Bioinsecticide Formulations Research Articles

A Native Bacillus Thuringiensis Strain with Higher Insecticidal Activity Against Adult Aedes Aegypti Than the Larvae

Mosquitoes of several species cause the deaths of millions of people annually, making them the major disease vectors. Bioinsecticide formulations derived from Bacillus thuringiensis are widely acknowledged as highly efficient, sustainable, environmentally friendly, and safe means of controlling insect pests. In this study, a strain of B. thuringiensis exhibiting significant efficacy in controlling Aedes aegypti was isolated from the local environment, identified, and physiologically characterized. The isolated B. thuringiensis strain exhibited positive insecticidal activity against the larvae and adult Aedes aegypti, causing mortality of the insect larvae at a rate of 3.33% and adult at 53.33 – 93.33% in 48 hours. Experiments conducted in a controlled laboratory setting showed that B. thuringiensis toxin exhibited higher levels of activity against the adult mosquito than the larvae. This study demonstrates that bioinsecticide formulation using B. thuringiensis as the active agent has the potential to effectively and sustainably manage larvae and adult Aedes aegypti.

Formulation of Neem Leaf and Croton Seed Essential Oils as a Natural Insecticide Tested on Mosquitoes and Cockroaches.

Essential oils are highly aromatic plant oils utilized as a new insect control alternative to synthetic insecticides because of environmental concerns. As a result, the purpose of this study was to evaluate the effects of essential oil extraction conditions on neem leaf and cotton seed, as well as to identify their chemical composition using gas chromatography-mass spectrometry. Moreover, both extracted oils were subjected to bioinsecticide formulation in individual as well as mixed forms, which were tested on mosquitoes and cockroaches using the conventional roach killer and a blank control as a comparison. The results revealed that the maximum yields of essential oils from neem leaf and croton seed were respectively 30.54 and 14.75%. The extraction process was greatly affected by the particle sizes because of the mass transfer limitation between solute-solvent interactions. The insecticidal evaluation showed that the blended form of essential oils at the 20% concentration has better efficiency than the individual oils, which accounted for 80% mosquito and 71.8% cockroach death rates within 4 h, whereas the synthetic roach killer completely killed all of the insects within the same time frame. Terpineol, α-terpinyl acetate, eucalyptol, and δ-cadinene are the active insecticidal compounds in neem leaf oil, whereas the active compounds of croton seed oil are epiglobulol, copaene, δ-cadinene, α-cubebene, and β-guaiene.

Pathogenicity of Indigenous Entomopathogen Liquid Formulation to Paddy Bugs Nymphs (Leptocorisa acuta Thunberg)

The purpose of this study was to determine the effectiveness of bio insecticide liquid formulations made from indigenous entomopathogens against paddy bugs nymphs. The study used a non-factorial Complete Randomized Design with 9 treatments and 1 control with five replications. The treatments studied consisted of E0: Control, E1: Beauveria sp Pky isolate + coconut water (CW), E2: Metarhizium sp isolate Jjt + AK, E3: Beauveria sp isolate Jts + CW, E4: Metarhizium sp isolate Lcc + CW, E5: Beauveria sp Pky isolate + shrimp shell extract (SSE), E6: Metarhizium sp isolate Jjt + SSE, E7: Beauveria sp isolate Jts + SSE, E8: Metarhizium sp isolate Lcc + SSE, E9: Synthetic insecticide b a Carbamate (Dharmabas 500 EC 2 ml L-1). The result showed that liquid bioinsecticide formulations were effective against mortality, infected nymphs and nymph death time. All entomopathogenic fungi isolates were able to cause infections in paddy bugs nymphs of 68% - 84%. Beauveria sp Pky isolate and Metarhizium sp isolate Jjt caused nymph mortality of 72% - 84% and the effectivity did not differ from insecticide b.a. Carbamate. Conidia viability of all entomopathogenic isolates in liquid media within 24 hours reached more than 80%. The fastest time to death of paddy bugs nymph occurred at 5.44 days ( Metarhizium sp. isolate) and 5.92 days (Beauveria sp. isolate) in coconut water media. Metarhizium sp. and Beauveria sp. in the medium of coconut water or shrimp shell extract has a very high potential to be developed as a bio insecticide, but it still needs to be further tested for its effectiveness in field conditions.Keywords: indigenous entomopathogen, coconut water, shrimp shell extract, paddy bugs nymph

Utilization of Soapberry as a Natural Surfactant in Cashew Nut Shell Liquid Bioinsecticide Formulation in Soybean Pest Management

The demand for soybean has been steadily increasing year after year. Pests are one of the most significant barriers to increasing soybean production. The goal of this study was to determine what concentration of soapberry fruit extract to use as an adjunctive ingredient in cashew nut shell liquid (CNSL) formulations to obtain the best pest control effect and soybean growth and yield. This study used a randomized complete block design with a single factor: soapberry extract concentration. The research included four treatments: application of CNSL formulations containing 4%, 3%, 2%, and 0% soapberry extract, application of CNSL formulations containing 1 g/liter detergent, synthetic pesticides (Fastac 1-2 liters/ha), and no pesticides as a control. Insecticide applications were effective against grasshopper and pod-sucking bugs in soybean cultivation; the CNSL with 3%, 4% soapberry and the CNSL with detergent had effectiveness comparable to the synthetic insecticides. Soybean growth was unaffected by any of the CNSL formulations or synthetic insecticide applications. However, using a CNSL formulation with 4% soapberry increased soybean yield as much as using a synthetic insecticide; it did not differ significantly from using a CNSL formulation with detergent. The soybean yield was highest in the CNSL formulation with detergent.
 Keywords: Keywords: soybean, soapberry, CNSL, pest

Isolation of Bacillus thuringiensis strains from Saudi Arabia soil and study of their potential efficiency against the lepidopteran pest Ephestia kuehniella

Insect pests represent a major threat to food crops and human health, and therefore have to be combated in several ways, including chemical methods. However, researchers demonstrated that these molecules are dangerous for the farmers, consumers and the environment in general. For this reason, scientists permanently searched environment friendly alternatives such as the use of the bacterium Bacillus thuringiensis classified as one of the best insect pathogens. This microorganism is known by its ability to produce two types of insecticidal proteins, Vegetative insecticidal proteins (Vip) and delta-endotoxins produced during vegetative and sporulation stages of growth, respectively. In the present study, 15 B. thuringiensis strains were isolated from soil collected from different regions in Saudi Arabia (Al Baha, Jeddah, Khulis and Yanbu). B. thuringiensis isolates were then classified according to the shape of their parasporal crystals identified under microscope and proteins content of these crystals. Delta-endotoxins efficiency of the different isolates was investigated and promising strains were identified as very active. After 5 days-treatment, B. thuringiensis isolates 14 and 7 killed Ephestia kuehniella larvae with low LC50 of about 59.18 and 65.67 mg/cm2, respectively. The results described in the present study proved that the new B. thuringiensis isolates could be of a great interest in the control of lepidopteran pests by using their delta-endotoxins in bioinsecticide formulations.

Efficacy Bacillus thuringiensis var. israelensis serotype H-14 (Bti H-14) for control Aedes spp. density in Denpasar, Bali

Background: A liquid bioinsecticide formulation containing Bacillus thuringiensis var. israelensis Serotype H-14 (Bti H-14) was tested in the field in household containers. The aim was to determine the effectiveness of Bti H-14 biolarvicide in controlling the density of Aedes spp. Larvae.Methods: This study was conducted in two phases of testing. First, to test the effective dose with 5 doses, namely (50 ul, 40 ul, 30 ul, 20 ul, and 10 ul) in 2.5 liters of water. Furthermore, the number of deaths was calculated after 24 hours of treatment and control by doing four repetitions. In the second phase, by conducting tests on containers in the household as many as 3171 containers were continuously observed every month, given Bti H-14 for 6 months. Observations were made before and after the application of Bti H-14 on larva density, mosquito density, and dengue cases. Data analysis was performed using paired t-test. Bti H-14 formulation to kill 50% of mosquito larvae (LC50) within 6 hours requires a concentration of 4 µl per liter.Results: Bti H-14 liquid formulation with delta-endotoxin and spores content of 600 ITU per ml or 1.2x109 CFU is effective in reducing larva density in household containers if done regularly.Conclusions: Bti H-14 liquid formulation is proven to be effective and easy to use for the control of Aedes larvae.

Biopesticidal Effect of Partitioned Extracts of Zanthoxylum zanthoxyloides (Lam.) Zepernick & Timler on Callosobruchus maculatus (Fab.)

Botanicals have become the focus for discovery of novel bioinsecticides for protection of crops most especially because of their biodegradability, non-toxicity, target organism specificity and environmental friendliness. Partitioned extracts of Zanthoxylum zanthoxyloides were investigated for biopesticidal effect on Callosobruchus maculatus with aim of identifying the most active components and increase ease of handling when compared to bulk crude plant material used by farmers. Crude powder of rootbark of Z. zanthoxyloides was made as well as partitioned extracts using Kupchan partition extraction methods with methanol, acetone, ethyl acetate and n-hexane as solvents. Analysis of the rootbark of the plant showed 19.87% ash content, 24% crude protein, 24.85% crude fibre, 3.13% oil extract, 15.42mg/100g alkaloids, 45.90mg/100g tannins, 1039.14mg/100g saponins, 26.47mg/100g flavonoids, 150.0mg/100g iron, 244.70mg/100g calcium, 36.65mg/100g magnesium, 449.00mg/100g potassium and 128.30mg/100g of phosphorus. The crude powder and extracts were bioassayed on C. maculatus in stored cowpea. Insecticidal activities of the partitioned extracts increased with increasing concentration; toxicity test revealed that n-hexane at 1%, 2% and 3%, acetone extract at 3%, and crude powder at 1.5g all have same effect on the insect as the positive control, achieving 100% mortality at 24 hours post treatment, number of adult emergence of 63.57 was recorded with 1% methanol extract against 28 in 3% of the extract. There was a significant difference in weight loss by the cowpea in the different fractional treatments while positive and negative controls also differed significantly (P>0.05) with all the partitioned treatments. Partition extracts from Z. zanthoxyloides were more bioactive than the crude powder, and n-hexane fractions contain the most active compounds against C. maculatus. n-hexane extract contain most active biomolecules to be explored for bioinsecticide formulation with high potency for development of new biopesticide, eliminate toxic components and increase ease of handling when compared to bulk crude plant material used by farmers.

Towards a development of novel bioinsecticides for organic control of Planococcus ficus in vineyards

The mealybug species, Planococcus ficus Signoret (Hemiptera: Pseudococcidae) is a pest that mainly affects grapevine plantations (Vitis vinifera L.), causing huge economic losses in the world. The numerous problems caused by the use of synthetic pesticides on the environment and human health have motivated the development and implementation of natural pesticides. The objective of this work was to develop effective and efficient contact bioinsecticide formulations for the control of P. ficus in vineyards. Anti-mealybug formulations were developed using (R)-(+)-pulegone as an active principle, TWEEN® 20 and soy lecithin powder as surfactants, and limonene and diatomaceous earth (DE) as insect waxy layer reducers. The insecticidal properties of formulations against vine mealybugs and their grapevine leaf phytotoxicity were evaluated in laboratory conditions. Pulegone+DE+Lecithin and Pulegone+Limonene+DE+Lecithin formulations showed the highest mortality rates of P. ficus (more than 70 % mortality) at 24 h and 48 h of treatment. This high mortality is probably due to an interaction between pulegone, DE and soy lecithin that allows more molecular mobility and increases the efficiency of the active substance, making it enter and act on the insect. In conclusion, the Pulegone+DE+Lecithin and Pulegone+Limonene+DE+Lecithin formulations can be an efficient tool for the organic control of P. ficus in vineyards.

Bioprocess plant design and economic analysis of an environmentally friendly insect controller agent produced with Azadirachta indica cell culture

Chemical pesticides cause serious problems to the environment, as well as human and animal health. A conceptual design and economic analysis was carried out for a hypothetical factory in order to produce an environmentally friendly insect controller agent from Azadirachta indica cell culture. The proposed factory is composed of a series of bioreactors that can produce 60.0 kg of active compound/batch. This designed plant had a capacity of 136 batches/year, which represents 40,800 kg of bioinsecticide, and it was located in Medellin (Colombia) with a total building cost of 12.2 million USD. Three bioinsecticide formulations, two equipment costs and variation in product/substrate yields were analyzed. To achieve profitability in the project, the minimum selling price of the formulated products was calculated between 76.9–330.0 USD/kg (Net Present Value > 0). This is the first report of a bioprocess factory design and economic analysis for an insect controller agent using plant cell culture.

Use of novel film forming starch complexes to directly and indirectly reduce insect damage to plants

Film-forming polymer technologies can contribute to insect-pest control by causing adverse effects directly to insects such as slowing development, causing mortality, and/or indirectly as a spray ingredient that improves pesticide efficacy by resisting environmental degradation such as wash-off by rain. Amylose complexes produced from either fatty acid [amylose sodium palmitate (Na-Palm)] or fatty ammonium salts [amylose-hexadecylammonium chloride complex (Hex-Am)], when blended with polyvinyl alcohol (PVOH), dry to form films that were significantly more water resistant then films formed by their constitutive components and adhered strongly to treated soybean [Glycine max (L.) Merrill] leaves with no impact on photosynthesis or yield in simulated field trials. As an experimental bioinsecticide formulation to resist wash-off, AfMNPV baculovirus was incorporated into both amylose complex formulations and sprayed on soybean. After a simulated rain event, soybean treated with the baculovirus/Na-Palm/PVOH (1:1) retained 93% of the original insecticidal activity against Trichoplusia ni (Hübner) larvae as compared with the baculovirus only treatment, 53% activity retained. Curiously, initial insecticidal efficacy of film-forming treatments was significantly lower against T. ni when compared with unformulated baculovirus treatments, thus prompting further investigations. Both amylose complexes reduced larval feeding when sprayed on soybean, corresponding with reduced insect mortality by the baculovirus, which must be ingested to initiate infection. Hex-Am film coating over artificial diet inhibited the growth and development of T. ni and caused increased T. ni mortality and developmental delay as film thickness of Hex-Am/PVOH increased. These investigations demonstrate the potential of a new modified starch complex formulation to serve as a protective film forming agent and contribute to control of insect pests.

Genetic Diversity among Transconjugants of Bacillus thuringiensis Used for Controlling Tomato Borer Tuta absoluta

This study aimed to chacterize transconjugants of Bacillus thuringiensis on the basis of genomic DNA patterns, plasmid curing and bioassay of toxicity against tomato borer, Tuta absoluta in the open field. Eight transconjugants generated from four matings were used in this study. The shortening of extrachromosomal genetic material lead to loss of antibiotic resistance genes in some Bt strains and their transconjugants , as well as, loss of chitin hydrolysis as a consequence. In contrast, some Bt transconjugants were not affected by elevated temperature because they are still harboring antibiotic resistance genes and encoded chitinase. The extrachromosomal genome of Serratia was more stable than Bacillus licheniformis which lost Cf resistance gene at elvevated temperature. Bt strains and their transconjugants were genetically different because some bands appeared and or disappeared between them. Two transconjugants out of eight generated additional bands than the other ones which showed a higher toxicity index in relation to the mid-parent and the other transconjugants. Tomato plants treated with bioinsecticide formulations showed significant increase in photosynthetic pigments. Furthermore, some Bt transconjugants significantly increased shoot dry weight compared to the negative and the positive controls. This study indicated that mixing of dissimilar toxin genes in addition to chitinase genes in the same transconjugant led to increase toxicity and delay of resistance in insect pest than single - toxin gene carried by single strain.

High-Throughput Production of Oxidized Animal Toxins in Escherichia coli.

High-throughput production (HTP) of synthetic genes is becoming an important tool to explore the biological function of the extensive genomic and meta-genomic information currently available from various sources. One such source is animal venom, which contains thousands of novel bioactive peptides with potential uses as novel therapeutics to treat a plethora of diseases as well as in environmentally benign bioinsecticide formulations. Here, we describe a HTP platform for recombinant bacterial production of oxidized disulfide-rich proteins and peptides from animal venoms. High-throughput, host-optimized, gene synthesis and subcloning, combined with robust HTP expression and purification protocols, generate a semiautomated pipeline for the accelerated production of proteins and peptides identified from genomic or transcriptomic libraries. The platform has been applied to the production of thousands of animal venom peptide toxins for the purposes of drug discovery, but has the power to be universally applicable for high-level production of various and diverse target proteins in soluble form. This chapter details the HTP protocol for gene synthesis and production, which supported high levels of peptide expression in the E. coli periplasm using a cleavable DsbC fusion. Finally, target proteins and peptides are purified using automated HTP methods, before undergoing quality control and screening.

Formulasi Insektisida Nabati Minyak Bungkil Mimba Dengan Surfaktan DEA

Neem cake oil has an active compound in form 242.2 ppm azadirachtin. The active compound has activity as feeding inhibitor, nesting rejection, growing inhibitor and mortality effects of many insect pests. Therefore, neem cake was extracted to produce oil. The purpose of this research was to obtain the best ratio of extraction oil from neem cake by maceration method and oil neem cake formulated by DEA surfactant. The oil extraction from neem cake was undertaken by maceration method using a solvent like n-hexane and produce the greatest yield at comparison neem cake and n-hexane 1:4 (w/v). The oil was produced from extraction of neem cake was solid form at room temperature, so it was added a general solvent as usually used in the pesticide industry to dissolved. i.e. Solvesso. The concentration of DEA surfactant used in neem cake oil formulation was 8%. The bioinsecticide formulations of this research was 8% DEA, 15% for oil neem cake and 15% for Solvesso in 30 g formulations . Keywords: DEA surfactant, insecticide formulation, maseration method, neem seed oil

Toxicity of an Annonin-Based Commercial Bioinsecticide Against Three Primary Pest Species of Stored Products.

The effects of a bioinsecticide formulation based on extract of Annona squamosa L. (Annonaceae) containing 10,000mgL-1 of acetogenin annonin as the main active ingredient were investigated against three primary pest species of stored grains in Brazil [maize weevil Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae), Mexican bean weevil Zabrotes subfasciatus (Boheman) (Coleoptera: Chrysomelidae: Bruchinae), and cowpea weevil Callosobruchus maculatus (Fabricius) (Coleoptera: Chrysomelidae: Bruchinae)] by means of residual contact bioassays. In a concentration-dependent manner, the annonin-based commercial bioinsecticide caused significant adult mortality of C. maculatus (LC50=6890μLkg-1), S. zeamais (LC50=2781μLkg-1), and Z. subfasciatus (LC50=2120μLkg-1) after 120h of residual contact exposure. In addition to acute toxicity, the tested bioinsecticide also promoted a significant reduction of the number of eggs laid by females of C. maculatus (EC50=5949.7μLkg-1) and Z. subfasciatus (EC50=552.7μLkg-1). Moreover, the bioinsecticide significantly reduced the number of emerged insects (F1 generation) of C. maculatus (EC50=2763.0μLkg-1), S. zeamais (EC50=1380.8μLkg-1), and Z. subfasciatus (EC50=561.5μLkg-1). The bioinsecticide also reduced the percentage of damaged grains for the three pest species studied, and its grain-protectant properties are comparable to or superior in efficacy in relation to a diatomaceous earth-based insecticide (Insecto® at 1000mgkg-1) used as a positive control. Thus, this standardized formulation has promising bioactivity against stored insect species and can be a useful component for IPM of stored grains in Brazil and elsewhere.

Evidence of recent interspecies horizontal gene transfer regarding nucleopolyhedrovirus infection of Spodoptera frugiperda.

BackgroundBaculoviruses are insect-associated viruses carrying large, circular double-stranded-DNA genomes with significant biotechnological applications such as biological pest control, recombinant protein production, gene delivery in mammals and as a model of DNA genome evolution. These pathogens infect insects from the orders Lepidoptera, Hymenoptera and Diptera, and have high species diversity which is expressed in their diverse biological properties including morphology, virulence or pathogenicity. Spodoptera frugiperda (Lepidoptera: Noctuidae), the fall armyworm, represents a significant pest for agriculture in America; it is a host for baculoviruses such as the Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) (Colombia strain, genotype A) having been classified as a Group II alphabaculovirus making it a very attractive target for bioinsecticidal use.ResultsGenome analysis by pyrosequencing revealed that SfMNPV ColA has 145 ORFs, 2 of which were not present in the other sequenced genotypes of the virus (SfMNPV-NicB, SfMNPV-NicG, SfMNPV-19 and SfMNPV-3AP2). An in-depth bioinformatics study showed that ORF023 and ORF024 were acquired by a recent homologous recombination process between Spodoptera frugiperda and Spodoptera litura (the Oriental leafworm moth) nucleopolyhedroviruses. Auxiliary genes are numerous in the affected locus which has a homologous region (hr3), a repetitive sequence associated with genome replication which became lost in SfColA along with 1 ORF. Besides, the mRNAs associated with two acquired genes appeared in the virus’ life-cycle during the larval stage. Predictive studies concerning the theoretical proteins identified that ORF023 protein would be a phosphatase involved in DNA repair and that the ORF024 protein would be a membrane polypeptide associated with cell transport.ConclusionsThe SfColA genome was thus revealed to be a natural recombinant virus showing evidence of recent horizontal gene transfer between different baculovirus species occurring in nature. This feature could be the cause of its high insecticidal power and therefore SfColA becomes a great candidate for bioinsecticide formulations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2218-5) contains supplementary material, which is available to authorized users.

In Vitro Compatibility of an Acetogenin-Based Bioinsecticide with Three Species of Entomopathogenic Fungi

Abstract Despite the promising acute and chronic toxicity of commercial acetogenin-based bioinsecticide formulations reported for different arthropod pests and their recent registration, we are unaware of any published study that examines the compatibility of these botanical derivatives with entomopathogenic fungi, which are important natural and applied biological control agents. We investigated the compatibility of Annona mucosa Jacq. (Magnoliales: Annonaceae) ethanolic seed extract (ESAM), an effective bioinsecticide previously characterized by its high acetogenin content, with 3 entomopathogenic fungal species, Beauveria bassiana (ESALQ-PL63 isolate), Isaria fumosorosea (ESALQ-1296 isolate) and Metarhizium anisopliae (ESALQ-E9 isolate) at various concentrations in different in vitro assays. The effects of the botanical insecticide varied according to the fungal isolate exposed, the biological variable analyzed, and the concentration used. However, based on the compatibility index adopted, ESAM was cla...

Field evaluation of the synergistic effects of neem oil with Beauveria bassiana (Hypocreales: Clavicipitaceae) and Bacillus thuringiensis var. kurstaki (Bacillales: Bacillaceae)

In the present study, the synergistic effects of Beauveria bassiana (Bals.-Criv. Vuill.) (isolate Bb11) and Bacillus thuringiensis var. kurstaki (Berliner) with neem oil were evaluated in three agroecological zones in Benin. Four bioinsecticide treatments (neem oil, neem oil and B. bassiana used separately for different target pests, neem oil mixed with B. bassiana and neem oil mixed with B. thuringiensis) were compared with a calendar-based treatment using synthetic insecticides and a control without insecticides. The bioinsecticide treatments were less effective than the calendar-based treatment at controlling cotton pests. There was no difference in yields and the number of damaged bolls in plots under treatments with the four bioinsecticide formulations, suggesting an absence of synergy between neem oil and B. bassiana and neem oil and B. thuringiensis. The numbers of natural enemies in all the bioinsecticide treatment plots and the control plots were similar and higher than those in the calendar-based treatment plots. The highest yield and profitability were obtained with the calendar-based treatment. Screening the compatibility of plant-based products and biopesticides through bioassays is essential for a successful application of their combinations in any integrated pest management strategy.

Polymer/Layered Silicate Nanocomposite as Matrix for Bioinsecticide Formulation

SummaryDue to current encouragement to the use of bioinsecticides for pest control and the susceptibility of biological agents to external factors, we investigated the use of a polymer nanocomposite (PLN, polymer/ layered silicate nanocomposite) as matrix to encapsulate an entomopathogenic fungus active against pest insects of palm trees. The beads were formed by extrusion and the following variables were assessed: fungus conidial concentration (series 1: 107; series 2: 108 and series 3: 109 conidia/mL) and nanolayered silicate concentration (0; 0.5; 1; 2 and 4%). The matrix was evaluated by X‐ray powder diffraction and Fourier transform infrared spectroscopy and the following characteristics of the products were assessed: percent of encapsulated conidia, size distribution and polydispersity index, swelling index, formulation's in vitro ability to release conidia and stability under different storage temperatures. PLN, whose interactions could be visualized by FTIR, proved to be a potential matrix for this fungus, because, while composed by natural substances non‐toxic to the environment, it succeeded to encapsulate high amounts of conidia (series 2). A barrier effect with bentonite increase was also demonstrated by increased fungus germination time and thermal stability.

Bio-insecticide Bacillus thuringiensis spores encapsulated with amaranth derivatized starches: studies on the propagation "in vitro".

Bacillus thuringiensis (Bt) is one of the bioinsecticides used worldwide due to its specific toxicity against target pests in their larval stage. Despite this advantage, its use is limited because of their short persistence in field when exposed to ultra violet light and changing environmental conditions. In this work, microencapsulation has been evaluated as a promising method to improve Bt activity. The objective of this study was to develop and characterize native and modified amaranth starch granules and evaluate their potential application as wall materials in the microcapsulation of B thuringiensis serovar kurstaki HD-1 (Bt- HD1), produced by spray drying. Native amaranth starch granules were treated by hydrolyzation, high energy milling (HEM) and were chemically modified by phosphorylation and succinylation. The size of the Bt microcapsules varied from 12.99 to 17.14 μm adequate to protect the spores of Bt from ultraviolet radiation. The aw coefficient of the microcapsules produced by the modified starches after drying was low (0.14-1.88), which prevent microbial growth. Microcapsules prepared with phosphorylated amaranth starch presented the highest bacterial count and active material yield. Different concentrations of the encapsulated Bt formulation in phosphorylated amaranth starch showed a high level of insecticidal activity when tested on M. sexta larvae and has great potential to be developed as a bioinsecticide formulation, also, the level of toxicity is much higher than that found in some of the products commercially available.

Effects of oral administration of Bacillus thuringiensis as spore-crystal strains Cry1Aa, Cry1Ab, Cry1Ac or Cry2Aa on hematologic and genotoxic endpoints of Swiss albino mice

Bacillus thuringiensis (Bt) as spore-crystal proteins (Cry) is widely used in bioinsecticide formulations to protect different crops by biological control. Bt insecticides are much more specific than broad-spectrum synthetic chemical insecticides, but after the advent of genetically modified plants expressing δ-endotoxins (transgenic crops), the bioavailability of Cry proteins has increased, and for biosafety reasons their adverse effects should be studied, mainly for non-target organisms. Thus, we evaluated, in Swiss albino mice, the hematotoxicity and genotoxicity of four strains of Bt spore-crystals, Cry1Aa, Cry1Ab, Cry1Ac and Cry2Aa, individually administered by gavage with a single dose of 27mg/kg, 136mg/kg or 270mg/kg, 24h, 72h or 7days before euthanasia. Binary combinations of these four spore-crystal proteins were also assayed at 270mg/kg with a single administration 24h before euthanasia. Control mice received filtered water or cyclophosphamide at 27mg/kg. For hematotoxicity evaluations, blood samples were drawn by cardiac puncture and processed in a multiple automated hematology analyzer; for genotoxicity analyses, micronucleus test was carried out in mice bone marrow cells. Spore-crystal administrations provoked selective hematotoxicity for the three exposure times, particularly for erythroid lineage. A significant reduction in bone marrow cell proliferation demonstrated cytotoxic but not genotoxic effects. These effects persisted for all exposure times, becoming more evident at 7days. Similar results were observed for binary combinations at 24h.