Bacillus Thuringiensis Strains Research Articles

Protatrans – growth biostimulants for centomopathogenic bacteria <i>Bacillus thuringiensis</i>

The study investigates the use of protatran compounds as growth stimulators for Bacillus thuringiensis bacteria, which are widely used as producers of biopesticides. Cultivation of the Bacillus thuringiensis strain subsp. kurstaki was carried out in a Luria-Bertani (LB) liquid medium. Protatrans (2-Me-C6H4OCH2COO- were added to the NN+(CH2CH2OH)3 (1), 4-Cl-C6H4 -SCH2COO-NN(CHCHOH) (2) and 4-Cl-CSOCHCOO NN+(CH2CH2OH)3 (3) media in concentrations of 1×10-4–1×10-8wt %. The LB medium without the addition of compounds 1–3 was used as a control. Cultures were incubated at a temperature of 30°C for 24 hours. The number of Bacillus thuringiensis cells was determined by serial dilution. The maximum growth was observed in a medium containing 1×10-4wt % of protatran 3. The number of cells was almost 10 times (966.7%) higher than in the control. In media with 1×10-5, 1×10-6, 1×10-7 and 1×10-8 wt % of compound 3, the number of cells was 4–7 times higher than in the control (by 371.7–666.7%). Protatrans 1 and 2 had a positive effect on Bacillus thuringiensis, increasing the number of cells by 83–292% compared to control. Therefore, it was demonstrated for the first time that commercially available non-toxic protatran compounds in microconcentrations are powerful growth stimulators for the entomopathogenic bacteria Bacillus thuringiensis. This indicates the potential for significant improvement and cost reduction of biotechnology for the production of bacterial insecticides based on Bacillus thuringiensis, used in agriculture, forestry and homesteads to control harmful insects.

Bioprospecting of Polyhydroxy butyrate (PHB) from Bacillus thuringiensis KUMBNGBT-41 by using Agro-industrial wastes

The diverse group of chemicals used in making of plastic is known to be highly toxic and poses a serious threat to the biosphere. These substances besides hitting hard to ecosystem cause an array of problems like birth defects, cancer, damage of nervous and immune systems. The present research work was aimed to isolate and characterize PHB producing bacteria from landfill soil sample collected from Sanehally, Chitradurga district and qualitatively screened by using viable staining techniques. Quantitative estimation was examined by using solvent extraction method. The PHB producing bacteria were characterized based on morphological, biochemical and molecular identification and confirmed as Bacillus thuringiensis strain KUMBNGBT-41 (Accession No. MW040075). Growth parameters were optimized for biomass production using various conditions such as media-nutrient broth, incubation time-72hrs, temperature-37˚C, pH-7.0, carbon source-glucose, nitrogen source-ammonium chloride and carbon to nitrogen ratio as 4:1. The low-cost production media was used for PHB production by cheaper substrates in submerged fermentation. PHB was analysed and quantified by using UV-Visible spectrophotometer at 200-600nm.

Bacillus thuringiensis strains to control Noctuidae pests

AbstractThis study aimed to select and characterize Bacillus thuringiensis strains with toxic activity on Helicoverpa armigera, Spodoptera cosmioides and Spodoptera eridania. Fifty B. thuringiensis (Bt) strains were used in selective bioassays for high mortality on H. armigera, and the four most efficient strains were tested on S. cosmioides and S. eridania. Twelve Bt strains were characterized by PCR to detect some cry, cyt and vip genes. Bioassays were also performed to detect the production of β‐exotoxins. Strain 237 caused a mortality rate of 95.6% due to β‐exotoxins. Strains 1368, 1521, 1814 and 3299 showed toxic activity on S. eridania. However, no strain was effective on S. cosmioides. The presence of β‐exotoxin was detected only in strain 237. The molecular characterization of the strains using specific cry, cyt and vip primers showed a great genetic diversity among the 12 strains that showed some mortality on H. armigera and S. eridania. Our results showed that detecting β‐exotoxin in Bt strains is important as it is toxic to insects and vertebrates and persistent in the environment. Strains 1368, 1521, 1814 and 3299 did not show toxicity to H. armigera, while they were toxic to S. eridania and did not produce β‐exotoxin. These strains may be useful for new formulations of biopesticides as well as for cloning and expression of plants' genes.

Characteristics of biological manganese oxides produced by manganese-oxidizing bacteria H38 and its removal mechanism of oxytetracycline

Oxytetracycline (OTC) is widely used in clinical medicine and animal husbandry. Residual OTC can affect the normal life activities of microorganisms, animals, and plants and affect human health. Microbial remediation has become a research hotspot in the environmental field. Manganese oxidizing bacteria (MnOB) exist in nature, and the biological manganese oxides (BMO) produced by them have the characteristics of high efficiency, low cost, and environmental friendliness. However, the effect and mechanism of BMO in removing OTC are still unclear. In this study, Bacillus thuringiensis strain H38 of MnOB was obtained, and the conditions for its BMO production were optimized. The optimal conditions were determined as follows: optimal temperature = 35 °C, optimal pH = 7.5, optimal Mn(Ⅱ) initial concentration = 10 mmol/L. The results show that BMO are irregular or massive, mainly containing MnCO3, Mn2O3, and MnO2, with rich functional groups and chemical bonds. They have the characteristics of small particle size and large specific surface area. OTC (2.5 mg/L) was removed when the BMO dosage was 75 μmol/L and the solution pH was 5.0. The removal ratio was close to 100 % after 12 h of culture at 35 °C and 150 r/min. BMO can adsorb and catalyze the oxidation of OTC and can produce ·O2−, ·OH, 1O2, and Mn(Ⅲ) intermediate. Fifteen products and degradation pathways were identified, and the toxicity of most intermediates is reduced compared to OTC. The removal mechanism was preliminarily clarified. The results of this study are convenient for the practical application of BMO in OTC pollution in water and for solving the harm caused by antibiotic pollution.

Use of agro-industrial bio-waste for the growth and production of a previously isolated Bacillus thuringiensis strain

BackgroundBacillus thuringiensis (Bt) is a widely used biopesticide. The bioinsecticide based on Bt is obtained by fermentation, but the substrates currently used for its production constitute ingredients of high commercial value. In this context, the use of agro-industrial residues as substrates is an alternative to make the fermentation process viable on a large scale, in addition to minimizing environmental problems and contributing to the destination of these residues for biotechnological purposes.ResultsIn the first part of this study, a previously isolated spore forming soil bacteria (Bv5) harboring and expressing a novel cry 8A gene was confirmed as B. thuringiensis based on its morphological characteristics, Gram staining, scanning electron microscopy (SEM) and genome sequencing. Bv5 was established as a Gram-positive spore forming bacteria with ellipsoidal spores and small round toxins. Bv5 genome comprised of the 5.30 Mb chromosome and two megaplasmids of 450 kb and 261 kb, respectively, with cry 8A gene located on the smallest megaplasmid. In the second part of the study, the physiological profile of the Bv5 strain during fermentation in different agro-industrial biowastes (cassava wastewater, orange pulp wash and whey) was analyzed. The fermentation experiment was divided into two stages. In the first stage, the agro-industrial waste with or without salts with the best results for biomass, spores and proteins production was selected. In the second stage, the effect of the selected medium in original and diluted form with the C:N balance was evaluated, in two different fermentation times (72 h and 96 h). Pulp wash enriched with salts was selected as the most suitable medium for the growth of Bv5 strain in the first stage. In the second stage pulp wash (without dilution) with the addition of salts, and with nitrogen supplementation, was considered the best for cell growth, spore and toxin production by Bv5.ConclusionsTo conclude, our study provide a new alternative for bio-waste from the orange juice industry, as well as potential culture medium for the Bt commercial scale production.

Morphological Features of Mosquito Larvicidal Bacillus Thuringiensis Isolated From Soil Samples of Nepal

Anopheles, Culex, and Aedes spp are the main vectors for transmitting malaria, Japanese encephalitis, lymphatics filariasis, dengue, zika, chikungunya, etc. Mosquito-borne diseases are increasing especially dengue in Nepal. Additional tools are required to decrease the disease rate. Biological control tools like Gambusia fish, Cyclopods, Bacillus thuringiensis, etc., have not been adopted and are unavailable in Nepal. The research aims to isolate mosquitocidal Bacillus thuringiensis a biological control tool from the soil samples of Nepal. Bacillus thuringiensis (Bt) was isolated from the soil samples of Nepal by the acetate selection method. The larvicidal bioassay of Bt was studied against the field-collected mosquito larvae. The isolate showing larvicidal activity was coded as Bacillus thuringiensis -14P2A (Bt-14P2A). The growth pattern of the positive control strain Bacillus thuringiensis var israelensis (Bti-IPS-82) and the test strain were studied in different types of culture media. The microscopic morphology was studied by using different staining techniques. Bt-14P2A and Bti -IPS-82 caused 100% mortality of field-collected larvae. Both the strains showed the same type of colony morphology in a standard culture media and uniform turbidity with sedimentation type of growth pattern in broth media. The strains were Gram-positive single rod-shaped of size 1.2X4.8µm in both negatively stained slides and Gram-stained slides. A slight difference in the endospore location was terminal in Bti-IPS-82 and subterminal in Bt-14P2A. The newly isolated Bt-14P2A differs from Bti-IPS-82 in arrangement and endospore location but harbors the same morphological characteristic and larvicidal activity as the positive control strain.

Channel Formation in Cry Toxins: An Alphafold-2 Perspective

Bacillus thuringiensis (Bt) strains produce pore-forming toxins (PFTs) that attack insect pests. Information for pre-pore and pore structures of some of these Bt toxins is available. However, for the three-domain (I-III) crystal (Cry) toxins, the most used Bt toxins in pest control, this crucial information is still missing. In these Cry toxins, biochemical data have shown that 7-helix domain I is involved in insertion in membranes, oligomerization and formation of a channel lined mainly by helix α4, whereas helices α1 to α3 seem to have a dynamic role during insertion. In the case of Cry1Aa, toxic against Manduca sexta larvae, a tetrameric oligomer seems to precede membrane insertion. Given the experimental difficulty in the elucidation of the membrane insertion steps, we used Alphafold-2 (AF2) to shed light on possible oligomeric structural intermediates in the membrane insertion of this toxin. AF2 very accurately (<1 Å RMSD) predicted the crystal monomeric and trimeric structures of Cry1Aa and Cry4Ba. The prediction of a tetramer of Cry1Aa, but not Cry4Ba, produced an ‘extended model’ where domain I helices α3 and α2b form a continuous helix and where hydrophobic helices α1 and α2 cluster at the tip of the bundle. We hypothesize that this represents an intermediate that binds the membrane and precedes α4/α5 hairpin insertion, together with helices α6 and α7. Another Cry1Aa tetrameric model was predicted after deleting helices α1 to α3, where domain I produced a central cavity consistent with an ion channel, lined by polar and charged residues in helix α4. We propose that this second model corresponds to the ‘membrane-inserted’ structure. AF2 also predicted larger α4/α5 hairpin n-mers (14 ≤n ≤ 17) with high confidence, which formed even larger (~5 nm) pores. The plausibility of these models is discussed in the context of available experimental data and current paradigms.

Microencapsulation of Bacillus thuringiensis strains for the control of Aedes aegypti

In this study, we investigated the microencapsulation of two strains of the entomopathogenic bacteria Bacillus thuringiensis (B. thuringiensis) (BtMA-750 and BtMA-1114), which are biopesticides of high toxicity for the mosquito vector Aedes aegypti. The encapsulation of different concentrations of microorganisms in starch microparticles was evaluated, and the inverse suspension polymerization technique was explored. It was possible to observe that the higher amounts of the biopesticide caused a slight decrease in the diameter of the particles; however, even when encapsulated, the biopesticide still presents an average diameter that is able to be consumed by the larvae of Aedes aegypti. Furthermore, it was noticed that the presence of both of the B. thuringiensis strains did not affect the thermal stability of the particles. The microencapsulated bacterial strains presented a high number of viable spores and preserved the expression of proteins with molecular masses corresponding to the insecticidal toxins Cry and Cyt, indicating that the encapsulation process was conducted satisfactorily. Finally, the encapsulated strains were tested against Ae. aegypti larvae and maintained 100% larval mortality even after 35 days. Therefore, microencapsulation of B. thuringiensis not only guarantees the bacterial activity, but also prolongs the action of the biopesticide. Collectively, such findings highlight the great potential of the new biopesticides, which may help to reduce the population indices of the mosquito vector Ae. aegypti via a sustainable and environment-friendly route.

Lactoferrin-based food supplements trigger toxin production of enteropathogenic Bacillus cereus.

Lactoferrin is an iron-binding glycoprotein exhibiting antibacterial, antiviral, antifungal, antiparasitic, antiinflammatory, antianaemic and anticarcinogenic properties. While its inhibitory effects against bacterial pathogens are well investigated, little is known about its influence on the production and/or mode of action of bacterial toxins. Thus, the present study aimed to determine the impact of food supplements based on bovine lactoferrin on Bacillus cereus enterotoxin production. First, strain-specific growth inhibition of three representative isolates was observed in minimal medium with 1 or 10 mg/mL of a lactoferrin-based food supplement, designated as product no. 1. Growth inhibition did not result from iron deficiency. In contrast to that, all three strains showed increased amounts of enterotoxin component NheB in the supernatant, which corresponded with cytotoxicity. Moreover, lactoferrin product no. 1 enhanced NheB production of further 20 out of 28 B. cereus and Bacillus thuringiensis strains. These findings again suggested a strain-specific response toward lactoferrin. Product-specific differences also became apparent comparing the influence of further six products on highly responsive strain INRA C3. Highest toxin titres were detected after exposure to products no. 7, 1 and 2, containing no ingredients except pure bovine lactoferrin. INRA C3 was also used to determine the transcriptional response toward lactoferrin exposure via RNA sequencing. As control, iron-free medium was also included, which resulted in down-regulation of eight genes, mainly involved in amino acid metabolism, and in up-regulation of 52 genes, mainly involved in iron transport, uptake and utilization. In contrast to that, 153 genes were down-regulated in the presence of lactoferrin, including genes involved in flagellar assembly, motility, chemotaxis and sporulation as well as genes encoding regulatory proteins, transporters, heat and cold shock proteins and virulence factors. Furthermore, 125 genes were up-regulated in the presence of lactoferrin, comprising genes involved in sporulation and germination, nutrient uptake, iron transport and utilization, and resistance. In summary, lactoferrin exposure of B. cereus strain-specifically triggers an extensive transcriptional response that considerably exceeds the response toward iron deficiency and, despite down-regulation of various genes belonging to the PlcR-regulon, ultimately leads to an increased level of secreted enterotoxin by a mechanism, which has yet to be elucidated.

The Imperative Use of Bacillus Consortium and Quercetin Contributes to Suppress Fusarium Wilt Disease by Direct Antagonism and Induced Resistance.

Fusarium wilt diseases severely influence the growth and productivity of numerous crop plants. The consortium of antagonistic rhizospheric Bacillus strains and quercetin were evaluated imperatively as a possible remedy to effectively manage the Fusarium wilt disease of tomato plants. The selection of Bacillus strains was made based on in-vitro antagonistic bioassays against Fusarium oxysporum f.sp. lycoprsici (FOL). Quercetin was selected after screening a library of phytochemicals during in-silico molecular docking analysis using tomato LysM receptor kinases "SILKY12" based on its dual role in symbiosis and plant defense responses. After the selection of test materials, pot trials were conducted where tomato plants were provided consortium of Bacillus strains as soil drenching and quercetin as a foliar spray in different concentrations. The combined application of consortium (Bacillus velezensis strain BS6, Bacillus thuringiensis strain BS7, Bacillus fortis strain BS9) and quercetin (1.0 mM) reduced the Fusarium wilt disease index up to 69%, also resulting in increased plant growth attributes. Likewise, the imperative application of the Bacillus consortium and quercetin (1.0 mM) significantly increased total phenolic contents and activities of the enzymes of the phenylpropanoid pathway. Non-targeted metabolomics analysis was performed to investigate the perturbation in metabolites. FOL pathogen negatively affected a range of metabolites including carbohydrates, amino acids, phenylpropanoids, and organic acids. Thereinto, combined treatment of Bacillus consortium and quercetin (1.0 mM) ameliorated the production of different metabolites in tomato plants. These findings prove the imperative use of Bacillus consortium and quercetin as an effective and sustainable remedy to manage Fusarium wilt disease of tomato plants and to promote the growth of tomato plants under pathogen stress conditions.

Assessment and Biodegradation of Polycyclic Aromatic Hydrocarbons in Soil and Water Around Petroleum Products Depot Suleja, Nigeria.

Petroleum contamination constitutes a frequent incidence in various petroleum depots in Nigeria. In this study, the polycyclic aromatic hydrocarbons (PAHs) present in soil and water in communities around Petroleum Products Marketing Company (PPMC) Suleja, Nigeria, were evaluated and degraded using indigenous microorganisms. The samples sites were divided into 7 plots from where samples of water and soil were obtained: one within the PPMC depot, five from communities surrounding the depot, and the control 93,000km from the depot. The microbial counts were determined using spread plate inoculation technique on minimal salt media. The microbial isolates were characterized and identified based on their cultural, biochemical, and molecular characteristics. The potential of the microbial isolates to utilize 0.05 mL of diesel, kerosene, engine oil, and crude oil was determined in a Bushnell Haas Broth, and the biodegradation was determined by total viable cell counts and spectrophotometry. The ability of the isolates to mineralize PAHs was also evaluated in a minimum salt media. The bacterial isolates were species of Streptococcus, Pseudomonas, Staphylococcus, Proteus, Escherichia, and Bacillus, while species of Penicillium, Aspergillus, Mucor, and Rhizopus were isolated among the fungi. Aspergillus niger strain ATCC 1015 and Bacillus thuringiensis strain M43 showed high capacity to utilize the 16 priority PAHs. The pahE1 gene was used by Bacillus thuringiensis, Pseudomonas aeruginosa and A. niger, while Penicillium notatum used pahE2 gene for the degradation of the PAH. The current study identified microbial isolates that can utilize priority PAHs, making them beneficial for oil spill bioremediation in tropical environments.

Endophytic establishment of native Bacillus thuringiensis strain in maize plants and its efficacy against Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae)

BackgroundBacillus thuringiensis (Bt) is known as the most successful microbial insecticide worldwide used against lepidopteran insect pests in agriculture. Native Bacillus isolate VKK5 showing insecticidal activity against Spodoptera frugiperda (FAW) (J.E. Smith) (Lepidoptera: Noctuidae) was characterized as B. thuringiensis (BtVKK5) on a morphological and molecular basis. Recent research has shown that Bt can be established as an endophytic organism for controlling insect pests. The present work aimed at assessing the colonization of BtVKK5 as an endophyte in five maize cultivars by seed treatment (ST), soil drenching (SD), foliar application (FA) and combination of all methods (ST + SD + FA) and its bioefficacy against neonates of FAW.ResultsEstablishment of inoculated BtVKK5 as endophytes in five maize cultivars, viz. Pusa HQPM7 Improved, Pusa Jawahar Hybrid Maize 1, Pusa Vivek Hybrid 27 Improved (PVH27I), Pusa HQPM5 Improved and DMRH 1301, was confirmed by re-isolating from the leaves of the plant on ampicillin-selected agar plates. Estimation of colony-forming units per gram of leaf showed that there was a significant difference in colonization of the Bt strain among maize cultivars by different inoculation methods. The colonies were further substantiated by the amplification of cry1A and cry1E genes. Bioefficacy studies showed the highest mortality (50%) in the ST + FA + SD inoculation method, followed by ST (40%) in PVH27I. Moreover, growth inhibition was observed in survived larvae on inoculated plants vis-a-vis control.ConclusionEstablishment of Bt strain as an endophyte in maize plants, complemented with insecticidal activity, could possibly lead to an innovative approach to the management of S. frugiperda and other borers.

Characterization of entomotoxic and nematotoxic genes from indigenous Bacillus thuringiensis strains and their biocontrol potential

BackgroundThe gram pod borer (Helicoverpa armigera Hubner), Bihar hairy caterpillar (Spilosoma obliqua Walker), tobacco caterpillar (Spodoptera litura Fabricius) and root knot nematode (Meloidogyne incognita (Kofoid and White) Chitwood) are the major insect pest and nematodes infesting mungbean, urdbean and other legumes. Bacillus thuringiensis (Bt) is a promising biocontrol agent that helps in managing the above pest. Deploying Bt biopesticides instead of chemical pesticides in legume cultivation enhances the soil health substantially. The present study envisages morphological, molecular cum biochemical characterization, entomotoxicity and nematotoxicity potential of four indigenous Bt strains (Ak2.IIPR, F8.IIPR, F5.IIPR and F6.IIPR) along with HD1 (standard check from BGSC).ResultsThe SEM micrograph analysis of above four Bt strains along with F1.IIPR revealed the presence of different combinations of insecticidal crystal proteins, viz. cuboidal crystal (CC), bipyramidal crystal (BC), spherical crystal (SC) and irregularly pointed crystal (IC). This was further confirmed by PCR amplification of Cry1, Cry2Aa, Cry2Ac, VIP3A and chi36 gene-specific primers and their phylogenetic analysis. F8.IIPR and Ak2.IIPR recorded the highest toxicity index against second-instar S. obliqua larvae (292.56 and 174.55), H. armigera (150.00 and 113.65) and S. litura (210.71 and 114.19) based on probit analysis. F5.IIPR (91.67%) and F8.IIPR (89.00%) recorded the highest juvenile mortality against M. incognita, followed by Ak2.IIPR (70.33%). The protein profiling of supernatant of terrific broth inoculated with F8.IIPR indicated the presence of VIP3A and Cry1I.ConclusionThe present study concluded that F8.IIPR and Ak2.IIPR were potential Bt strains isolated from Indo-Gangetic plains which is having both entomocidal and nematicidal properties. Further, these strains were deposited at national repository, ICAR-NBAIM with the following accession no.: NAIMCC-SB-065 and NAIMCC-SB-066, respectively. These strains can be formulated as microbial biopesticide.

Relative activity of 15 bacterial strains against the larvae of Helicoverpa armigera, Spodoptera exigua, and Spodoptera litura (Lepidoptera: Noctuidae).

Crystal toxins produced by different strains of entomopathogenic Bacillus thuringiensis (Bt) have been characterized and widely applied as commercial biological pesticides owing to their excellent insecticidal properties. This study aimed to identify novel bacterial strains effective in controlling Spodoptera exigua Hübner, Helicoverpa armigera Hübner, and Spodoptera litura Fabricius. Fifteen culturable bacterial strains were isolated from 60 dead larvae (H. armigera and S. exigua) collected in the field. The biochemical characteristics and 16S rRNA sequences of these strains indicated that one strain (B7) was Lysinibacillus sp., 12 strains (B1, B3, B4, B5, B6, B8, P2, P3, P4, P5, P6, and DW) were Bt kurstaki, and P2-2 and B2 were Bacillus velezensis subsp. Laboratory bioassays indicated that strains B3, P6, B6, and P4 showed high toxicity to second-instar larvae of S. exigua, with LC50 values of 5.11, 6.74, 205.82, and 595.93 µg/ml, respectively; while the strains P5, B5, B6, and P6, were the most efficient against second-instar larvae of H. armigera with LC50 values of 725.82, 11,022.72, 1,282.90, 2,005.28, respectively, and strains DW, P3, P2, and B4 had high insecticidal activity against second-instar larvae of S. litura with LC50 values of 576.69, 1,660.96, 6,309.42, and 5,486.10 µg/ml, respectively. In conclusion, several Bt kurstaki strains with good toxicity potential were isolated and identified in this study. These strains are expected to be useful for biointensive integrated pest management programs to reduce the use of synthetic insecticides.

Assessment of bio-formulations of indigenous strains of Bacillus thuringiensis, Metarhizium robertsii and Metarhizium majus for management of the rhinoceros beetle, Oryctes rhinoceros L., in field

BackgroundAmong all the coconut pests, rhinoceros beetle causes acute and serious damage to coconut palm. Management of this pest is very difficult due to its nocturnal activity, and also, it damages the emerging leaf inside the bud. Management of rhinoceros beetle using entomopathogens will be of great importance as it is economical and ecofriendly. Studies were carried out to decipher the biocontrol potential of indigenous entomopathogenic bacterium (Bacillus thuringiensis) and entomopathogenic fungi (Metarhizium robertsii and M. majus) against Oryctes rhinoceros in the field, individually as well as in combination, by soil drench as well as by topical spray method.ResultsThe study showed that B. thuringiensis strain NBAIR-BTAN4 showed 24.8% mortality at 5th week in soil drench method and 24% mortality in topical spray method. M. robertsii showed 24% mortality at 5th week in soil drench method as well as topical spray method. Similarly, M. majus showed 24% mortality in soil drench method and 23.2% in topical spray method at 5th week. In combination, NBAIR-BTAN4 + M. robertsii showed 40.8 and 44% in soil drench and topical spray method at 5th week, respectively. Combination of NBAIR-BTAN4 + M. majus showed 44.8 and 40.8% in soil drench and topical spray method at 5th week, respectively. Combination of NBAIR-BTAN4 + M. robertsii + M. majus showed 52.8 and 57.6% mortality at 5th week in soil drench and topical spray method, respectively.ConclusionThe present study showed application B. thuringiensis in combination with M. robertsii and M. majus is effective in management rhinoceros beetle in coconut orchard. Study also indicated that soil drench method is more promising strategy than topical spray method in managing larval population of the beetles and also confirmed that B. thuringiensis, M. robertsii and M. majus are compatible and they seemed to have a synergistic effect in controlling the pest in coconut orchard.

Distribution and Genetic Diversity of Genes from Brazilian Bacillus thuringiensis Strains Toxic to Agricultural Insect Pests Revealed by Real-Time PCR.

Bacillus thuringiensis is a Gram-positive aerobic bacterium and the most used biopesticide worldwide. Given the importance of B. thuringiensis strain characterization for the development of new bioinsecticides or transgenic events and the identification and classification of new B. thuringiensis genes and strains to understand its distribution and diversity, this work is aimed at creating a gene identification system based on qPCR reactions utilizing core B. thuringiensis genes cry1, cry2, cry3, cry4, cry5, app6, cry7, cry8, cry9, cry10, cry11, vpb1, vpa2, vip3, cyt1, and cyt2 for the characterization of 257 strains of B. thuringiensis. This system was based on the Invertebrate Bacteria Collection from Embrapa Genetic Resources and Biotechnology and analyzed (a) the degree of correlation between the distribution of these strains and the origin of the substrate from which the strain was isolated and (b) between its distribution and geoclimatic conditions. This study made it possible to observe that the cry1, cry2, and vip3A/B genes occur homogeneously in the Brazilian territory, and some genes are found in specific regions. The biggest reservoir of variability is within B. thuringiensis strains in each region, and it is suggested that both geoclimatic conditions and regional crops interfere with the genetic diversity of the B. thuringiensis strains present in the region, and B. thuringiensis strains can constantly exchange genetic information.

Identification, phylogenetic analysis, and genome mining of the tetracycline-resistant Bacillus thuringiensis strain m401 reveal its potential for biotechnological and biocontrol applications

Bacillus thuringiensis is an entomopathogen belonging to the Bacillus cereus clade. We isolated a tetracycline-resistant strain called m401, recovered it from honey, and identified it as Bacillus thuringiensis sv. kumamotoensis based on the average nucleotide identity calculations (ANIb) comparison and the analysis of the gyrB gene sequences of different B. thuringiensis serovars. Sequences with homology to virulence factors [cytK, nheA, nheB, nheC, hblA, hblB, hblC, hblD, entFM, and inhA] and tetracycline resistance genes [tet(45), tet(V), and tet(M)/tet(W)/tet(O)/tet(S) family] were identified in the bacterial chromosome. The prediction of plasmid-coding regions revealed homolog sequences to the MarR and TetR/AcrR family of transcriptional regulators, toxins, and lantipeptides. The genome mining analysis revealed 12 regions of biosynthetic gene clusters responsible for synthesizing secondary metabolites. We identified biosynthetic gene clusters coding for bacteriocins, siderophores, ribosomally synthesized post-translationally modified peptide products, and non-ribosomal peptide synthetase clusters that provide evidence for the possible use of Bt m401 as a biocontrol agent. Furthermore, Bt m401 showed high inhibition against all Paenibacillus larvae genotypes tested in vitro. In conclusion, Bt m401 owns various genes involved in different biological processes, such as transductional regulators associated with antibiotic resistance, toxins, and antimicrobial peptides with potential biotechnological and biocontrol applications.

Isolation, Genomic, and Proteomic Characterization of a Novel Neotropical Strain of Bacillus thuringiensis with Mosquitocidal Activities

The combination of genomic and proteomic analyses is a useful tool for the study of novel Bacillus thuringiensis (Bt) strains, as these approaches allow the accurate identification of pesticidal proteins and virulence factors produced. Here, we isolated and evaluated the potential of a novel Neotropical Bt strain (TOD651) for controlling larvae of Aedes aegypti and Culex quinquefasciatus mosquitoes. Aiming for the full comprehension of the TOD651 larvicidal potential, we further evaluated the whole TOD651 genome and conducted the proteomic analysis of the TOD651 spore–crystal mixtures. Our results showed that Bt TOD651 similarly killed both A. aegypti (0.011 µg/mL) and C. quinquefasciatus (0.023 µg/mL) larvae, exhibiting similar potency to the commercial Bt strain. The genome sequence revealed that Bt TOD651 harbors cry11Aa3, cry10Aa4, cry4Aa4, cry4Ba5, cyt1Aa5, cyt1Ca1, cyt2Ba13, mpp60Aa3, and mpp60Ba3. The proteomic analysis revealed no expression of Mpp60Aa3, while all the other pesticidal proteins were expressed (Cry4Ba5 was more abundant than Cyt1Aa5). The expression of the Mppe showed the major proportions between proteases. The virulent factor neutral protease B and spore coat proteins were also expressed. The expression of relevant pesticidal proteins (e.g., Cry, Cyt, Mpp, and other pathogenic factors), whose actions can occur in a synergic relation, indicates that the biocontrol using Bt TOD651 may contribute to delaying the selection of resistant individuals.

Estimation of acute toxicity of bioinsecticide Turinbash-G for honey bee <i>Apis mellifera mellifera L.</i>

Introduction. Pesticide poisoning is one of the main causes of mass death of bee colonies observed in different countries of the world. However, at present, abandoning the use of plant protection products is not possible, since insects and crop weeds are responsible for the loss of at least half of the world's food supply. An increase in insect pest activity due to global warming will lead to even greater losses. All this dictates the need for a wider introduction of biological methods of plant protection. Bioinsecticides based on entomopathogens can become an alternative to chemicals that have a pronounced toxic effect on nontarget organisms.Methods. The object of the study was honey bee Apis mellifera mellifera L. of the Carpathian breed, subjected to oral and contact exposure to Turinbash-G, the active ingredient of which is the bacterium Bacillus thuringiensis subsp. aizawai strain 12K and Bacillus thuringiensis subsp. thuringiensis, strain VNIIVEA-177. As a guide, the work used «Methodological recommendations for assessing the effect and potential hazard of pesticides for honeybees», approved by the Russian Academy of Agricultural Sciences and the Department of Veterinary Medicine of the Ministry of Agriculture of the Russian Federation, as well as GOST 33038-2014 and GOST 33039-2014 «Mеthods for testing chemical products posing a danger to the environment.» In the course of the studies, the acute oral toxicity and acute contact toxicity of the active substance of Turinbash-G.Results. Studies have not revealed signs of acute toxicity of the Turinbash-Zh bioinsecticide for honey bees both with oral and with contact exposure to the drug. The maximum mortality rate of bees in the experimental groups 96 hours after the end of exposure to the studied drug is 16.7%. LD50 in determining the acute oral toxicity of the active substance of the drug Turinbash-Zh — above the value of 1·106 CFU/bee, LK50 — above 100 ml/l; in determining the acute contact toxicity LD50 above the value of 1·105 CFU/bee, LK50 — above 100 ml/l.

Are Bacillus thuringiensis strains like any other Bacillus cereus strains? Phenotypic-based tools to locate Bacillus thuringiensis in the diversity of the Bacillus cereus sensu lato group

Some Bacillus thuringiensis (Bt) strains are used as pesticide agent. This species belongs to Bacillus cereus (Bc) group which contains many species with a high phenotypic diversity, and could be pathogenic like B. cereus. The aim of this study was to characterize the phenotype of 90 strains belonging to Bc group, half of which were Bt. Knowing that Bt strains belong to different phylogenetic Bc groups, do Bt strains have the same phenotype than other Bc group strains?Five phenotypic parameters were estimated for 90 strains in the Bc group, of which 43 were Bt strains: minimal, maximal and optimal growth temperature, cytotoxicity on Caco-2 cells, heat resistance of spores. The dataset was processed by principal component analysis, showing that 53% of the variance of the profiles corresponded to factors linked to growth, heat resistance and cytotoxicity. The phenotype followed the phylogenetic groups based on panC. Bt strains showed similar behavior to other strains in the Bc group, in our experimental conditions. Commercial bio-insecticide strains were mesophilic with low heat resistance.