LC50 values for potential Bt strains against S. litura varied from 0.87 μg/gm (VKK-AC1) to 9.36 μg/gm (VKK-AG2) while, against S. exigua ranged from 1.00 μg/g (VKKAC2) to 13.95 μg/g (VKK-SO) of diet. Gene profiling of potential Bt strains revealed the presence of cry1A, cry1D, cry1I, and cry2 gene. Further studies on characterization of these novel cry genes from potential native Bt strains will be valuable for management of Spodoptera spp.

Molecular cloning of a new cry2A-type gene from Bacillus thuringiensis strain Nn10 and its expression studies

In the present study, eight indigenous Bacillus thuringiensis isolates of Western Ghats of India with more than 90% toxicity against Helicoverpa armigera were characterized for cry2A gene sub families. Seven of the eight isolates harboured cry2Aa, cry2Ab and cry2Ac genes alone and or in combination. Further, the indigenous cry2Aa gene(s) from Bacillus thuringiensis isolate Nn10 which showed 100% mortality against Helicoverpa armigera was cloned and expressed into recombinant Bt strains for management of resistance development in insects. The ORF of cry2Aa (∼1.9 kb) gene(s) from Nn10 isolate was ligated with T/A vector (pTZ57 R/T) and expressed in E. coli, DH5α. Automated sequence analysis of newly cloned recombinant cry2Aa revealed 99% homology to 916 bases in the 3′ region of minus strand and 100% homology with 720 bases in the 5’ region of holotype cry2Aa1 gene. The partial Cry2Aa amino acid sequence of Bt strain, Nn10, deduced from the nucleotide sequence generated by M13F primer showed four amino acid variation in comparison to Cry2Aa1 holotype, at 338, 345, 346 and 489th position of ORF and the sequence was submitted to the NCBI. Further the expression of ORF of cry2Aa of Nn10 into acrystalliferous Bt strain, 4Q7 using expression vector pHT3P2T under the transcriptional control of cry3Aa promoter and cry2Aa terminator. SDS PAGE analysis of recombinant protein exhibited a prominent band of about 65 kDa. Bioassay studies revealed that recombinant proteins, Cry2Aa of Nn10 was toxic to Helicoverpa armigera with LC50 value of 7.26 μg ml−1.

Biofabricated nanoparticles: Their delivery and utility in Plutella xylostella management

The diamondback moth (DBM), Plutella xylostella (L.) (Lepidoptera: Plutellidae) is one of the most serious cosmopolitan pests of cruciferous plants. It causes severe damage to crop quality and production, with estimated global losses of 20-40% per year. Consumer preference regarding pest-free and uncontaminated crop, unavailability of highly competitive natural enemies as well as migratory nature of DBM has obligated for the mandatory use of insecticides. The over reliance and indiscriminate use of chemical insecticides to control DBM has lead to development of resistance against almost all the modern insecticides. In addition, it has also developed resistance against most of the Bt strain. To overcome the problem of resistance, there is an urgent need for new and highly efficient method to control DBM which must be eco-friendly, cost effective and has minimum impact on the environment. One of such alternatives is nanotechnology which fulfil all the criteria to become an ideal insecticide or delivery system for insect pest management especially DBM. Nanotechnology offers great improvement in the field of pesticides as it is less toxic, shelf-life enhancement and higher water solubility. Loading of Indoxacarb on nanoparticles are reported to be highly effective they suppressed the activity of detoxification enzymes such as GST, CarE, and P450. Green synthesis of AgNPs, Single walled carbon nano tube (SWCNT) also exhibits larvicidal and pupicidal effect. These are also reported to adversely affect food consumption, growth, pupation and fecundity of P. xylostella.These nanoparticles have been found to be more stable, also the controlled release of active ingredient for a long time and provides target specific control of P. xylostella for solving adverse situations ofthe crop fields like less food security, lesser food productivity and environmental imbalance.

The Combination of Bacillus Thuringiensis and Its Engineered Strain Expressing dsRNA Increases the Toxicity against Plutella Xylostella.

RNA interference (RNAi) has been developed and used as an emerging strategy for pest management. Here, an entomopathogen Bacillus thuringiensis (Bt) was used to express the dsRNA for the control of Plutella xylostella. A vector containing a 325-bp fragment of the conserved region of P. xylostella arginine kinase gene (PxAK) flanking in two ends with the promoter Pro3α was developed and transferred into Bt 8010 and BMB171, and consequently engineered Bt strains 8010AKi and BMB171AKi expressing dsRNA of PxAK were developed. The two engineered Bt strains were separately mixed with Bt 8010 in a series of ratios, and then fed to the P. xylostella larvae. We found that 8010:8010AKi of 9:1 and 8010:BMB171AKi of 7:3 caused a higher mortality than Bt 8010. PxAK expression levels in the individuals treated with the mixtures, 8010AKi and BMB171Aki, were lower than that in the control. The intrinsic rate of increase (r) and net reproductive rate (R0) of the population treated with 8010:8010AKi of 9:1 were lower than those of the population treated with Bt 8010 or 8010AKi. We developed a Bt-mediated insect RNAi for the control of P. xylostella and demonstrated a practical approach to integrating the entomopathogen with RNAi technique for the pest management.

Genetic Modification Approaches for Parasporins Bacillus thuringiensis Proteins with Anticancer Activity.

Bacillus thuringiensis (Bt) is a bacterium capable of producing Cry toxins, which are recognized for their bio-controlling actions against insects. However, a few Bt strains encode proteins lacking insecticidal activity but showing cytotoxic activity against different cancer cell lines and low or no cytotoxicity toward normal human cells. A subset of Cry anticancer proteins, termed parasporins (PSs), has recently arisen as a potential alternative for cancer treatment. However, the molecular receptors that allow the binding of PSs to cells and their cytotoxic mechanisms of action have not been well established. Nonetheless, their selective cytotoxic activity against different types of cancer cell lines places PSs as a promising alternative treatment modality. In this review, we provide an overview of the classification, structures, mechanisms of action, and insights obtained from genetic modification approaches for PS proteins.

Solid Fermentation of Bacillus thuringiensis: Method Development

Bacillus thuringiensis (Bt) is a significant bioinsecticidal agent that has been solely produced by liquid fermentation. With the increasing demand of bioinsecticides for organic agriculture, solid fermentation is preferred to enable low-cost and large-scale production of Bt. Therefore, in this study, we attempted to develop a solid state fermentation method to produce Bt by testing different solid substrates and also the effects of water percentage, fermentation time, temperature and seeding rate on the productions of biomass and parasporal crystal bodies (crystal proteins) of the Bt strain PAM33. Corn bran, rice bran and soybean meal are suitable to be utilized as substrates for biomass production. However, crystal proteins are only observed in solid fermentation using soybean meal as the substrate after 6 days of culture. The temperature range of 25-30°C and 10% seeding rate are the most suitable conditions for solid fermentation of strain PAM33 to obtain the bacterial density of approximate 109 CFU/g and the highest quantity of crystal proteins. This study shows that solid fermentation of Bt is feasible and effective to obtain bacterial biomass and crytal proteins by using cheap and available agricultural wastes. Thus, it can be a potential approach to produce the important Bt-based insecticide products for use in organic agriculture.

Efficacy of local Bacillus thuringiensis isolates against tomato leaf miner (Tuta absoluta) larvae on tomato plants under screenhouse conditions

Abstract. The negative impact of chemical pesticides on the environment and the increased resistance of tomato leafminer (Tuta absoluta) field populations to chemical pesticides have promoted research on alternative control measures. Biological control with Bacillus thuringiensis (Bt) may be an alternative, especially against larval instars of T. absoluta. A total of five B. thuringiensis strains were isolated from soil sampled from two different Cow range lands in Zaria, Nigeria; and they were screened for the presence of the cry1 genes using polymerase chain reaction. Of the five isolates, two (40%) showed the presence of the cry1 genes. Results of the bioassay conducted against 2nd instar larvae of T. absoluta at 28±2°C indicated that each of the concentrations (25, 50, 75 and 100 ppm) of the spore crystal mixtures derived from the isolates harbouring cry1 genes caused significant mortality to larvae of T. absoluta after 72 hours in comparison to the control (0 ppm). Probit analysis was used to determine the LC50 and LT50 values. When the treatments were assessed at 48 and 72 hours, LC50 values against larvae were 74.1 and 25.3 ppm for isolate F3, while the LT50 values of that same isolate F3 at 100 ppm and 75 ppm were 36.3 and 42.7 hours, respectively. B. thuringiensis strain F2 achieved 68.7% reduction in T. absoluta damage on tomato plants, while B. thuringiensis isolate F3 achieved 71.3% reduction. Therefore, the spore crystal mixture derived from indigenous Bt strains is the candidate to be used for foliar application against T. absoluta and it is recommended into integrated pest control strategies for the management of T. absoluta.

The Endophytism of Bacillus thuringiensis in Cotton Plants at Acquisition and Oviposition by Bemisia tabaci

Beneficial microorganisms can be applied on the soil or seeds and can protect plants against insect pests or pathogens. It is known which Bacillus thuringiensis (Bt) strains that, when introduced into the soil can be absorbed by the plant and translocate throughout this with insecticidal activity against insect pests that feed on them. For this reason, the ability of Bemisia tabaci ‘B biotype’ (Hemiptera: Aleyrodidae) to ingest Bt sap was evaluated in cotton plants inoculated with the Bt strain, which encodes the GFP gene, Btk-gfp (B. thuringiensis subsp. kurstaki - green fluorescent protein), and tested for oviposition, comparing untreated plants to those treated with Bt strains. The recovery of Btk-gfp was observed in the plant along with its ability to be acquired by nymphs and adults of B. tabaci. Plants treated with Bt strain S1806 had a lower number of B. tabaci eggs than plants that received only sterile water.

Whole Genome Sequencing Analysis of Bacillus thuringiensis GR007 Reveals Multiple Pesticidal Protein Genes.

Bacillus thuringiensis (Bt) are soil ubiquitous bacteria. They produce a great variability of insecticidal proteins, where certain of these toxins are used worldwide for pest control. Through their adaptation to diverse ecosystems, certain Bt strains have acquired genetic mobile elements by horizontal transfer, harboring genes that encode for different virulent factors and pesticidal proteins (PP). Genomic characterization of Bt strains provides a valuable source of PP with potential biotechnological applications for pest control. In this work, we have sequenced the complete genome of the bacterium Bt GR007 strain that is toxic to Spodoptera frugiperda and Manduca sexta larvae. Four replicons (one circular chromosome and three megaplasmids) were identified. The two largest megaplasmids (pGR340 and pGR157) contain multiple genes that codify for pesticidal proteins: 10 cry genes (cry1Ab, cry1Bb, cry1Da, cry1Fb, cry1Hb, cry1Id, cry1Ja, cry1Ka, cry1Nb, and cry2Ad), two vip genes (vip3Af and vip3Ag), two binary toxin genes (vpa2Ac and vpb1Ca), five genes that codify for insecticidal toxin components (Tc’s), and a truncated cry1Bd-like gene. In addition, genes that codify for several virulent factors were also found in this strain. Proteomic analysis of the parasporal crystals of GR007 revealed that they are composed of eight Cry proteins. Further cloning of these genes for their individual expression in Bt acrystalliferous strain, by means of their own intrinsic promoter showed expression of seven Cry proteins. These proteins display differential toxicity against M. sexta and S. frugiperda larvae, where Cry1Bb showed to be the most active protein against S. frugiperda larvae and Cry1Ka the most active protein against M. sexta larvae.

Characterization of native Bacillus thuringiensis strains against storage pest Tribolium castaneum (Coleoptera: Tenebrionidae)

Fifty one Bacillus thuringiensis (Bt) like bacteria were isolated from diverse sources, viz. soil, silo dusts, insect cadaver, and phyllosphere and evaluated by feeding bioassays against larvae and adults of coleopteran pest red flour beetle, Tribolium castaneum (Herbst) in Division of Entomology, ICAR-IARI, New Delhi during 2014-15. It is a common pest known for attacking and infesting stored grains or products. Fifteen potential Bt strains were shortlisted on the basis of preliminary screening by single dose assays at 100 μg/g of diet against larvae and adults of T. castaneum. The virulence (LC50) of potential isolates was further assessed. The most effective Bt isolate was VKK-GJ4 (LC50=7.02 μg/g of diet) against neonates followed by VKK-GA6 (LC50=19.03 μg/g of diet) against adults of T. castaneum. Gene profiling of potential Bt strains revealed the presence of cry1, cry3, cry7, cry8, cry9, cry11, cry24 and cry28 gene. However, in three Bt strains, viz. JK-5, Tri5-5 and JKII3-2 no cry gene was found but they showed insecticidal activity against T. castaneum. Thus, quantification of the toxicity by insect bioassay is the only way to assess the potential of a strain for the pest control.

Cloning and characterization of the Cry79Aa1 gene from a lepidopteran active strain of Bacillus thuringiensis

Bacillus thuringiensis (Bt) has been used globally as a biopesticide for effective and environmentally friendly pest control. Research has intensified following the development of resistance by lepidopteran species to Bt insecticidal crystal proteins. Discovering new Bt strains with novel toxin properties which can overcome resistance is one of the strategies to improve pesticide sustainability. The genome of the Bacillus thuringiensis LTS290 strain was sequenced and assembled in 252 contigs containing a total of 6,391,328 bp. The novel cry79Aa1 gene from this strain was identified and cloned. Cry79Aa1 contains 729 amino acid residues and a molecular mass of 84.8 kDa by SDS-PAGE analysis. Cry79Aa1 was found to be active against the lepidopteran larvae of Spodoptera exigua, Helicoverpa armigera, and Plutella xylostella with LC50 values of 13.627 µg/mL, 42.8 µg/mL, and 38.086 µg/mL, respectively. However, Cry79Aa1 protein showed almost no insecticidal activity against Leguminivora glycinivorella, although some degree of growth retardation was observed.

Structure characteristics and function of a novel extracellular polysaccharide from Bacillus thuringiensis strain 4D19

Bacillus thuringiensis (Bt) are entomopathogenic bacteria that produce different kinds of insecticidal proteins. However, studies on Bt exopolysaccharides are lacking. Here, we aimed to explore the characteristics and insecticidal synergism of EPS, an exopolysaccharide from Bt strain 4D19. The molecular weight of EPS-2 was 58.0 kDa, which consisted of mannose (44.2%), GlcN (35.5%), D-GalN (8.0%), glucose (5.5%), arabinose (5.1%), galactose (0.9%), Man-UA (0.3%) and Glc-UA (0.2%). The toxicity of insecticidal proteins against Plutella xylostella was increased by adding EPS. EPS-2 bound to Cry1Ac protoxin and promoted the binding of Cry1Ac protoxin to the gut membrane of P. xylostella, but did not bind to activated toxins. These results suggested that EPS-2 may bind to the protoxin C-terminal region to enhance insecticidal activity. Our findings indicated that Bt strains produce exopolysaccharide to enhance the toxicity of insecticidal crystal proteins, which could be applied in biopesticide research and product development.

Bacillus thuringiensis RZ2MS9, a tropical plant growth-promoting rhizobacterium, colonizes maize endophytically and alters the plant's production of volatile organic compounds during co-inoculation with Azospirillum brasilense Ab-V5.

The beneficial features of Bacillus thuringiensis (Bt) are not limited to its role as an insecticide; it is also able to promote plant growth interacting with plants and other plant growth-promoting rhizobacterium (PGPR). The PGPR Bt strain RZ2MS9 is a multi-trait maize growth promoter. We obtained a stable mutant of RZ2MS9 labelled with green fluorescent protein (RZ2MS9-GFP). We demonstrated that the Bt RZ2MS9-GFP successfully colonizes maize's roots and leaves endophytically. We evaluated whether RZ2MS9 has an additive effect on plant growth promotion when co-inoculated with Azospirillum brasilense Ab-V5. The two strains combined enhanced maize's roots and shoots dry weight around 50% and 80%, respectively, when compared to the non-inoculated control. However, non-differences were observed comparing RZ2MS9 alone and when co-inoculated with Ab-V5, In addition, we used co-inoculation experiments in glass chambers to analyse the plant's volatile organic compounds (VOCs) production during the maize-RZ2MS9 and maize-RZ2MS9-Ab-V5 interaction. We found that the single and co-inoculation altered maize's VOCs emission profile, with an increase in the production of indoles in the co-inoculation. Collectively, these results increase our knowledge about the interaction between the Bt and maize, and provide a new possibility of combined application with the commercial inoculant A. brasilense Ab-V5.

Identification of Bacillus thuringiensis Strains for the Management of Lepidopteran Pests

Bacillus thuringiensis (Bt)-based bioinsecticides and transgenic plants expressing proteins with insecticidal activity (Cry and Vip) have been successfully used in several integrated pest management programs worldwide. Lepidoptera comprise some of the most economically important insect pests of the major agricultural crops. In this study, the toxicity of 150 Bt strains was evaluated against Helicoverpa armigera (Hübner) larvae. Eight strains (426, 520B, 1636, 1641, 1644, 1648, 1657 and 1658) showed high insecticide activity against H. armigera and were thereforetested against Anticarsia gemmatalis (Hübner), Spodoptera cosmioides (Walker), Chrysodeixis includens (Walker), and Diatraea saccharalis (Fabricius) larvae. Our results showed that most of the Bt strains were also toxic to these lepidopteran species. The biochemical and molecular analyses of these strains revealed that they had a similar protein profile; however, their cry and vip gene contents were variable. In addition, the median lethal concentration (LC50) of the selected strains indicated that the strains 1636, 1641, and 1658 were the most effective against H. armigera, showing LC50 values of 185.02, 159.44, and 192.98 ng/cm2, respectively. Our results suggest that the selected Bt strains have great potential to control the lepidopteran pests H. armigera, A. gemmatalis, D. saccharalis, S. cosmioides, and C. includes.

Polyurethane foam as an inert support using concentrated media improves quality and spore production from Bacillus thuringiensis.

Bacillus thuringiensis (Bt) (Bacillales:Bacillaceae) is a gram-positive bacterium that produces spores, several virulence factors and insecticidal toxins, making this microorganism the most used biopesticide worldwide. The use of inert supports such as polyurethane foam (PUF) in solid cultures has been a great alternative to produce various metabolites, including those produced by Bt. In this study we compared the yields, productivity and quality of the spores by two wild strains of Bt, (Y15 and EA3), grown in media with high substrate concentration in both culture systems: liquid and solid (PUF as solid inert support). Both strains showed 2.5- to 30-fold increases in spore production and productivity in solid culture, which showed an even greater increase when considering the spores retained in the PUF observed by scanning electron microscopy. Moreover, spore produced in solid culture showed up to sevenfold higher survival after a heat-shock treatment, relative to spores from liquid culture. The infectivity against larvae of Galleria mellonella (Lepidoptera:Pyralidae) improved also in spores from solid cultures. This comparison showed that the culture of Bt on solid support has clear advantages over liquid culture in terms of the production and quality of spores, and that those advantages can be attributed only to the culture system, as the same media composition was used in both systems.