Cry Gene Content Research Articles

Molecular Characterisation and Toxicity Analysis of Indigenous Bacillus thuringiensis Berliner Isolates against Cucurbit Fruit Fly Maggots, Zeugodacus cucurbitae (Coquilett) (Diptera: Tephritidae)

Background: Indiscriminate use of insecticides against fruit flies (Tephritidae: Diptera) has led to the development of residue and resistance and there is a need to try alternate eco-friendly management practices. Bacillus thuringiensis (Bt) is the most widely used bacterium forming proteins active against insects of different orders. Thus, the present study was framed to characterize and evaluate 50 indigenous Bt isolates against cucurbit fruit fly maggots, Zeugodacus cucurbitae. Methods: Colony morphology was observed visually, while crystal morphology was observed through a microscope. The cry gene content of the Bt isolates was screened by Polymerase Chain Reaction and the proteins harbored were assessed by SDS- PAGE analysis. Full diet contamination method was used to evaluate the toxicity of Bt isolates against cucurbit fruit fly maggots. Result: Uniform full white, off-white and creamy white colony colours were found except one isolate each, exhibiting full white at centre surrounded by off white and off white at centre surrounded by full white. Spherical shape crystal (45.10%) was predominant followed by cuboidal (29.41%), bipyramidal (17.65%), rectangular (3.92%) and minute crystal attached to spore (3.92%). Insecticidal proteins varied from ~15 to greater than 200kDa in size with one to four and more distinct bands per isolate. PCR screening revealed cry4Aa in 2 isolates, cry4Ba and cry11Aa in one isolate each, cyt1 in 1 isolate. The standard strain Bti 4Q2 (100% mortality of maggots) and the indigenous Bt isolates, T166 (92.59%), T184 (91.11%) and T60 (90.48%) were found to be significantly toxic.

Molecular Characterization of Native Bacillus thuringiensis Strains from Root Nodules with Toxicity Against the Fall Armyworm (FAW, Spodoptera frugiperda) and Brinjal Ash Weevil (Myllocerus subfasciatus).

The fall armyworm is an exotic pest which destroys a wide variety of crops Querywhereas the brinjal ash weevil is a serious pest of eggplant and other solanaceous vegetables. The goal of this research is to find a sustainable and ecologically friendly bio-control agent for managing FAW and brinjal ash weevils. Twelve natural Bacillus thuringiensis strains were isolated from cowpea root nodules, and the Gram-positive cells with characteristic Bt crystal structures were discovered using phase contrast and scanning electron microscopy. There were bipyramidal, cuboidal, rhombus, and spherical crystals. The Bt cry gene content was characterized by PCR analysis, which revealed the presence of cry1, cry1I, cry3, cry7, cry7,8, cry14, cry26, and cry55 genes. The identity of Bt was confirmed by cloning and sequencing the cry genes. In the nucleotide sequences, no pseudo genes or indels were found in cry sequences. SDS-PAGE examination indicated the presence of bands ranging in size from 13 to 130kDa, with 50-60kDa being the most common. When compared to the control, the new native Bt strains were lethal, with pathogenicity ranging from 93 to 100% against S. frugiperda larvae and M. subfasciatus adults. The studies revealed that the native strains with conserved regions of 16S rRNA genes were compared to NCBI database sequences and classified as native Bt strains with 99-100% similarity to known Bt strains. In conclusion, native Bt strains from cowpea root nodules were shown to have bio-insecticidal activity against fall armyworm larvae and brinjal ash weevil adults.

Bio controlled efficacy of Bacillus thuringiensis cry protein protection against tomato fruit borer Helicoverpa armigera in a laboratory environment

Bio-pesticides that utilize beneficial microbial agents represent a new option that can potentially reduce reliance on chemical pesticides. To facilitate control of crop pests, this study includes both selection and characterization of Bacillus thuringiensis strains based on Cry gene content and toxicity evaluation. The result is an effective and non-hazardous polyphagous lepidopteran pest controller, Helicoverpa armigera. To achieve this objective, we selected ten soil bacteria samples selected from 10 different agricultural regions in Queretaro State, Mexico. Certainly, 65 and 130 kDa protein bands were observed in SDS-PAGE analysis of spore-crystal mixtures. Efficacy of bio-pesticides, 4 g/L effective water yielded maximum mortality; a mean of 64.88% of tomato fruit borer larvae were eliminated. The PCR sequences of bacterial strain were characterized and showed 98–99% similarity shows the distance from comparison of the 16S rRNA gene sequences of Bacillus thuringiensis.

Molecular characterization of an indigenous lepidopteran toxic Bacillus thuringiensis strain T532

Bacillus thuringiensis Berliner (Bt) being an eco-friendly bioinsecticide is effectively used in pest management strategies and, therefore, isolation and identification of new strains effective against a broad range of target pests is important. In the present study, four indigenous Bt isolates (T489, T491, T495 and T532) were taken and investigated for their properties such as crystal morphology, cry gene(s) content and insecticidal activity. Bipyramidal, cuboidal and spherical crystals were observed. Among the four isolates only one isolate was found positive for lepidopteran specific cry genes. The isolate T532 which showed the presence of cry1, cry2Aa and cry2Ab genes was selected for protein profiling and bioassay. The isolate showed the presence of two proteins of molecular weights at ~135 and ~65 kDa sizes. Artificial diet based bioassay resulted in 100% mortality of the neonate larvae of H. armigera and S. litura for the isolate T532 which was comparable to that of the positive control (HD1). The full length cry2A gene was amplified and cloned into E. coli DH5α strain. The recombinant plasmids were sequenced and the sequence was deposited in GenBank nucleotide database. The accession number of the gene is MH475905. The gene was named by the Bacillus thuringiensis delta endotoxin nomenclature committee as cry2Aa23 .

Diversity of Bacillus thuringiensis Strains From Qatar as Shown by Crystal Morphology, δ-Endotoxins and Cry Gene Content.

Bacillus thuringiensis (Bt) based insecticidal formulations have been recognized as one of the most successful, environmentally safe and sustainable method of controlling insect pests. Research teams worldwide are in search of Bt diversity giving more choices of bio-insecticides and alternatives to address insect resistance. In fact, there are many unexplored ecologies that could harbor novel Bt strains. This study is the first initiative to explore Bt strain diversity in Qatar. A collection of 700 Bt isolates was constructed. Scanning electron microscopy of Bt crystals showed different crystal forms, with a high abundance of spherical crystals compared to the bipyramidal ones. Among the spherical crystals, four different morphologies were observed. The δ-endotoxin content of parasporal crystals from each Bt isolate revealed that there are 16 different protein profiles among the isolates of the collection. On the other hand, plasmid pattern analysis showed seven different plasmid profiles. Their insecticidal activity was predicted by exploring the δ-endotoxin coding genes and conducting qualitative insecticidal bioassays. 19 smooth spherical crystal producing isolates have been identified that could be possible candidates for endotoxin production targeting Dipteran insects. Another group of 259 isolates producing bipyramidal and cuboidal crystals could target Lepidopteran and Coleopteran insects. The remaining 422 isolates have novel profiles. In conclusion, Qatari soil ecology provides a good collection and diversity of Bt isolates. In addition to strains harboring genes encoding common endotoxins, the majority are different and very promising for the search of novel insecticidal endotoxins.

Toxin Gene Contents and Activity of Bacillus thuringiensis Strains Against Two Sugarcane Borer Species, Diatraea saccharalis (F.) and D. flavipennella (Box).

Bacillus thuringiensis (Berliner) bears essential characteristics in the control of insect pests, such as its unique mode of action, which confers specificity and selectivity. This study assessed cry gene contents from Bt strains and their entomotoxicity against Diatraea saccharalis (F.) and Diatraea flavipennella (Box) (Lepidoptera: Crambidae). Bioassays with Bt strains were performed against neonates to evaluate their lethal and sublethal activities and were further analyzed by PCR, using primers to identify toxin genes. For D. saccharalis and D. flavipennella, 16 and 18 strains showed over 30% larval mortality in the 7th day, respectively. The LC50 values of strains for D. saccharalis varied from 0.08×105 (LIIT-0105) to 4104×105 (LIIT-2707) spores+crystalsmL-1. For D. flavipennella, the LC50 values of strains varied from 0.40×105 (LIIT-2707) to 542×105 (LIIT-2109) spores+crystalsmL-1. For the LIIT-0105 strain, which was the most toxic to D. saccharalis, the genes cry1Aa, cry1Ab, cry1Ac, cry1B, cry1C, cry1D, cry1F, cry1I, cry2Aa, cry2Ab, cry8, and cry9C were detected, whereas for the strain LIIT-2707, which was the most toxic to D. flavipennella, detected genes were cry1Aa, cry1Ab, cry1Ac, cry1B, cry1D, cry1F, cry1I, cry2Aa, cry2Ab, and cry9. The toxicity data and toxin gene content in these strains of Bt suggest a great variability of activity with potential to be used in the development of novel biopesticides or as source of resistance genes that can be expressed in plants to control pests.

Selection and characterization of Bacillus thuringiensis strains toxic against pyralid stored‐product pests

AbstractIn this study, we present the selection and the characterization of Bacillus thuringiensis strains toxic against pyralid pests of stored products. Among 201 new B. thuringiensis strains isolated from different countries, two strains (BLB249 and BLB384) showed greater toxicity than the commercialized strain B. thuringiensis kurstaki HD1 against Ephestia kuehniella larvae. Morphological, molecular and biochemical investigations revealed that these strains were similar to HD1 but presented different cry gene content. Additional bioassays revealed that only strain BLB249 displayed higher toxicity than HD1 against Plodia interpunctella larvae. The study of Cry protoxin activation by midgut proteases of E. kuehniella and P. interpunctella larvae supported that higher toxicity of BLB249 and BLB384 strains compared to HD1 was not due to differential protoxin activation. Moreover, the toxic strains produced δ‐endotoxins and spores in similar amounts to HD1. Interestingly, the δ‐endotoxin production and the yield of BUPM26 strain were 32.87% and 35.12% greater than that of HD1. The potent insecticidal activities of BLB249 and BLB384 strains and the high level of δ‐endotoxin production by BUPM26 strain make them excellent candidates for use against E. kuehniella and P. interpunctella larvae.

Selection and characterization of Bacillus thuringiensis mutants over-producing δ-endotoxins

Improvement of the microbial strain by subjecting the genetic material to mutagenic agents offers an opportunity to reduce the production cost. This study aims to enhance the δ-endotoxin production of Bacillus thuringiensis strain S24 that is 2-fold more toxic than the commercialized strain HD1 against Ephestia kuehniella (Lepidoptera: Pyralidae) larvae through random mutagenesis. B. thuringiensis mutants were generated from the S24 strain by using nitrous acid, as chemical mutagenic agent. Three mutants (M29, M12 and M20) proved to be the most promising δ-endotoxin producer among 30 mutants analyzed in a screening program performed in shake flasks using a complex medium based on starch and soya bean. These mutants demonstrated an improvement of δ-endotoxin production of up to 21 and 74% compared with the parental wild strain S24 and HD1, respectively. The characterization of the over-producing mutants showed that their colony morphology, crystal shape, plasmid and protein patterns, cry gene content and toxicity are not affected by mutagenesis treatment. However, the crystal protein patterns of three under-producing mutants (M21, M14 and M11) were different to that of the parental wild strain. The high potentiality of the over-producing mutants makes them excellent candidates as biological agents for the control of pyralid stored-product pests.

Distribution, occurrence of cry genes, and lepidopteran toxicity of native Bacillus thuringiensis isolated from fig tree environments in Aydın Province

Bacillus thuringiensis (Bt) has a significant impact on biological pest control because of the insecticidal activity through its parasporal inclusion bodies (crystal proteins). Fig is an economically important plant in Turkey; agricultural pests result in a considerable economic loss in fig quality and cultivation. The aim of this work was to isolate, characterize, and determine the lepidopteran toxicity of Bt obtained from fig groves in Aydın Province. A total of 606 colonies (out of 1167) obtained from 380 samples were identified as Bt based on parasporal crystal formation. The highest Bt index of 0.60 was observed in the Kuyucak region. A total of 288 Bt isolates were characterized in terms of cry gene content by PCR analysis. It was found that the cry1 plus cry2 genotype was the most abundant (40%) in our collection. Bioactivity tests indicated that 6 isolates exhibited high mortalities against Cadra cautella and 3 isolates were found to exhibit high toxicity against Carpophilus hemipterus. Moreover, 13 Bt isolates exhibiting toxic activity against fig pests were further characterized based on specific cry gene content, protein profiles, and PCR-RFLP analysis. Among cry1 genes, the cry1Aa, cry1Ab, cry1Ac, cry1B, cry1C, cry1D, and cry1Ea genes were the most frequent (100%). Protein profiles of isolates toxic to C. cautella were different from those of isolates toxic to C. hemipterus. PCR-RFLP analysis indicated that toxic isolates differed from the reference strain with respect to cry1 type gene. Finally, it was concluded that Bt strains isolated from fig groves showed high level of toxicity against fig pests. These strains can serve as potential biopesticides for the control of C. cautella in the region as well as alternative biopesticides in the case of pesticide resistance in insects.

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.

Diversity analysis and characterization of Coleoptera-, Hemiptera- and Nematode-active cry genes in native isolates of Bacillus thuringiensis

Applications to combat non-lepidopteran insects are not as common as applications against lepidopteran insects. The aim of the present work was to isolate and identify Bacillus thuringiensis isolates from soil samples using five approaches, viz., analysis of crystal protein production by microscopy; detection of cry gene content by PCR, SDS-PAGE profiling; cloning and sequencing; phylogenetic analysis; and toxicity testing. Two hundred soil samples were used for isolation of B. thuringiensis and a total of 69 putative isolates of B. thuringiensis that produce parasporal crystalline inclusions were isolated from 5,267 Bacillus-like colonies. A bipyramidal inclusion was predominant in 32.2 % of the B. thuringiensis isolates compared to other shapes. Crystal protein profiling of B. thuringiensis isolates by SDS-PAGE analysis showed the presence of bands of 130, 73, 34, 25 and 13 kDa, among which 50–60 kDa bands were present abundantly. PCR analysis revealed the predominance of Coleopteran-active cry genes in these isolates. Variation in nucleotide sequences, crystal morphology and mass of crystal protein(s) purified from the isolates of B. thuringiensis revealed genetic and molecular diversity. Four strains containing Coleopteran-active cry genes showed higher toxicity against Myllocerus undecimpustulatus undatus Marshall (Coleoptera: Curculionidae) adults when compared with B. thuringiensis subsp. morrisoni pathovar tenebrionis. These results are useful in emphasizing the distribution of cry genes and for prognostication of toxicity, and may contribute to the identification of novel candidate genes for bioengineered crop protection.

Cloning and Sequencing of a Coleopteran specific Novel cry gene of a local isolate of Bacillus thuringiensis

A cry gene, obtained from a locally isolated Bt. strain, CAMB # 30382 (isolated from grain dust of Shakargarh, Punjab, Pakistan), highly effective against Tribolium castaneum (Commonly known as red flour beetle), was amplified through Polymerase chain reaction (PCR) by using specific primers. The gene was ligated to a cloning vector pGEM-T and was cloned into an E.coli strain DH5α. The positive clones were screened for the cry gene content. The partial sequencing of the cloned gene from this strain was accomplished and sequence homology was analyzed. The alignment of the sequenced gene with gene sequences present in gene data bank showed two silent mutations, at nucleotide 676 and 701. However, two other nucleotide changes at position 2887 and 2902 of holotype cryIIIf (cry8Ca) gene were resulting from amino acid change near 3 end were observed. These changes were causing change in the stretch of amino acids from valine, tyrosine, serine & glutamine in the holotype cryIIIf to glycine, phenylalanine, alanine & asparginine respectively in the clone from CAMB isolate 30382. This change may have drastic effect on the toxicity spectrum of the Cry protein isolated from this local isolate in comparison with the Cry protein from the Buibui. serovar japonesis. Partial sequencing has shown it to be a varient of cryIII gene, which may be a novel cry gene.

A novel Bacillus thuringiensis strain and its pathogenicity against three important pest insects

A highly pathogenic Bacillus thuringiensis (Bt) strain was isolated from a soil sample in Turkey and characterized in terms of both its 16S-ITS rDNA region and cry gene content. This strain (SY49.1) harboured several cry genes producing crystalline inclusions known to have toxicity on lepidopteran, dipteran and coleopteran pests. The 16S-ITS sequence analysis of Bt SY49.1 showed 98% similarity to Bt serovar andalousiensis BGSC 4AW1. The larvae of Ephestia kuehniella and Plodia interpunctella were treated with a spore-crystal mixture of this strain in the dose range of 50–1000 μg g−1. Mortality rates were higher than 90% at the highest concentration for these pests. For Tribolium castaneum much higher concentrations were needed than with lepidopteran pests larvae. At the highest concentration tested of 10 mg g−1, about 62% mortality was obtained. It was concluded that this native strain of Bt could be used as an effective biocontrol agent against various lepidopteran pests.

Isolation of Bacillus thuringiensis strains that contain Dipteran-specific cry genes from Ilha Bela (São Paulo, Brazil) soil samples

The entomophatogenic bacterium Bacillus thuringiensis produces crystal proteins, named Cry proteins which are encoded by the cry genes. This bacterium is used on biological control of important economical pests, as well as in the control of disease´s vectors, such as Aedes aegypti, a mosquito that transmits the dengue viruses. Isolates of this bacterium can be characterized by the content of cry genes and this prediction helps target different insect orders. In this research, we isolated 76 colonies of B. thuringiensis from 30 soil samples that were taken from Ilha Bela (SP, Brazil), a place where simulids are already biologically controlled by B. thuringiensis, to find bacterial isolates that were capable of controlling A. aegypti. The 16S ribosomal subunit genes of the selected isolates were sequenced, and the isolates were molecularly characterized based on their Dipteran-specific cry gene contents. Eight of the 76 isolates (10.52%) contained the cry4Aa, cry4Ba or cry10Aa genes, these isolates were carried out against A. aegypti larvae on bioassay. The presence or absence of specific cry genes was associated with the observed average larval mortalities. From the 76 isolates, seven (9.2%) were potentially able to control A. aegypti larvae. Therefore these are promising isolates for the biological control of A. aegypti larvae.

Caracterização de novos isolados de Bacillus thuringiensis para o controle de importantes insetos-praga da agricultura

A bactéria Bacillus thuringiensis Berliner produz um corpo de inclusão paraesporal (cristal) de natureza proteica, formado durante a esporulação, que atua de forma eficiente no controle de insetos-praga de culturas economicamente importantes. Esse cristal é constituído de proteínas Cry, que são codificadas pelos genes cry; um isolado pode ser caracterizado pelo conteúdo de genes cry que apresenta. Visando caracterizar novos isolados no combate de insetos-praga pertencentes às ordens Lepidoptera e Coleoptera, 76 isolados bacterianos foram analisados molecularmente e tiveram seu potencial de controle avaliado por meio de bioensaios com larvas de Spodoptera frugiperda (J.E. Smith), Sphenophorus levis Vaurie e Tenebrio molitor Linnaeus. As análises moleculares indicaram 11 isolados (14,5% da coleção), contendo genes lepidóptero-específicos e 17 (22,37%) com genes coleóptero-específicos. As análises de patogenicidade revelaram dois isolados com alto potencial de controle para lagartas de S. frugiperda, um para larvas de S. levis e seis prejudiciais ao desenvolvimento das larvas de T. molitor. Esses isolados de B. thuringiensis podem ser promissores no controle biológico das referidas pragas.

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 27kDa, isolate UP1 were of 85, 60 and 40kDa and isolate GN24 were of 130, 90, 66 and 45kDa. 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.

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

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

Characterisation and toxicity ofBacillus thuringiensisstrains from hazelnut pests and fields

In order to find and identify more toxic insecticidal Bacillus thuringiensis Berliner (Bt) strains, a survey was carried out of B. thuringiensis isolate pests belonging to Coleoptera, Lepidoptera and Diptera and from soils in hazelnut fields. Of 16 isolates having Bacillus cereus-B. thuringiensis morphology, eight were classified as B. thuringiensis because of the production of parasporal delta-endotoxin crystals. In this study, eight isolates of B. thuringiensis from hazelnut pests (isolates Bn1, Mm2, Mnd and Xd3) and from hazelnut soils (isolates 6, 27, 40 and 46) have been characterised in detail. These isolates were compared with reference strains by electron microscopy, SDS-PAGE analysis, cry gene content, serological test and insecticidal activity. Results indicate that Bn1 and MnD are B. thuringiensis subsp. kurstaki, and Mm2 and Xd3 are B. thuringiensis subsp. tenebrionis. In addition, isolate 6 is B. thuringiensis subsp. israelensis, isolates 27 and 46 are B. thuringiensis subsp. kumamotoensis and isolate 40 is B. thuringiensis subsp. indiana. The four B. thuringiensis isolates from hazelnut pests may be valuable as biological control agents against coleopteran and lepidopteran insects.

Biochemical and molecular characterization of δ-endotoxins in Bacillus thuringiensis

The delta-endotoxins (delta-ETX) of four native strains (RT7, RT19, RT25, and RT25), and one reference strain (4L1) of Bacillus thuringiensis were biochemically and molecularly characterized to determine their potential toxic activity against lepidopteran larvae. Crystals of delta-ETX were purified through a two-phase system to determine their morphology, molar mass, solubility, and resistance to proteinases. Toxic activity and cry gene content were also determined. Crystals from native strains exhibited polyhedral, irregular and cuboidal shapes, while those from 4L1 were bipyramidal. Seven proteins with estimated molar mass approximately 30-134 kDa were detected as the main components of the native delta-ETX. Only crystals from 4L1, RT24, and RT25 underwent complete solubilization at pH >12.0. Crystals from all strains produced trypsin-resistant peptides. None of the cry genes associated with toxicity in lepidopterans (cry1, cry2, cry9) was found in the native strains; however, 4L1 strain harbors cry1 and cry2 genes. Strains RT19 and RT25 caused significant mortality against Trichoplusia ni larvae with partial solubilization at pH 10, strain 4L1 caused 100 % mortality. Toxicity of native strains may come from a novel cry gene.

Characterization of two Bacillus thuringiensis ssp. morrisoni strains isolated from Thaumetopoea pityocampa (Lep., Thaumetopoeidae)

The pine processionary moth Thaumetopoea pityocampa Den. and Schiff. (Lep., Thaumetopoeidae) is one of the most harmful insect pests for pine species in Mediterranean countries including Turkey. Two Bacillus thuringiensis isolates obtained from T. pityocampa were identified and characterized in terms of crystal shape using electron microscopy, SDS–PAGE analysis, cry gene contents, H-serotype and insecticidal activity. Examination by a scanning electron microscope showed that Tp6 and Tp14 isolates have flat square and bipyramidal crystal shapes, respectively. PCR analysis showed that Tp6 contains cry3 gene and Tp14 isolate contains cry1 and cry2 genes. On the other hand, the presence of Cry3 and Cry1 proteins were confirmed by observation of approximately 65- and 130-kDa proteins by SDS–PAGE in Tp6 and Tp14 isolates, respectively. According to H-serotype results, these isolates were identified as Bacillus thuringiensis ssp. morrisoni (H8a8b). Toxicity tests were performed against six insect species belonging to Lepidoptera and Coleoptera. The highest insecticidal activity was 100% for Tp6 isolate on larvae of Agelastica alni and Leptinotarsa decemlineata and 100% for Tp14 isolate on larvae of Malacosoma neustria. Our results indicate that isolates Tp6 and Tp14 may be valuable biological control agents for various coleopteran and lepidopteran pests.