PCR-based identification of Bacillus thuringiensis isolated from soil samples in Nigeria

Six isolates of Bacillus thuringiensis isolated from soil samples confirmed to be toxic to mosquito larvae were differentiated using a PCR-Based technique. Three of these isolates initially identified using a serological technique were further differentiated with the PCR amplification of the delta-endotoxin target sequences. Using the total DNA of isolates as template, at least four isolates yielded amplicons one or all the crystal protein genes, cryI a, b, c, or II with sizes ranging from 238-1070 bp. None of these isolates yielded an amplicon for any of Cry IV A, B and D tested. Of the four isolates identified by PCR technique one isolate remained unidentified by serology.     

Modified method for selective enrichment and isolation of Bacillus thuringiensis from soil

Many colonies of Bacillus thuringiensis (Bt) were found to be lost using the isolation technique described by Martin and Travers modified by Morris (Morris method). Hence, a modified method for isolation of B. thuringiensis is described and compared with the Morris method. Screening methodology adopted by this method delivers an immensely rich and potentially more useful library of Bt colonies with ‘presumptive-positive’ morphology and 10-fold higher per cent frequency of Bt colonies as compared to the Morris method.     

Metalloprotease from Bacillus thuringiensis

Bacillus thuringiensis var. kurstaki was shown to produce an extracellular, metal chelator-sensitive protease during the early stages of sporulation. Protease production in nutrient broth was dependent upon supplementation with Mn2+ or Ca2. The addition of Ca24 was required for enzyme stabilization...     

Trichostrongylus colubriformis: isolation and characterization of ovicidal activity from Bacillus thuringiensis israelensis

Bioassay of media fractions from cultivation of Bacillus thuringiensis israelensis revealed that ovicidal activity for eggs of the ruminant nematode Trichostrongylus colubriformis was found in microbial crystals, but was not released into culture medium. The purified delta-endotoxin of B. t. israelensis, composed of two 25 kDa proteins, had no effect on nematode eggs. A fraction that had high ovicidal activity for eggs of T. colubriformis was isolated by high performance liquid chromatography from crystals of B. t. israelensis. Retention of the compound(s) on size exclusion columns indicated a mol wt of 1510 when compared to standards. The LD50 of this fraction for nematode eggs was not altered significantly by 5 mM calcium chloride with and without ethylenediaminetetraacetic acid or the enzyme inhibitor phenylmethylsulfonyl fluoride (10(-6) M). The fraction was susceptible to proteolytic hydrolysis by bacterial protease VI whereas the fungal protease XIII slightly decreased the ovicidal effects of the fraction. The ovicidal activity was stable for 2 days at ambient temperature or 2 months at 0 C, but declined after 7 days at ambient temperature. Little activity was lost after heating to 100 C for 60 min. The ovicidal effects were also pH dependent with increased toxicity at alkaline pH values. The fraction, however, had no effect on larvae of the mosquito Aedes aegypti or mice after intraperitoneal injection.     

cry Genes profiling and the toxicity of isolates of Bacillus thuringiensis from soil samples against American bollworm,Helicoverpa armigera

Aims: The aim of this study was to search for Bacillus thuringiensis (Bt) harbouring cry1A gene which could effectively control cotton pest, American bollworm, Helicoverpa armigera. Methods and Results: cry gene profiling of 50 Bt isolates showed the presence of cry1, cry2, cry3, cry4, cry7, cry8 and cry9 genes. None of the isolates harboured cry1 gene alone. It was always found in combination with cry3. There was no isolate positive for cry10 gene. Considering isolates with single cry genes, the frequency of cry4 was predominant (22%) followed cry2 (6%), cry3 (4%) and cry8 (2%). Isolates having two cry genes in combination had 14% incidence for cry2 + cry4, 12% for cry3 + cry4 and 10% for cry1 + cry3. The most dominant three gene linkage was cry1 + cry3 + cry4. Further profiling of cry1 gene showed that cry1K gene was abundantly present in all combinations such as cry1A, cry1D, cry1F and cry1I. However, cry1C existed independent of other subtypes. Finally, the Bt isolates with cry1A were analyzed for 16S rRNA gene, which showed two distinct groups of isolates on the basis of sequence homology. Bioassays of spore–crystal mixtures of SBS‐Bt4, 8, 17, 21 and 26 harbouring cry1 against neonate larvae of H. armigera showed LC₅₀ 1288, 1202, 467·7, 524·8 and 108·5 μg ml^?1. The SBS‐Bt26 showed fourfold higher toxicity than the cry 1Ac harbouring positive control, HD‐73. Conclusions: None of the isolates harboured single cry 1 gene. They were always in combination of two or three genes. A Bt isolate (Bt26) had fourfold higher toxicity against H. armigera larvae compared with the positive control HD 73 and hence can be commercially exploited to control insect pest. Significance and Impact of the Study: The inter relationship between the cry genes content and the toxicity may allow better understanding of Bt ecology.     

DNA isolation from soil samples for cloning in different hosts

Many protocols to extract DNA directly from soil samples have been developed in recent years. We employed two extraction methods which differed in the method of lysis and compared these methods with respect to yield, purity and degree of shearing. The main focus was on the specific isolation of DNA from different microorganisms, especially DNA from actinomycetes, as these cells are very difficult to lyse, in contrast to non-actinomycetes. Thus, we used both methods to isolate DNA from Pseudomonas, Arthrobacter and Rhodococcus and from soil spiked with the respective microorganisms. Both methods rendered high DNA yields with a low degree of shearing, but differed in the type of cells that were lysed. By one protocol (utilizing enzymatic lysis) only DNA from the Gram-negative Pseudomonas strain could be obtained whereas, by the other protocol (utilizing mechanical lysis), all microorganisms that were used could be lysed and DNA extracted from them. Using a combination of both protocols, DNA from those organisms could be obtained selectively. Furthermore, one of the protocols was modified, resulting in higher DNA yield and purity.     

Extracellular ribonuclease from Bacillus thuringiensis

The ability of the strain Bacillus thuringiensis var. subtoxicus to produce extracellular ribonuclease (ribonuclease Bt) was studied. It was found that the culture medium possesses a RNA-depolymerizing activity whose maximum is observed 4-5 hours after the beginning of the linear growth phase. A three-step chromatography of the culture extract on phosphocellulose resulted in a homogeneous enzyme with a molecular mass of 12000 Da. The enzyme showed the maximum activity towards RNA at pH 8.5, catalyzed the hydrolysis of polyribonucleotides and guanosine-2',3'-cyclophosphate. Hence, the enzyme can be related to base-nonspecific cyclizing ribonucleases showing the guanylic specificity towards nucleoside-2',3'-cyclophosphates.     

Malachite green pre-enrichment medium for improved salmonella isolation from heavily contaminated samples

V an S chothorst , M. & R enaud , A. M. 1985. Malachite green pre-enrichment for improved salmonella isolation from heavily contaminated samples. Journal of Applied Bacteriology 59 , 223–230.
Large numbers of competitive bacteria may hinder the isolation of salmonellas from food and environmental samples when a pre-enrichment method is used. The addition of 0.1 g/1 of malachite green (MG) to buffered peptone water (BPW) inhibited the multiplication of Gram-positive bacteria. Brilliant green had a similar effect but only when the normal recommended concentration of 0.02 g/1 was raised to 0.05 g/1. Pure strains of salmonellas were inhibited by MG in BPW, but addition of non fat dried milk (NFDM) (5 g/1 or more) counteracted this effect. MG did not affect the recovery of salmonellas injured by heat, freezing, low water activity or acidity in BPW with NFDM. It was concluded that addition of MG to BPW may improve the possibility of isolating salmonellas from heavily contaminated materials by limiting the competitive growth of Gram-positive bacteria and the subsequent lowering of the pH of the broth.     

Malachite green pre-enrichment medium for improved salmonella isolation from heavily contaminated samples

Large numbers of competitive bacteria may hinder the isolation of salmonellas from food and environmental samples when a pre-enrichment method is used. The addition of 0.1 g/l of malachite green (MG) to buffered peptone water (BPW) inhibited the multiplication of Gram-positive bacteria. Brilliant green had a similar effect but only when the normal recommended concentration of 0.02 g/l was raised to 0.05 g/l. Pure strains of salmonellas were inhibited by MG in BPW, but addition of non fat dried milk (NFDM) (5 g/l or more) counteracted this effect. MG did not affect the recovery of salmonellas injured by heat, freezing, low water activity or acidity in BPW with NFDM. It was concluded that addition of MG to BPW may improve the possibility of isolating salmonellas from heavily contaminated materials by limiting the competitive growth of Gram-positive bacteria and the subsequent lowering of the pH of the broth.     

Simple method for the isolation of the antilepidopteran toxin from Bacillus thuringiensis subsp. kurstaki

A protein with a molecular mass of 66 kDa was isolated by a simple, rapid, and inexpensive method, using 3-N-morpholinopropanesulfonic acid, potassium thiocyanate, and dithiothreitol, from a mixture of spores, parasporal crystals, and cell debris of Bacillus thuringiensis subsp. kurstaki. The protein was active against the third instar larvae of Trichoplusia ni, was soluble in 19 mM Na2CO3, and was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and confirmed as the insecticidal component of the 132-kDa protoxin of B. thuringiensis subsp. kurstaki by an enzyme-linked immunosorbent assay using antibodies prepared against the protoxin.     

A technique for the effective enrichment and isolation of Bacillus thuringiensis

Abstract The Neurospora crassa exo -1 mutant produced maximum extracellular glucoamylase activity in media supplemented with starch as the sole carbon source. The apparent molecular mass of the enzyme was 82 kDa (SDS-PAGE and gel filtration). The enzyme was a glycoprotein with 5.1 % carbohydrate content and exhibited a temperature optimum of 60 °C. The pH optima were 5.4 and 5.0 for glucoamylase and maltase activities, respectively. Cu²⁺ inhibited maltase activity while Mn²⁺ stimulated glucoamylase activity. The purified enzyme hydrolyzed branched substrates more efficiently than linear substrates. Starch was the best substrate utilized and amylose was hydrolyzed faster than maltose. Kinetic experiments suggested that maltose and starch were hydrolyzed at the same catalytic site.     

Bacillus thuringiensis in fecal samples from greenhouse workers after exposure to B. thuringiensis-based pesticides

In a study of occupational exposure to Bacillus thuringiensis, 20 exposed greenhouse workers were examined for Bacillus cereus-like bacteria in fecal samples and on biomonitoring filters. Bacteria with the following characteristics were isolated from eight individuals: intracellular crystalline inclusions characteristic of B. thuringiensis, genes for and production of B. cereus enterotoxins, and positivity for cry11 as determined by PCR. DNA fingerprints of the fecal isolates were identical to those of strains isolated from the commercial products used. Work processes (i.e., spraying) correlated with the presence of B. thuringiensis in the fecal samples (10(2) to 10(3) CFU/g of feces). However, no gastrointestinal symptoms correlated with the presence of B. thuringiensis in the fecal samples.     

Optimization of growth medium for the production of spores from Bacillus thuringiensis using response surface methodology

The effects of cultivation medium compositions including tapioca, fishmeal, CaCO₃ and (NH₄)₂SO₄ for the growth of Bacillus thuringiensis YMB 96-1988 were accessed by using response surface methodology (RSM). The two-level (2^4–1) fractional factorial designs (FFD) which involve two concentrations of each nutrient, and the paths of steepest ascent were effective in searching for the major factors of the bacteria growth. This allows the fitting of a first order linear model to the data. In this study, supplementary CaCO₃ showed a negative effect on the spore production based on the first order regression coefficients derived from SAS programme. Subsequently, a 2³ central composite design (CCD) was used for allocation of treatment combinations. Preliminary studies showed that tapioca and fishmeal is believed to be the major factors for the growth of B. thuringiensis. Estimated optimum compositions for the production of spores by B. thuringiensis are as follows: tapioca, 5.01%; fishmeal 5.86%; (NH₄)₂SO₄ 0.06% and resulted in a maximum spore count of 8.56?×?10⁸/ml was obtained. This value is close to the 8.35?×?10⁸/ml spore density as counted from actual experimental observations.     

Characterization of Bacillus thuringiensis soil isolates from Cuba, with insecticidal activity against mosquitoes

Chemical insecticides may be toxic and cause environmental degradation. Consequently, biological control for insects represents an alternative with low ecological impact. In this work, three soil isolates (A21, A51 and C17) from different regions of the Cuban archipelago were identified, characterized and evaluated against Aedes aegypti and Culex quinquefasciatus. The new isolates were compared with reference IPS82 strain and two strains isolated from biolarvicides Bactivec and Bactoculicida, respectively. The differentiation was done by morphological, biochemical, bioassays activity and molecular methods (SDS-PAGE, plasmid profile and random amplified polymorphic analysis). All isolates were identified as Bacillus thuringiensis. The A21, A51 and C17 isolates showed higher larvicide activity than Bactivec's isolated reference strain, against both A. aegypti and C. quinquefasciatus. A21 isolate had a protein profile similar to IPS82 and Bactivec strain. A51 and C17 isolates produced a characteristic proteins pattern. A21 and A51 isolates had plasmid patterns similar to IPS82 standard strain, while C17 isolate had different both plasmid profile and protein bands. All the studied isolates showed a diverse RAPD patterns and were different from the strains previously used in biological control in Cuba.     

Survival and conjugation of Bacillus thuringiensis in a soil microcosm

The survival and conjugation ability of sporogenic and asporogenic Bacillus thuringiensis strains were investigated in broth, in non-amended sterile clay soil monoculture and in mixed soil culture. The 75 kb pHT73 plasmid carrying an erythromycin resistance determinant and a cry1Ac gene was transferred in mating broth and soil microcosm. Survival of strains was assessed in soil monoculture and in mixed soil culture for up to 20 days. Sporogenic strains rapidly formed viable spores which were maintained until the end of the experiment. The asporogenic strains were no longer recovered after 8 days of incubation. This study shows that the environmental impact of asporogenic B. thuringiensis strains is lower than that of sporogenic B. thuringiensis strains. Thus, the use of asporogenic strains may significantly reduce any potential risk (gene transfer, soil and plant contamination) due to the dissemination of B. thuringiensis-based biopesticides in the environment.     

Immigration of Bacillus thuringiensis to bean leaves from soil inoculum or distal plant parts

AIMS: We addressed the process of immigration of Bacillus thuringiensis from soil to leaves and its capacity to grow on bean diffusate medium (BDM), a medium designed to simulate the nutrient composition of the phylloplane. METHODS AND RESULTS: Two different B. thuringiensis strains were inoculated into soils, onto seeds or onto lower leaves of bean plants to determine if they were able to disperse to upper leaves under controlled conditions. While B. thuringiensis isolates were commonly recovered from leaves exposed to such inocula, populations were very low (<10 CFU cm(-2) of leaf). In addition, the number of cells of B. thuringiensis recovered decreased with increasing distance from the soil or from the inoculated leaves. Moreover, B. thuringiensis colonies did not grow well on BDM. CONCLUSIONS: This indicates that B. thuringiensis disperses poorly from the soil or the seed to the leaves or between leaves of the same plant under controlled conditions. Bacillus thuringiensis apparently has greater nutrient requirements than other bacterial species that are prominent inhabitants of the phylloplane. SIGNIFICANCE AND IMPACT OF THE STUDY: Finding the mechanisms that favour bacteria colonization on leaves will in turn help to improve the efficacy of biocontrol agents against the target pests.     

Assessment of microencapsulated formulations for improved residual activity of Bacillus thuringiensis

Bacillus thuringiensis Berliner is a highly efficacious bioinsecticide used to control lepidopteran pests in the field. Unfortunately, it has limited residual activity on plants because sunlight inactivates spores and crystals and they can be washed off by rain. To minimize loss of activity, formulations must contain UV protectants, stickers, or both. We tested approximately 80 formulations and determined optimal combinations of ingredients and spray drying conditions for improving B. thuringiensis residual activity after simulated rain and simulated sunlight. B. thuringiensis stability, after simulated sunlight (xenon light/8 h) and rain (5 cm/50 min), was improved using formulations based on lignin, corn flours, or both, with up to 20% of the active ingredient, when compared with technical powder or Dipel 2x in laboratory assays. Two formulations, made with corn flours or lignin + pregelatinized corn flour (PCF), killed 51.6 and 75.3% of Ostrinia nubilalis (Hübner) neonates after rain, respectively, versus 27% for technical powder. When the insecticidal activity was tested after simulated sunlight, corn flour-based formulations killed 78.5% of test larvae, and the lignin + PCF formulation killed 70.4%, in contrast to technical powder which caused an average of 29% mortality. Formulations made with Dipel 2x rather than technical powder, caused 62.5% mortality (corn flour-based formulations), and 72.3% mortality (lignin + PCF), versus 53.4% for Dipel 2x after rain. When tested after simulated sunlight, formulations killed 95% of the larvae (average of both formulations) versus 82% for Dipel 2x. In a field test, formulations were applied to cabbage and insecticidal activity was determined against Trichoplusia ni (Hübner) neonates exposed to treated leaves. Insecticidal activity of the corn flour-based formulations was comparable to Dipel 2x for 4 d after treatment, but was significantly better than Dipel 2x 7 d after application. A lignin and PCF-based formulation showed significantly higher residual activity than Dipel 2x, 4 and 7 d after application.