New strategy for identification of novel Cry-type genes from Bacillus thuringiensis strains

We designed five degenerate primers for detection of novel cry genes from Bacillus thuringiensis strains. An efficient strategy was developed based on a two-step PCR approach with these primers in five pair combinations. In the first step, only one of the primer pairs is used in the PCR, which allows amplification of DNA fragments encoding protein regions that include consensus domains of representative proteins belonging to different Cry groups. A second PCR is performed by using the first-step amplification products as DNA templates and the set of five primer combinations. Cloning and sequencing of the last-step amplicons allow both the identification of known cry genes encoding Cry proteins covering a wide phylogenetic distance and the detection and characterization of cry-related sequences from novel B. thuringiensis isolates.     

Screening of Bacillus thuringiensis serotypes by polymerase chain reaction (PCR) for insecticidal crystal genes toxic against coffee berry borer

Using PCR,257 isolates of Bacillus thuringiensis(Bt) were screened for cry-type genes. Of 257 isolates/strains, 60 isolates were identified as cry7/8, 10 isolates as cry3 and 36 isolates as cry 1I. One specific strain of B. thuringiensis (sumiyoshiensis; T03B 001) was investigated for the presence of cry7 and cry8 genes. Genes Cry7 and cry8 were first detected in this strain using family primers prior to analysis by exclusion polymerase chain reaction (E-PCR) using specific type primers. E-PCR conducted with the above said primers led to the identification by agarose gel electrophoresis of a remaining 1.5 Kb family band indicating a potentially novel gene. This PCR product, (1.5 Kb), was purified from the gel and cloned in pGEM-T Easy vector. Twenty recombinant colonies bearing 1.5 Kb insert were identified and three randomly selected representatives of the group, clones 7, 8 and 10, were sequenced and compared to all cry7 and cry8 sequences available from Gene Bank. Alignments with available DNA and protein sequences showed that all these clones contained a gene related to cry8Aa1. Analysis using protein sequence alignment showed that the sequence from clone 7 differed from the closest relative, known under the new nomenclature as cry 8Aa1, by 44%. The crystal proteins from B. thuringiensis sumiyoshiensis (T03B 001) was toxic to coffee berry borer larvae.     

Strategy for amplification and sequencing of insecticidal <Emphasis Type="Italic">cry1A</Emphasis> genes from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

A feasible and fully described strategy, with a detailed list of primers, for amplifying, cloning and sequencing known and potentially novel cry1A genes harboured by a Bacillus thuringiensis strain was successfully established. Based on the analysis of conserved regions of the cry1A genes, the 1AF and 1UR oligonucleotide primers were designed to amplify the whole open reading frame of these genes. The PCR products obtained revealed the successful amplification of cry1A genes from 13 B. thuringiensis strains. These bacteria were previously known to harbour at least one cry1A gene. An Argentinean B. thuringiensis isolate INTA Mo1-12 was randomly chosen for cloning and sequencing of cry1A genes by using a primer set developed in this study. Both nucleotide and amino acid sequences similarity analysis revealed that cry1Aa and cry1Ac from B. thuringiensis INTA Mo1-12 are new natural variants, showing several differences with the other known cry1A subclasses. These genes were named by the B. thuringiensis Pesticidal Crystal Protein Nomenclature Committee as cry1Aa15 and cry1Ac21 respectively.     

苏云金芽孢杆菌4.0718菌株的杀虫晶体蛋白基因分析

根据苏云金杆菌(Bacillus thuringiensis)cry1、cry2和cry3型基因的保守区分别设计了3对通用引物Un1(d)/Un1?、Un2(d)/Un2?和Un3(d)/Un3?,以Bt4.0718菌株质粒DNA为模板进行PCR扩增,通过扩增产物片段的分子量大小来确定该菌株所含有的杀虫晶体蛋白基因类型。随后根据上述3类cry基因的高变区设计特异引物再次进行PCR鉴定。结果表明:Bt4.0718菌株含有cry1Aa、cry1Ab、cry1Ac、cry1Cb、cry2Ac和新基因cry4.5等6种基因类型。这一结果为利用该菌株构建高效广谱杀虫工程菌提供了客观依据。     

Isolation and toxicity of Bacillus thuringiensis from potato-growing areas in Bolivia

Bacillus thuringiensis was isolated from 116 samples collected in high altitude potato-growing areas in Bolivia. In these regions, main potato pests are the potato tuberworm Phthorimaea operculella, and the Andean weevils Premnotrypes latithorax and Rhigopsidius tucumanus. B. thuringiensis was found in 60% of the samples. The main percentage of samples with B. thuringiensis was found in larvae of R. tucumanus (78%). Bioassays were performed with 112 isolates. None resulted toxic to either larvae or adults of the two Andean weevils. However, 18 isolates from this study showed more toxicity against the beet armyworm Spodoptera exigua than the standard strain var. kurstaki isolated from DELFIN. Among these isolates, three were also effective against P. operculella, conferring better or equal protection to the tubers than the reference strain HD-1 isolated from DIPEL. The most toxic strains against S. exigua and P. operculella were characterized in terms of serotyping, crystal morphology, protein profile, and cry gene content. PCR was performed with primers amplifying genes from the cry1, cry2, cry3, cry4, cry7, 8, and cry9Aa families. The toxic strains presented bipyramidal crystals, at least a band of 130kDa in SDS-PAGE, and showed an amplification product with cry1 family primers. One of the isolates did not amplify with any specific primer belonging to known cry1 genes. Restriction Fragment Length Polymorphism (RFLP) confirmed the presence of a novel gene and sequence comparison showed that this gene had homology to cry1G.     

Composition and ecological distribution of cry proteins and their genotypes of Bacillus thuringiensis isolates from warehouses in China

The composition and distribution of insecticidal crystal proteins (Cry proteins) and their genotypes of Bacillus thuringiensis isolates from warehouses were evaluated through SDS-PAGE and PCR techniques. The results showed that the electrophoretic patterns of delta-endotoxin crystal preparations were divided into five types. The isolates containing approximately 135 kDa with a 65-kDa protein or only a approximately 135-kDa protein, which amounted to 55.74 and 35.25% of all isolates respectively, were the two major profiles of Cry protein isolated. The distribution of cry genes of B. thuringiensis from warehouses was highly variable. Cry protein genotypes detected in B. thuringiensis isolates included cry1Aa5, cry1Ab9, cry1Ac5, cry1Ba, cry1Ca1, cry1Da1, cry1Ea3, cry2, and cry3 genes, but not cry1Fa2. Among them, cry2, cry1Ac5, and cry1Ab9 genes were the most common in our B. thuringiensis isolates. Most B. thuringiensis isolates contained several cry genes in a total of 18 profiles. Among them, cry1Ac5 with cry1Ea3; cry1Aa5, cry1Ab9, cry1Ac5 with cry1Ea3; and cry1Aa5, cry1Ab9 with cry1Ac5 were the three principal profiles. The distribution of the Cry proteins and cry genes in isolates depended on geography and type of warehouses. Gene profiles may be used as markers for insecticidal activity of B. thuringiensis strains, but they did not directly reflect the toxic level of B. thuringiensis strains. The serotype of B. thuringiensis strains did not directly reflect the specific cry gene profiles in the strains, but certain relationships can be established between the serotype and cry genotype.     

Screening and characterization of Bacillus thuringiensis isolates from Brazil for the presence of coleoptera-specific cry genes

Forty-three Bacillus thuringiensis isolates from Brazil and 3 from Argentina were screened, using the polymerase chain reaction (PCR), for various coleoptera-specific cry genes. Seven isolates produced specific and/or nonspecific DNA fragments in a PCR reaction with primers specific for two coleopteran cry genes and 4 of these produced DNA fragments with primers specific for 7 known coleopteran cry genes. These isolates showed, by electron microscopy, the presence of spherical crystals. They also showed proteins of around 70 kDa which were immunologically similar to the Cry3Aa protein from B. thuringiensis subsp. tenebrionis. The 3 isolates from Argentina were toxic to T. molitor, and although no isolate from Brazil showed toxicity, they might show toxicity to another insect species.     

Diversity of cry genes and genetic characterization of Bacillus thuringiensis isolated from Brazil

Two hundred and eighteen Bacillus thuringiensis isolates from Brazil were characterized by the presence of crystal protein genes by PCR with primers specific to different cry and cyt genes. Among these isolates, 95 were selected according to their geographic origin for genetic characterization with the 16S rRNA gene, RAPD, and plasmid profile. Isolates containing cry1 genes were the most abundant (48%) followed by the cry11 and cyt (7%) and cry8 genes (2%). Finally, 40.3% of the isolates did not produce any PCR product. The plasmid profile and RAPD analysis showed a remarkable diversity among the isolates of B. thuringiensis not observed in the 16S rRNA gene. These results suggest that the genetic diversity of B. thuringiensis species results from the influence of different ecological factors and spatial separation between strains generated by the conquest of different habitats.     

Identification of cry1I-type genes from Bacillus thuringiensis strains and characterization of a novel cry1I-type gene

A PCR-restriction fragment length polymorphism method for identification of cry1I-type genes from Bacillus thuringiensis was established by designing a pair of universal primers based on the conserved regions of the genes to amplify 1,548-bp cry1I-type gene fragments. Amplification products were digested with the Bsp119I and BanI enzymes, and four kinds of known cry1I-type genes were successfully identified. The results showed that cry1I-type genes appeared in 95 of 115 B. thuringiensis isolates and 7 of 13 standard strains. A novel cry1I-type gene was found in one standard strain and six isolates. The novel cry1I gene was cloned from B. thuringiensis isolate Btc007 and subcloned into vector pET-21b. Then it was overexpressed in Escherichia coli BL21(DE3). The expressed product was shown to be toxic to the diamondback moth (Plutella xylostella), Asian corn borer (Ostrinia furnacalis), and soybean pod borer (Leguminivora glycinivorella). However, it was not toxic to the cotton bollworm (Helicoverpa armigera), beet armyworm (Spodoptera exigua), or elm leaf beetle (Pyrrhalta aenescens) in bioassays. Subsequently, the Cry protein encoded by this novel cry gene was designated Cry1Ie1 by the B. thuringiensis delta-endotoxin nomenclature committee.     

Assessment of cry1 gene contents of Bacillus thuringiensis strains by use of DNA microarrays

A single Bacillus thuringiensis strain can harbor numerous different insecticidal crystal protein (cry) genes from 46 known classes or primary ranks. The cry1 primary rank is the best known and contains the highest number of cry genes which currently totals over 130. We have designed an oligonucleotide-based DNA microarray (cryArray) to test the feasibility of using microarrays to identify the cry gene content of B. thuringiensis strains. Specific 50-mer oligonucleotide probes representing the cry1 primary and tertiary ranks were designed based on multiple cry gene sequence alignments. To minimize false-positive results, a consentaneous approach was adopted in which multiple probes against a specific gene must unanimously produce positive hybridization signals to confirm the presence of a particular gene. In order to validate the cryArray, several well-characterized B. thuringiensis strains including isolates from a Mexican strain collection were tested. With few exceptions, our probes performed in agreement with known or PCR-validated results. In one case, hybridization of primary- but not tertiary-ranked cry1I probes indicated the presence of a novel cry1I gene. Amplification and partial sequencing of the cry1I gene in strains IB360 and IB429 revealed the presence of a cry1Ia gene variant. Since a single microarray hybridization can replace hundreds of individual PCRs, DNA microarrays should become an excellent tool for the fast screening of new B. thuringiensis isolates presenting interesting insecticidal activity.     

RFLP analysis of cry1 and cry2 genes of Bacillus thuringiensis isolates from India

The PCR-RFLP method has been useful for detection of known genes and identification of novel genes. In the present study, degenerate primers were designed from five groups of cry1 genes for PCR-RFLP analysis. Bacillus thuringiensis (Bt) isolates from different regions were evaluated for PCR amplification of various cry1 genes using newly designed primers and cry2 genes using reported primers. PCR analysis showed an abundance of cry1A genes and especially cry1Ac genes in isolates from all regions. RFLP analysis revealed the presence of multiple cry1A genes in isolates from central and southern regions. Unique digestion patterns of cry1A genes were observed in isolates from each region. Few of the isolates represented a digestion pattern of cry1A genes that did match to any of the known cry1A genes. RFLP analysis suggested an abundance of cry2Ab along with a novel cry2 gene in Bt isolates from different regions of India. Sequence analysis of the novel cry2 gene revealed 95% sequence identity to cry2Ab and cry2Ah genes. Phylogenetic analysis revealed that the novel cry2 gene could have diverged earlier than the other cry2 genes. Our results encourage finding of more diverse cry2 genes in Bt isolates. Rarefaction analysis was used to compare cry1A gene diversity in isolates from different soil types. It showed a higher degree of cry1A gene diversity in isolates from central region. In the present study, we propose the use of novel degenerate primers for cry1 genes and the PCR-RFLP method using a single enzyme to distinguish multiple cry1A and cry2 genes as well as identify novel genes.     

Molecular detection of nematicidal crystalliferous Bacillus thuringiensis strains of Iran and evaluation of their toxicity on free-living and plant-parasitic nematodes

The characterization of nematode-effective strains and cry genes in the Iranian Bacillus thuringiensis (Bt) collection (70 isolates) is presented. Characterization was based on PCR analysis using 12 specific primers for cry5, cry6, cry12, cry13, cry14, and cry21 genes encoding proteins active against nematodes, crystal morphology, and protein band patterns as well as their nematicidal activity on root-knot nematode (Meloidogyne incognita) and two free-living nematodes (Chiloplacus tenuis and Acrobeloides enoplus). PCR results with primers for these genes showed that 22 isolates (31.5%) contain a minimum of one nematode-active cry gene. Strains containing the cry6 gene were the most abundant and represent 22.8% of the isolates. Bt strains harboring cry14 genes were also abundant (14.2%). cry21 and cry5 genes were less abundant, found in 4.2% and 2.8% of the strains, respectively. In total, six different nematode-active cry gene profiles were detected in this collection. Four isolates did not show the expected PCR product size for cry5, cry6, and cry21 genes; they might contain potentially novel insecticidal crystal protein genes. Twenty-two Bt isolates containing nematode-active cry genes were selected for preliminary bioassays on M. incognita. Based on these bioassays, four isolates were selected for detailed bioassays. Isolates YD5 and KON4 at 2 x 10(8) CFU/mL concentrations showed 77% and 81% toxicity on M. incognita, respectively. The free-living nematodes C. tenuis and A. enoplus were more susceptible and the highest mortality was observed within 48 h of incubation at all of the concentrations tested. Maximum mortality was recorded for isolates SN1 and KON4 at 2 x 10(8) CFU/mL concentrations and resulted in 68% and 77% adults deaths of C. tenuis and 68% and 72% for A. enoplus, respectively. Our results showed that PCR is a useful technique for toxicity prediction of nematicidal Bt isolates.     

The clonal structure of Bacillus thuringiensis isolates from north-east Poland does not correlate with their cry gene diversity

The genetic relationship among 103 natural Bacillus thuringiensis isolates was investigated on the basis of polymerase chain reaction amplification of their specific crystal (cry) protein type genes and chromosomal DNA profiling by pulsed-field gel electrophoresis (PFGE). The strains were recovered from the intestines of small wild rodents and insectivores from the Biebrza National Park and the Lomza Landscape Park of the Narew River Valley in north-east Poland. The percentage of B. thuringiensis strains harbouring genes coding for toxins active against Lepidoptera (cry1, cry2, cry9) was very high (64%) compared with that of Diptera-specific strains (cry4, 14%). No strain with cry genes coding for proteins directed against coleopteran larvae and nematodes was found. After digestion with NotI and AscI, only nine PFGE pulsotypes were observed among all isolates, indicating a clonal structure for the B. thuringiensis population from NE Poland. Interestingly, no correlation was observed between the DNA pulsotype strains and their crystal gene content and diversity. These results therefore emphasize the importance of cry gene horizontal transfer occurring among natural isolates of B. thuringiensis.     

Two δ-Endotoxin Genes, cry9Da and a Novel Related Gene, Commonly Occurring in Lepidoptera-Specific Bacillus thuringiensis Japanese Isolates That Produce Spherical Parasporal Inclusions

Six Lepidoptera-specific Bacillus thuringiensis isolates, which belong to the four H serovars (sotto, fukuokaensis, canadensis, and galleriae) and produce spherical parasporal inclusions, were examined for assignment of the classes of the delta-endotoxin genes. Gene analysis was conducted by PCR technique with primers designed to probe the genes cry9Ca and cry9Da. The data revealed that the delta-endotoxin of a serovar canadensis isolate is encoded by the gene cry9Da, while those of the five other strains are encoded by an undescribed delta-endotoxin gene. DNA fragments from five strains had an identical 1917-bp nucleotide sequence, covering the four conserved regions and a partial sequence of the block 5 region. The deduced amino acid sequence exhibited a 70.6% homology to that of the corresponding region of the Cry9Ea delta-endotoxin protein which is active on the order Lepidoptera, and a 63.1% homology to the Cry9Ca protein highly toxic to the noctuid lepidopterans. The results showed that Japanese isolates of B. thuringiensis producing spherical parasporal inclusions with Lepidoptera-specific activity are categorized into two groups: one produces the class Cry9Da protein and the other a novel delta-endotoxin allied to the class Cry9. It also appeared that heterogeneous multiple H serovars are involved in each group.     

排除法PCR加变性梯度凝胶电泳鉴别未知基因

通过鉴别未知的cry4亚组基因来确定排除法PCR加变性梯度凝脉电泳新方法。方法：应用排除法PCR的组基因和亚组基因引物扩增杀蚊毒素基因,cry4。这些引物是根据已知的cyr4基因的共有或特有DNA片段来设计的。组基因引物扩增产物被用于变性梯度凝胶电泳。平行变性梯度凝胶是由8％的聚丙烯酰胺加上20％到80％的变性剂组成。结果：已知的和未知的cry4来组基因被组基因引物扩增的产物在变性梯度凝胶电泳被分离开。尽管它们之间仅有两个碱基对不同（T在位置224和G在位置394）。应用组基因和亚组基因引物扩增,新发现的基因可被分类到次亚组基因水平。从5个苏云金芽孢杆菌亚菌中发现三个未发表的cry4亚组基因。结论：排除法PCR加变性梯度凝胶电泳是一个高度敏感、特异性和可靠性强的新方法,可用于鉴别各种未知的亚组基因。     

Screening of cry gene contents of Bacillus thuringiensis strains isolated from avocado orchards in Mexico, and their insecticidal activity towards Argyrotaenia sp. (Lepidoptera: Tortricidae) larvae

Aims:  To screen for Bacillus thuringiensis strains from avocado orchards in two Mexican states with lepidopteran-specific cry gene content and evaluate their insecticidal activity against Argyrotaenia sp., an undescribed species present in avocado orchards.
Methods and Results:  Lepidopteran-active cry 1, cry 2 and cry 9 genes were detected by PCR analysis in 37 isolates. cry 1 genes were more frequent in Michoacán, but were undetected in Nayarit isolates. cry 9 and cry 2 genes were detected in isolates from both states, although cry 2 genes were less frequent. A variety of crystal shapes were observed among the isolates. According to gene profile, eight isolates were selected and tested against 2-day old Argyrotaenia sp. larvae. Standard strain HD-125 caused the highest mortality followed by strain MR-26 from Michoacán at a concentration of 500 μg ml⁻¹, respectively.
Conclusions:  Bacillus thuringiensis strains isolated from avocado orchards exhibit a low toxic activity towards Argyrotaenia sp. larvae, in spite of their specific cry gene content.
Significance and Impact of the Study:  Toxic activity of B. thuringiensis is not necessarily related to insect pest habitat and neither to specific cry gene content associated to other lepidopterans.     

Toxic activity of Bacillus Thuringiensis isolates to Aedes Aegypti (L.) (Diptera: Culicidae) larvae

Aedes aegypti (L.), the main vector of dengue fever in Brazil, has been controlled with the use of massive chemical products, contributing to the development of resistance and decreasing the insect control efficiency. The control of dipterans with bioinsecticides based on Bacillus thuringiensis has been satisfactory, due to the production of insecticidal proteins denominated Cry (crystal), Cyt (cytolytic) toxins and Chi (chitinase), and to the synergistic effects among them. The present work aimed to select B. thuringiensis isolates efficient against A. aegypti larvae. A bacterial collection containing 1,073 isolates of B. thuringiensis, obtained from different locations of Brazilian territory, had the DNA isolated and submitted to PCR amplifications using specific primers for cry4Aa, cry4Ba, cry11Aa, cry11Ba, cyt1Aa, cyt1Ab, cyt2Aa and chi genes. For the LC50 and LC90 determination, the entomopathogenic isolates were evaluated by selective and quantitative bioassays. Only 45 isolates (4.2%) presented amplicons for the cry and cyt genes. The chi gene sequence was detected in 25 (54.3%) of those isolates. From the 45 isolates submitted to the selective bioassays, 13 caused 100% mortality of A. aegypti larvae. The identification of cry, cyt and chi genes of B. thuringiensis and the toxicity analysis on A. aegypti led to the selection of a set of isolates that have the potential to be used in the formulation of new bioinsecticides.     

Phenotypic and genotypic comparisons of 23 strains from the Bacillus cereus complex for a selection of known and putative B. thuringiensis virulence factors

Sixteen Bacillus thuringiensis, four Bacillus cereus and three Bacillus anthracis isolates were screened for a selection of known and putative B. thuringiensis virulence factors. PCR primers were designed to detect genes for phosphatidylcholine specific phospholipase C, phosphatidylinositol specific phospholipase C, immune inhibitor A, vegetative insecticidal protein 3A, a protein proposed to be involved in capsule synthesis, a newly identified Ser/Thr kinase homologue and enterotoxin entS. Motility, the presence of flagella, haemolysis, chitinase and lecithinase production were also evaluated. The widely varying profiles of the 23 strains from the complex provide a pool of different genotypes that can help to identify factors involved in pathogenicity.     

Diversity of Bacillus thuringiensis strains from Latin America with insecticidal activity against different mosquito species

The characterization of selected Bacillus thuringiensis strains isolated from different Latin America countries is presented. Characterization was based on their insecticidal activity against Aedes aegypti, Culex quinquefasciatus, and Anopheles albimanus larvae, scanning electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and plasmid profiles as well as PCR analysis using novel general and specific primers for cry and cyt genes encoding proteins active against mosquitoes (cyt1, cyt2, cry2, cry4A, cry4B, cry10, cry11, cry17, cry19, cry24, cry25, cry27, cry29, cry30, cry32, cry39, and cry40). Strains LBIT315, LBIT348, and IB604 showed threefold higher mosquitocidal activity against A. aegypti and C. quinquefasciatus larvae than B. thuringiensis subsp. israelensis and displayed high similarities with the B. thuringiensis subsp. israelensis used in this study with regard to protein and plasmid profiles and the presence of cry genes. Strain 147-8906 has activity against A. aegypti similar to that of B. thuringiensis subsp. israelensis but has different protein and plasmid profiles. This strain, harboring cry11, cry30, cyt1, and cyt2 genes, could be relevant for future resistance management interventions. Finally, the PCR screening strategy presented here led us to identify a putative novel cry11B gene.     

Specific PCR primers directed to identify cryI and cryIII genes within a Bacillus thuringiensis strain collection.

In this paper we describe a PCR strategy that can be used to rapidly identify Bacillus thuringiensis strains that harbor any of the known cryI or cryIII genes. Four general PCR primers which amplify DNA fragments from the known cryI or cryIII genes were selected from conserved regions. Once a strain was identified as an organism that contains a particular type of cry gene, it could be easily characterized by performing additional PCR with specific cryI and cryIII primers selected from variable regions. The method described in this paper can be used to identify the 10 different cryI genes and the five different cryIII genes. One feature of this screening method is that each cry gene is expected to produce a PCR product having a precise molecular weight. The genes which produce PCR products having different sizes probably represent strains that harbor a potentially novel cry gene. Finally, we present evidence that novel crystal genes can be identified by the method described in this paper.