Bacillus Thuringiensis Isolates Research Articles

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.

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

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

&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.

Bacillus thuringiensis VP4: A potential keratinase producer isolated from poultry waste soils of Himachal Pradesh

Keratinase enzymes are protein hydrolyzing enzymes belonging to the serine hydrolase group and act specifically on keratin proteins to degrade them. The current study was conducted to isolate best keratinase producer bacterial strains. Four Bacillus species (B. cereus PP1- OP748781, B. cereus JP1- OP748782, B. cereus TP1- OP748783 and B. thuringiensis VP4- OP420549), were isolated from the poultry farms of Himachal Pradesh. These strains were evaluated for keratinase production by using chicken feathers in shake flask culture. Among four isolates, Bacillus thuringiensis VP4 showed highest enzymatic activity 343 U/ml. The molecular weight of the keratinase was observed 45 KDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The optimization of enzyme production was done with isolate Bacillus thuringiensis VP4 at various parameters such as temperature, pH and effect of metal ions. The maximum keratinase production was observed at 35°C temperature and pH 10.0. The isolated strain Bacillus thuringiensis VP4 might be used to produce poultry compost through degradation of chicken feathers and poultry waste in short time which may also be helpful for farmers and overcoming environmental pollution.

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.

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.

Statistical improvement of protease production from a new isolate Bacillus thuringiensis strain-MA8 and its application in the production of enzyme-modified cheese

Enzyme-modified cheese (EMC) is a concentrated cheese flavor that is produced enzymatically from dairy substrates to provide an intense source of cheese flavor with broad applications. In this study, EMC was produced by enzymatic biotransformation from a new bacterial isolate described and molecularly identified as Bacillus thuringiensis strain-MA8. Optimization of protease production conditions using one-variable-at-a-time followed by multi-factorial (Plackett-Burman and Box-Behnken) designs increased production by 7-fold. Protease was used at different concentrations (300 and 900 U/100 g curd) as a cost-effective source of concentrated cheese flavor in the EMC preparation. Sensorial evaluation of EMC revealed that the overall acceptability, flavor, and texture were improved from the 2nd day compared to the control, and then decreased on the 4th day without any apparent bitterness. The chemical characteristics of EMC showed that the addition of protease extracts increased the total volatile fatty acids, water-soluble nitrogen, and acidity of EMC significantly (p≤0.05) compared to the control. The amino acids profile revealed that EMC1 which was treated with (300 U/100 g curd) protease had the highest essential amino acids (EAA) and EAA/total amino acids ratio. Nutritional parameters including protein efficiency ratio, biological value, and chemical score of EMC were higher than control based on Val, Met + Cys, Ile, Leu, and Phe + Tyr amino acids. Also, Scanning Electron Microscopy showed significant changes in EMC compared to the control. In conclusion, the addition of (300 U/100g curd) of protease revealed good EMC characteristics without any apparent defect.

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.

The potential mosquitocidal activity of cry4A toxic region crystal protein gene from local isolates of Bacillus thuringiensis against Aedes aegypti

The present study was conducted to isolate Bacillus thuringiensis (B.t.) strains from different ecological regions of Pakistan and determined the mosquitocidal activity of cry4A crystal protein gene against the dipteran insects. Out of 25 B.t. isolates, 5 isolates were selected on the basis of toxicity and 4 were identified as Bacillus thuringiensis GCU-DAB-TK-04 (MN922746), GCU-DAB-TK-06 (MT032397), GCU-DAB-TK-09 (MN922747), and GCU-DAB-TK-13 (MT032398). GCU-DAB-TK-04 was found to be the most toxic strain against the 3rd instar larvae of Aedes aegypti. The presence of Cry 4A protein was confirmed by the amplification of cry4A gene (accession number MT001910) from GCU-DAB-TK-04. The highest spores toxicity was shown by GCU-DAB-TK-04 (LC50 = 104 μg/ml) while LC50 values of other strains were GCU-DAB-TK-13 (LC50 = 602 ± 0.43 μg/ml) GCU-DAB-TK-06 (LC50 = 812 ± 0.63 μg/ml), GCU-DAB-TK-12 (LC50 = 1230 ± 1.14 μg/ml), and GCU-DAB-TK-09 (LC50 = 7585 ± 1.17 μg/ml). Similarly, the highest toxicity of total cell protein was also shown by GCU-DAB-TK-04 (LC50=676 ± 0.53 μg/ml while LC50 values of other strains were GCU-DAB-TK-13 (LC50 = 724 ± 1.12 μg/ml), GCU-DAB-TK-06 (LC50 = 741 ± 0.64 μg/ml), GCU-DAB-TK-12 (LC50 = 912 ± 0.65 μg/ml), and GCU-DAB-TK-09 (LC50 = 1621 ± 1.13 μg/ml). The order of toxicity of B.t. strains was GCU-DAB-TK-04 > GCU-DAB-TK-13 > GCU-DAB-TK-06 > GCU-DAB-TK-12 > GCU-DAB-TK-09. Protein analysis showed that 130 kDa (probably Cry4A, B), 70 kDa (Cry4C, D) and 20 kDa (Cyt enhancer protein of Cry4) proteins were present in all B.t. strains. These B.t. strains have found great potential to grow into bio-insecticidal formulation for the eco-friendly control of mosquitoes.

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.

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

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

Isolation, Purification, and Toxicity Test of Bacillus thuringiensis from Cows Cage Soil Againts Drosophila melanogaster

Bacillus thuringiensis is one of the bacteriae species that be able to kill insects. The latest research showed that the cell plasm of this bacteria contains crystalline protein with natural insectisides properties. Therefore the insecticidal effect of this species on insects which is decreasing cultivation plant production is interest to be examined. One of the insect species that are well-known causes of loss of farm production in fruit flies (Drosophila melanogaster). Therefore this research is designed to isolate, purify, and thenassess Bacillus thuringiensis toxicity against Drosophila melanogaster as animal treatment. Bacillus thuringiensis was successfully isolated from soil of cows cage by using Lauria Bertani medium contain 0,25 M buffer solution Acetic, pH 6.8. The result shows that Bacillus thuringiensis isolate is a gram-positive bacteria. Position of endospore is located between the middle and the end of the cell (subterminal spore). Toxicity test was conducted by mixing 0.5 g/L, 1 g/L, 2 g/L, 4 g/L of pure Bacillus thuringiensis isolate into Drosophila melanogaster medium. The treatments effect were monitored for 15 consecutive days. Toxicity test shows that the treatment has no significant effect on the treated animal mortality. This result shows that crystal-producing Bacillus thuringiensis strain isolated from cows cage soil do not have insecticidal properties for Drosophila melanogaster. It is concluded that Drosophila melanogaster is a non-target species of Bacillus thuringiensis.

Evaluation of Cuban Bacillus thuringiensis (Berliner, 1911) (Bacillales: Bacillacea) isolates with larvicidal activity against Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae)

Objective: To evaluate 11 Cuban native Bacillus (B.) thuringiensis isolates in order to select one with the best larvicidal activity against Aedes (Ae.) aegypti and low cytotoxicity.Methods: The cry and cyt genes of the isolates (A21, A51, L95, L910, M29, R84, R85, R87, R89, U81 and X48) were amplified by PCR. The influence of organic matter and NaCl on the larvicidal activity was tested by bioassays. Cytotoxicity was assayed on peritoneal macrophages of BALB/c mice.Results: The cyt1 (Aa, Ab, Ba), cyt2, cry4aA, cry4Ba, cry11 (Aa, Ba, Bb) and cry10 genes were identified in all native Cuban isolates. The larvicidal activity (LC90) of seven isolates was affected by the presence of organic matter in the water, while A21, A51, L910, R84, U81 and X48 had better LC50, LC90, LC95 than the 266/2 9-VII-98 control strain. The LC50 of two isolates was affected by the presence of NaCl and A21, A51, R85 isolate had better larvicidal activity than the 266/2 9-VII-98 control strain. In terms of toxicity against macrophages, the extracts of nine isolates were less cytotoxic than the control strains.Conclusions: Native isolate A21 had the main virulence factors against Ae. aegypti larvae, displayed a good larvicidal activity in presence of different factors related with Ae. aegypti breeding sites, and had low citotoxicity against macrophages. These results can contribute to the improvement of existing biological control strategies and the development of new biolarvicides.

Whole genome analysis and functional characterization of a novel Bacillus thuringiensis (Bt 62) isolate against sugarcane white grub Holotrichia serrata (F)

In this study, we report the whole genome assembly of Bt 62, a novel isolate harbouring cry8 holotype gene identified by us earlier. Sequencing was carried out using a combination of Illumina NextSeq 500 and Oxford Nanopore sequencing Technologies (ONT). The final assembled genome was 6.13 Mb comprising a circular chromosome and four plasmids. The bioassay studies against Holotrichia serrata (F.) (Coleoptera: Scarabaeidae), a polyphagous pest infesting sugarcane and other crops, indicated significant toxicity to first instar grubs over untreated larvae achieving a highest mean mortality of 91.11% for various doses tested. In vitro proteolytic assay and histopathological studies of the midgut of infected white grubs revealed proteolytic processing of the protoxin and extensive degeneration of larval midgut epithelial cells. The results demonstrate that this novel isolate could be used as a biopesticide or its crystal toxin genes could be expressed in sugarcane and other crops for resistance against H. serrata.

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

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

Biosynthesis of 2-Heptanone, a Volatile Organic Compound with a Protective Role against Honey Bee Pathogens, by Hive Associated Bacteria.

Beehives are populated by bacterial species with a protective role against honey bee pathogens thanks to the production of bioactive metabolites. These compounds are largely unexploited despite their high potential interest for pest management. This study evaluated the capability of bacterial species associated with honey bees to produce 2-heptanone, a volatile organic compound with anesthetic properties of the parasitic mite Varroa destructor. The production of this compound was quantified by SPME-GC-MS in a culture filtrate of nine bacterial strains isolated from the surface of honey bees, and the biosynthetic potential was evaluated in bacterial species associated with apiaries by searching for protein homologs putatively involved in its biosynthesis by using biocomputational tools. The findings pointed out that 2-heptanone was produced by Acetobacteraceae bacterium, Bacillus thuringiensis and Apilactobacillus kunkeei isolates in concentrations between 1.5 and 2.6 ng/mL and that its production was strain-specific. Putative methylketone synthase homologs were found in Bacillus, Gilliamella, Acetobacteraceae, Bartonella and Lactobacillaceae, and the protein sequence results were distributed in nine Sequence Similarity Network (SSN) clusters. These preliminary results support the hypothesis that 2-heptanone may act as a mediator of microbial relationships in hives and provide contributions to assess the role and biosynthetic potential of 2-heptanone in apiaries.

Isolation and Use of Bacillus thuringiensis for the Production of Bio-Insecticide in Control of Mosquito Larvae

Bacillus thuringiensis is a widely studied bacterium and it is known for its use in pest management. It is selectively active on pests and less likely to cause resistance; hence it is considered a suitable replacement to chemical pesticides. The study assessed the potential of Bacillus thuringiensis in controlling mosquito larvae. Bacillus thuringiensis isolates selected were tested against secondary stage larvae of mosquito. Thirty-six larvae (6 each) were transferred into each test tubes (7 x 9) cm with 30ml sterile distilled water. The stock suspension of cultures of Bacillus thuringiensis in broth was diluted to 107, 106, 10 5, 104, 103 and 102 in sterile water, following the McFarland standard method for microbial load count. The test tubes were kept at room temperature, larval mortality was observed over time within 24hrs. The results showed that all mosquito larvae died at the 107 and 106 dilutions but at dilutions 105, 104 and 103 though affecting mosquito larvae, it was highly dependent on time because there was a decrease in concentration. The study showed that B. thuringiensis is safe for use in aquatic environments, including drinking-water reservoirs, for the control of mosquito, black fly and nuisance insect larvae. The products should contain the ICPs and be free from other microorganisms and biologically active metabolites.