Thuringiensis Isolates Research Articles

Pathogenicity of Syrian Isolates of Bacillus Thuringiensis ( Berliner ) against the Cereal Leafminer Syringopais Temperatella Led. ( Lep. , Scythrididae ) under Laboratory Conditions

This study investigates the efficacy of 22 Syrian isolates of Bacillus thuringiensis (Berliner) (Bt) against Syringopais temperatella under laboratory conditions by feeding larvae on bacteria-contaminated barley leaves.The results indicated that all Bt isolates caused concentration and time related mortality, in which the highest mortalities were recorded at the highest concentration (original concentration), and at the latest time after Bt application(72 h).Mortality caused by all Bt isolates was significantly higher than mortality in the control. Isolate no. 1 gave significantly (P<0.05) the highest mortality percentage (73.3%) against the early larval instars. For the late larval instars, the isolates no. 21 and 22 were at bar with each other and gave significantly (P<0.05) the highest percentage of mortality (53.3%). Early larval instars were significantly (P<0.05) more susceptible to all concentrations of Bt isolates used than the late larval ones.In conclusion, the study showed that some of B. thuringiensis isolates tested in this study were pathogenic to S. temperatella.

Bio Efficacy and Cry Profile of Native Bacillus thuringiensis Isolates against Cabbage Leaf Webber, Crocidolomia binotalis and Spodoptera litura Fabricius

An investigation was carried out to assess the bio-efficacy and cry profile of hundred native Bacillus thuringiensis isolates against Cabbage Leaf Webber viz., Crocidolomia binotalis and Spodoptera litura at the Department of Entomology, University of Agricultural Sciences, Dharwad. Among the hundred native isolates used, isolates DBT-763, DBT-787, DBT-754 and DBT-2370 reported 86.67 per cent mortality against Crocidolomia binotalis and DBT-772 showed 73.33 per cent mortality against Spodoptera litura . Lepidopteron specific cry genes viz., cry1, cry2, cry8, and cry9 genes amplified in native isolates viz ., DBT-2367, DBT-2366, DBT-2299, DBT-2299, respectively and none of the studied native isolates shown amplification for the cry20 gene.

Diversity of &lt;i&gt;Bacillus thuringiensis&lt;/i&gt; strains isolated from coffee plantations infested with the coffee berry borer &lt;i&gt;Hypothenemus hampei&lt;/i&gt;

The coffee berry borer Hypothenemus hampei Ferrari (Coleoptera: Scolytidae) was first reported infecting Costa Rican coffee plantations in the year 2000. Due to the impact that this plague has in the economy of the country, we were interested in seeking new alternatives for the biological control of H. hampei, based on the entomopathogenic bacteria Bacillus thuringiensis. A total of 202 B. thuringiensis isolates obtained from Costa Rican coffee plantations infested with H. hampei were analyzed through crystal morphology of the crystal inclusions and SDS-PAGE of 6-endotoxins, while 105 strains were further evaluated by PCR for the presence cry, cyt and vip genes. Most of the Bt strains showed diverse crystal morphologies: pleomorphic (35%), oval (37%), bipyramidal (3%), bipyramidal and oval (12%), bipyramidal, oval and pleomorphic (10%) and bipyramidal, oval and cubic (3%). The SDS-PAGE analyses of the crystal preparations showed five strains with delta-endotoxin from 20 to 40 kDa, six from 40 to 50 kDa, seven from 50 to 60 kDa, 19 from 60 to 70 kDa, 29 from 70 to 100 kDa and 39 from 100-145 kDa. PCR analyses demonstrated that the collection showed diverse cry genes profiles having several genes per strain: 78 strains contained the vip3 gene, 82 the cry2 gene, 45 the cry1 and 29 strains harbored cry3-cry7 genes. A total of 13 strains did not amplified with any of the cry primers used: cry1, cry2, cry3-7, cry5, cry11, cry12 and cry14. Forty-three different genetic profiles were found, mainly due to the combination of cry1A genes with other cry and vip genes. The genetic characterization of the collection provides opportunities for the selection of strains to be tested in bioassays against H. hampei and other insect pests of agricultural importance.

Comparative analysis of quantitative reverse transcription real-time PCR and commercial enzyme imunoassays for detection of enterotoxigenicBacillus thuringiensisisolates

Entomopathogenic Bacillus thuringiensis is closely related to Bacillus cereus, a human pathogen known to cause emesis and diarrhea. Standard detection methods do not distinguish these bacilli. Hemolysin BL (hbl) and non-hemolytic enterotoxin (nhe) genes that encode, respectively, HBL and NHE enterotoxins, are known to be harbored in both bacterial species, suggesting that differentiation of these bacilli is clinically and epidemiologically relevant. In this study the reliability of quantitative reverse transcription real-time PCR (qRT-PCR) and enzyme immunoassays (EIAs) in detecting hbl and nhe transcripts and corresponding toxins in environmental B. thuringiensis isolates was assessed. At least one enterotoxin gene was present in each isolate, and nhe or hbl genes were found in 85% and 55% of the strains, respectively. Based on statistical analyses, both BCET-RPLA and Duopath detected HBL at similar levels, and TECRA and Duopath can be used interchangeably for the detection of NHE, although TECRA has significantly lower sensitivity than Duopath. Thus, as potential enterotoxic B. thuringiensis strains occur in the natural environment, and EIA results may not correspond with the presence of enterotoxin genes and their expression, we suggest that reliable interpretation will be significantly enhanced by including qRT-PCR to support inferences based on EIAs.

FAFLP analysis of Brazilian Bacillus thuringiensis isolates.

A total of 65 Bacillus thuringiensis (Bt) isolates were subjected to analysis of genetic relationship using fAFLP (fluorescent Fragment Length Polymorphism), in order to determine the genetic diversity within a group of Bt strains. 26 strains from different subspecies were identified as it follows: 9 kindly provided by the USDA (United States Department of Agriculture), 9 kindly provided by the Institute Pasteur and eight from Embrapa Maize and Sorghum Bt Collection, and 39 strains with no subspecies information also from Embrapa’s Bt Collection. DNA sample was double digested with restriction enzymes EcoRI and MseI, and the fragments were linked to adapters. Selective amplification reactions were performed using five primer combinations and the amplified fragments were separated by gel electrophoresis on an ABI377 sequencer. Genetic distances were obtained by the complement of the Jaccard coefficient and the groups were performed by the UPGMA method. Five primer combinations generated 495 scorable fragments and 483 were found to be polymorphic. Out of 26 subspecies, strains 344 and T09 (B. thuringiensis subsp. tolworthi) showed the highest similarity (15%), while isolates HD3 B. thuringiensis subsp finitimus and T24 B. thuringiensis subsp neoleonensis were the most genetically distant (92%). B. thuringiensis isolates with no subspecies identification, found in samples from Goiás State showed higher similarity forming a group with an average distance of 6%, and the closest subspecies to this group was B. thuringiensis subsp thuringiensis (HD2) with 52% of similarity. This similarity may be due to the fact that these organism exchange genetic material by conjugation, and it is relatively common to have evolutionary characteristics of their ancestors.

In vitro susceptibility of Varroa destructor and Apis mellifera to native strains of Bacillus thuringiensis

Fifty-four Bacillus-like strains were isolated from dead Varroa destructor collected in 24 colonies of bees from seven apiaries. The total proteins of 22 isolates were evaluated against adult female V. destructor (100 μg/mL), but only nine caused over 80 % mortality. The LC50 of EA26.1, EA3 and EA11.3 isolates were calculated. The most virulent isolate was EA26.1, as 96.7 % of the mites were killed 36 h after treatment, and the LC50 was 1.50 μg/mL. EA3 produced 93.3 % mite mortality after 48 h treatment, with an LC50 of 7.1 μg/mL. Finally, EA11.3 produced 93.3 % mite mortality after 60 h treatment, with an LC50 of 22.8 μg/mL. Additionally, the EA3 and EA26.1 isolates were used to determine mortality on adults and larvae of worker bees of A. mellifera. When the 16S rRNA gene was amplified and sequenced, the three strains were found to belong to the species B. thuringiensis. The results showed that honey bee adults and larvae are not negatively affected by identified B. thuringiensis isolates, indicating that they could be successfully used for the biological control of V. destructor without adverse effects on adults and larvae of A. mellifera, and reduce the negative impact of this mite on colonies.

Acquisition of Insect-Resistant Transgenic Maize Harboring a Truncated cry1Ah Gene via Agrobacterium-Mediated Transformation

A novel insecticidal gene cry1Ah was cloned from Bacillus thuringiensis isolate BT8 previously for plant genetic engineering improvement. Truncated active Cry1Ah toxin has a toxicity level similar to that of the full-length Cry1Ah toxin. In this study, plant expression vector pMhGM harboring truncated cry1Ah gene was transformed into maize (Zea mays L.) immature embryos by Agrobacterium tumefaciens-mediated transformation at which maize alcohol dehydrogenase matrix attachment regions (madMARs) were incorporated on both sides of the gene expression cassette to improve gene expression. A total of 23 PCR positive events were obtained with a transformation efficiency of 5% around. Bioassay results showed that events 1-4 and 1-5 exhibited enhanced resistance to the Asian corn borer (Ostrinia furnacalis). These two events were further confirmed by molecular analysis. Southern blot suggested that a single copy of the cry1Ah gene was successfully integrated into the maize genome. Western blot and ELISA showed that the foreign gene cry1Ah was expressed stably at high level in maize and could be inherited stably over generations. The results of a bioassay of T1-T4 transgenic maize plants indicated that the transgenic plants were highly toxic to the Asian corn borer and their resistance could be inherited stably from generation to generation. Thus, events 1-4 and 1-5 are good candidates for the breeding of insect-resistant maize.

Bacillus cereus and Bacillus thuringiensis spores in Korean rice: Prevalence and toxin production as affected by production area and degree of milling

We determined the prevalence of and toxin production by Bacillus cereus and Bacillus thuringiensis in Korean rice as affected by production area and degree of milling. Rough rice was collected from 64 farms in 22 agricultural areas and polished to produce brown and white rice. In total, rice samples were broadly contaminated with B. cereus spores, with no effect of production area. The prevalence and counts of B. cereus spores declined as milling progressed. Frequencies of hemolysin BL (HBL) production by isolates were significantly (P ≤ 0.01) reduced as milling progressed. This pattern corresponded with the presence of genes encoding the diarrheal enterotoxins. The frequency of B. cereus isolates positive for hblC, hblD, or nheB genes decreased as milling progressed. Because most B. cereus isolates from rice samples contained six enterotoxin genes, we concluded that B. cereus in rice produced in Korea is predominantly of the diarrheagenic type. The prevalence of B. thuringiensis in rice was significantly lower than that of B. cereus and not correlated with production area. All B. thuringiensis isolates were of the diarrheagenic type. This study provides information useful for predicting safety risks associated with B. cereus and B. thuringiensis in rough and processed Korean rice.

English

Biological control may reduce nematode populations to acceptable levels using Bacillus thuringiensis. Thus, the search for new cry proteins that are active on this type of target is highly relevant. To identify novel cry genes, B. thuringiensis isolates were analysed using molecular biology. The isolates with DNA sequences resembling genes that are effective against nematodes were fully sequenced through primer walking. The characterised novel cry gene was cloned in an expression vector, and the target protein was expressed at in an appropriate E. coli strain. The highest levels of protein expression were expressed at 18°C. To discern the nematotoxicity of this protein, bioassays were conducted using free living nematodes as targets. The bioassays revealed that the assessed free living nematodes were susceptible to the analysed cry protein. These results indicate that nematodes can be targets of cry protein produced by isolates of this bacterium. Key words: Biological control, primer walking, cry proteins, bioassays.

Characterization of eight Bacillus thuringiensis isolates originated from fecal samples of Fuzhou Zoo and Fuzhou Panda Center

In eight fecal samples of 5 herbivorous animals from Fuzhou Zoo and Fuzhou Panda Center were found eight Bacillus thuringiensis isolates. Obtained isolates were characterized by crystal microscopy, cry identification, and assay against the development of Eimeria tenella oocyst in chicken embryo. Bt WCB1, WCB2, WCB3 and WCB8, originated from horse, spotted deer, giant panda and lesser panda, respectively, all exhibited bipyramidal and cuboidal crystal and harbored the same cry genes, which were cry1Cb, cry1Db, cry1Fa, cry1Ib and cry2Ab. WCB7, a Bt isolate with bipyramidal crystal from zebra, showed a different cry pattern, cry1Cb, cry1Db, cry1Fa and cry1Ia. Bt WCB6, the third Bt isolate from giant panda, also contained bipyramidal crystal, however, harbored cry1Ib only. It was noted that Bt isolate WCB5 contained cuboidal crystal and harbored an unknown cry3 gene. It was very interesting that no cry genes were detected from Bt isolate WCB4 with dot-like crystal. Parasporal crystal proteins of WCB3, WCB4 and WCB7 obviously inhibited the development of E. tenella oocyst in chicken embryo.

Screening ofBacillus thuringiensisIsolates Recovered from Diverse Habitats in India for the Presence of Insect and Nematode-activecryGenes

Novel cry genes with different structure and mode of action are potential candidates for resistant management strategies. Isolation of novel genes needs thorough screening of large number of native Bacillus thuringiensis (Bt) isolates collected from diverse habitats and ecotypes. In the present study, 63 Bt isolates recovered from different habitats in India have been investigated for their insecticidal and nematicidal cry gene content. Bt isolates were screened through Polymerase Chain Reaction (PCR) using degenerate primers for the presence of cry1, cry1A and nematode-active cry genes. Among the collection of 63 Bt isolates 21 were found positive for the presence of cry1-type genes, 49 for cry1A-type genes and 20 for nematode-active cry genes. Nine Bt isolates were found positive for all three types of cry genes. The study also show that PCR based screening method is rapid and highly useful for characterization of Bt isolates to reveal their insecticidal spectrum.

Screening &lt;i&gt;Bacillus thringiensis&lt;/i&gt; Isolates from Soil Samples of Wuzhishan Primitive Tropic Rainforest in Hainan and Identifying Novel cry-type Genes

The Wuzhishan, located at the center of Hainan Island, is the largest Primitive Tropic Rainforest in Hainna island. In this study, we collected 234 soil samples from Wuzhishan at the different heights above sea level from 700 m to 1 650 m. Following the approach of selection culture medium with sodium acetate and treatment of high temperature, we o Bt ained 886 Bacillus isolates and further detected 21 Bacillus thuringiensis (Bt) isolates through observation of microscopy, of which the isolating rate reached 2.3%. In order to mine the insecticidal genes from the identified Bt isolates, the PCR strategy with universal primer pairs, combined with DNA sequencing technology was used to detected cry genes. The result showed that the Bt isolates from Wuzhishan include cry1-like , cry4-like , cry39-like , cry40-like , cry31-like , cry46-like genes and so on, presenting that the o Bt ained Bt isolates are abundant in novel cry genes, which may facilitate to clone novel Bt crystal toxin genes.

Occurrence of Bacillus thuringiensis and their phages in Yemen soil.

Bacillus thuringiensis (Bt) isolates were found in all samples of soil in nine Governorates of Yemen. From 384 isolates of Bacillus recovered from these soil samples after acetate selection, 104 isolates (27.1%) were Bt. Five isolates of Bt were selected and insecticidal activity was tested against Culex pipiens, Callosobruchus maculatus and Spodoptera littoralis. The Bt isolate YH18 gave toxicity to all tested insects larvae. This study extended to isolate phages active against the selected Bt isolates. Five phages were isolated and classified into two groups of tailed phages. Four phages with long non-contractile tails and hexagonal heads (Siphoviridae) and one phage with very short tail and isometric head (Podoviridae). Susceptibility of selected Bt to infect by these phages was studied by spot-test technique. Also the Bt isolate no YH18 was resistant to all tested phages. This is the first report illustrates the diversity and the abundance of Bt and Bt phage in Yemen soil.

Proteomics study of silver nanoparticles toxicity on Bacillus thuringiensis

Emerging technologies in functional genomics and proteomics provide a way of achieving high-throughput analyses, understanding effects on protein populations and sub-populations and follow up environmental stresses. To accomplish these, the action of homemade spherical Silver nanoparticles colloidal suspension (AgNPs) against Bacillus thuringiensis (isolate from Oryza sativa L. rhizosphere) was investigated by a proteomic approach (2-DE and NanoLC/FT-ICR MS identification). Thirty four responsive (up/down regulated) proteins were identified. Proteomic results revealed that an exposure of B. thuringiensis cells with different concentrations of AgNPs resulted in an accumulation of envelope protein precursors, indicative of the dissipation of a proton motive force. Identified proteins are involved in oxidative stress tolerance, metal detoxification, transcription and elongation processes, protein degradation, cytoskeleton remodeling and cell division. The expression pattern of these proteins and their possible involvement in the nontoxicity mechanisms were discussed.

Selection and characterisation of an HD1‐like Bacillus thuringiensis isolate with a high insecticidal activity against Spodoptera littoralis (Lepidoptera: Noctuidae)

Spodoptera littoralis (Boisduval) larvae are known by their susceptibility to Bacillus thuringiensis subsp. aizawai strains. In order to prevent the appearance of B. thuringiensis (Bt) resistance and to develop economical Bt-based biopesticides, the selection and the characterisation of a B. thuringiensis isolate toxic against S. littoralis larvae and overproducing δ-endotoxins were investigated. Among 124 Tunisian B. thuringiensis isolates assessed against S. littoralis larvae, four isolates showed toxicity similar to and higher than the toxicity of the aizawai strain HD133 and the kurstaki strain HD1 respectively. The plasmid pattern of the selected isolates was similar to that of HD1. Polymerase chain reaction (PCR) analysis using specific primers revealed that these isolates present different gene contents. The only detected gene encoding Spodoptera-specific toxin was cry9. The selected isolates were found to produce bipyramidal and cubic crystals. The assessment of δ-endotoxin production by these isolates showed that BUPM28 produced 43.71 and 80.81% more δ-endotoxin than HD1 and HD133 respectively. The application of osmotic or heat shock stress on the BUPM28 isolate made it possible to enhance δ-endotoxin production by 22 and 23% respectively. On the basis of its potent insecticidal activity and its high level of δ-endotoxin production, the BUPM28 isolate can be considered to be an effective alternative for the control of S. littoralis.

Virulence of some native Bacillus thuringiensis isolates against Ephestia kuehniella (Zeller) (Lep., Pyralidae) and Pieris brassicae (Lep., Pieridae) larvae isolated from stored products of Urmia city

Bacillus thuringiensis isolates were recovered from numerous sources including soil, grain dust, plant leaves, diseased insect larvae from insectariums and sericulture environments. B. thuringiensis strains were isolated using acetate selection method with 0.025 M. concentration. The morphology of crystals was studied using light microscopy. Bioassay tests were conducted on Ephestia kuehniella (Zeller) (L.) as well as Pieris brassicae (L.). Based on the results, 35 B. thuringiensis strains were isolated from 140 samples. Majority of strains (%31.42) had bipyramidal crystals. There was a significant difference in toxicity to insects among B. thuringiensis isolates; 28.57 and 14.28% of the isolates were toxic to the larvae of P. brassicae and E. kuehniella, respectively, causing more than 50% mortality. Results indicated that B. thuringiensis isolates with insecticidal activity could be used in integrated pest management to control farm and stored product pests.

Effects of Bacillus thuringiensis isolates and single nuclear polyhedrosis virus in combination and alone on Helicoverpa armigera

Cotton bollworm is a key pest of cotton and other crops in many parts of Asia. Evaluation of three native Bacillus thuringiensis isolates and HaSNPV on cotton bollworm was studied. Results revealed that KD-2 isolate was the superior isolate, that its LC50 was 1.2 × 107 spore ml−1 and LC50 of HaSNPV equal to 9.2 × 103 OB ml−1. Interactions on the mortality and debilitating effects between B. thuringiensis KD-2 isolate (3 × 106, 6 × 106, 1.21 × 107 and 1.8 × 107 spore ml−1) and HaSNPV (2.3 × 103, 4.6 × 103 and 9.2 × 103 OB ml−1) on second instars larvae of Helicoverpa armigera were evaluated in laboratory. The larvae of cotton bollworm were fed for 48 h on the treated diets. The results revealed that the Bt at 1.2 × 107 spore ml−1, mixed with all above-mentioned concentrations of HaSNPV, had antagonistic effects. However, the Bt at 1.8 × 107 spore ml−1, mixed with HaSNPV at 9.2 × 103 OB ml−1, had additive effect. The Bt at 6 × 106 spore ml−1 mixed with all above-mentioned concentrations of HaSNPV demonstrated additive effects. The Bt at 3 × 106 spore ml−1 had additive effect with HaNPV at 2.3 × 103 and 4.6 × 103 OB ml−1. The mortality observed in larvae infected with the combination of both Bt (3 × 106 spore ml−1) and HaSNPV (9.2 × 103 OB ml−1) was interpreted as synergist effect. Therefore, it could be concluded that combination of the lowest Bt concentration and the highest HaSNPV concentration had synergist effect on H. armigera second instar larvae.

Bioinsecticide activity of Bacillus thuringiensis isolates on tomato borer, Tuta absoluta (Meyrick) and their molecular identification

Twelve (12) bacterial isolates were isolated from dead larvae of Tuta absoluta (4th instar) from tomato cultivated fields at Fayoum Governorate, Egypt. All isolates were preliminarily identified as members of the genus Bacillus based on morphological and biochemical characteristics. According to the results of the pathogenicity of Bacillus isolates against different larval instars of T. absoluta , the 12 isolates revealed varying efficiencies and the isolates B1, B2, B3 and B4 showed high mortality of 93.3, 90, 86.7 and 80% on day 7, respectively, on the 4th instar larvae. Also, protecto ( Bacillus thuringiensis subsp. kurstaki) recorded the highest mortality when the 4th instar larvae were treated with 2 g / 2 liter of water (96.7%) on the 5th day post treatment. Isolate B12 recorded the lowest percentage mortality of the 4th instar larvae (13.3%). In addition, there was a significant decrease in egg hatching percentage reaching 44.12% compared with the control, meanwhile, the adult emergence decreased after treatment and only 38 and 30 adults emerged from the cages containing tomato plants infested by eggs with B1 and protecto compared with the control which gave 253 adults that emerged. Further, genetic identification of 12 isolates was performed using randomly amplified poylmorphic DNA (RAPD) markers to determine their genetic diversity pattern. Different random primers were used for RAPD amplification, which generated a total of 52 fragments; of these 42 were polymorphic and 10 monomorphic. The primers OPA02, OPA04, and OPA07 produced 100% polymorphic fragments, whereas primers OPA1, OPA3, OPA05, OPA06, OPA08 and OPA09 produced 1, 3, 1, 2, 1 and 2 monomorphic fragments, respectively. When the RAPD banding pattern data was subjected to dendrogram construction, the 4 isolates fell into two separate clusters, clusters I cluster II, which includes 1 and 3 B. thuringiensis isolates, respectively. The RAPD technique was shown to be effective in differentiating closely related isolates and applied to confirm the identification of Bacillus isolates by API system which was used to reveal the phylogenetic relationships between the isolates. Keywords : Bacillus thuringiensis , bioinsecticide, randomly amplified poylmorphic DNA-polymerase chain reaction (RAPD-PCR), instar larvae, Tuta absoluta African Journal of Biotechnology Vol. 12(23), pp. 3699-3709

Characterisation of lepidopteran-active Bacillus thuringiensis isolates recovered from infected larvae

As a part of an ongoing nationwide programme focused on finding novel strains of Bacillus thuringiensis (Bt) that are toxic to some of the major pests that impact economically important crops, we initiated a search for Bt isolates native to Syria. We succeeded in assembling a collection of 40 Bt isolates recovered from infected larvae of Galleria mellonella, Helicoverpa armigera and Ephestia kuehniella. Light microscopy showed that all isolates produce bipyramidal and cuboidal crystal proteins. The 50% lethal concentration of the spore-crystal mixture of the 40 isolates against E. kuehniella larvae varied from 3 to more than 200 µg g−1. A comparison of the LC50 values of the tested isolates with the reference strain Bt kurstaki HD-1 (20.55 µg g−1), showed that some of these isolates have a similar or up to six times higher toxicity potential. PCR screening revealed that all obtained isolates contain cry1 and cry2 genes, whereas only four contain cry9. Moreover, the proteins of 130 and 65/70 Kda encoded by these genes were detected in the SDS-PAGE of the purified parasporal bodies. Flagellar serotyping classified 30 as serovar kurstaki, six isolates serovar aizawai, one isolate cross-reacted with more than one H3 antisera and three were not typeable. Assays of toxicity of the aizawai isolates against third instar of G. mellonella showed that four, which contain cry9, have almost similar toxicity to the commercial strain Bt aizawai B401. Therefore, these isolates could be adopted for future applications to control G. mellonella. Moreover, this study contributes to our knowledge of Bt diversity in Syria where to date very few collections have been described.

Profile of cry from native Bacillus thuringiensis isolates and expression of cry1I

The characterization of 255 Bacillus thuringiensis isolates of Coorg, Sharavatti and BR hills, containing genes known to be active against coleopteran and lepidopteran insect species was done through PCR amplification using the specific and degenerate primers. The isolates were also tested for their insecticidal activity against Plutella xylostella. Among the coleopteran specific genes screened, the most predominant was cry1I gene which was present in 18 isolates at a frequency of 7.05%. cry1 gene was found to be most abundant (35.39%) among the lepidopteran specific genes. A variant of cry1I gene based on amplicon restriction fragment length polymorphism (ARFLP) was cloned into pTZ57R/T and subcloned in an expression vector pQE-30 after amplification of a 2169 bp DNA fragment of cry1I gene from B. thuringiensis DBT189, the sequence which showed 99% homology with known cry1Ia gene from B. thuringiensis subsp. kurstaki. There were six mismatches between the two amino acid sequences. The cry1I- type gene consisted of an open reading frame of 2124 bp that would encode for 720 amino acids. An expected band size of 81 kDa was observed after sodium dodecyl sulphate- polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicating the expression of cry1I gene.