Throwing Brazilian strains into the melting pot of P. xylostella resistance to Bacillus thuringiensis.

The resistance of pest insects to biopesticides based on the bacterium Bacillus thuringiensis (Bt) is normally associated with changes to the receptors involved in the mechanism of action of the pesticidal proteins produced by Bt. In some strains of Plutella xylostella (the diamondback moth) resistance has evolved through a signalling mechanism in which the genes encoding the receptor proteins are downregulated whereas in others it has been linked to structural changes in the receptors themselves. One such well characterized mutation is in the ABCC2 gene indicating that changes to this protein can result in resistance. However other studies have found that knocking out this protein does not result in a significant level of resistance. In this study we wanted to test the hypothesis that constitutive receptor downregulation is the major cause of Bt resistance in P. xylostella and that mutations in the now poorly expressed receptor genes may not contribute significantly to the phenotype. To that end we investigated the expression of a receptor (ABCC2) and the major regulator of the signalling pathway (MAP4K4) in two resistant and four susceptible strains. No correlation was found between expression levels and susceptibility; however, a frameshift mutation was identified in the ABCC2 receptor in a newly characterized resistant strain.

Performance insights into spray-dryer microencapsulated Bacillus thuringiensis cry pesticidal proteins with gum arabic and maltodextrin for effective pest control.

Bacillus thuringiensis (Bt) produces crystals composed mainly of Cry pesticidal proteins with insecticidal activity against pests but are highly susceptible to degradation by abiotic factors. In this sense, encapsulation techniques are designed to improve their performance and lifetime. However, the effects of polymeric matrix encapsulation such as gum arabic and maltodextrin by spray-dryer in the mechanisms of action of Bt kurstaki and Bt aizawai are unknown. We analyzed crystal solubilization, protoxin activation, and receptor binding after microencapsulation and compared them with commercial non-encapsulated products. Microencapsulation did not alter protein crystal solubilization, providing 130 kDa (Cry1 protoxin) and 70 kDa (Cry2 protoxin). Activation with trypsin, chymotrypsin, and larval midgut juice was analyzed, showing that this step is highly efficient, and the protoxins were cleaved producing similar ~ 55 to 65 kDa activated proteins for both formulations. Binding assays with brush border membrane vesicles of Manduca sexta and Spodoptera frugiperda larvae provided a similar binding for both formulations. LC50 bioassays showed no significant differences between treatments but the microencapsulated treatment provided higher mortality against S. frugiperda when subjected to UV radiation. Microencapsulation did not affect the mechanism of action of Cry pesticidal proteins while enhancing protection against UV radiation. These data will contribute to the development of more efficient Bt biopesticide formulations. KEY POINTS: • Microencapsulation did not affect the mechanisms of action of Cry pesticidal proteins produced by Bt. • Microencapsulation provided protection against UV radiation for Bt-based biopesticides. • The study's findings can contribute to the development of more efficient Bt biopesticide formulations.

Beauveria bassiana biogenic nanoparticles for the control of Noctuidae pests.

BACKGROUND: Biogenic metallic and oxide metal nanoparticles have potential as alternatives for several current problems in agriculture, such as the control of caterpillars which cause huge losses in the production of important crops. In the present study, capped and uncapped silver, iron oxide and titanium dioxide nanoparticles were synthesized from the filtrate of Beauveria bassiana and evaluated in regard to physico-chemical characteristics, capping composition, cytotoxicity, genotoxicity and biological activity on Helicoverpa armigera and Spodoptera frugiperda caterpillars. RESULTS: A difference in the physico-chemical parameters of the capped and uncapped nanoparticles was observed, with larger aggregation and lower stability of the uncapped. In regard to the study of the capping, the presence of functional groups of biomolecules as well as the activity of B. bassiana hydrolytic enzymes were observed. Cytotoxic effects on the tested cell lines were not observed and DNA damage levels increased with more intense effects of the uncapped nanoparticles. In regard to the biological activity against Noctuidae pests, the uncapped and capped iron oxide, and uncapped titanium dioxide nanoparticles occasioned higher mortality (76%, 60% and 51%, respectively) but no differences in LC50 were recorded. Moreover, sublethal effects were reported on Helicoverpa armigera whereas Spodoptera frugiperda showed low susceptibility to the nanoparticles. CONCLUSION: The results demonstrate that biogenic metallic and oxide metal nanoparticles might show promising effects for the control of caterpillars which cause damage on important agricultural crops. Further investigations are necessary to understand the mechanisms of action and optimize the biological activity of these new nanomaterials. © 2023 Society of Chemical Industry.

Bacillus thuringiensis-based bioinsecticides affect predation of Euborellia annulipes on diamondback moth larvae.

Interactions between earwigs and entomopathogens, such as Bacillus thuringiensis (Bt), are still poorly understood. This study tested whether Bt-based bioinsecticides have any effect on the predation of Euborellia annulipes (Lucas) (Dermaptera: Anisolabididae) on Plutella xylostella (L.) (Lepidoptera: Plutellidae), one of the pests with the largest number of cases of use and resistance to Bt. Fourth instar larvae were Bt infected by feeding on collard green leaves treated with Dipel®WG and XenTari®WG at the manufacturer-recommended doses. We used one no-choice condition, in which the predator had access to uninfected or Bt-infected larvae separately, and four free-choice conditions: uninfected vs Dipel®-infected larvae, uninfected vs XenTari®-infected larvae, Dipel®-infected vs XenTari®-infected larvae, and uninfected vs Bt-infected larvae with both bioinsecticides. Uninfected larvae were less consumed than those infected by both Bt-bioinsecticides in the no-choice condition. There was a higher consumption of uninfected over Dipel®-infected larvae in the free-choice condition. Overall, uninfected larvae were preferred over both Bt-based bioinsecticides infected larvae. We also used six different prey densities. The ringlegged earwig's predation rate enhanced as the prey population density increased, but the functional response was not affected by Bt-infection, being type II. The predator invested a low amount of handling time on Bt-fed prey and increased the maximum predation rate. Bt-based bioinsecticides cause effects on E. annulipes predation by altering their feeding preference and some aspects of its predatory behavior. The results of our study provide an important background for understanding interactions between earwigs and Bt. In addition, they can be used for decision making during approaches to integrated P. xylostella management.

Increasing ecological heterogeneity can constrain biopesticide resistance evolution.

Microbial biopesticides containing living parasites are valuable emerging crop protection technologies against insect pests, but they are vulnerable to resistance evolution. Fortunately, the fitness of alleles that provide resistance, including to parasites used in biopesticides, frequently depends on parasite identity and environmental conditions. This context-specificity suggests a sustainable approach to biopesticide resistance management through landscape diversification. To mitigate resistance risks, we advocate increasing the range of biopesticides available to farmers, whilst simultaneously encouraging other aspects of landscape-wide crop heterogeneity that can generate variable selection on resistance alleles. This approach requires agricultural stakeholders to prioritize diversity as well as efficiency, both within agricultural landscapes and the biocontrol marketplace.

Genetic diversity of Brazilian Bacillus thuringiensis isolates with toxicity against Aedes aegypti (Diptera: Culicidae).

Bacillus thuringiensis (Bt) isolates native to Maranhão (BtMA) that are highly toxic to Aedes aegypti larvae and seven standard subspecies of Bt were analyzed for genetic diversity using the rep-PRC technique with BOX, ERIC, REP, MB1, and GTG5 markers. The rep-PCR technique is considered an extremely reliable, reproducible, fast and highly discriminatory technique that may be used even among populations of the same species. These five markers revealed a total of 38 polymorphic DNA fragments for 30 BtMA isolates. Eight groups were obtained with the dendrogram generated through Pearson's correlation analysis, with four groups formed only with BtMA isolates and four comprised of isolates of BtMA and the standard subspecies toxic to dipterans and lepidopterans. Despite the high genetic diversity of BtMA, a low correlation between the collection site, gene content and mortality against A. aegypti larvae was evidenced. The clustering of the standard subspecies of Bt that were toxic against dipterans with BtMA isolates confirm the mosquitocidal action of the native isolates from Maranhão, and they can be used as an alternative for A. aegypti control and other insects of medical importance and for the control of agricultural pests.

Sub-lethal effects of a Bt-based bioinsecticide on the biological conditioning of Anticarsia gemmatalis.

Bioinsecticides based on Bacillus thuringiensis (Bt) Berliner, 1915 are widely used to control lepidopteran in several crops. However, surviving insects exposed to the sub-lethal concentration of Bt-based bioinsecticides can suffer a multitude of effects on the biological conditioning known as hormesis. Here, we aimed to provide a clearer understanding of the biological conditioning of Anticarsia gemmatalis (Hübner, 1818), exposed to different concentrations of a Bt-based bioinsecticide, by assessing life table parameters over three generations. We defined five sub-lethal concentrations (LC5, LC10, LC15, LC20, and LC25) from the response curve estimate of A. gemmatalis. Deionized water was used as a control. We assessed the parameters of eggs-viability and the duration of the stages, incubation, larval, pre-pupal, pupal, adult, pre-oviposition and total biological cycle. Data were used to construct the fertility life table using the two-sex program. The survival curves showed greater variation in the proportion of individuals at each development stage using the LC25. The sub-lethal concentrations did not influence the incubation-eggs period, pre-pupal and pupal. However, the larval and adult stages using LC25 and LC10 were the most affected. Changes in sex ratio were observed using LC20 and LC5. The toxic effect of Bt-based bioinsecticide interfered mainly in the parameters of fertility, sex ratio, net reproduction rate (R0), and gross reproduction rate (GRR).

Zein based-nanoparticles loaded botanical pesticides in pest control: An enzyme stimuli-responsive approach aiming sustainable agriculture.

Nanoencapsulation of biopesticides is an important strategy to increase the efficiency of these compounds, reducing losses and adverse effects on non-target organisms. This study describes the preparation and characterisation of zein nanoparticles containing the botanical compounds limonene and carvacrol, responsive to proteolytic enzymes present in the insects guts. The spherical nanoparticles, prepared by the anti-solvent precipitation method, presented in the nanoparticle tracking analysis (NTA) a concentration of 4.7 × 1012 ± 1.3 × 1011 particles.mL-1 and an average size of 125 ± 2 nm. The formulations showed stability over time, in addition to not being phytotoxic to Phaseolus vulgaris plants. In vivo tests demonstrated that formulations of zein nanoparticles containing botanical compounds showed higher mortality to Spodoptera frugiperda larvae. In addition, the FTIC probe (fluorescein isothiocyanate) showed wide distribution in the larvae midgut, as well as being identified in the feces. The trypsin enzyme, as well as the enzymatic extract from insects midgut, was effective in the degradation of nanoparticles containing the mixture of botanical compounds, significantly reducing the concentration of nanoparticles and the changes in size distribution. The zein degradation was confirmed by the disappearance of the protein band in the electrophoresis gel, by the formation of the lower molecular weight fragments and also by the greater release of FTIC after enzymes incubation. In this context, the synthesis of responsive nanoparticles has great potential for application in pest management, increasing the selectivity and specificity of the system and contributing to a more sustainable agriculture.

Encapsulation Strategies for Bacillus thuringiensis: From Now to the Future.

Bacillus thuringiensis (Bt) has been recognized for its high potential in the control of various agricultural pests. Developments in micro/nanotechnology have opened new perspectives for the production of more efficient formulations that can overcome some obstacles associated with its use in the field, such as formulation instability and loss of activity as a result of the degradation of pesticidal protein by its exposure to ultraviolet radiation, among other problems. This review describes current studies and recent discoveries related to Bt and processes for the encapsulation of Bt derivatives, such as Cry pesticidal proteins. Different techniques are described, such as extrusion, emulsion, spray drying, spray cooling, fluidized bed, lyophilization, coacervation, and electrospraying to obtain micro- and nanoparticulate systems. It is noteworthy that products based on microorganisms present less risk to the environment and non-target organisms. However, systematic risk assessment studies of these new Bt biopesticides are necessary, considering issues, such as interactions with other organisms, the formation of toxic secondary metabolites, or the interspecific transfer of genetic material. Given the great potential of these new formulations, a critical assessment is provided for their future use, considering the technological challenges that must be overcome to achieve their large-scale production for efficient agricultural use.

Compatibility of Bt biopesticides and adjuvants for Spodoptera frugiperda control.

Spodoptera frugiperda is a pest of economic importance for several crops with resistance reports to Bt crops and pesticides. Eco-friendly Bt biopesticides may be an alternative to chemical insecticides due to their selectivity and specificity. However, the efficacy of Bt biopesticides may be influenced by the association with other chemicals, such as adjuvants. This study evaluated the compatibility and toxicity of Bt biopesticides mixed with adjuvants for the control of S. frugiperda. The treatments included the association of Dipel SC and Dipel PM with adjuvants. Compatibility tests were used to evaluate the Bt mixture. Bt suspensions obtained from mixtures of Bt and adjuvants at 106 and 3 × 108 spores/mL-1 were used to evaluate S. frugiperda mortality and distilled water was used as the control. The addition of the adjuvant LI increased growth and sporulation, indicating compatibility with Bt biopesticides. The other adjuvants were toxic to reducing Bt growth and sporulation. Only the mixture of Bt with LI and Bt alone was effective to S. frugiperda. The addition of adjuvants to Bt biopesticide affect the Bt sporulation, growth and mortality.

Bacillus thuringiensis Cry1Ab Domain III ß-16 Is Involved in Binding to Prohibitin, Which Correlates with Toxicity against Helicoverpa armigera (Lepidoptera: Noctuidae).

Helicoverpa armigera is a major insect pest of several crops worldwide. This insect is susceptible to some Bacillus thuringiensis (Bt) Cry insecticidal proteins expressed in transgenic crops or used in biopesticides. Previously, we identified H. armigera prohibitin (HaPHB) as a Cry1Ac-binding protein. Here, we further analyzed the potential role of PHB as a Cry toxin receptor in comparison to cadherin (CAD), well recognized as a Cry1Ac receptor. HaPHB-2 midgut protein and HaCAD toxin-binding region (TBR) fragment from H. armigera were expressed in Escherichia coli cells, and binding assays with different Cry1 toxins were performed. We demonstrated that Cry1Ab, Cry1Ac, and Cry1Fa toxins bound to HaPHB-2 in a manner similar to that seen with HaCAD-TBR. Different Cry1Ab mutant toxins located in domain II (Cry1AbF371A and Cry1AbG439D) or domain III (Cry1AbL511A and Cry1AbN514A), which were previously characterized and found to be affected in receptor binding, were analyzed regarding their binding interaction with HaPHB-2 and toxicity against H. armigera One ß-16 mutant (Cry1AbN514A) showed increased binding to HaPHB-2 that correlated with 6-fold-higher toxicity against H. armigera, whereas the other ß-16 mutant (Cry1AbL511A) was affected in binding to HaPHB-2 and lost toxicity against H. armigera Our data indicate that ß-16 from domain III of Cry1Ab is involved in interactions with HaPHB-2 and in toxicity. This report identifies a region of Cry1Ab involved in binding to HaPHB-2 from a Lepidoptera insect, suggesting that this protein may participate as a novel receptor in the mechanism of action of the Cry1 toxins in H. armigeraIMPORTANCEHelicoverpa armigera is a polyphagous pest that feeds on important crops worldwide. This insect pest is sensitive to different Cry1 toxins from Bacillus thuringiensis In this study, we analyzed the potential role of PHB-2 as a Cry1 toxin receptor in comparison to CAD. We show that different Cry1 toxins bound to HaPHB-2 and HaCAD-TBR similarly and identify ß-16 from domain III of Cry1Ab as a binding region involved in the interaction with HaPHB-2 and in toxicity. This report characterized HaPHB-Cry1 binding interaction, providing novel insights into potential target sites for improving Cry1 toxicity against H. armigera.

Interactions of Bacillus thuringiensis strains for Plutella xylostella (L.) (Lepidoptera: Plutellidae) susceptibility.

Bacillus thuringiensis (Bt) biopesticides are an environmentally safe alternative to the management of Plutella xylostella pesticide resistance evolution. We evaluated P. xylostella susceptibility to six Bt strains cultivated and applied individually, and 15 combinations of Bt strains mixed after cultivation. Three combinations resulted in synergism and one in antagonism. Promising results of larval mortality with synergistic effects were obtained with the combinations Bt var. thuringiensis strain HD-2 + Bt finitimus strain HD-3, Bt var. thuringiensis strain HD-2 + Bt dendrolimus strain HD-7 and Bt var. thuringiensis strain HD-2 + Bt var. aizawai strain HD-11.

Effect of addition of adjuvants on physical and chemical characteristics of Bt bioinsecticide mixture.

Bacillus thuringiensis (Bt) is the main bacterium used in the formulation of bioinsecticides because it produces toxins and spores that are toxic to several orders of insects. The efficacy of Bt bioinsecticide is influenced by the quality of its application. The association with other crop protection products, such as adjuvants, can affect the physical and chemical parameters of the mixture. This study evaluated the physical and chemical parameters, volume median diameter (VMD), uniformity coefficient of droplets (SPAN), percentage of volume in drift droplets (%V <100 µm), contact angle, surface tension, potential of hydrogen (pH) and electrical conductivity (E.C.) of Bt bioinsecticides in concentrated suspension (SC), and wettable powder (WP) formulations associated with adjuvants. The largest droplet diameter and smallest values of drift droplets were found in the WP formulation with lower drift potential. The addition of mineral oil and surfactant to the mixtures of bioinsecticide reduced contact angle values and surface tension of the droplets, resulting in greater spreading of droplets in leaves. The addition of lecithin and propionic-acid-based adjuvants lowered the pH in both formulations. The adjuvants used in this study affected the physical and chemical characteristics of the mixtures, improving or impairing the quality of Bt bioinsecticide applications.

Immune response and susceptibility to Cotesia flavipes parasitizing Diatraea saccharalis larvae exposed to and surviving an LC25 dosage of Bacillus thuringiensis.

Biological control using entomopathogens and natural enemies is an ecofriendly method for pest management in agriculture. Biological control agents often can be simultaneously employed and compatibility between agents may improve pest suppression. We investigated the influence of the entomopathogen Bacillus thuringiensis (Bt) on the immune system of the sugarcane borer Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae) to determine if such changes impact parasitization by Cotesia flavipes Cameron, 1891 (Hymenoptera: Braconidae). The immune response of surviving D. saccharalis larvae fed with an LC25 dosage of a Bt-based biopesticide (Dipel®) was analyzed (total hemocyte count, hemocyte adhesion, and activities of phenoloxidase and lysozyme). Furthermore, the suitability of surviving Bt-fed larvae as hosts for C. flavipes was assessed by measuring parasitoid attributes such as number and size of teratocytes, weight of pupae, length of adult female tibia and number of emerged adults. Total hemocyte count, but not hemocyte adhesion, total protein content and phenoloxidase activity increased in the hemolymph of non-parasitized Bt-fed larvae (Bt-NP) compared to control larvae (NBt-NP). Lysozyme activity increased only after parasitization without Bt exposure (NBt-P). After parasitization, the immunological parameters activated in Bt-NP larvae decreased to levels at or below those observed in control larvae, showing that C. flavipes can regulate the activated immune response of Bt-fed larvae. The development of C. flavipes was not impaired in Bt-fed larval hosts (Bt-P); no changes were observed for teratocyte size, weight of pupal mass, length of hind tibia and number of adults emerged.

Association of zein nanoparticles with botanical compounds for effective pest control systems.

BACKGROUND: Botanical compounds from plant species are known to have pesticidal activity and have been used in integrated pest management programs. The varied spectrum of the pesticidal action of these compounds can also avoid selection of resistance in pest populations. In this study, mixtures of the botanical compounds geraniol, eugenol and cinnamaldehyde were encapsulated in zein nanoparticles to improve their stability and efficiency. Biological effects of the nano-scale formulations of the botanical compounds were evaluated against two agricultural pests: the two-spotted spider mite (Tetranychus urticae) and the soybean looper (Chrysodeixis includes). RESULTS: The formulations were stable over time (120 days) with a high encapsulation efficiency (>90%). Nanoencapsulation also provided protection against degradation of the compounds during storage and led to a decrease in toxicity to non-target organisms. The release of the compounds (especially eugenol and cinnamaldehyde) from the nanoparticles was directly influenced by temperature, and the main mechanism of release was through a diffusion-based process. Nanoencapsulated compounds also showed superior efficiency compared to the emulsified compounds in terms of repellency and insecticidal activity. CONCLUSION: The findings of this study indicate that the convergence of botanical compounds with nano-scale formulation has the potential to improve efficacy for their sustainable use in integrated pest management in agriculture. © 2019 Society of Chemical Industry.

Identification of midgut membrane proteins from different instars of Helicoverpa armigera (Lepidoptera: Noctuidae) that bind to Cry1Ac toxin.

Helicoverpa armigera is a polyphagous pest sensitive to Cry1Ac protein from Bacillus thuringiensis (Bt). The susceptibility of the different larval instars of H. armigera to Cry1Ac protoxin showed a significant 45-fold reduction in late instars compared to early instars. A possible hypothesis is that gut surface proteins that bind to Cry1Ac differ in both instars, although higher Cry toxin degradation in late instars could also explain the observed differences in susceptibility. Here we compared the Cry1Ac-binding proteins from second and fifth instars by pull-down assays and liquid chromatography coupled to mass spectrometry analysis (LC-MS/MS). The data show differential protein interaction patterns of Cry1Ac in the two instars analyzed. Alkaline phosphatase, and other membrane proteins, such as prohibitin and an anion selective channel protein were identified only in the second instar, suggesting that these proteins may be involved in the higher toxicity of Cry1Ac in early instars of H. armigera. Eleven Cry1Ac binindg proteins were identified exclusively in late instar larvae, like different proteases such as trypsin-like protease, azurocidin-like proteinase, and carboxypeptidase. Different aminopeptidase N isofroms were identified in both instar larvae. We compared the Cry1Ac protoxin degradation using midgut juice from late and early instars, showing that the midgut juice from late instars is more efficient to degrade Cry1Ac protoxin than that of early instars, suggesting that increased proteolytic activity on the toxin could also explain the low Cry1Ac toxicity in late instars.

Spodoptera albula susceptibility to Bacillus thuringiensis-based biopesticides.

Single concentration and virulence (mean lethal concentration) bioassays were performed to evaluate the susceptibility of S. albula second instar larvae to seven Bacillus thuringiensis-based biopesticides. Bioassays were conducted using three replicates and repeated three times at 25 °C, 70 ±â€¯10% RH, and a 12:12 (light/dark) photoperiod; mortality was recorded seven days after treatment. The results were subjected to a Tukey's test and Probit analysis. Agree, DiPel SC, and XenTari achieved mortality rates of up to 80%, with the first of these being the most virulent against S. albula. Different Dipel formulations showed different degrees of larvicidal activity.

Geraniol Encapsulated in Chitosan/Gum Arabic Nanoparticles: A Promising System for Pest Management in Sustainable Agriculture.

The nanoencapsulation of botanical compounds (such as geraniol) is an important strategy that can be used to increase the stability and efficiency of these substances in integrated pest management. In this study, chitosan/gum arabic nanoparticles containing geraniol were prepared and characterized. In addition, evaluation was made of the biological activity of geraniol encapsulated in chitosan/gum arabic nanoparticles toward whitefly ( Bemisia tabaci). The optimized formulation showed a high encapsulation efficiency (>90%) and remained stable for about 120 days. The formulation protected the geraniol against degradation by UV radiation, and the in vitro release was according to a diffusion mechanism that was influenced by temperature. An attraction effect was observed for Bemisia tabaci, indicating the potential of this type of system for use in pest management, especially in trap devices.

Isolation and molecular characterization of Bacillus thuringiensis found in soils of the Cerrado region of Brazil, and their toxicity to Aedes aegypti larvae

ABSTRACT This study investigated the potential of Bacillus thuringiensis isolates obtained in the Cerrado region of the Brazilian state of Maranhão for the biological control of Aedes aegypti larvae. The isolates were obtained from soil samples and the identification of the B. thuringiensis colonies was based on morphological characteristics. Bioassays were run to assess the pathogenicity and toxicity of the different strains of the B. thuringiensis against third-instar larvae of A. aegypti. Protein profiles were obtained by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Polymerase chain reaction assays were used to detect the toxin genes found in the bacterial isolates. Overall, 12 (4.0%) of the 300 isolates obtained from 45 soil samples were found to present larvicidal activity, with the BtMA-104, BtMA-401 and BtMA-560 isolates causing 100% of mortality. The BtMA-401 isolate was the most virulent, with the lowest median lethal concentration (LC50) (0.004 × 107 spores/mL), followed by the Bacillus thuringiensis var. israelensis standard (0.32 × 107 spores/mL). The protein profiles of BtMA-25 and BtMA-401 isolates indicated the presence of molecular mass consistent with the presence of the proteins Cry4Aa, Cry11Aa and Cyt1, similar to the profile of Bacillus thuringiensis var. israelensis IPS-82. Surprisingly, however, none of the cry and cyt genes analyzed were amplified in the isolate BtMA-401. The results of the present study revealed the larvicidal potential of B. thuringiensis isolates found in the soils of the Cerrado region from Maranhão, although further research will be necessary to better elucidate and describe other genes associated with the production of insecticidal toxins in these isolates.

Two methods for isolation of endophytic and edaphic Bacillus spp. from sugarcane fields / Dois métodos para isolamento de Bacillus spp. endofíticos e edáficos de canaviais

Bacillus has been widely studied and used for the control of pests and diseases. The adapted protocol proposed by POLANCZYK (2004) proved to be more efficient than the one by the World Health Organization (WHO, 1985) to isolate edaphic strains of Bacillus. However, it has not been assessed for isolation of endophytic strains, which are much less abundant in the nature and more difficult to be isolated. This study aimed to compare two methodological procedures for isolation of Bacillus, established by the WHO (1985) and by POLANCZYK (2004), regarding their efficiency for isolation of endophytics and edaphics Bacillus strains from inside the root tissue of sugarcane, as well as from the associated soil sample, collected from 11 locations; and to compare the density of bacteria in both environments. Endophytic and edaphic strains of Bacillus were isolated by both procedures. However, the isolation protocol performed by POLANCZYK (2004) made more efficient by having a greater number of colony forming units (CFU) per gram of soil and root indicating that this procedure is more useful, especially for isolation of endophytic strains of Bacillus, which are much less abundant in the nature than edaphic strains, being therefore more difficult to be isolated. Using the Polanczyk protocol (2004), Bacillus strains were recovered from all roots (endophytic) and soil (edaphic) samples of all the 11 fields, suggesting that the plant root may be another important source for isolation of Bacillus besides the soil. Higher densities of Bacillus were isolated from the edaphic environment compared with the endophytic environment, with significant differences when isolated by Polanczyk method (2004).(AU)

Bacillus tem sido amplamente estudado e usado para o controle de pragas e doenças. O protocolo adaptado proposto por POLANCZYK (2004) mostrou-se mais eficiente do que o da Organização Mundial de Saúde (WHO, 1985) para isolar cepas edáficas de Bacillus. No entanto, não foi avaliado quanto ao isolamento de estirpes endofíticas, que são muito menos abundantes na natureza e mais difíceis de isolar. Este estudo teve como objetivos comparar dois procedimentos metodológicos para o isolamento de Bacillus, o estabelecido pela OMS (WHO, 1985) e o de POLANCZYK (2004), quanto a sua eficiência para o isolamento de estirpes endofitas e edáficas de Bacillus originárias do interior do tecido radicular de cana-de-açúcar, bem como de amostras de solos associados, coletada de 11 locais; e comparar a densidade de bactérias em ambos os ambientes. As cepas endofíticas e edáficas de Bacillus foram isoladas por ambos os procedimentos. No entanto, o protocolo de isolamento realizado por POLANCZYK (2004) demonstrou-se mais eficiente por gerar maior número de unidades de formação de colônias (CFU) por grama de solo e raiz, indicando que esse procedimento é mais útil, especialmente para isolamento de estirpes endofíticas de Bacillus, que são muito menos abundantes na natureza do que as cepas edáficas, sendo, portanto, mais difíceis de serem isoladas. Usando o protocolo de POLANCZYK (2004), as cepas de Bacillus foram isoladas de todas as amostras de raízes (endofíticas) e de solo (edáficas) de todos os 11 campos, sugerindo que a raiz da planta pode ser outra fonte importante de isolamento de Bacillus além do solo. As densidades mais altas de Bacillus foram isoladas do ambiente edáfico em comparação com o ambiente endofítico, com diferenças significativas quando isoladas pelo método de POLANCZYK (2004).(AU)