Characterization of a novel <Emphasis Type="Italic">cry8</Emphasis> gene specific to Melolonthidae pests: <Emphasis Type="Italic">Holotrichia oblita</Emphasis> and <Emphasis Type="Italic">Holotrichia parallela</Emphasis>

A new polymerase chain reaction–restriction fragment length polymorphism method for the identification of cry8-type genes from Bacillus thuringiensis has been established by designing a pair of new universal primers. By this method, a novel gene, cry8Ga1, encoding a polypeptide of 1,157 amino acids with a deduced molecular mass of 131.2 kDa was identified and cloned from B. thuringiensis HBF-18. Recombinant B. thuringiensis strain HD8G, harboring cry8Ga1, has insecticidal activity against larvae of Melolonthidae pests: Holotrichia oblita and Holotrichia parallela. This is the first report of a Cry toxin that has insecticidal activity to Melolonthidae pest H. oblita.     

Construction of a Bacillus thuringiensis engineered strain with high toxicity and broad pesticidal spectrum against coleopteran insects

A newly engineered strain, denominated BIOT185, was constructed through integrating the cry8Ca2 gene into the endogenous plasmid of BT185 (contains cry8Ea1) by homologous recombination. The thermosensitive plasmid vector was eliminated by the rising temperature of recombinant cultures. No antibiotic gene or other unnecessary genes were introduced to the new strain. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis demonstrated that the cry8Ca2 gene was expressed normally and produced a 130-kDa protein in the BIOT185 strain. Bioassay results showed that the new strain had high toxicity to the pests Anomala corpulenta and Holotrichia parallela, which often damage the same fields.     

Characterization of Two Novel <Emphasis Type="Italic">cry8</Emphasis> Genes from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strain BT185

Two novel cry8-type genes, cry8Ea1 and cry8Fa1, obtained from a Holotrichia parallela–specific Bacillus thuringiensis strain, BT185, were characterized. Findings showed that cry8Ea1 and cry8Fa1 encoded polypeptides of 1164 and 1174 amino acid residues, respectively. The deduced amino acid sequences of both Cry8Ea1 and Cry8Fa1 polypeptides are the most similar to that of Cry8Ba1. Eight conserved blocks (blocks 1–8) exist in Cry8Ea1 and Cry8Fa1 polypeptides compared with known Cry proteins. Cry8Ea1 and the Cry8Fa1 toxins could form spheric crystals when they were expressed in the acrystalliferous mutant strain HD73⁻. The spores and crystals from the recombinant strain containing cry8Ea1 were toxic to Holotrichia parallela, with an LC₅₀ of 0.0875 × 10⁸ colony-forming units (CFU)/g. However, Cry8Fa1 expressed in the recombinant strain was not toxic to H. parallela, Anomala corpulenta, or H. oblita.     

The cry3Aa gene of Bacillus thuringiensis Bt886 encodes a toxin against long-horned beetles

This report describes the identification of a new toxigenic strain of Bacillus thuringiensis specific for long-horned beetles. B. thuringiensis Bt866 encodes a cry3Aa-like gene (Bt886cry3Aa) that is 1,956 bp in length and is predicted to encode an 85.78-kDa protein. The gene is highly similar to cry3Aa1, differing in only six nucleotides and four amino acids. The four disparate amino acids occur within the conserved domains of the Cry3Aa toxin. The expression of Bt866cry3A in Escherichia coli cells resulted in a high level of toxicity toward Apriona germari Hope larvae. More than 75% of the larvae were killed; and the remaining survivors exhibited slower growth. These results indicate that the toxigenic strain Bt886cry3Aa encodes a protein that is specific against long-horned beetles. Genetic engineering of the Bt866cry3Aa gene into poplar plantations may provide resistance to long-horned beetles.     

Characterization of one novel cry8 gene from Bacillus thuringiensis strain Q52-7

Bacillus thuringiensis (Bt) is the most widely used insecticidal microbe due to its specific toxicity and safe use with respect to animals and the environment. In this study, we isolated Bt strain Q52-7 from a soil sample collected in the Qian Shan District, Liao Ning Province, China. We observed that the Q52-7 strain produced spherical crystals. The Bt Q52-7 strain had high toxicity against Asian Cockchafer (Holotrichia parallela), exhibiting an LC₅₀ of 3.80 × 10⁹ cfu/g, but is not toxic for Anomala corpulenta Motschulsky and Holotrichia oblita. Using general cry8 primers, we amplified a 1.3 kb fragment with the polymerase chain reaction. Specific primers were designed for the amplified fragment to clone the full-length coding region. A novel gene, cry8Na1, had 69 % sequence similarity with cry8Ca1. cry8Na1 gene was successfully expressed in the HD-73^– acrystalliferous mutant of Bt subsp. Kurstaki HD-73. Bioassays demonstrated that the Cry8Na1 protein is highly toxic for the H. parallela, with a 50 % lethal concentration of 8.18 × 10¹⁰ colony forming units per gram.     

Molecular Cloning of a New Crystal Protein Gene cry1Af1 of Bacillus thuringiensis NT0423 from Korean Sericultural Farms

A new cry1Ab-type gene encoding the 130 kDa protein of Bacillus thuringiensis NT0423 bipyramidal crystals was cloned, sequenced, and expressed in a crystal-negative B. thuringiensis host. Hybridization experiments revealed that the crystal protein gene is located on a 44 MDa plasmid of B. thuringiensis NT0423. A strong positive signal detected on the 6.6 kb HindIII fragment from B. thuringiensis NT0423 plasmid DNA was cloned and sequenced. The cry1Ab-type gene, designated cry1Af1, consisted of open reading frame of 3453 bp, encoding a protein of 1151 amino acid residues. The polypeptide has the deduced amino acid sequences predicting molecular masses of 130,215 Da. With both Bt I and Br II promoter sequences were found, the B. thuringiensis NT0423 crystal protein gene promoter closely aligned with those of cry1A-type crystal protein gene. When compared with known sequences of other Cry and Cyt proteins, the Cry1Af1 protein showed maximum 93% sequence identity to Cry1Ab protein of B. thuringiensis subsp. kurstaki. The expressed Cry1Af1 protein in a crystal-negative B. thuringiensis host appears to have strong insecticidal activity against lepidopteran larvae (Plutella xylostella). Crystals containing Cry1Af1 were about six times more toxic than the wild-type crystals of B. thuringiensis NT0423. Received: 20 February 2001 / Accepted: 17 April 2001     

Toxicity of a <Emphasis Type="Italic">Bacillus thuringiensis israelensis</Emphasis>-like strain against <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis>

Bacillus thuringiensis (Bt) Berliner is a promising agent for microbial control of agriculturally and medically important insects. This study aimed at searching for Bt strains encoding Cry proteins that act more efficiently against fall armyworm. Thirty Bt strains were isolated from soil samples in Pernambuco State and evaluated through bioassays. Among these, strain I4A7 was the most efficient against the fall armyworm, Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera: Noctuidae), and thus it was characterized by biochemical sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and molecular (polymerase chain reaction (PCR) and sequencing reaction) methods. The protein pattern of this strain on a SDS–PAGE was similar to that of B. thuringiensis israelensis (Bti). Moreover, I4A7 cry DNA sequence showed high identity (99–100%) to genes cry4Aa, 4Ba, 10Aa, 11Aa, cyt1Aa and cyt2B from Bti. The toxicity of the newly isolated Bti-like strain upon S. frugiperda should be considered as this strain might be used in combination with other Bt strains, such as B. thuringiensis var. kurstaki (Btk). Handling Editor: Helen Roy.     

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.     

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.     

Evidence of the Involvement of E358, A498 and C571 of a New Cry1Ac δ-endotoxin of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in its High Insecticidal Activity Against <Emphasis Type="Italic">Ephestia kuehniella</Emphasis>

A new cry1Ac-type gene was cloned from Bacillus thuringiensis strain BLB1, sequenced and expressed. The deduced amino acid sequence of the polypeptide has a predicted molecular mass of 132.186 kDa. The amino acid sequence alignment of BLB1 Cry1Ac with those of the published ones showed that this is a new delta-endotoxin. When compared with Cry1Ac of Bacillus thuringiensis strain HD1, it was found that BLB1 Cry1Ac harbours three mutations: V358E localized in domain II and V498A and Y571C localized in domain III. When the BLB1 Cry1Ac toxin was expressed in an acrystalliferous strain of B. thuringiensis (HD1CryB), bipyramidal crystals were produced. The spore–crystal mixture of this recombinant strain was at least two-fold more active against larvae of the lepidopteran Ephestia kuehniella than that of the recombinant strain expressing Cry1Ac of HD1. The study of the structural effect of these mutations suggested that they may stabilize key regions involved in the binding of the domains II and III to insect receptors.     

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.     

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

Aims: 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. Methods and Results: 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 LC₅₀ 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. Conclusions: 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. Significance and Impact of the Study: The inter relationship between the cry genes content and the toxicity may allow better understanding of Bt ecology.     

A chimeric cry8Ea1 gene flanked by MARs efficiently controls Holotrichia parallela

Key message

Peanuts transformed with the synthetic cry8Ea1 gene flanked by MARs are a potentially effective control strategy against white grubs. Cry8Ea1 protein levels of the construct containing MARs were increased by 2.5 times.

Abstract

White grubs are now recognized as the most important pests of peanut worldwide. A synthetic cry8Ea1 gene, which was toxic to Holotrichia parallela larvae, was expressed in chimeric peanut roots using an Agrobacterium rhizogenes-mediated transformation system. The relative mRNA and protein levels of the cry8Ea1 gene were confirmed by quantitative real-time PCR and ELISA, respectively. The effects of matrix attachment regions (MARs) on the expression and activity of the cry8Ea1 gene were analyzed. The average expression level of cry8Ea1 in peanut roots was higher for the plants harboring constructs flanked by MARs from tobacco. Moreover, differing from previous studies, the synthetic cry8Ea1 gene flanked by MARs showed more variation in protein levels than mRNA levels. These composite plants containing cry8Ea1 gene flanked by MARs exhibited a high toxicity against Holotrichia parallela larvae as shown by bioassay analysis, thus offering a potential effective combination to control subterranean insects in peanuts.     

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.     

Characterization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> ser. <Emphasis Type="Italic">jordanica</Emphasis> (Serotype H71), a Novel Serovariety Isolated in Jordan

The novel strain of Bacillus thuringiensis J112 isolated from a soil sample in Jordan was classified and characterized in terms of toxicity against dipteran and nematode larvae, crystal protein pattern, plasmid profile, and cry gene content. A new name, Bacillus thuringiensis serovariety jordanica (H serotype 71), is proposed for the reference strain J112. The parasporal crystal proteins were toxic to 3^rd instar larvae of Drosophila melanogaster and to 2^nd stage juveniles of root knot nematodes Meloidogyne javanica and M. incognita, but showed poor mosquitocidal activity towards Culex pipiens molestus and Culiseta longiareolata larvae. Solubilized and trypsin-digested crystal proteins possessed moderate hemolytic activity against sheep erythrocytes. SDS-polyacrylamide gel electrophoresis revealed that crystals are composed of several polypeptides ranging from 24 to 170 kDa, of which the 20-, 42-, 140-, and 170-kDa proteins were the major components. Analysis of the plasmid pattern of J112 revealed the presence of two large plasmidic bands of about 160 and 205 kbp. PCR with total DNA from strain J112 and specific primers for cry1, cry2, cry3, cry4, and cyt2A genes revealed that cry1, cry3A, cry4, cry5 and cyt2a genes are present. Received: 9 August 2002 / Accepted: 4 September 2002     

An Improved PCR-Restriction Fragment Length Polymorphism (RFLP) Method for the Identification of cry1-Type Genes

The cry1-type genes of Bacillus thuringiensis represent the largest cry gene family, which contains 50 distinct holotypes. It is becoming more and more difficult to identify cry1-type genes using current methods because of the increasing number of cry1-type genes. In the present study, an improved PCR-restriction fragment length polymorphism (PCR-RFLP) method which can distinguish 41 holotypes of cry1-type genes was developed. This improved method was used to identify cry1-type genes in 20 B. thuringiensis strains that are toxic to lepidoptera. The results showed that the improved method can efficiently identify single and clustered cry1-type genes and can be used to evaluate cry1-type genes in novel strain collections of B. thuringiensis. Among the detected cry1-type genes, we identified four novel genes, cry1Ai, cry1Bb, cry1Ja, and cry1La. The bioassay results from the expressed products of the four novel cry genes showed that Cry1Ai2, Cry1Bb2, and Cry1Ja2 were highly toxic against Plutella xylostella, whereas Cry1La2 exhibited no activity. Moreover, Cry1Ai2 had good lethal activity against Ostrinia furnacalis, Hyphantria cunea, Chilo suppressalis, and Bombyx mori larvae and considerable weight loss activity against Helicoverpa armigera.     

Cloning and characterization of a novel cry8Ab1 gene from Bacillus thuringiensis strain B-JJX with specific toxicity to scarabaeid (Coleoptera: Scarabaeidae) larvae

Isolation of Bacillus thuringiensis (Bt) strain or its cry gene encoding insecticidal crystal protein (ICP) with specific toxicity is of great importance to biological control of insect pests. In this study, by screening 66 strains of Bt isolated from soil samples collected in Shandong Province, China, a new cry8-type gene from Bt strain B-JJX was identified via PCR-RFLP method. This novel gene, cry8Ab1, was cloned from the Bt strain B-JJX and expressed in an acrystalliferous mutant strain HD-73^?. The open reading frame of the cry8Ab1 gene consists of 3543 bp with a G + C content of 37.99% and encodes a protein of 1180 amino acids with a putative MW of 133.3 kDa which was confirmed by SDS-PAGE analysis. The Cry8Ab1 protein was expressed and released as spherical parasporal crystals from Bt acrystalliferous mutant strain HD-73^? along with the presence of spores. In bioassays, this protein was toxic to 3-day-old larvae of the scarabaeid pests, Holotrichia oblita and H. parallela, with an LC₅₀ of 5.72 and 2.00 μg toxin g^?1 soil, respectively. The results are in accordance with the insecticidal activities of the original Bt strain B-JJX, which had an LC₅₀ of 1.72 and 0.96 μg toxin g^?1 soil against H. oblita and H. parallela, respectively.     

Characterization of the cry1Ac17 Gene from an Indigenous Strain of Bacillus thuringiensis subsp. kenyae

An indigenously isolated strain of Bacillus thuringiensis subsp. kenyae exhibited toxicity against lepidopteran as well as dipteran insects. The lepidopteran active cry1Ac protoxin gene coding sequence of 3.5 kb from this strain was cloned into vector pET28a(+). However, it could not be expressed in commonly used Escherichia coli expression hosts, BL21(DE3) and BL21(DE3)pLysS. This gene is classified as cry1Ac17 in the B. thuringiensis toxic nomenclature database. The coding sequence of this gene revealed that it contains about 3% codons, which are not efficiently translated by these expression hosts. Hence, this gene was expressed in a modified expression host, Epicurian coli BL21-Codonplus (DE3)-RIL. The expression of gene yielded a 130-kDa Cry1Ac17 protein. The protein was purified and its toxicity was tested against economically important insect pests, viz., Helicoverpa armigera and Spodoptera litura. LC₅₀ values obtained against these insects were 0.1 ng/cm³ and 1231 ng/cm², respectively. The higher toxicity of Cry1Ac17 protein, compared to other Cry1Ac proteins, toward these pests demonstrates the potential of this isolate as an important candidate in the integrated resistance management program in India.     

Characterization of a novel cry2Aa-type gene from Bacillus thuringiensis subsp. kurstaki

A 4 kb BamHI-HindIII fragment, corresponding to the cry2A operon of Bacillus thuringiensis subsp. kurstaki strain BNS3, was cloned. The sequencing of the corresponding cry2Aa-type gene, termed crybns3-4, revealed an open reading frame of 1902 bp, encoding a protein of 633 amino-acid residues. Both nucleotide and amino-acid sequences similarity analysis revealed that crybns3-4 is a new cry2Aa-type gene which has several differences from the reported cry2Aa-type genes. The transfer of the cloned operon to an acrystalliferous mutant of BNS3, revealed an expression of the new cry2Aa-type gene and a production of parasporal crystal inclusions in the transformants.