Development of a Bacillus thuringiensis-based assay on Lygus hesperus

Lygus hesperus Knight (Hemiptera: Miridae) is an economically important insect pest controlled primarily by chemical pesticides. Bacillus thuringiensis Berliner is a gram-positive bacterium, which upon sporulation produces a parasporal inclusion body, the crystal. The latter, in some strains, exhibits specific insecticidal activities against lepidopteran, coleopteran, or dipteran pests. The aim of the present work was to develop a Bacillus thuringiensis-based assay on L. hesperus. Several factors involved in the expression of the B. thuringiensis insecticidal activity were assayed for their effects on L. hesperus mortality. We show that the choice of the L. hesperus diet type, the L. hesperus developmental stages, the B. thuringiensis crystal alkaline solubilization buffers, the dialysis buffers, the dialyzed solubilization buffers, and the proteolytic activation can all significantly affect the L. hesperus viability. This work provides essential information in pinpointing key steps before the establishment of a thorough screening program for B. thuringiensis strains expressing antihemipteran activity.     

A Bacillus thuringiensis S-layer protein involved in toxicity against Epilachna varivestis (Coleoptera: Coccinellidae)

The use of Bacillus thuringiensis as a biopesticide is a viable alternative for insect control since the insecticidal Cry proteins produced by these bacteria are highly specific; harmless to humans, vertebrates, and plants; and completely biodegradable. In addition to Cry proteins, B. thuringiensis produces a number of extracellular compounds, including S-layer proteins (SLP), that contribute to virulence. The S layer is an ordered structure representing a proteinaceous paracrystalline array which completely covers the surfaces of many pathogenic bacteria. In this work, we report the identification of an S-layer protein by the screening of B. thuringiensis strains for activity against the coleopteran pest Epilachna varivestis (Mexican bean beetle; Coleoptera: Coccinellidae). We screened two B. thuringiensis strain collections containing unidentified Cry proteins and also strains isolated from dead insects. Some of the B. thuringiensis strains assayed against E. varivestis showed moderate toxicity. However, a B. thuringiensis strain (GP1) that was isolated from a dead insect showed a remarkably high insecticidal activity. The parasporal crystal produced by the GP1 strain was purified and shown to have insecticidal activity against E. varivestis but not against the lepidopteran Manduca sexta or Spodoptera frugiperda or against the dipteran Aedes aegypti. The gene encoding this protein was cloned and sequenced. It corresponded to an S-layer protein highly similar to previously described SLP in Bacillus anthracis (EA1) and Bacillus licheniformis (OlpA). The phylogenetic relationships among SLP from different bacteria showed that these proteins from Bacillus cereus, Bacillus sphaericus, B. anthracis, B. licheniformis, and B. thuringiensis are arranged in the same main group, suggesting similar origins. This is the first report that demonstrates that an S-layer protein is directly involved in toxicity to a coleopteran pest.     

Cotton plants expressing a hemipteran-active Bacillus thuringiensis crystal protein impact the development and survival of Lygus hesperus (Hemiptera: Miridae) nymphs

The plant bugs Lygus hesperus Knight (Hemiptera: Miridae) and L. lineolaris (Palisot de Beauvois) have emerged as economic pests of cotton in the United States. These hemipteran species are refractory to the insect control traits found in genetically modified commercial varieties of cotton. In this article, we report the isolation and characterization of a 35 kDa crystal protein from Bacillus thuringiensis, designated TIC807, which causes reduced mass gain and mortality of L. hesperus and L. lineolaris nymphs when presented in an artificial diet feeding assay. Cotton plants expressing the TIC807 protein were observed to impact the survival and development of L. hesperus nymphs in a concentration-dependent manner. These results, demonstrating in planta activity of a Lygus insecticidal protein, represent an important milestone in the development of cotton varieties protected from Lygus feeding damage.     

Specific primers for the detection of vip3A insecticidal gene within a Bacillus thuringiensis collection

A PCR-based method was developed for the rapid detection of vip3A gene encoding a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects. Specific primer combinations (three primers for the normal strand and two primers for the complementary strand) were capable of generating diagnostic fragments that successfully predicted the presence of the gene encoding the Vip3A insecticidal toxin in various B. thuringiensis strains. Specificity of amplicons generated by primer pairs was confirmed by restriction endonuclease digestion and DNA sequence analysis. The evaluation of B . thuringiensis strains for biological activity against insect pests of rice is thus aided by the grouping of strains based on their potential insecticidal spectrum.     

Identification and characterization of the new bacillus thuringiensis serovars pirenaica (serotype H57) and iberica (serotype H59)

Two new Bacillus thuringiensis strains have been classified by the H antigen of the cells and differentiated by their morphological, biochemical and molecular characteristics. The flagellar agglutination showed that both strains bore specific H antigens which allowed their classification as the new serotypes H57 and H59. The serovar names proposed for the type strains characterized in this work are B. thuringiensis ser. pirenaica, for the H serotype 57, and B. thuringiensis ser. iberica, for the H serotype 59. Further characterization of these strains, by means of SDS-PAGE, Western inmunodetection, plasmid profile and cry -gene identification by polymerase chain reaction, confirmed the originality of the two novel serotypes. Toxicity tests carried out against several insect species, belonging to the orders Lepidoptera, Diptera and Coleoptera, showed no detectable insecticidal activity for either of the B. thuringiensis strains.     

Specificity and efficacy of purified Bacillus thuringiensis proteins against agronomically important insects

The host range and relative efficacy of three purified Bacillus thuringiensis insect control proteins were determined against 17 different agronomically important insects representing five orders and one species of mite. The three B. thuringiensis proteins were single gene products from B. thuringiensis ssp. kurstaki HD-1 (CryIA(b)) and HD-73 (CryIA(c)), both lepidopteran-specific proteins, and B. thuringiensis ssp. tenebrionis (CryIIIA), a coleopteran-specific protein. Seven insects showed sensitivity to both B. thuringiensis ssp. kurstaki proteins, whereas only 1 of the 18 insects was sensitive to B. thuringiensis ssp. tenebrionis protein. The level of B. thuringiensis ssp. kurstaki protein required for 50% mortality (LC50) varied by 2000-fold for these 7 insects. A larval growth inhibition assay was developed to determine the amount of B. thuringiensis ssp. kurstaki protein required to inhibit larval growth by 50% (EC50). This extremely sensitive assay enabled detection of B. thuringiensis ssp. kurstaki HD-73 levels as low as 1 ng/ml.     

Isolation of a non-insecticidal Bacillus thuringiensis strain belonging to serotype H8a8b

Bacillus thuringiensis strains non-toxic to Lepidoptera, Bombyx mori and Diptera, Culex pipiens pallens larvae were isolated from Korean soil samples during an investigation of B. thuringiensis isolates highly toxic to insect pests. One of these isolates, NTB-88, produces parasporal inclusions about 138 kDa in size and is non-toxic to 19 insect species of three orders, Lepidoptera, Diptera and Coleoptera, even though it is highly susceptible to tryptic cleavage. Study of flagellar (H) antibodies of 33 B. thuringiensis strains revealed that NTB-88 has an H antigen identical with that of subsp. morrisoni (serotype 8a8b). Comparison of parasporal inclusion proteins and plasmid DNA patterns of strain NTB-88 with B. thuringiensis subsp. morrisoni HD-12 and B. thuringiensis subsp. morrisoni PG-14 showed that the isolate is a novel non-insecticidal B. thuringiensis strain belonging to serotype 8a8b.     

Increase of the Bacillus thuringiensis Secreted Toxicity Against Lepidopteron Larvae by Homologous Expression of the vip3LB Gene During Sporulation Stage

The Vegetative insecticidal Vip3A proteins display a wide range of insecticidal spectrum against several agricultural insect pests. The fact that the expression of vip3 genes occurs only during the vegetative growth phase of Bacillus thuringiensis is a limiting factor in term of production level. Therefore, extending the synthesis of the Vip proteins to the sporulation phase is a good alternative to reach high levels of toxin synthesis. In this study, we have demonstrated that the maximal production of the secreted Vip3LB (also called Vip3Aa16) during the growth of the wild-type strain B. thuringiensis BUPM 95 is reached at the end of the vegetative growth phase, and that the protein remains relatively stable in the culture supernatant during the late sporulation stages. The vip3LB gene was cloned and expressed under the control of the sporulation dependant promoters BtI and BtII in B. thuringiensis BUPM 106 (Vip3(-)) and BUPM 95 (Vip3(+)) strains. The examination of the culture supernatants during the sporulation phase evidenced the synthesis of Vip3LB and its toxicity against the second-instars larvae of the Lepidopteron insect Spodoptera littoralis for the recombinant BUPM 106. Moreover, there was an increase of the Vip3LB synthesis level and an enhancement of the oral toxicity for the recombinant BUPM 95 resulting from the expression of the vip3LB gene during both the vegetative and sporulation phases and the relative stability of the Vip3LB protein.     

Chitinolytic activities in Bacillus thuringiensis and their synergistic effects on larvicidal activity

AIMS: To investigate the distribution of chitinase in Bacillus thuringiensis strains, and the enhancing effects of the chitinase-producing B. thuringiensis strains on insecticidal toxicity of active B. thuringiensis strain against Spodoptera exigua larvae. METHODS AND RESULTS: The chitinolytic activities of B.thuringiensis strains representing the 70 serotypes were investigated by the whitish opaque halo and the colorimetric method. Thirty-eight strains produced different levels of chitinase at pH 7.0, and so did 17 strains at pH 10.0. The strain T04A001 exhibited the highest production, reaching a specific activity of 355 U ml(-1) in liquid medium. SDS-PAGE and Western blotting showed that the chitinase produced by some B. thuringiensis strains had a molecular weight of about 61 kDa. The bioassay results indicated that the chitinase-producing B. thuringiensis strains could enhance the insecticidal activity of B. thuringiensis strain DL5789 against S. exigua larvae, with an enhancing ratio of 2.35-fold. CONCLUSION: This study demonstrated that chitinase was widely produced in B. thuringiensis strains and some of the strains could enhance the toxicity of active B. thuringiensis strain. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first investigation devoted exclusively to analyse the distribution of chitinase in B. thuringiensis. It infers that the chitinase produced by B. thuringiensis might play a role in the activity of the biopesticide.     

Olfactory Responses of <Emphasis Type="Italic">Anaphes iole</Emphasis> (Hymenoptera: Mymaridae) to Volatile Signals Derived from Host Habitats

Anaphes iole Girault is a frequent parasitoid of Lygus spp. eggs in the United States, and has potential as a biological control agent against Lygus hesperus Knight in different crops. Feeding and oviposition by L. hesperus induce emission of plant volatiles, but studies to date do not address the role of plant volatiles in the host-searching behavior of A. iole. In this study, a four-arm olfactometer was used to test the responses of female parasitoids to odors emanating from cotton (Gossypium hirsutum L., Malvaceae) plants damaged by L. hesperus females, L. hesperus males, larvae of the nonhost Spodoptera exigua Hubner, or mechanically, or to odors from L. hesperus females alone. In addition, various plants damaged by L. hesperus females were evaluated in the olfactometer: cotton, alfalfa (Medicago sativa L., Fabaceae), common groundsel (Senecio vulgaris L., Asteraceae), annual ragweed (Ambrosia artemisifolia L., Asteraceae), and redroot pigweed (Amaranthus retroflexus L., Amaranthaceae). In all olfactometry bioassays, treatment odors were compared against three controls (humidified air). Results showed that A. iole females were consistently attracted to odors derived from different plant–L. hesperus complexes, while odors from plants subjected to nonhost (S. exigua) or mechanical damage and L. hesperus females alone were not attractive or only variably attractive. These findings suggest that while searching for hosts A. iole females use specific volatiles induced by L. hesperus feeding and oviposition to locate hosts inhabiting a wide variety of plants, including annual and perennial species from four plant families. It was suggested that future research should seek to identify the chemical elicitors involved in the release of plant volatiles attractive to A. iole females.     

Identification of vip3A-type genes from Bacillus thuringiensis strains and characterization of a novel vip3A-type gene

AIMS: To search for novel Vip3A proteins for controlling insect pests. METHODS AND RESULTS: A pair of universal primers was designed based on the conserved regions of five vip3A genes. Amplified products were digested with the HindIII and EcoR enzymes so as to confirm different restriction fragment length polymorphism (RFLP) patterns used to identify vip3A-type genes. The vip3A gene types of 606 Bacillus thuringiensis strains were screened and three patterns of RFLP were successfully identified. Two novel vip3A genes were found and one of these, vip3Aa19, was further characterized and its product was confirmed toxic to Spodoptera exigua, Helicoverpa armigera and Plutella xylostella larvae. Partial sequences of another novel vip3A-type gene were obtained that shared 83% homology with that of the vip3Af1 gene. CONCLUSIONS: A polymerase chain reaction (PCR)-RFLP system we developed could be used for identifying novel vip3A-genes from B. thuringiensis strains. A novel Vip3A protein was found to have a broader insecticidal spectrum. SIGNIFICANCE AND IMPACT OF THE STUDY: The reported method is a powerful tool to find novel Vip3A proteins from large-scale B. thuringiensis strains. The novel Vip3A protein may be used to control insect pests or resistant insect pests by constructing genetically engineered strains or transgenic plants.     

Genetic variability of Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) populations from Latin America is associated with variations in susceptibility to Bacillus thuringiensis cry toxins

Bacillus thuringiensis strains isolated from Latin American soil samples that showed toxicity against three Spodoptera frugiperda populations from different geographical areas (Mexico, Colombia, and Brazil) were characterized on the basis of their insecticidal activity, crystal morphology, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of parasporal crystals, plasmid profiles, and cry gene content. We found that the different S. frugiperda populations display different susceptibilities to the selected B. thuringiensis strains and also to pure preparations of Cry1B, Cry1C, and Cry1D toxins. Binding assays performed with pure toxin demonstrated that the differences in the toxin binding capacities of these insect populations correlated with the observed differences in susceptibility to the three Cry toxins analyzed. Finally, the genetic variability of the three insect populations was analyzed by random amplification of polymorphic DNA-PCR, which showed significant genetic diversity among the three S. frugiperda populations analyzed. The data presented here show that the genetic variability of S. frugiperda populations should be carefully considered in the development of insect pest control strategies, including the deployment of genetically modified maize in different geographical regions.     

A phylogenetic analysis of Bacillus thuringiensis serovars by RFLP-based ribotyping

AIMS: To determine the 23S and 5S rRNA gene fingerprints in order to reveal phylogenetic relationships among Bacillus thuringiensis strains. METHODS AND RESULTS: Eighty-six B. thuringiensis strains which include 80 serovar type strains, five intraserovar strains and a non-serotypeable strain, wuhanensis, were tested. Total DNA was digested with EcoRI and HindIII. The 23S and 5S rRNA gene restriction fragment length polymorphisms showed 82 distinctive ribopatterns. The dendrogram generated by numerical analysis showed 10 phylogenetic groups and six ungrouped serovars at the 95.5% DNA relatedness rate. A second dendrogram was constructed using a combination of the data from this study and from a previous study on 16S rRNA gene fingerprinting. It revealed eight distinct phylogenetic groups and three ungrouped serovars at the 94% DNA relatedness rate. CONCLUSION: This method permitted the classification and positioning of a wide variety of B. thuringiensis strains on a phylogenetic tree. Bacillus thuringiensis strains appear to be relatively homogeneous and to share a high degree of DNA relatedness. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes a further step to the definition of valid taxonomic sublevels for the B. thuringiensis species.     

Isolation of Bacillus thuringiensis from intertidal brackish sediments in mangroves

Intertidal brackish sediments in mangroves were examined for isolation of Bacillus thuringiensis strains with novel toxicity spectra. A total of 18 B. thuringiensis isolates were recovered from eight sediment samples (36.4%) out of 22 samples tested. The frequency of B. thuringiensis was 1.3% among the colonies of Bacillus cereus/B. thuringiensis group. While five isolates were allocated to the four H serogroups, the majority of the isolates were serologically untypable or untestable. Two isolates belonging to the serovar israelensis/tochigiensis (H14/19) exhibited strong toxicities against larvae of the mosquito, Culex pipiens molestus, and mammalian cells (sheep erythrocyte and two human cancer cell lines) in vitro. The other 16 isolates showed no toxicity against the mosquito and mammalian cells. None of the isolates showed larvicidal activity against the diamondback moth, Plutella xylostella. Strong lectin activities against sheep erythrocytes were associated with two serologically untestable isolates and an H3 isolate.     

黑龙江省苏云金杆菌的分离及对夜蛾科昆虫高毒力菌株的筛选

【目的】为了发掘新的苏云金杆菌(Bacillus thuringiensis)的资源,在黑龙江省不同地区采集不同类型的土壤样品分离出对夜蛾科具有高毒力的菌株。【方法】采用醋酸钠选择性筛选法筛选Bt菌株,利用10对通用引物对分离株进行基因型分析,SDS-PAGE进行杀虫晶体蛋白分析,同时测定苏云金杆菌分离株对棉铃虫Helicoverpa armigera(Hübner)、甘蓝夜蛾Mamestra brassicae(Linnaeus)、斜纹夜蛾Spodoptera litura(Fabricius)的杀虫活性。【结果】从黑龙江省不同地区采集的352份不同类型的土壤样品中,共分离出46株苏云金芽孢杆菌野生菌株,出菌率为13.06%。油镜下可观察到伴孢晶体的形态有菱形、球形、镶嵌形及不规则形。结果表明产菱形晶体的菌株多含有cry1类基因,而同时产生菱形、球形及不规则形晶体的菌株则含有多种基因型。SDS-PAGE蛋白分析发现这些菌株主要表达130、90、60 ku蛋白。对其中的部分菌株进行毒力测定,结果表明有4株菌株对3种夜蛾科昆虫具有高毒力。【结论】黑龙江省苏云金芽孢杆菌分布广泛,类型多样,已获得对夜蛾科昆虫有高毒力的菌株,这对夜蛾科害虫的绿色防控及延缓其抗性具有重要意义。     

Evidence of oral toxicity of Photorhabdus temperata strain K122 against Prays oleae and its improvement by heterologous expression of Bacillus thuringiensis cry1Aa and cry1Ia genes

Photorhabdus temperata strain K122 exhibited oral toxicity against Prays oleae with an LC50 of 58.1 x 10(6) cells ml(-1). Recombinant P. temperata strains expressing the cry1Aa and/or cry1Ia genes of Bacillus thuringiensis have been constructed. The two cry genes, encoding delta-endotoxins, were placed under the control of the lac promoter and IPTG dependent expression in P. temperata was demonstrated. The presence of the cry genes in K122 resulted in a clear improvement of oral toxicity. This improvement was of 6.2-, 6.6-, and 14.6-fold for the strains K122(pBCcry1Aa), K122(pBScry1Ia), and K122(pBCcry1Aa + pBScry1Ia), respectively. Furthermore, determination of the Synergistic Factor between Cry1Aa and Cry1Ia showed that they act synergistically. This work demonstrates that the heterologous expression of B. thuringiensis cry genes in P. temperata can be used to improve and broaden its host range for insect control.     

Control of resistant pink bollworm (Pectinophora gossypiella) by transgenic cotton that produces Bacillus thuringiensis toxin Cry2Ab

Crops genetically engineered to produce Bacillus thuringiensis toxins for insect control can reduce use of conventional insecticides, but insect resistance could limit the success of this technology. The first generation of transgenic cotton with B. thuringiensis produces a single toxin, Cry1Ac, that is highly effective against susceptible larvae of pink bollworm (Pectinophora gossypiella), a major cotton pest. To counter potential problems with resistance, second-generation transgenic cotton that produces B. thuringiensis toxin Cry2Ab alone or in combination with Cry1Ac has been developed. In greenhouse bioassays, a pink bollworm strain selected in the laboratory for resistance to Cry1Ac survived equally well on transgenic cotton with Cry1Ac and on cotton without Cry1Ac. In contrast, Cry1Ac-resistant pink bollworm had little or no survival on second-generation transgenic cotton with Cry2Ab alone or with Cry1Ac plus Cry2Ab. Artificial diet bioassays showed that resistance to Cry1Ac did not confer strong cross-resistance to Cry2Aa. Strains with >90% larval survival on diet with 10 microg of Cry1Ac per ml showed 0% survival on diet with 3.2 or 10 microg of Cry2Aa per ml. However, the average survival of larvae fed a diet with 1 microg of Cry2Aa per ml was higher for Cry1Ac-resistant strains (2 to 10%) than for susceptible strains (0%). If plants with Cry1Ac plus Cry2Ab are deployed while genes that confer resistance to each of these toxins are rare, and if the inheritance of resistance to both toxins is recessive, the efficacy of transgenic cotton might be greatly extended.     

Correspondence of high levels of beta-exotoxin I and the presence of cry1B in Bacillus thuringiensis

Examination of 640 natural isolates of Bacillus thuringiensis showed that the 58 strains (9%) whose supernatants were toxic to Anthonomus grandis (Coleoptera: Curculionidae) produced between 10 and 175 micro g of beta-exotoxin I per ml. We also found that 55 (46%) of a sample of 118 strains whose culture supernatants were not toxic to A. grandis nevertheless produced between 2 and 5 micro g/ml. However, these amounts of beta-exotoxin I were below the threshold for detectable toxicity against this insect species. Secretion of large amounts of beta-exotoxin I was strongly associated with the presence of cry1B and vip2 genes in the 640 natural B. thuringiensis isolates studied. We concluded that strains carrying cry1B and vip2 genes also possess, on the same plasmid, genetic determinants necessary to promote high levels of production of beta-exotoxin I.     

Fate of Bacillus thuringiensis strains in different insect larvae

In favorable conditions Bacillus thuringiensis spores germinate and vegetative cells multiply, whereas in unfavorable conditions Bacillus thuringiensis sporulates and produces insecticidal crystal proteins. The development of B. thuringiensis strains was investigated in the larvae of insects belonging to the orders Lepidoptera and Diptera. Bacillus thuringiensis strains able to kill the insects did not always multiply in cadavers. Strains with no specificity to kill the insect sometimes multiplied when the insects were killed mechanically. These results indicate that some insect larvae represent an environment that favors the germination of B. thuringiensis spores and the multiplication of vegetative cells; however, there was no correlation between the toxin specificity and the specificity of the host.     

Bacillus thuringiensis serovar israelensis is highly toxic to the coffee berry borer, Hypothenemus hampei Ferr. (Coleoptera: Scolytidae)

A native collection of Bacillus thuringiensis strains was screened, once a reliable bioassay technique to assess the toxicity against the coffee berry borer (CBB) first-instar larvae was developed. A first round of bioassays with 170 strains indicated that the great majority of them showed no or very little insecticidal activity and that very few showed significant levels of toxicity. Interestingly, only those strains that had previously been associated with mosquitocidal activity were also toxic to CBB. Qualitative bioassays (using one high dose) were carried out only with those native mosquitocidal strains, corroborating their significant toxicity towards the CBB first-instar larvae. Most of these strains belong to serovar israelensis. In a second approach, strains from the Institut Pasteur type collection, whose mosquitocidal activity had been previously demonstrated, were also subjected to bioassays. Only those strains that showed a comparable protein content in their parasporal crystals to the israelensis type strain also showed high levels of toxicity towards CBB. Finally, an accurate LC(50) was estimated, using purified parasporal crystals from B. thuringiensis serovar israelensis type strain, at 219.5 ng cm(-2) of diet. All the statistical requirements for a reliable estimator were fulfilled. This is the first report of B. thuringiensis serovar israelensis being active against a coleopteran species.