Cry11 Genes 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.

Selection and Characterization of Bacillus thuringiensis (Berliner) (Eubacteriales: Bacillaceae) Strains for Ecdytolopha aurantiana (Lima) (Lepidoptera: Tortricidae) Control.

The citrus fruit borer, Ecdytolopha aurantiana (Lima, 1927) (Lepidoptera: Tortricidae), is responsible for major losses to the citrus industry because it causes rot and drop of fruits. The current study aimed to select and characterize Bacillus thuringiensis (Berliner, 1911) strains toxic to E. aurantiana. For this purpose, 47 B. thuringiensis strains were evaluated in selective bioassays using first instar larvae of E. aurantiana. The lethal concentration (LC50) of the most toxic strains was estimated, and the strains were characterized by morphological, biochemical, and molecular methods. Of the 47 strains tested, 10 caused mortality above 85% and showed mean lethal concentrations between 1.05E+7 and 1.54E+8sporesmL-1. The lowest LC50 values were obtained for the HD-1 standard strain and the BR145, BR83, BR52, and BR09 strains. The protein profile showed the presence of Cry proteins of 60, 65, 70, 80, and 130kDa. The molecular characterization showed the presence of cry1, cry2, cry3, and cry11 genes. The morphological analysis identified three different crystalline inclusions: bipyramidal, round, and cuboidal. The cry1 and cry2 genes were the most frequent among the B. thuringiensis strains evaluated and encode Cry proteins toxic to insects of the order Lepidoptera, which agree with the toxicity results obtained by the selective bioassays against E. aurantiana. The results showed four different B. thuringiensis strains toxic to E. aurantiana at the same level as the HD-1 standard strain, and these strains have biotechnological potential for E. aurantiana control through the production of transgenic plants or the formulation of biopesticides.

Screening of new isolates of Bacillus thuringiensis (Bt) and cloning of the cry genes

Nine new indigenous isolates of Bacillus thuringiensis (Bt) were characterized for their colony type, crystal inclusion and toxicity analysis with Helicoverpa armigera Hubner and Spodoptera litura Linn. Genomic deoxyribonucleic acid (DNA) isolated from all the new isolates were subjected to screening for cry1 , cry2 , cry4 , cry10 and cry11 genes and predicted possible potential DNA amplicons were cloned and sequenced. Partial cry1 gene fragment (~1.5 kb) amplified by degenerate primers and about 450 bp DNA fragment amplified by cry10 gene specific primers from two isolates T109 and T136 were cloned in to T/A cloning vector. DNA sequencing of about 1.5 kb amplicon showed 99% homology to the holotype sequence of cry1Ac1 . Nucleotide sequence of about 450 bp fragments of isolate T109 and T136 showed homology to a hypothetical protein and serine/threonine phosphatase respectively. Keywords: Bacillus thuringiensis (Bt), cloning, cry genes, polymerase chain reaction (PCR), toxicity analysis

Detection of the mosquitocidal toxin genes encoding Cry11 proteins from Bacillus thuringiensis using a novel PCR-RFLP method.

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for detection of cry11 genes from Bacillus thuringiensis was established. Based on the analysis of conserved regions of the cry11 genes, 2 oligonucleotide primers were designed to amplify a 1459-bp fragment of the cry11Aa gene, and a 1471-bp of the cry11Ba and cry11Bb genes. The amplification products were digested with restriction endonuclease HinfI. Exotic B. thuringiensis strains and native isolates collected from soils, leaves and stored product dust of Argentina were analyzed to study the distribution of cry11 genes. The PCR-RFLP patterns revealed the detection of cry11 genes in 3 of 64 exotic strains and in 10 of 107 native B. thuringiensis isolates tested. Just the cry11Aa gene subclass was detected among these bacteria. Since the methodology was also developed to detect cry11Ba and cry11Bb genes, an experimental future confirmation will be required. Based on the results obtained, the PCR-RFLP method presented may be a valuable tool for specific detection of the mosquitocidal toxin genes encoding Cry11 proteins from B. thuringiensis.

Biological characteristics of Bacillus thuringiensis strain Bt11 and identification of its cry-type genes

A novel strain of Bacillus thuringiensis Bt11, isolated from soil samples in China, was classified and characterized in terms of its crystal proteins, cry genes content. The Bt11 strain showed high toxicity against Spodoptera exigua and Helicoverpa armigera neonates. Bt11 strain shares morphological and biochemical characteristics with the previously described Bacillus thuringiensis subsp. kurstaki. SDS-polyacrylamide gel electrophoresis revealed that crystals were composed of several polypeptides ranging from 20 to 130 kDa, of which the 35, 80, and 130 kDa proteins were the major components. PCR-RFLP with total DNA from strain Bt11 and specific primers for cry1, cry2, cry3, cry4/10, cry7, cry8, cry9, and cry11 genes revealed that cry1Aa, cry1Ab, cry1Ia, and cry9Ea genes were present.

Identification and Distribution of Bacillus thuringiensis Isolates from Primeval Forests in Yunnan and Hainan Provinces and Northeast Region of China

Ninety-two Bacillus thuringiensis isolates were screened from 683 soil samples collected from tropical and semitropical primeval forests in Yunnan and Hainan provinces of China. Several shapes of crystals, including bipyramidal, square, ovoid, spherical, and amorphous, were observed in the B. thuringiensis isolates. Twenty-six pairs of primers were used to identify 31 holotype cry genes at primary rank of the B. thuringiensis cry gene nomenclature system. The cry gene-types of 92 B. thuringiensis isolates and 33 B. thuringiensis isolates screened from Northeast region of China were identified by PCR-RFLP and SDS-PAGE methods. Fifty-eight isolates harbored cry1 genes, 32 isolates cry2 genes, 12 isolates cry8 genes, 3 isolates cry9 genes, 12 isolates cry11 genes, and 13 isolates cry30 genes. Of the tested isolates, 42 produced no reaction product with 26 pairs of primers and also exhibited no toxicity against 8 insect species tested. The isolate Z2-34 harbored a novel cry30 gene, exhibited insecticidal activity against Aedes albopictus of Dipterans. The accession number of the novel genes in this study is AY916046. Isolation and identification of B. thuringiensis and cry gene are important for investigating the diversity of B. thuringiensis resources and cloning new cry gene.

Diversity of Colombian strains of Bacillus thuringiensis with insecticidal activity against dipteran and lepidopteran insects

To evaluate the genetic and molecular diversity and insecticidal activity of Bacillus thuringiensis isolates from all the natural regions of Colombia. A total of 445 isolates from a collection of B. thuringiensis were characterized. The parasporal crystal morphology that was most abundant was bipyramidal (60%). Almost 10% of the isolates were toxic to Spodoptera frugiperda and 5.6% against Culex quinquefasciatus larvae. cry gene content determined by PCR indicated that 10.6% of the isolates contained cry1 genes and 1.1% contained cry2, cry4 or cry11 genes. Protein content of the parasporal crystal was determined by SDS-PAGE; 25 and 18 different protein profiles were found in isolates active against S. frugiperda and C. quinquefasciatus, respectively. Bacillus thuringiensis presents great genetic and molecular diversity even in isolates from the same soil sample. Moreover, the diversity and activity of the isolates might have a relationship with the geographical origin of the samples. The results obtained here indicate that some of the B. thuringiensis isolates characterized in this study are potential control agents that could be used in programmes against mosquitoes and S. frugiperda.

Diversity of Bacillus thuringiensis strains in the maize and bean phylloplane and their respective soils in Colombia.

To evaluate the distribution of Bacillus thuringiensis strains from maize and bean phylloplane and their respective soils. B. thuringiensis was isolated from the phylloplane and soil of maize and bean from three municipalities in Antioquia, Colombia. Ninety six samples of phylloplane and 24 of soil were analyzed. A total of 214 isolates were obtained from 96 phylloplane samples while 59 isolates were recovered from 24 soil samples. Sixty five per cent and 12% of the phylloplane and soil isolates, respectively, showed activity against Spodoptera frugiperda. These isolates contained delta-endotoxin proteins of 57 and 130 kDa. The most toxic isolates against S. frugiperda had the genotype cry1Aa, cry1Ac, cry1B, and cry1D. In contrast, 27% of the phylloplane isolates and 88% of the soil isolates were active against Culex quinquefasciatus and had protein profiles similar to B. thuringiensis serovar. medellin and B. thuringiensis serovar. israelensis. The most active isolates contain cry4 and cry11 genes. The predominant population of B. thuringiensis on the phylloplane harbored the cry1 gene and was active against S. frugiperda, whereas in soil, isolates harboring cry11 gene and active against C. quinquefasciatus were the majority. The predominance of specific B. thuringiensis populations, both on the leaves and in the soil, suggests the presence of selection in B. thuringiensis populations on the studied environment.

Distribution and characterization of Bacillus thuringiensis on the phylloplane of species of piper (Piperaceae) in three altitudinal levels.

Bacillus thuringiensis is found naturally on the phylloplane. In this study 35 samples from 13 species of the genus Piper (Piperaceae) were collected from three altitudinal levels located between 1800 and 2900 m above sea level in the Colombian Andean forest of Central Cordillera. Two hundred and fifty-six isolates of B. thuringiensis were obtained from 74% of the samples studied. B. thuringiensis index (number of isolates of B. thuringiensis/number of isolates of sporulated bacilli) was 0.2. The isolates were characterized by crystal morphology, the presence of cry genes by PCR, and toxicity against insects. Fifty-five percent of the isolates found presented bipyramidal-crystal morphology, and 42% had round-crystal morphology. Seventy percent of the isolates amplified cry1 [cry one] genes (generally toxic to lepidopterans); 41.4% amplified cry4 and/or cry11 [cry eleven] genes (generally toxic to dipterans), and none of the isolates amplified cry3 genes (generally toxic to coleopterans). The most abundant genotype of cry genes (54.7% of the total) was cry1Aa, cry1Ab, cry1Ac, cry1Ad, and cry1B. From the total isolates found, 7.8% presented both cry1 and cry11 genes, and five isolates (2.0%) harbored cry1, cry4, and cry11 genes; all these isolates were toxic to Culex quinquefasciatus (Diptera) but not to Spodoptera frugiperda (Lepidoptera). To our knowledge, these genotypes have not been previously reported. Overall, almost 60% of the isolates were toxic to S. frugiperda, and a little more than 40% of the isolates were toxic to C. quinquefasciatus. The populations of viable vegetative cells and spores per unit area were estimated and studied statistically. No significant differences in the number of B. thuringiensis isolates per cm2 of leaf among the three altitudinal levels were found, nor were they found among the different Piper species evaluated. This study increases the knowledge of the ecology of B. thuringiensis.