Thuringiensis Isolates Research Articles

In Vitro Evaluation of Native <i>Bacillus thuringiensis</i> (Berliner) Isolates against Different Insects

Evaluation of native strains of Bacillus thuringiensis (Berliner) isolates against different orders of insects and characterization of responsible Cry genes was carried out. Among the native isolates, BGC-1 showed the least LC50 value of 5.24 μg/ ml and was comparable to the reference strain HD1 (2.89 μg/ ml) against tobacco caterpillar Spodoptera litura (Fabricius). The isolate RCM-1 recorded least LC50 value of 4.69 μg/ ml against the housefly Musca domestica Linnaeus. One isolate viz., GHB-1 was found to be potential against both the larvae of S. litura and M. domestica. The isolate RCM-2 registered the least LC50 value of 8.21 μg/ ml against grubs of red flour beetle Tribolium castaneum (Herbst). Several isolates had more than one Cry gene in them. Among them, 12 isolates (63.15%) were found to contain Cry3 genes, 11 isolates harbour Cry4 genes, five isolates had Cry1, Cry2 and Cry11 genes each and four isolates contain Cry7 genes each. Native isolate, GHB-1 had Cry1, Cry3, Cry4 and Cry11 genes.

Potential risk of biofilm-forming Bacillus cereus group in fresh-cut lettuce production chain

Bacillus cereus and Bacillus thuringiensis, which belong to the B. cereus group, are widely distributed in nature and can cause food poisoning symptoms. In this study, we collected 131 isolates belonging to the B. cereus group, comprising 124B. cereus and seven B. thuringiensis isolates, from fresh-cut lettuce production chain and investigated their potential risk by analyzing genotypic (enterotoxin and emetic toxin gene profiles) and phenotypic (antibiotic susceptibility, sporulation, and biofilm formation) characteristics. Enterotoxin genes were present only in B. cereus, whereas the emetic toxin gene was not detected in any of the B. cereus isolates. All isolates were susceptible to vancomycin, which is a last resort for treating B. cereus group infection symptoms, but generally resistant to β-lactam antimicrobials, and had the ability to form spores (at an average sporulation rate of 24.6 %) and biofilms at 30 °C. Isolates that formed strong biofilms at 30 °C had a superior possibility of forming a dense biofilm by proliferating at 10 °C compared to other isolates. Additionally, confocal laser scanning microscopy (CLSM) images revealed a notable presence of spores within the submerged biofilm formed at 10 °C, and the strengthened attachment of biofilm inner cells to the substrate was further revealed through biofilm structure parameters analysis. Collectively, our study revealed the prevalence and contamination levels of B. cereus and B. thuringiensis at fresh-cut lettuce production chain and investigated their genotypic and phenotypic characteristics, aiming to provide valuable insights for the development of potential risk management strategies to ensure food safety, especially along the cold chain.

Native Bacillus thuringiensis isolates against the old-world bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in Ethiopia

ABSTRACT Chickpea ( Cicer arietinum L.) production in Ethiopia has been seriously constrained by the insect pest, Helicoverpa armigera. Insecticides have been used for decades to control this noxious pest. However, the restriction on chemical insecticides necessitates biological pest control alternatives. Therefore, the present study investigated the effectiveness of native Bacillus thuringiensis (Bt) isolates against Helicoverpa armigera 3rd instar larvae. The treatments consisted of Bt concentrations at the LD90 values, whereas the control plots were sprayed with distilled water and a commercial Bt. var. thuringiensis product. The results revealed statistically (p < 0.05) significant differences between the native Bt isolates and the control plots. Post 1st, pre 2nd and post 2nd Bt sprayed larval populations counted per plant ranged from 0.8 ± 0.3–3.1 ± 0.1, 0.6 ± 0.1–3.6 ± 0.0 and 0.5 ± 0.1–3.6 ± 0.4, respectively. Pod damage (%) and grain yield (t/ha) ranged between 9.7 ± 3.1 and 47.5 ± 9.5, 1.0 ± 0.0 and 2.6 ± 0.5, respectively compared to 47.5 ± 9.5% pod damage and 1.0 ± 0.0 (t/ha) grain yield from the control plots. These results indicate the potential of these bio-insecticides in the management of chickpea pod borer. Future research in Ethiopia should focus on additional collection, screening, characterisation, and testing in field trials.

Evaluation and Identification of Novel Insecticidal Toxin Genes From &lt;i&gt;Bacillus Thuringiensis&lt;/i&gt; (Berliner) Isolates

The present study evaluated fifteen Bacillus thuringiensis (Berliner) isolates against second instar larvae of tobacco leaf eating caterpiller Spodoptera litura (F) and Vip genes were identified in the effective isolates. Four NCIM isolates (5111, 5112, 5116 and 5117) were found promising, and their LC50 ranged from 2.5x105 to 3.33x105 ppm and 2.20x105 to 2.85x105 ppm in diet incorporation and surface contamination methods, respectively. The NCIM-5111 isolate exhibited the lowest LC50 of 2.5x105 ppm in diet incorporation and 2.2x105 ppm in the surface contamination method. Surface contamination method revealed more mortality of 6.66 to 10.00% across the NCIM isolates. All the isolates were further used for identification of insecticidal toxin (Vip, Cyt) genes, and it was observed that four NCIM isolates harbour the Vip3Aa1 gene and one viz., NCIM-5112 isolate carries the Vip1/ Vip2 gene, which has distinct motifs from the Vip toxins that are currently in use.

Interactions between Bacillus thuringiensis and selected plant extracts for sustainable management of Phthorimaea absoluta

Phthorimaea absoluta is a global constraint to tomato production and can cause up to 100% yield loss. Farmers heavily rely on synthetic pesticides to manage this pest. However, these pesticides are detrimental to human, animal, and environmental health. Therefore, exploring eco-friendly, sustainable Integrated Pest Management approaches, including biopesticides as potential alternatives, is of paramount importance. In this context, the present study (i) evaluated the efficacy of 10 Bacillus thuringiensis isolates, neem, garlic, and fenugreek; (ii) assessed the interactions between the most potent plant extracts and B. thuringiensis isolates, and (iii) evaluated the gut microbial diversity due to the treatments for the development of novel formulations against P. absoluta. Neem recorded the highest mortality of 93.79 ± 3.12% with an LT50 value of 1.21 ± 0.24 days, Bt HD263 induced 91.3 ± 3.68% mortality with LT50 of 2.63 ± 0.11 days, compared to both Bt 43 and fenugreek that caused < 50% mortality. Larval mortality was further enhanced to 99 ± 1.04% when Bt HD263 and neem were combined. Furthermore, the microbiome analyses showed that Klebsiella, Escherichia and Enterobacter had the highest abundance in all treatments with Klebsiella being the most abundant. In addition, a shift in the abundance of the bacterial genera due to the treatments was observed. Our findings showed that neem, garlic, and Bt HD263 could effectively control P. absoluta and be integrated into IPM programs after validation by field efficacy trials.

High-throughput screening for novel Bacillus thuringiensis insecticidal proteins revealed evidence that the bacterium exchanges Domain III to enhance its insecticidal activity.

Approximately 3000 Bacillus thuringiensis (Bt) isolates were screened to discover novel three-domain (3D) Cry proteins active against Helicoverpa zea (corn earworm). From 400 active isolates found during the primary screening, Cry1Ac and Cry2A, which are known to be active against H. zea, were removed using multiplex-primer PCR and high-throughput column chromatography. This process reduced the number of active cultures to 48. DNA segments encoding Domain III of these 48 cultures were amplified by PCR and sequenced. Sequencing revealed two novel Cry1B-type Domain IIIs. Further sequencing of the flanking regions of these domains revealed that one was part of Cry1Bj (GenBank: KT952325). However, the other Domain III lacked Domains I and II. Instead, this Domain III was associated with two open reading frames, ORF1 and ORF2. ORF1 was identified as an ATP-binding protein, and ORF2 as an ATPase, suggesting that Bt exchanges Domain III among homologous Cry proteins.

&lt;i&gt;In vitro&lt;/i&gt; evaluation of microencapsulated &lt;i&gt;Bacillus thuringiensis&lt;/i&gt; exposed to different temperatures against &lt;i&gt;Helicoverpa armigera&lt;/i&gt; (Hubner)

The experiment was conducted to prepare and evaluate microencapsulation of lyophilized Spore Crystal Aggregate (SCA) of native Bacillus thuringiensis isolate BGC-1 and standard isolate HD-1 against second instar larvae of Helicoverpa armigera at the Department of Agricultural Entomology, Bheemarayanagudi. The zetasizer analyzer results revealed that the microcapsule diameter ranged from 3.2 to 8.3 µm. Median lethal concentrations of the BGC-1 and Bt-HD1 were 0.66 g/l and 0.50 g/l, respectively. UV protectants viz., melanin and (PABA) para-amino benzoic acid were evaluated by exposing microencapsulated Bacillus thuringiensis to temperature regimes of 25°C, 30°C, 35°C, 40°C and 45°C in the B.O.D at different intervals of time. Among four microencapsulated formulations, BGC-1 with melanin recorded the highest mortality of 95 % at zero h exposure. As time increased, the mortality decreased and HD-1 was on par with BGC-1. HD-1 melanin showed significantly higher mortality of 62.50 to 92.50 % followed by BGC-1 (melanin) which ranged from 70 to 90 %. Even though formulations were exposed to different temperatures, because of encapsulation, potential to cause insect mortality was retained.

Isolation and identification of bacteria from the invasive pest Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) and evaluation of their biocontrol potential.

As an alternative to chemical insecticides, gut bacteria of insects could be used to control insect pests. In this study, bacteria associated with Tuta absoluta, an invasive species that has developed resistance to chemical insecticides, were isolated, and their potential for pest control was investigated. We isolated 13 bacteria from larvae of the pest and identified the isolates on the basis of their morphological, physiological, biochemical, and molecular characteristics as Bacillus thuringiensis (Ta1-8), Staphylococcus petrasii (Ta9), Citrobacter freundii (Ta10), Chishuiella changwenlii (Ta11), Enterococcus casseliflavus (Ta12), and Pseudomonas tremae (Ta13). A laboratory screening test at 109 cfu/ml showed that B. thuringiensis (Bt) isolates caused more than 90% mortality after 3 days. Among the isolates, Bt-Ta1 showed the highest mortality in a short time. The LC50 and LC90 values for Bt-Ta1 were estimated to be 1.2 × 106 and 2 × 109 cfu/ml, respectively. Detailed characterization of Bt-Ta1 revealed that it is one of the serotypes effective on lepidopterans and contains the genes cry1Aa, cry2Aa, and vip3Aa, which encode lepidopteran toxic proteins. Bt-Ta1 isolate has been shown to have the potential to be used in the integrated management of Tuta absoluta.

Isolation of two indigenous Bacillus thuringiensis isolates from Natore District in Bangladesh

Isolation of two indigenous Bacillus thuringiensis isolates from Natore District in Bangladesh

Genomic sequencing of fourteen Bacillus thuringiensis isolates: insights into geographic variation and phylogenetic implications

ObjectiveThis work was performed in support of a separate study investigating the activity of pesticidal proteins produced by Bacillus thuringiensis against the Asian citrus psyllid, Diaphorina citri. The fourteen Bacillus isolates chosen were selected from a large, geographically diverse collection that was characterized only by biochemical phenotype and morphology of the parasporal crystal, hence, for each isolate it was desired to determine the specific pesticidal proteins produced, assign each to a Bacillus cereus multilocus sequence type (ST), and predict their placement within the classical Bt serotyping system. In addition, phylogenetic distances between the isolates and Bacillus thuringiensis serovar type strains were determined by calculating digital DNA-DNA hybridization (dDDH) values among the isolates.ResultsBased on the assembled sequence data, the isolates were found to be likely representatives of the Bt serovars kurstaki (ST 8), pakistani (ST 550), toumanoffi (ST 240), israelensis (ST 16), thuringiensis (ST 10), entomocidus (ST 239), and finitimus (ST 171). In cases where multiple isolates occurred within a predicted serovar, pesticidal protein profiles were found to be identical, despite the geographic diversity of the isolates. As expected, the dDDH values calculated for pairwise comparisons of the isolates and their apparent corresponding Bt serovar type strains were quite high (> 98%), however dDDH comparisons of the isolates with other serovar type strains were often surprisingly low (< 70%) and suggest unrecognized taxa within Bt and the Bacillus cereus sensu lato.

Interspecies Horizontal Transfer and Specific Integration of the Mosquitocidal Toxin-Encoding Plasmid pTAND672-2 from Bacillus thuringiensis subsp. israelensis to Lysinibacillus sphaericus.

pTAND672-2, a 144-kb resident plasmid of Bacillus thuringiensis serovar israelensis strain TAND672, was sequenced and characterized. This extrachromosomal element carries mosquitocidal toxin-, conjugation-, and recombinase-encoding genes, together with a putative arbitrium system, a genetic module recently discovered in temperate phages controlling lysogeny-lysis transition and in mobile genetic elements (MGEs) where its function remains clarified. Using conjugation experiments, pTAND672-2 is shown to be a novel integrative and conjugative element (ICE), which can horizontally transfer from B. thuringiensis serovar israelensis to Lysinibacillus sphaericus, another mosquitocidal bacterium, where it integrates into the chromosome. Its integration and circularization are reversible and involve a single-cross recombination between 33-bp specific sites, attB in the chromosome of L. sphaericus and attP in pTAND672-2. CDS143, coding for the putative tyrosine integrase Int143 distantly related to site-specific tyrosine Xer recombinases and phage integrases, can mediate the integration of pTAND672-2 to attB. The B. thuringiensis mosquito-killing genes carried by pTAND672-2 are efficiently transcribed and expressed in L. sphaericus, displaying a slight increased toxicity in this bacterium against Aedes albopictus larvae. The occurrence of pTAND672-2-like plasmids within the Bacillus cereus group was also explored and indicated that they all share a similar genetic backbone with diverse plasmid sizes, ranging from 58 to 225 kb. Interestingly, among them, the pEFR-4-4 plasmid of Bacillus paranthracis EFR-4 and p5 of B. thuringiensis BT-59 also display conjugative capability; moreover, like pTAND672-2 displays a chimeric structure between the pCH_133-e- and pBtoxis-like plasmids, pBTHD789-3 also appears to be mosaic of two plasmids. IMPORTANCE Horizontal transfer of mobile genetic elements carrying mosquitocidal toxin genes may play a driving role in the diversity of mosquitocidal bacteria. Here, the 144-kb mosquitocidal toxin-encoding plasmid pTAND672-2 is the first verified integrative and conjugative element (ICE) identified in Bacillus thuringiensis serovar israelensis. The key tyrosine integrase Int143, involved in the specific integration, is distantly related to other tyrosine recombinases. The study also reports the occurrence and potential interspecies transmission of pTAND672-2-like plasmids with varied sizes in B. thuringiensis, Bacillus paranthracis, and Bacillus wiedmannii isolates belonging to the Bacillus cereus group. This study is important for further understanding the evolution and ecology of mosquitocidal bacteria, as well as for providing new direction for the genetic engineering of biopesticides in the control of disease-transmitting mosquitoes.

Intraspecific Diversity and Pathogenicity of Bacillus thuringiensis Isolates from an Emetic Illness.

This study describes an emetic food-borne intoxication associated with a Bacillus cereus group species and the characterization of the bacterial isolates from the incident in aspects of molecular tying, genetic factors, cytotoxicity, and pathogenic mechanisms relating to emetic illness. Through the polyphasic identification approach, all seven isolates obtained from food and clinical samples were identified as Bacillus thuringiensis. According to multilocus sequence typing (MLST) analysis, intraspecific diversity was found within the B. thuringiensis isolates. Four allelic profiles were found, including two previously known STs (ST8 and ST15) and two new STs (ST2804 and ST2805). All isolates harbored gene fragments located in the cereulide synthetase (ces) gene cluster. The heat-treated culture supernatants of three emetic B. thuringiensis isolates, FC2, FC7, and FC8, caused vacuolation and exhibited toxicity to Caco-2 cells, with CC50 values of 56.57, 72.17, and 79.94 µg/mL, respectively. The flow cytometry with the Annexin V/PI assay revealed both apoptosis and necrosis mechanisms, but necrosis was the prominent mechanism that caused Caco-2 cell destruction by FC2, the most toxic isolate.

Toxicity Evaluation and Genetic Improvement of Bacillus thuringiensis Isolated from Different Regions in Assiut, Egypt against Mosquito Larvae

This study aims to evaluate the toxicity and genetic improvement of Bacillus thuringiensis isolates. Isolates were obtained from soil, insect and water samples from different regions of Assiut, Egypt for biological control of mosquito larvae. B. thuringiensis colonies were identified based on morphological and then by PCR which detect the Cry toxic genes in the isolates. Bioassays were performed to evaluate the toxicity of different strains of B. thuringiensis against mosquito larvae such as (Culex spp). In general, 36 B. thuringiensis isolates were obtained (31 from soil, 4 from insects, and 1 from water). And they were all toxic to mosquito larvae with different mortality percentages from 7 to 97% after 48 hours. Isolate Am2 recorded the highest mortality percentage 97% and Mn3 lowest mortality percentage 7%. PCR revealed that Am2 isolate which caused the highest mortality encodes three different types of Cry toxins, Cyt1AA, Cry1Ac and Cry2Aa. This isolate Am2 was examined by scanning electron microscopy to observe the shape of the Cry proteins. The results showed that the Am2 isolate contained of spherical and cuboidal toxic proteins. Then UV-mutagenesis was performed on the Am2 isolate to improve its toxicity. Out of 30 obtained UV-mutants, only one mutant showed improvement in the mortality of mosquito larvae since it caused a mortality rate of 100%. The results of the present study revealed the larvicidal efficacy of B. thuringiensis (Am2) isolate found in the soil of Assiut, could be used in biological control program of mosquito larvae.

Optimization of feather degradation by a Bacillus thuringiensis isolate using response surface methodology and investigation of the feather protein hydrolysate structure.

Valorization of chicken feather is a long-sought approach for its sustainable disposal. Being protein rich, hydrolyzed chicken feather has a wide range of applications, not limited to formulation of microbiological culture media, animal feed, and biofertilizers, but extends to synthesis of bioplastic films, cosmetics, and biomedicals. In this study, a potent keratinolytic isolate was recovered from soil and identified by 16S rRNA as Bacillus thuringiensis. Feather degradation by the isolate was optimized through response surface methodology. First, one-variable-at-a-time technique to assign the factors that affect feather degradation, then Box-Behnken central composite design model were employed. The model, involving three independent variables (initial pH, inoculum size, and concentration of supplementary glucose), was significant (R2 =0.9716). According to the model, complete feather degradation is obtained at an inoculum size of B. thuringiensis B4 equal to 1×1010 CFU/ml, when feather meal broth is supplemented with 1.5% (w/v) glucose and pH adjusted to 8.5. Protein content of the lysate was 327.8±25μg/ml, and no carbohydrates were detected. SEM/EDX analysis has shown that the hydrolysate consisted mainly of O, P, S, and Se in addition to carbon, while FTIR images assured the presence of carboxyl and amino groups characteristic of peptides and amino acids.

Characterization of Bacillus thuringiensis from Cotton Fields and its Effectiveness against Spodoptera litura

Cotton is a cash crop of many countries as it serves as a source of fiber, edible oil, and seedcake. It is grown in a diverse range of environment. Bacillus thuringiensis (Bt) is a gram positive bacterium found in different habitats but mostly found in soil. This bacterium produces endotoxin which is harmful for various insects. Numerous crystal-forming strains of Bt exist in nature, but not all of them are efficient against insect pests. Bt cotton is prevalent internationally because of less insect attack. The key objective of this research was to analyze different Bacillus thuringiensis isolates residing in soil of different cotton fields of Multan and Bahawalpur districts and to identify them on the basis of morphology. To attain this, different soil samples were collected from Multan (Gup wala, Mosey Wala, Taloki Wala, Hafiz Wala, Choudary Wala, Basti Mangla Mari, Sharkha Wala, Lawain Wala, Ghulamo Wala) and Bahawalpur district (Basti Deewan Wali, Check No 8, Basti Tariqabad, Basti Khaji Wala, Basti Khandin, Basti Rammo Wali, Sultanpur, Khurampur, Jahanpur). About 178 colonies of different bacteria from eighteen soil samples appeared on Nutrient Agar plates. The screening of colonies based on morphology yielded 18 colonies of Bt. We performed endo-toxin production experiment and confirmed the presence of parasporal crystals in identified B. thuringiensis colonies. Colony forming units of soil samples collected from Multan and Bahawalpur represented statistically significant results. Based on morphology, eight colonies of B. thuringiensis were confirmed out of eighteen colonies. Results of the current study revealed that out of 18 isolates, 8 bacterial isolates that were grown on the media had an increased elevation, a round, white, slimy morphology, and smooth edges. Toxicity test for resistance to cotton leaf worm second instar larvae revealed that two strains (BtS2, BtS7) of B. thuringiensis were toxic and showed 39.25% mortality. This study will help to identify the local Bt isolates that can be used for the indigenous insect pest control.

Molecular characterization and toxicity evaluation of indigenous Bacillus thuringiensis isolates against key lepidopteran insect pests

BackgroundThe indiscriminate use of synthetic insecticides is not affordable and poses collateral damage to humans, non-target organisms, and environmental health. Bacillus thuringiensis (Bt) is a cosmopolitan, antagonistic soil bacterium employed as an alternative to chemical insecticides in pest management. In this study, genetic diversity and insecticidal activity of 50 indigenous Bt isolates were investigated in order to find the effective Bt isolates with a broad spectrum of insecticidal activity against lepidopteran insects.ResultsMost of the Bt isolates investigated appeared as creamy white in colour and fried egg or mucoid-type colonies having a flat or raised elevation with entire or undulated margins. Out of 50 isolates investigated, crystal shapes varied from cuboidal (40.00%), spherical (36.00%), bipyramidal (30.00%), rectangular (10.00%), and minute crystal attached to the spores (28.00%). SDS-PAGE analysis revealed that the molecular weight of the crystal proteins of Bt isolates ranged from ~ 30 to ~ 200 kDa in size. Among the Bt isolates screened, ~ 135 kDa size representing Cry1 protein was observed in 12 isolates (24.00%) and 65 kDa representing Cry2 was observed in 14 isolates (28.00%). PCR analysis was performed for cry1, cry2, cry3, cry4, cry9, vip1, vip2, and vip3 genes, which revealed the presence of cry1 gene alone in 5 isolates, and cry2 alone in 7 isolates, whereas 7 isolates were positive for cry1, cry2, and vip3 genes together. None of the Bt isolates showed the presence of cry3, cry4, cry9, vip1, and vip2 genes. Toxicity of Bt isolates was tested against four species of lepidopteran larvae, viz. Plutella xylostella Linnaeus, Helicoverpa armigera Hubner, Spodoptera litura Fabricius, and S. frugiperda Smith. Among them, 6 isolates (T29, T30, T31, T357, T381, and T388) produced 100% larval mortality against all four species of insects.ConclusionThe present study showed the diversity of Bt isolates and confirmed the significance of relentless exploration of Bt isolates for novel genes. Further investigations need to be carried out to disclose the hidden potential of these toxic isolates.

Prevalence and characterization of toxigenic Bacillus cereus group isolated from low-moisture food products.

The present study was conducted to determine the occurrence of B. cereus group members in low-moisture food products by phenotypic and genetic assessment and to evaluate the toxigenic potential of B. cereus group isolates. According to the results of their morphological shape, growth temperature range, strain-specific gene distribution, 79.5% and 20.5% among 112 isolates were identified as B. cereus sensu stricto (s.s.) and B. thuringiensis, respectively and other toxigenic B. cereus group members was not found. All B. cereus group isolates possessed nheABC, hblACD, cytK, entFM genes, and the most frequent gene was nheA. Only three B. cereus s. s. isolates exhibited as emetic toxin gene-harboring B. cereus group. Several B. cereus s.s. and B. thuringiensis isolates from a low-moisture food products were moderate biofilm formers and showed resistance to rifampicin, tetracycline, or clindamycin. The existence of B. cereus s.s. and B. thuringiensis in low-moisture food products indicates the possible risk of foodborne infections due to their virulence potential.

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.

Assessment of the Larvicidal Potency of Bacillus thuringiensis Against the Moth of Spoladea recurvalis on Amaranths Plant

The control of Spoladeare curvalis, a major pest of amaranth plant, has been through the use of synthetic pesticides which have been reported to have detrimental effects. This study was aimed at assessing the larvicidal potency of B. thuringiensis against the larvae of Spoladea recurvalis on amaranth plant. The assessment of the larvicidal potency of B. thuringiensis isolates from cow range land and refuse dump sites in Zaria metropolis habouringCry1 gene (CR2, CR4, RD1) was carried out by spraying spore crystal mixture of B. thuringiensis isolates on amaranth plants infested with Spoladea recurvalis larvae. The mortality of S. recurvalis larvae, under a screen house were assessed after 24, 48 and 72hours of exposure to the spore crystal mixture of B. thuringiensis isolates. Spore crystal mixtures of B. thuringiensis isolates were effective in causing larvae mortality with more than 60% mean mortality rate of S. recurvalis larvae after 72hours of exposure. The mortality rate of Spoladea rescurvalis increased with increase in exposure period. Spore crystal mixture of Bacillus thuringiensis isolates should be considered as an alternative to synthetic pesticides for the control of Spoladea recurvalis larvae on amaranth plant. Keywords: Bacillus thuringiensis; spore; larvae; amaranth; larvicidal.

Characterisation of native &lt;i&gt;Bacillus thuringiensis&lt;/i&gt; isolates toxicity to fall armyworm, &lt;i&gt;Spodoptera frugiperda&lt;/i&gt; (J.E. Smith)

Toxicity of nine indigenous Bacillus thuringiensis (Bt) isolates collected from Tamil Nadu, India were tested against fall armyworm, Spodoptera frugiperda. At 30 ?g/ml concentration, two Bt isolates viz., T350 and T532 recorded 100 per cent mortality whereas isolates T527 and T532 registered 96 per cent mortality against neonate larvae of S. frugiperda in leaf dip bioassay. SDS PAGE analysis of spore crystal mixture revealed the presence of Cry1 and Cry2 proteins with visible bands at 130 kDa and 65 kDa. PCR screening results showed the presence of cry1 (cry1A, cry1Aa, cry1Ab), cry2 (cry2Aa, cry2Ab) in four isolates and vip3A genes in three isolates but cry9 gene was not present in any of the isolates tested.