Diversity of Bacillus thuringiensis strains from soil in China and their pesticidal activities

A large number of Bacillus thuringiensis (Bt) isolates have been obtained from soil samples in China. The flagellar antigen serotypes, cry genes and crystal proteins of 570 Bt isolates were determined, and the pesticidal activity was assayed against the insects, Plutella xylostella, Heliothis armigera, Phaedon brassicae and Locusta migratoria manilensis, and the snail, Oncomelania hupensis. The results indicated that the Bt isolates were distributed within 35 H-serotypes, in which isolates of H3 were the most abundant (20%) followed by H5 (13%), H7 (9%) and H4 (8.7%), whereas isolates of other H-serotypes were less than 6%. The percentage of isolates containing the genes cry1Ac, cry1Aa/cry1Ac, cry1Aa/cry1Ac/cry1Ab, and cry1Aa/cry1Ac/cry1C was 14.7%, 6.6%, 5.6%, and 7.0%, respectively, while 265 isolates, representing 46.5% of the 570 Bt isolates, did not show any amplification product for the genes cry1Aa, cry1Ac, cry1C, cry2, cry3, cry4, cry7Aa. Some of the 570 Bt isolates caused high mortality of the assayed pests with 14.9%, 6%, 1.6%, 1.1%, and 0.2% of the isolates killing more than 90% of P. xylostella, H. armigera, P. brassicae, O. hupensis, and L. migratoria manilensis, respectively. The remaining 76.2% of the 570 isolates caused no mortality or less than 90% mortality against the tested insect and snail species.     

Detection and characterisation of a Bacillus thuringiensis crystal protein with nematicidal activity against the pinewood nematode Bursaphelenchus xylophilus

Nematicidal Bacillus thuringiensis (Bt) strains were isolated from forests in Zhejiang, China for further characterisation. PCR analysis was performed with nine pairs of primers specific for cry1, cry2, cry3, cry4, cry5, cry6, cry9, cry11 and cry13 to characterise and classify cry gene groups from Bt isolates. The isolates from individual cry groups were tested for nematicidal activity against the pinewood nematode Bursaphelenchus xylophilus, which is implicated in pine wilt disease. PCR identified 14 different categories of cry gene combinations, indicating a large diversity of cry genes. The cry1 gene was by far the most abundant in Bt isolates and was found in 68% of samples. The Bt isolates zjfc85 and zjfc392 were from two distinct classes, but shared the same cry5 amplification profile and the same ~130 kDa protein; they had the highest nematicidal activity against pinewood nematode during the 48 h exposure tests, resulting in 90 and 59% mortality (9% of mortality under control conditions), respectively. The ~130 kDa Cry protein from isolate zjfc85 was purified and named as Cry5Ba3. Bioassay results indicated pinewood nematode was highly susceptible to Cry5Ba3 and exhibited profound growth abnormalities after exposure to Cry5Ba3. Our results are a novel finding and provide a potential strategy to manage pine wilt disease caused by B. xylophilus based on a nematicidal Bt.     

Characterization of Bacillus thuringiensis isolates from soil and small mammals that harbour vip3A gene homologues

The insecticidal and psychrotropic potential of 132 new isolates of Bacillus thuringiensis from northeastern Poland (74 from animals and 58 from soil) were determined by screening these for vip and cry genes encoding, respectively, vegetative insecticidal proteins (Vip) and Cry proteins, and cspA that encoded the CspA cold shock protein that confers psychrotropy in Bacillus species. The vip3A gene, encoding Vip3A toxic to lepidopterans, was found in ~5% of the isolates from animals and ~17% the isolates from soil, whereas coleopteran-specific vip1 and vip2 genes were present in 8% of the isolates from soil. Nucleotide sequences of vip3A-specific amplicons were highly conserved, with only a few containing minor differences from vip3A. Despite the high level of vip3A conservation, isolates harbouring the gene demonstrated a high level of heterogeneity based on whole-cell genomic DNA RFLP analysis with pulsed-field gel electrophoresis (PFGE) and plasmid profiling. Eight isolates positive for vip3A contained cry1 and six also harboured the cry2 gene, which encodes an endotoxin toxic to lepidopteran insects. However, none of these isolates contained cry genes coding for proteins toxic to coleopteran or dipteran insects. Due to the known potential for synergistic interactions between Vip and Cry proteins, the isolates positive for vip3A and cry genes may be used in resistance management strategies directed against lepidopteran larvae. Finally, all of the B. thuringiensis vip3A-positive isolates harboured the cspA gene, but only two were confirmed to be psychrotrophs.     

Isolation and characterization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains from different grain habitats in Turkey

Bacillus thuringiensis (Bt) is a gram-positive, spore-forming bacterium and it produces insecticidal crystal (cry) proteins during sporulation. Because the genetic diversity and toxic potential of Bt strains differ from region to region, strains have been collected and characterized all over the world. The aim of this study is to isolate Bt strains in grain-related habitats in Turkey and to characterize them on the basis of crystal morphology, cry gene content, and chromosomal and plasmid DNA profiles. Four approaches were taken analysis with phase contrast (PC) microscopy, polymerase chain reaction (PCR), pulsed field gel electrophoresis (PFGE) and plasmid isolation. Ninety-six samples were collected from Central Anatolia and the Aegean region. Bt was isolated from 61 of 96 samples (63.5) and 500 Bt-like colonies were obtained. One hundred and sixty three of the colonies were identified as Bt based on cry protein formation using PC microscopy. Among the examined colonies, the overall proportion identified (as Bt index) was 0.33. We found that 103 isolates were positive for the five different cry genes (cry1, cry2, cry3, cry4 and cry9) examined with PCR. In addition, plasmid profiling of 37 cry gene-positive isolates indicated that the 15 kb plasmid band was present in all isolates; however, 11 of 37 isolates had more than one plasmid band at different sizes. Finally, chromosomal DNA profiling by PFGE gave rise to different DNA patterns for isolates containing the same cry gene which suggests a high level of diversity among the Bt strains isolated.     

Cloning and characterization of truncated <Emphasis Type="Italic">cry1Ab</Emphasis> gene from a new indigenous isolate of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

The insecticidal crystal protein(s) encoded by cry gene(s) of Bacillus thuringiensis (Bt) have been used for insect control both as biopesticides and in transgenic plants. A new 3′-truncated cry1Ab gene was cloned from an indigenous isolate of Bt, A19-31. Nucleotide sequencing and homology search revealed that the deduced amino acid sequence of Cry1Ab toxin of Bt strain A19-31 had a variation of two amino acid residues with the holotype sequence, Cry1Ab1. Expression of the 3′-truncated cry1Ab gene was studied in an acrystalliferous strain of Bt (4Q7). SDS-PAGE and immunostrip analysis of spore-crystal mixture revealed a low level expression of the 3′-truncated cry1Ab gene. Insecticidal activity assay showed that the recombinant 3′-truncated cry1Ab gene product was toxic to larvae of both Helicoverpa armigera and Spodoptera litura.     

Distribution and diversity of Dipteran-specific cry and cyt genes in native Bacillus thuringiensis strains obtained from different ecosystems of Iran

One hundred and twenty-eight Bacillus thuringiensis isolates from fields of different ecological regions of Iran were collected to study the distribution and diversity of Dipteran-specific cry and cyt genes. The percentage of samples with Bt showed significant differences between different regions and also between different fields. The most Bt frequency was observed in the soil samples collected from Caspianic zone (7%) and soils of cotton (17%). Characterization of isolates was based on morphological characteristics of crystals, plasmid profiles and protein band patterns as well as PCR analysis using general and specific primers for 22 different cry and cyt genes encoding proteins active against mosquitoes. Thirty-eight different cry gene profiles were detected in this collection. Several of them were found to be different from all previously published profiles and none of the previous researches reported these numbers of profiles. Strains containing cry2-type genes were the most abundant and represent 57.1% of the isolates. Strains harboring cry24 and cry10 genes were also highly abundant (38.7 and 32.8%, respectively). cry11, cry4, cry17, cry19, cry21, cry29, cyt1, and cry9 genes were less abundant, found in 25.7, 14.3, 11.4, 1.4, 4.3, 1.4, and 10% of the strains, respectively. Among the cry2 gene containing isolates, 37.5% strains harbored cry2Aa, 55% cry2Ab, 2.5% cry2Ac, and 5% other or novel cry2-type genes. Among the cry4 gene containing isolates, 0% strains harbored cry4A, 60% cry4B, and 40% cry4C, cry4D or novel cry4 type genes. Finally, based on crystal morphology, protein patterns and PCR, 21 strains were selected as potentially high Dipteran-active for bioassays. Also our results showed that some of the isolates may harbor minimum a putative novel cry gene.     

Cloning,Characterization and Diversity of Insecticidal Crystal Protein Genes of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Native Isolates from Soils of Andaman and Nicobar Islands

Bt strains were isolated from soils of Andaman and Nicobar Islands and characterized by microscopic and molecular methods. Diversity was observed both in protein and cry gene profiles, where majority of the isolates showed presence of 65 kDa protein band on SDS-PAGE while rest of them showed 130, 72, 44, and 29 kDa bands. PCR analysis revealed predominance of cry1I and cry7, 8 genes in these isolates. The PCR screening strategy presented here led us to identify putative novel cry genes which could be active against Coleoptera insects. Variation in the nucleotide sequences of cry genes from the isolates suggests that the genetic diversity of Bt isolates results from the influence of different ecological factors and spatial separation between strains generated by the conquest of different habitats in the soils of Andaman and Nicobar islands. The implications of our studies are important from the point of view of identifying novel cry genes that could be toxic to insects other than lepidoptera.     

Molecular Characterization of Novel Serovars of Bacillus thuringiensis Isolates from India

Novel Bacillus thuringiensis isolates GS4, GN24 and UP1 were isolated and characterized by determination of serotyping, insecticidal protein by SDS-PAGE, plasmid composition, cry gene content and insect toxicity. Serologically two isolates GS4 and UP1 were allocated to the H3abce which is a new serovar while isolate GN24 was of H3ab type. Isolate GS4 produced flat crystal inclusions while UP1 produced cuboidal crystals. PCR analysis found that both isolates contained cry1 and cry1Ac genes. The major protein bands found of isolate GS4 were of molecular weights 175, 135, 97, 88, 66, 54 and 27 kDa, isolate UP1 were of 85, 60 and 40 kDa and isolate GN24 were of 130, 90, 66 and 45 kDa. Though isolates GS4 and UP1 belonged to a new serovar H3abce, they showed different crystal inclusions and cry gene content. Isolate GS4 was toxic to lepidopteran insect larvae of Helicoverpa armigera but UP1 did not showed any toxicity.     

Expression of a cry2Aa gene in an acrystalliferous Bacillus thuringiensis strain and toxicity of Cry2Aa against Helicoverpa armigera

In the recent past research has been mainly focused on the expression of cry1 genes of Bacillus thuringiensis (Bt) to engineer lepidopteran insect resistance in plants. Search for structurally different toxins is necessary for the management of resistance development in insects. The intact cry2Aa operon (3.95 kb) of a new isolate of Bt, 47-8, was subcloned into a Bt shuttle vector, pHT3101 (6.7 kb). Recombinant pHT3101 containing the cry2Aa operon of Bt strain 47-8 was named as pTN2Aa and used to transform acrystalliferous Bt strain 4Q7 by electroporation. Phase contrast microscopic observation revealed the presence of crystalline inclusions in the transformants of Bt strain 4Q7 harbouring pTN2Aa. SDS–PAGE of a spore–crystal mixture prepared from transformants of acrystalliferous Bt strain 4Q7 harbouring pTN2Aa showed a single band of about 65 kDa alone confirming the expression of the cloned cry2Aa. Bioassay with Helicoverpa armigera showed 71.4% mortality caused by the proteins encoded by the newly cloned cry2Aa gene (at the concentration of 2.3 g/l) on the seventh day and all the survivors that escaped from Cry2Aa toxicity showed severe (81–99%) inhibition in larval growth.     

Expression of a synthetic cry1AcF gene in transgenic Pigeon pea confers resistance to Helicoverpa armigera

Pigeon pea is an important legume. Yield losses due to insect pests are enormous in the cultivation of this crop. Expression of cry proteins has led to increased resistance to pests in several crops. We report in this paper, expression of a chimeric cry1AcF (encoding cry1Ac and cry1F domains) gene in transgenic pigeon pea and its resistance towards Helicoverpa armigera. PCR, Southern hybridization, RT‐PCR and Western analysis confirmed stable integration and expression of the cry1AcF gene in pigeon pea transgenics. When screened for efficacy of the transformants for resistance against H. armigera, the transgenics showed not only high mortality of the larva but could also resist the damage caused by the larvae. Analysis for the stable integration, expression and efficacy of the transgenics resulted in the identification of four T3 plants arising from two T1 backgrounds as highly promising. The results demonstrate potentiality of the chimeric cry1AcF gene in developing H. armigera‐resistant pigeon pea.     

Dissection of <Emphasis Type="Italic">cry</Emphasis> Gene Profiles of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Isolates in Taiwan

On the basis of the newly revised nomenclature system of cry genes, the PCR amplification method has been adopted to resolve the cry gene combinations of 294 Bacillus thuringiensis isolates from five selected areas of Taiwan. Our results indicate that cry1 (especially cry1A + 1B + 1F) and cry2 were the most abundant cry genes in Taiwan. In contrast, cry3 and cry6 genes were detected only on Yang Ming Mountain, while the cry13 gene was found only on Snow Mountain. In addition, some distinctive combinations of cry genes were detected in distinct areas of Taiwan, such as cry1C, cry1D, cry1C + 1D, cry4, cry1 + 4, cry1 + 11, cry4 + 11, and cry1 + 4 + 11 in the Taipei area; cry1A + 1C + 1F in the Taichung area; cry1E and cry1A + 1B + 1I on Yang Ming Mountain; cry1 + 13, cry1 + 2 + 11, and cry1 + 2 + 13 on Snow Mountain; and cry1 + 5 and cry1 + 2 + 5 on Jade Mountain. These data clearly indicate that the distribution of cry gene combinations of B. thuringiensis isolates seems to be geographically related.     

Characterization of a Bacillus thuringiensis strain with a broad spectrum of activity against lepidopteran insects

The insect pathogen Bacillus thuringiensis is suitable for use in biological control, and certain strains have been developed as commercial bioinsecticides. The molecular and biological characterization of a Bacillus thuringiensis subsp. aizawai strain, named HU4‐2, revealed its potential as a bioinsecticide. The strain was found to contain eight different cry genes: cry1Ab, cry1Ad, cry1C, cry1D, cry1F, cry2, cry9Ea1, and a novel cry1I‐type gene. Purified parasporal crystals from strain HU4‐2 comprised three major proteins of 130–145 kDa, which were tested for their insecticidal potency to four species of Lepidoptera (Helicoverpa armigera, Spodoptera exigua, S. littoralis, and S. frugiperda) and three species of mosquito (Culex pipiens pipiens, Aedes aegypti, and Anopheles stephensi). The crystal proteins were highly toxic against all the species of Lepidoptera tested, moderately toxic against two of the mosquito species (C. pipiens and Ae. aegypti), but no toxicity was observed against a third species of mosquito (An. stephensi) at the concentrations used in our study. The LC₅₀ values of the HU4‐2 Bt strain against H. armigera larvae (5.11 µg/ml) was similar to that of HD‐1 Bt strain (2.35 µg/ml), the active ingredient of the commercial product Dipel®. Additionally, the LC₅₀ values of the HU4‐2 Bt strain against S. littoralis, S. frugiperda, and S. exigua (2.64, 2.22, and 3.38 µg/ml, respectively) were also similar to that of the Bt strain isolated from the commercial product Xentari® for the same three species of Spodoptera (1.94, 1.34, and 2.19 µg/ml, respectively). Since Xentari® is significantly more toxic to Spodoptera spp. than Dipel® and, reciprocally, Dipel® is significantly more toxic against H. armigera than Xentari®, we discuss the potential of the HU4‐2 strain to control all these important lepidopteran pests.     

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.     

Cloning and characterisation of a novel cry1H gene effective against lepidopterous larvae from a Bacillus thuringiensis strain

We characterised a novel holotype cry gene (cry1Hc1) harboured in BN23-5 Bt strain which was isolated from soil samples in Sichuan, China. In this study, the full length of the cry1Hc1 gene was cloned from the strain. The cry1Hc1 gene was inserted into a shuttle vector (pSTK) and expressed in an acrystalliferous mutant Bacillus thuringiensis HD73^?. In this transformant, cry1Hc1 was expressed and diamond-shaped parasporal crystals were formed. The resulting insecticidal activity showed that the Cry1Hc1 protein exhibited high larvicidal activity against larvae of Plutella xyllostella and Ostrinia furnacalis with lethal concentration 50 of 55.21 and 118.44?μg/ml, respectively. Sequence analysis of the gene was also performed; the Cry1Hc1 protein retained eight conserved regions commonly found in the existing Cry proteins.     

Characterisation of the cry54Ab1 operon of Bacillus thuringiensis subsp. sichuansis strain MC28

A novel cry gene operon harbouring two open reading frames (ORFs) was discovered in a large plasmid of Bacillus thuringiensis (Bt) subsp. sichuansis MC28 (pMC189). The first ORF encoded 743 amino acids. It had a deduced molecular mass of 84.5 kDa and 73.2% identity with Cry54Aa1 protein as indicated by amino acid homology. This cry gene was designated as cry54Ab1 by the Bt Toxin Nomenclature Committee. The second ORF encoded 503 amino acids and had a deduced molecular mass of 58.2 kDa. It is here called as orf2. The whole cry54Ab1 operon, cry54Ab1 and orf2 were digested with BamHI/SalI and inserted into a shuttle vector, pSTK. The recombinant plasmids were transferred into an acrystalliferous mutant Bt HD73^?. Parasporal inclusions were observed only under expression of the whole cry54Ab1 operon. The bioassay experiments showed that expressed products of the cry54Ab1 and the whole cry54Ab1 operon were all toxic to Culex quinquefasciatus (Diptera).     

Analysis of <Emphasis Type="Italic">cry</Emphasis> Gene Profiles in <Emphasis Type="Italic">Bacillus Thuringiensis</Emphasis> Strains Isolated During Epizootics in <Emphasis Type="Italic">Cydia pomonella</Emphasis> L.

The crystal morphology and the profiles of genes encoding protein toxins (Cry and Cyt) were analyzed in 12 Bacillus thuringiensis strains isolated during epizootics in laboratory culture lines of Cydia pomonella, 2 isolates cultured from Leucoma salicis larvae, and 9 reference strains. Epizootic isolates produced crystals of the same bipyramidal shape; however, they revealed a variety of number and type of cry genes. Genes cry1I, cry2Ab, and cry9B were the most frequently observed in epizootic strains. Gene cry1I was noted in of 50% epizootic isolates. Eighty-three percent of them harbored gene cry2Ab. Gene cry9B was found for 42% of strains isolated during epizootics. Three isolates showed the largest number of cry genes and their variety; hence, they were chosen for the toxicity assay of their crystals and spores on C. pomonella larvae. One of them had approximately sixfold higher insecticidal activity than the reference strain B. thuringiensis subsp. kurstaki BTK STANDARD.     

Recombinant Bacillus thuringiensis strain shows high insecticidal activity against Plutella xylostella and Leptinotarsa decemlineata without affecting nontarget species in the field

Aims: To construct a recombinant Bacillus thuringiensis (Bt) strain with broad insecticidal spectrum and investigate its impact on nontarget organisms in field. Method and Results: The cry-type gene of wild Bt strain UV17 was identified and a novel cry1Ba gene was cloned. The cry3Aa7 gene, which was highly toxic to coleopteran pests, was introduced into UV17, and a recombinant strain designated as UV173A was obtained. Bioassay results showed that UV173A was not only highly toxic against Plutella xylostella (50% lethal concentration [LC₅₀] = 18·03 μg ml^–1), but also against coleopteran Leptinotarsa decernlineata (LC₅₀ = 0·19 mg ml^–1). The recombinant strain was then tested in field trials to monitor its spatial variation of population and to investigate the impact on nontarget invertebrates. Conclusions: A recombinant Bt stain UV173A with broad insecticidal spectrum was obtained, and it did not cause adverse effects on the population of nontarget organisms. Significance and Impact of the Study: The results obtained here indicated that cry1Ba3 gene may be useful for the resistance management of P. xylostella, and the recombinant stain UV173A was potential for field application against some crucifer vegetable pests as well as L. decemlineata.     

Coleopteran-specific and putative novel cry genes in Iranian native Bacillus thuringiensis collection

The characterization of the strains containing Coleopteran-specific and also putative novel cry genes in Iranian native Bacillus thuringiensis collection is presented. Characterization was based on PCR analysis using 31 general and specific primers for cry1B, cry1I, cry3A, cry3B, cry3C, cry7A, cry8A, cry8B, cry8C, cry14, cry18, cry26, cry28, cry34 and cry35 genes, protein band patterns as well as their insecticidal activity on Xanthogaleruca luteola Mull. larvae. Forty six isolates (65.7%) contained minimum one Coleopteran-active cry gene. Based on universal primers, strains containing cry18 and cry26 genes were the most abundant and represent 27.1% and 24% of the isolates, respectively, whereas cry14, cry3, cry28, cry34, cry35, cry7, cry8 genes were less abundant, found in 14.2, 12.5, 10, 7, 7 and 5.6% of the strains, respectively. Based on specific primers, isolates containing cry1I were the most abundant (48.5%). Two strains containing Coleopteran-active cry genes showed higher activity against X. luteola larvae than B. thuringiensis subsp. morrisoni pathovar tenebrionis. Thirty isolates, when assayed for cry1C, cry5, cry6, cry8b, cry9, cry10, cry11, cry18, cry24 and cry35 genes, showed unexpected size bands. Cloning and sequencing of the amplicons allowed both the identification of known cry genes and the detection of putative novel cry1C sequences.