A selective insecticidal protein from Pseudomonas mosselii for corn rootworm control

The coleopteran insect western corn rootworm (WCR, Diabrotica virgifera virgifera) is an economically important pest in North America and Europe. Transgenic corn plants producing Bacillus thuringiensis (Bt) insecticidal proteins have been useful against this devastating pest, but evolution of resistance has reduced their efficacy. Here, we report the discovery of a novel insecticidal protein, PIP‐47Aa, from an isolate of Pseudomonas mosselii. PIP‐47Aa sequence shows no shared motifs, domains or signatures with other known proteins. Recombinant PIP‐47Aa kills WCR, two other corn rootworm pests (Diabrotica barberi and Diabrotica undecimpunctata howardi) and two other beetle species (Diabrotica speciosa and Phyllotreta cruciferae), but it was not toxic to the spotted lady beetle (Coleomegilla maculata) or seven species of Lepidoptera and Hemiptera. Transgenic corn plants expressing PIP‐47Aa show significant protection from root damage by WCR. PIP‐47Aa kills a WCR strain resistant to mCry3A and does not share rootworm midgut binding sites with mCry3A or AfIP‐1A/1B from Alcaligenes that acts like Cry34Ab1/Cry35Ab1. Our results indicate that PIP‐47Aa is a novel insecticidal protein for controlling the corn rootworm pests.     

Isolation and characterization of EG2158, a new strain of Bacillus thuringiensis toxic to coleopteran larvae, and nucleotide sequence of the toxin gene

Summary A novel strain of Bacillus thuringiensis was isolated from soybean grain dust from Kansas and found to be toxic to larvae of Leptinotarsa decemlineata (Colorado potato bectle). The strain (EG2158) synthesized two parasporal crystals: a rhomboid crystal composed of a 73115 dalton protein and a flat, diamond-shaped crystal composed of a protein of approximately 30 kDa. Plasmid transfer and gene cloning experiments demonstrated that the 73 kDa protein was encoded on an 88 MDa plasmid and that the protein was toxic to the larvae of Colorado potato beetle (CPB). The sequence of the 73 kDa protein, as deduced from the sequence of its gene (cryC), was found to have regions of similarity with several B. thuringiensis crystal proteins: the lepidopteran-toxic P1 proteins of var. kurstaki and berliner, the lepidopteran- and dipteran-toxic P2 (or CRYB1) protein of var. kurstaki, and the dipteran-toxic 130 kDa protein of var. israelensis. While B. megaterium cells harboring the cryC gene from EG2158 synthesized significant amounts of the 73 kDa CRYC protein, Escherichia coli cells did not. The cryC-containing B. megaterium cells produced rhomboid crystals that were toxic to CPB larvae.     

Sip1Ab gene from a native Bacillus thuringiensis strain QZL38 and its insecticidal activity against Colaphellus bowringi Baly

In this study, we collected 540 soil samples from northeast China and isolated the wild-type strain of Bacillus thuringiensis (Bt) by identifying and cloning 9 Bt strains that expressed the secreted insecticidal protein (Sip) gene. We selected the strain QZL38 for further study. The sip gene was identified from the Bt strain QZL38 using polymerase chain reaction (PCR). We sequenced a 1095-base pair fragment of DNA that encodes 364 amino acid residues of a 41.18?kDa pro-toxin and compared it with the registered Sip1Ab protein amino acid residue sequence. The sequence was submitted to GenBank with the accession no. KP231523, and the gene was named sip1Ab. The Sip1Ab protein expressed in Escherichia coli showed insecticidal activity against Colaphellus bowringi Baly, with an LC50 of 1.051?μg?mL^?1. To identify the active fragment of the Sip1Ab toxin, four pairs of primers with different truncation positions were designed, and the recombinant proteins were expressed in E. coli. The truncated Sip protein expressed in E. coli showed insecticidal activity against C. bowringi Baly. The insecticidal activity of the recombinant proteins against C. bowringi Baly from the Sip1Ab signal peptide after removal of 30 amino acid residues showed an LC50 of 1.078?μg?mL^?1. Sip proteins may play an important role in the prevention and control of the C. bowringi Baly.     

Binary toxins from Bacillus thuringiensis active against the western corn rootworm, Diabrotica virgifera virgifera LeConte

The western corn rootworm, Diabrotica virgifera virgifera LeConte, is a significant pest of corn in the United States. The development of transgenic corn hybrids resistant to rootworm feeding damage depends on the identification of genes encoding insecticidal proteins toxic to rootworm larvae. In this study, a bioassay screen was used to identify several isolates of the bacterium Bacillus thuringiensis active against rootworm. These bacterial isolates each produce distinct crystal proteins with approximate molecular masses of 13 to 15 kDa and 44 kDa. Insect bioassays demonstrated that both protein classes are required for insecticidal activity against this rootworm species. The genes encoding these proteins are organized in apparent operons and are associated with other genes encoding crystal proteins of unknown function. The antirootworm proteins produced by B. thuringiensis strains EG5899 and EG9444 closely resemble previously described crystal proteins of the Cry34A and Cry35A classes. The antirootworm proteins produced by strain EG4851, designated Cry34Ba1 and Cry35Ba1, represent a new binary toxin. Genes encoding these proteins could become an important component of a sustainable resistance management strategy against this insect pest.     

Binary insecticidal crystal protein from Bacillus thuringiensis,strain PS149B1: effects of individual protein components and mixtures in laboratory bioassays

A family of novel binary insecticidal crystal proteins, with activity against western corn rootworm, Diabrotica virgifera virgifera LeConte, was identified from Bacillus thuringiensis Berliner. A binary insecticidal crystal protein (bICP) from B. thuringiensis strain PS149B1 is composed of a 14-kDa protein (Cry34Abl) and a 44-kDaprotein (Cry35Ab1). These proteins have been co-expressed in transgenic maize plants, Zea mays L., and effectively control western corn rootworm larvae under field conditions. Laboratory experiments were conducted to better understand the contribution of each component protein to the in vivo activity of the bICP. The 14-kDa protein is active alone against southern corn rootworm, Diabrotica undecimpunctata howardi Barber, and was synergized by the 44-kDa protein. In mixtures, the concentration of the 14-kDa protein had a greater impact on efficacy than the 44-kDa component. Although both proteins are clearly required for maximal insecticidal activity, laboratory results did not support the formation of a stable, fixed-ratio complex of the two component proteins.     

Enhancement of Bacillus thuringiensis Cry3Aa and Cry3Bb Toxicities to Coleopteran Larvae by a Toxin-Binding Fragment of an Insect Cadherin

The Cry3Aa and Cry3Bb insecticidal proteins of Bacillus thuringiensis are used in biopesticides and transgenic crops to control larvae of leaf-feeding beetles and rootworms. Cadherins localized in the midgut epithelium are identified as receptors for Cry toxins in lepidopteran and dipteran larvae. Previously, we discovered that a peptide of a toxin-binding cadherin expressed in Escherichia coli functions as a synergist for Cry1A toxicity against lepidopteran larvae and Cry4 toxicity against dipteran larvae. Here we report that the fragment containing the three most C-terminal cadherin repeats (CR) from the cadherin of the western corn rootworm binds toxin and enhances Cry3 toxicity to larvae of naturally susceptible species. The cadherin fragment (CR8 to CR10 [CR8-10]) of western corn rootworm Diabrotica virgifera virgifera was expressed in E. coli as an inclusion body. By an enzyme-linked immunosorbent microplate assay, we demonstrated that the CR8-10 peptide binds α-chymotrypsin-treated Cry3Aa and Cry3Bb toxins at high affinity (11.8 nM and 1.4 nM, respectively). Coleopteran larvae ingesting CR8-10 inclusions had increased susceptibility to Cry3Aa or Cry3Bb toxin. The Cry3 toxin-enhancing effect of CR8-10 was demonstrated for Colorado potato beetle Leptinotarsa decemlineata, southern corn rootworm Diabrotica undecimpunctata howardi, and western corn rootworm. The extent of Cry3 toxin enhancement, which ranged from 3- to 13-fold, may have practical applications for insect control. Cry3-containing biopesticides that include a cadherin fragment could be more efficacious. And Bt corn (i.e., corn treated with B. thuringiensis to make it resistant to pests) coexpressing Cry3Bb and CR8-10 could increase the functional dose level of the insect toxic activity, reducing the overall resistance risk.The Cry3 class of Bacillus thuringiensis Cry proteins is known for toxicity to coleopteran larvae in the family Chrysomelidae. Cry3Aa and Cry3Bb proteins are highly toxic to Colorado potato beetle (CPB) Leptinotarsa decemlineata (Coleoptera: Chrysomelidae), and both were used for the development of Bt crops (crops treated with B. thuringiensis to make them resistant to pests) and Bt biopesticides. Due to the limited efficacy of Cry3-based biopesticides/plants and the success of competing chemical pesticides, these biopesticides have had limited usage and sales (12). Cry3Bb is toxic to corn rootworms (8, 17), and a modified version is expressed in commercialized MON863 corn hybrids (26).Cry3 toxins have a mode of action that is similar to, yet distinct from, the action of lepidopteran-active Cry1 toxins. The Cry3A protoxin (73 kDa) lacks the large C-terminal region of the 130-kDa Cry1 protoxins, which is removed by proteases during activation to toxin. The Cry3A protoxin is activated to a 55-kDa toxin and then further cleaved within the toxin molecule (5, 18). Activated Cry3A toxin binds to brush border membrane vesicles with a K_d (dissociation constant) of ∼37 nM (19) and recognizes a 144-kDa binding protein in brush border membrane vesicles prepared from the yellow mealworm Tenebrio molitor (Coleoptera: Tenebrionidae) (2). Recently, Ochoa-Campuzano et al. (20) identified an ADAM metalloprotease as a receptor for Cry3Aa toxin in CPB larvae.Structural differences between Cry3Bb and Cry3Aa toxins must underlie the unique rootworm activities of Cry3Bb toxin. As noted by Galitsky et al. (11), differences in toxin solubility, oligomerization, and binding are reported for these Cry3 toxins. Recently, Cry3Aa was modified to have activity against western corn rootworm (WCRW) Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) (27). Those authors introduced a chymotrypsin/cathepsin G cleavage site into domain 1 of Cry3Aa that allowed the processing of the 65-kDa form to a 55-kDa toxin that bound rootworm midgut.Cadherins function as receptors for Cry toxins in lepidopteran and dipteran larvae. A critical Cry1 toxin binding site is localized within the final cadherin repeat (CR), CR12, of cadherins from tobacco hornworm Manduca sexta (Lepidoptera: Sphingidae) and tobacco budworm Heliothis virescens (Lepidoptera: Noctuidae) (14, 28). Unexpectedly, a fragment of B. thuringiensis R₁ cadherin, the Cry1A receptor from M. sexta, not only bound toxin but enhanced Cry1A toxicity against lepidopteran larvae (6). If the binding residues within CR12 were removed, the resulting peptide lost the ability to bind toxin and lost its function as a toxin synergist. Recently, we identified a cadherin from mosquito Anopheles gambiae (Diptera: Culicidae) that binds Cry4Ba toxin and probably functions as a receptor. We discovered a similar effect where a fragment of a cadherin from A. gambiae enhanced the toxicity of the mosquitocidal toxin Cry4Ba to mosquito larvae (15). Sayed et al. (22) identified a novel cadherin-like gene in WCRW and proposed this protein as a candidate Bt toxin receptor. The cadherin-like gene is highly expressed in the midgut tissue of larval stages. The encoded protein is conserved in structure relative to that of other insect midgut cadherins.In this study, we hypothesized that a fragment from a beetle cadherin that contains a putative Bt toxin binding region might enhance the insecticidal toxicities of Cry3Aa and Cry3Bb toxins. The region spanning CR8 to CR10 (CR8-10) of the WCRW cadherin (22) was cloned and expressed in E. coli. This cadherin fragment significantly enhanced the toxicities of Cry3Aa and Cry3Bb toxins to CPB and rootworms.     

Two novel strains of Bacillus thuringiensis toxic to coleopterans.

Two novel strains of Bacillus thuringiensis were isolated from native habitats by the use of genes coding for proteins toxic to coleopterans (cryIII genes) as hybridization probes. Strain EG2838 (isolated by the use of the cryIIIA probe) contained a cryIIIA-hybridizing plasmid of approximately 100 MDa and synthesized crystal proteins of approximately 200 (doublet), 74, 70, 32, and 28 kDa. Strain EG4961 (isolated by the use of a cryIIIA-related probe) contained a cryIIIA-hybridizing plasmid of approximately 95 MDa and synthesized crystal proteins of 74, 70, and 30 kDa. Structural relationships among the crystal proteins of strains EG2838 and EG4961 were detected; antibodies to the CryIIIA protein toxic to coleopterans reacted with the 74- and 70-kDa proteins of EG2838 and EG4961, antibodies to the 32-kDa plus 28-kDa proteins of EG2838 reacted with the 30-kDa protein of EG4961, and antibodies to the 200-kDa proteins of EG2838 reacted with the 28-kDa protein of EG2838. Experiments with B. thuringiensis flagella antibody reagents demonstrated that EG2838 belongs to H serotype 9 (reference strain B. thuringiensis subsp. tolworthi) and that EG4961 belongs to H serotype 18 (reference strain B. thuringiensis subsp. kumamotoensis). A mixture of spores plus crystal proteins of either EG2838 or EG4961 was toxic to the larvae of Colorado potato beetle (Leptinotarsa decemlineata), and significantly, the EG4961 mixture was also toxic to the larvae of southern corn rootworm (Diabrotica undecimpunctata howardi). DNA restriction blot analysis suggested that strains EG2838 and EG4961 each contained a unique gene coding for a protein toxic to coleopterans.     

Two novel strains of Bacillus thuringiensis toxic to coleopterans.

Two novel strains of Bacillus thuringiensis were isolated from native habitats by the use of genes coding for proteins toxic to coleopterans (cryIII genes) as hybridization probes. Strain EG2838 (isolated by the use of the cryIIIA probe) contained a cryIIIA-hybridizing plasmid of approximately 100 MDa and synthesized crystal proteins of approximately 200 (doublet), 74, 70, 32, and 28 kDa. Strain EG4961 (isolated by the use of a cryIIIA-related probe) contained a cryIIIA-hybridizing plasmid of approximately 95 MDa and synthesized crystal proteins of 74, 70, and 30 kDa. Structural relationships among the crystal proteins of strains EG2838 and EG4961 were detected; antibodies to the CryIIIA protein toxic to coleopterans reacted with the 74- and 70-kDa proteins of EG2838 and EG4961, antibodies to the 32-kDa plus 28-kDa proteins of EG2838 reacted with the 30-kDa protein of EG4961, and antibodies to the 200-kDa proteins of EG2838 reacted with the 28-kDa protein of EG2838. Experiments with B. thuringiensis flagella antibody reagents demonstrated that EG2838 belongs to H serotype 9 (reference strain B. thuringiensis subsp. tolworthi) and that EG4961 belongs to H serotype 18 (reference strain B. thuringiensis subsp. kumamotoensis). A mixture of spores plus crystal proteins of either EG2838 or EG4961 was toxic to the larvae of Colorado potato beetle (Leptinotarsa decemlineata), and significantly, the EG4961 mixture was also toxic to the larvae of southern corn rootworm (Diabrotica undecimpunctata howardi). DNA restriction blot analysis suggested that strains EG2838 and EG4961 each contained a unique gene coding for a protein toxic to coleopterans.     

Timing of the Initial Spray Application of Bacillus thuringiensis for Control of the Colorado Potato Beetle (Coleoptera: Chrysomelidae) in Potatoes

Field experiments were conducted in 1989 and 1990 to evaluate the timing of the initial spray application of Bacillus thuringiensis subsp. tenebrianis, relative to egg development in the field, for control of the Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say) in potatoes. Effective potato beetle control was achieved with B. thuringiensis if the initial spray was applied during the period beginning when an average of 1-30% of marked egg masses had hatched, extending to 4 days after 30% egg hatch, of the initial flush of eggs. Significantly greater defoliation, numbers of third and fourth instar CPB and lower yields occurred in plots when the initial B. thuringiensis application was delayed until 6 days after 30% egg hatch, compared with plots treated at 30% egg hatch. In 1990, no improvement in CPB control was gained by application of the initial B. thuringiensis spray at first observation of CPB egg deposition or at first egg hatch, compared with application at 30% egg hatch. Recommendations for proper timing of B. thuringiensis sprays for effective CPB management are presented.     

Independent Action between DvSnf7 RNA and Cry3Bb1 Protein in Southern Corn Rootworm,Diabrotica undecimpunctata howardi and Colorado Potato Beetle,Leptinotarsa decemlineata

In recent years, corn rootworm (CRW)-resistant maize events producing two or more CRW-active Bt proteins have been commercialized to enhance efficacy against the target pest(s) by providing multiple modes of action (MoA). The maize hybrid MON 87411 has been developed that produces the CRW-active Cry3Bb1 Bt protein (hereafter Cry3Bb1) and expresses a RNAi-mediated MoA that also targets CRW. As part of an environmental risk assessment for MON 87411, the potential for an interaction between the CRW-active DvSnf7 RNA (hereafter DvSnf7) and Cry3Bb1 was assessed in 12-day diet incorporation bioassays with the southern corn rootworm (SCR, Diabrotica undecimpunctata howardi). The potential for an interaction between DvSnf7 and Cry3Bb1 was evaluated with two established experimental approaches. The first approach evaluated each substance alone and in combination over three different response levels. For all three response levels, observed responses were shown to be additive and not significantly different from predicted responses under the assumption of independent action. The second approach evaluated the potential for a fixed sub-lethal concentration of Cry3Bb1 to decrease the median lethal concentration (LC₅₀) of DvSnf7 and vice-versa. With this approach, the LC50 value of DvSnf7 was not altered by a sub-lethal concentration of Cry3Bb1 and vice-versa. In addition, the potential for an interaction between the Cry3Bb1 and DvSnf7 was tested with Colorado potato beetle (CPB, Leptinotarsa decemlineata), which is sensitive to Cry3Bb1 but not DvSnf7. CPB assays also demonstrated that DvSnf7 does not alter the activity of Cry3Bb1. The results from this study provide multiple lines of evidence that DvSnf7 and Cry3Bb1 produced in MON 87411 have independent action.     

Large-scale production and characterization of Bacillus thuringiensis subsp. tenebrionis insecticidal protein from Escherichia coli

Bacillus thuringiensis subsp. tenebrionis insecticidal protein was produced in recombinant Escherichia coli and purified to near homogeneity to provide quantities of protein for safety-assessment studies associated with the registration of transgenic potato plants. The 68-kDa protein is produced naturally by Bacillus thuringiensis subsp. tenebrionis by translation initiation at an internal initiation site in the native DNA sequence. The gene sequence specific for this truncated protein was expressed in E. coli strain JM 101 and fermented at the 1000-l scale. The protein accumulated as insoluble inclusion bodies, and was purified by extraction at pH␣10.8 with carbonate buffer, selective precipitation at pH 9.0, and differential centrifugation. No chromatography steps were required to produce over 50 g purified protein as a lyophilized powder with a purity greater than 95 % and demonstrating full insecticidal activity against Colorado potato beetle larvae. The protein was further characterized to assure identity and suitability for use in safety-assessment studies. Received: 31 May 1996 / Received revision: 11 September 1996 / Accepted: 13 October 1996     

Tritrophic interactions among Bt maize,an insect pest and entomopathogens: effects on development and survival of western corn rootworm

Agricultural systems often provide a model for testing ecological hypotheses, while ecological theory can enable more effective pest management. One of the best examples of this is the interaction between host‐plant resistance and natural enemies. With the advent of crops that are genetically modified to produce insecticidal toxins from the bacterium Bacillus thuringiensis (Bt), a new form of host‐plant resistance has been introduced to agroecosystems. How Bt crops interact with natural enemies, especially insect pathogens in below‐ground systems, is not well understood, but provides a unique opportunity to study below‐ground tritrophic interactions. In this study, we used two species of entomopathogenic fungi and three species of entomopathogenic nematodes to determine how this community of soil‐borne natural enemies might interact with Bt maize (event 59122, expressing the insecticidal protein Cry34/35Ab1) to affect survival and development of western corn rootworm (Diabrotica virgifera virgifera), which is an obligate root feeder and a serious pest of maize. We ran two experiments, one in a greenhouse and one in a growth chamber. Both experiments consisted of a fully crossed design with two maize treatments (Bt maize and non‐Bt maize) and two entomopathogen treatments (present or absent). The community of entomopathogens significantly increased mortality of western corn rootworm, and Bt maize increased larval developmental time and mortality. Entomopathogens and Bt maize acted in an independent and additive manner, with both factors increasing the mortality of western corn rootworm. Results from this study suggest that entomopathogens may complement host‐plant resistance from Bt crops.     

Development of insect resistance in tomato plants expressing the δ-endotoxin gene ofBacillus thuringiensis subsp.tenebrionis

A crystal -endotoxin gene ofBacillus thuringiensis subsp.tenebrionis (B.t.t.) encoding a coleopteran insect-specific toxin was used to construct a chimeric gene which expressed the toxin in plant cells. Via anAgrobacterium tumefaciens binary vector system, the toxin gene was transferred into tomato cells. From leaf disks recombinant plants were regenerated. Hybridization experiments demonstrated that these plants synthesized toxin-specific mRNA of the expected size. Transgenic tomato plants with the chimericB.t.t. toxin gene contained a 74 kDa protein which cross-reacted with toxin antibodies. The expression caused a significant insecticidal activity of the transgenic tomato plants against Colorado potato beetle larvae.     

Bacillus thuringiensis Cry34Ab1/Cry35Ab1 Interactions with Western Corn Rootworm Midgut Membrane Binding Sites

Background

Bacillus thuringiensis (Bt) Cry34Ab1/Cry35Ab1 are binary insecticidal proteins that are co-expressed in transgenic corn hybrids for control of western corn rootworm, Diabrotica virgifera virgifera LeConte. Bt crystal (Cry) proteins with limited potential for field-relevant cross-resistance are used in combination, along with non-transgenic corn refuges, as a strategy to delay development of resistant rootworm populations. Differences in insect midgut membrane binding site interactions are one line of evidence that Bt protein mechanisms of action differ and that the probability of receptor-mediated cross-resistance is low.

Methodology/Principal Findings

Binding site interactions were investigated between Cry34Ab1/Cry35Ab1 and coleopteran active insecticidal proteins Cry3Aa, Cry6Aa, and Cry8Ba on western corn rootworm midgut brush border membrane vesicles (BBMV). Competitive binding of radio-labeled proteins to western corn rootworm BBMV was used as a measure of shared binding sites. Our work shows that ¹²⁵I-Cry35Ab1 binds to rootworm BBMV, Cry34Ab1 enhances ¹²⁵I-Cry35Ab1 specific binding, and that ¹²⁵I-Cry35Ab1 with or without unlabeled Cry34Ab1 does not share binding sites with Cry3Aa, Cry6Aa, or Cry8Ba. Two primary lines of evidence presented here support the lack of shared binding sites between Cry34Ab1/Cry35Ab1 and the aforementioned proteins: 1) No competitive binding to rootworm BBMV was observed for competitor proteins when used in excess with ¹²⁵I-Cry35Ab1 alone or combined with unlabeled Cry34Ab1, and 2) No competitive binding to rootworm BBMV was observed for unlabeled Cry34Ab1 and Cry35Ab1, or a combination of the two, when used in excess with ¹²⁵I-Cry3Aa, or ¹²⁵I-Cry8Ba.

Conclusions/Significance

Combining two or more insecticidal proteins active against the same target pest is one tactic to delay the onset of resistance to either protein. We conclude that Cry34Ab1/Cry35Ab1 are compatible with Cry3Aa, Cry6Aa, or Cry8Ba for deployment as insect resistance management pyramids for in-plant control of western corn rootworm.     

Effectiveness of recombinant soybean cysteine proteinase inhibitors against selected crop pests

Three recombinant soybean cysteine proteinase inhibitors (rSCPIs), L1, R1 and N2, were assessed for their potential to inhibit the growth and development of three major agricultural crop pests known to utilize digestive cysteine proteinases: Western corn rootworm (Diabrotica virgifera virgifera, WCR), Colorado potato beetle (Leptinotarsa decemlineata, CPB) and cowpea weevil (Callosobruchus maculatus, CW). In vitro experiments showed that cysteine proteinase activities in the crude gut extracts of the WCR, CPB, and CW were inhibited to various degrees by the three rSCPIs. Of the three rSCPIs tested, N2 was most effective in inhibiting the crude gut extract of WCR, CPB, and CW (50% inhibition at 5 x 10(-8), 5 x 10(-8), and 3 x 10(-7) M, respectively). The L1 was the least potent of the three CPIs tested, with 50% inhibition at 5 x 10(-6) M of the crude gut extracts of WCR. Results of in vivo experiments conducted to assess the effect of the three rSCPIs on the vital growth parameters of WCR, CPB and CW were consistent with results of the in vitro experiments.     

Construction and analysis of Sip1Aa insecticidal protein random recombination library

The Sip1Aa protein from Bacillus thuringiensis is highly toxic to Colaphellus bowringi Baly. In order to obtain mutant proteins with higher insecticidal activity, a random recombinant library of Sip1Aa protein was constructed using error-prone PCR. A total number of 100 positive transformants were randomly selected for sequence determination, and 25 mutants (M1 to M25) were selected and expressed the respective Sip1Aa mutants. These Sip1Aa variants had a total of 29 base mutations, with an average of 1.2 base mutations per mutant. Compared with that of the wild-type Sip1Aa protein, the insecticidal activity of the mutants M1 (A31G, Y118C, D227E), M5 (K168R) and M21 (I307T) was significantly decreased, with and LC₅₀ values 4 to 6 times higher than the Sip1Aa protein. The mutant M8 (R174S) showed increase in the insecticidal activity against the Colaphellus bowringi Baly was obtained, with an LC₅₀ value 4-fold less than the Sip1Aa protein. The results of this study provide reference for the molecular modification of Sip1Aa protein and the study of key sites of its insecticidal activity.     

Novel Bacillus thuringiensis Binary Insecticidal Crystal Proteins Active on Western Corn Rootworm, Diabrotica virgifera virgifera LeConte

A new family of insecticidal crystal proteins was discovered by screening sporulated Bacillus thuringiensis cultures for oral activity against western corn rootworm (WCR) larvae. B. thuringiensis isolates PS80JJ1, PS149B1, and PS167H2 have WCR insecticidal activity attributable to parasporal inclusion bodies containing proteins with molecular masses of ca. 14 and 44 kDa. The genes encoding these polypeptides reside in apparent operons, and the 14-kDa protein open reading frame (ORF) precedes the 44-kDa protein ORF. Mutagenesis of either gene in the apparent operons dramatically reduced insecticidal activity of the corresponding recombinant B. thuringiensis strain. Bioassays performed with separately expressed, biochemically purified 14- and 44-kDa polypeptides also demonstrated that both proteins are required for WCR mortality. Sequence comparisons with other known B. thuringiensis insecticidal proteins failed to reveal homology with previously described Cry, Cyt, or Vip proteins. However, there is evidence that the 44-kDa polypeptide and the 41.9- and 51.4-kDa binary dipteran insecticidal proteins from Bacillus sphaericus are evolutionarily related. The 14- and 44-kDa polypeptides from isolates PS80JJ1, PS149B1, and PS167H2 have been designated Cry34Aa1, Cry34Ab1, and Cry34Ac1, respectively, and the 44-kDa polypeptides from these isolates have been designated Cry35Aa1, Cry35Ab1, and Cry35Ac1, respectively.     

A freeze-dried diet to test pathogens of Colorado potato beetle

The Colorado potato beetle is an important pest on potato, eggplant, and tomato. Because Colorado potato beetles develop resistance to insecticides quickly, new methods are needed for control. Bacillus thuringiensis is the only bacterium to successfully control Colorado potato beetle. Until recently, one of the drawbacks to testing bacteria against the Colorado potato beetle has been the lack of an artificial diet for screening. Previous artificial diets will only be consumed by Colorado potato beetle larvae when fresh. To improve storage, we developed a freeze-dried diet, based on a 96-well plate, suitable to feed larvae for the duration of a bioassay. Individual diet components were tested both for their effect on insect growth and on pathogen toxicity. When the preservatives, methylparaben and sorbic acid, were removed from the diet, the average weight of second instar larvae increased from 7.9 mg to greater than 9.8 mg. The preservatives inhibited the growth of two of the bacteria tested, Photorhabdus luminescens HM and Chromobacterium sp. PRAA. The removal of these preservatives also allowed for fungal growth and reduced survival from 94 to 38%. Removing diet preservatives, that inhibited the growth of Chromobacterium sp. PRAA, increased the total mortality of the larvae as well as reducing the time needed to kill 50% of the larvae. Compared to incorporation of bacteria into molten diet, the total mortality of Colorado potato beetle fed either P. luminescens HM or Chromobacterium sp. PRAA on freeze-dried diet doubled. Preparation of freeze-dried diet need not be synchronized with the insect or the pathogen. The freeze-dried diet gave consistent results as measured by low control mortality and pathogen toxicity over time.