Spore-crystal Preparations Research Articles

Impact of Antibiotics on Efficacy of Cry Toxins Produced in Two Different Genetically Modified Bt Maize Varieties in Two Lepidopteran Herbivore Species, Ostrinia nubilalis and Spodoptera littoralis.

The insecticidal crystal proteins from Bacillus thuringiensis (Bt) are widely-used biopesticides that are used both as Bt spore-crystal preparations in sprayable formulations and as activated toxins in genetically modified (GM) plants. Models for their modes of action have been proposed but many issues remain unresolved. Among those is the role of commensal gut bacteria in target insect death: previous studies showed that antibiotics attenuate the toxicity of Bt sprays. We tested whether antibiotics interfere with the effects of GM plant-produced Bt toxins in larvae of two Lepidopteran species, the European corn borer Ostrinia nubilalis and the cotton leafworm Spodoptera littoralis. The larvae were reared on artificial diet with or without antibiotics and, thereafter, fed two varieties of Bt GM maize in comparison to conventional non-Bt maize leaves sprayed with antibiotic solution and/or with a Bt formulation. Antibiotics significantly reduced or delayed the toxicity of Cry toxins, although to a lesser extent than previously reported for Bt-sprays. This supports the hypothesis that Cry toxins induce mortality by themselves in the absence of Bt bacteria and spores, and of commensal gut bacteria. However, larvae that were not treated with antibiotics died faster and at a higher rate which was further compounded by plant variety and species sensitivity. These findings support a hypothesis that toxicemia alone can inflict significant mortality. However, in the absence of antibiotics, the gut bacteria likely enhance the Cry toxin effect by inflicting, additionally, bacterial septicemia. This has important implications in field situations where antibiotic substances are present—e.g., from manure of animals from conventional production systems—and for ecotoxicological testing schemes of Bt toxins and nontarget organisms that are often using artificial diets enriched with high concentrations of antibiotics.

Synergistic Activity Between S-Layer Protein and Spore-Crystal Preparations from Lysinibacillus sphaericus Against Culex quinquefasciatus Larvae.

Lysinibacillus sphaericus is used for the biological control of mosquitoes. The main toxicity mechanism of pathogenic strains is a binary toxin produced during sporulation. S-layer is a proteinaceous structure on the surface of bacteria; its functions have been involved in the interaction between bacterial cells and the environment, for example, as protective coats, surface recognition, and biological control. In L. sphaericus, S-layer protein (SlpC) is expressed in vegetative cells, and is also found in spore-crystal preparations; it has larvicidal activity in Culex spp. In this study, partial and completed sporulated culture toxicities were compared; also, S-layer protein and spore-crystal proteins were tested against Culex quinquefasciatus larvae for possible interactions. Larvicidal activity obtained with a combination of SlpC and spore-crystal proteins from strain III(3)7 showed no significant interaction, whereas, combinations of both preparations from strain 2362 showed synergistic effect. The highest synergistic activity observed was between spore protein complex from strain 2362 and SlpC from III(3)7. S-layer protein could be considered a good alternative in formulation improvement, for biological control of mosquitoes.

Characterization of Cry Proteins in Native Strains ofBacillus thuringiensisand Activity AgainstAnastrepha ludens1

Abstract. Bacillus thuringiensis (Berliner) strains isolated from soil of citrus orchards were tested for insecticidal activity against the Mexican fruit fly, Anastrepha ludens (Loew), a key citrus pest in Mexico. From a total of 55 soil samples, 201 isolates were selected, for a total B. thuringiensis index of 0.66. The collection was characterized through light microscopy, polyacrylamide gel electrophoresis (SDS-PAGE), and PCR analysis detecting cry2, cry4, cry10, cry11, and cry19 genes. Of the 201 isolates, 51% produced ovoid crystals, 28% adhered to the spore, 15% were pleomorphic, 3% were bipyramidal, 2% cubic, and 1% was pyramidal type. Six colonies were positive for the cry10 gene and one for the cry19 gene. SDS-PAGE of spore-crystal preparations revealed seven electrophoresis patterns. These were bioassayed against Mexican fruit fly adults, obtaining maximum mortality of 28%.

Molecular docking and site-directed mutagenesis of a Bacillus thuringiensis chitinase to improve chitinolytic, synergistic lepidopteran-larvicidal and nematicidal activities.

Bacterial chitinases are useful in the biocontrol of agriculturally important pests and fungal pathogens. However, the utility of naturally occurring bacterial chitinases is often limited by their low enzyme activity. In this study, we constructed mutants of a Bacillus thuringiensis chitinase with enhanced activity based on homology modeling, molecular docking, and the site-directed mutagenesis of target residues to modify spatial positions, steric hindrances, or hydrophilicity/hydrophobicity. We first identified a gene from B. thuringiensis YBT-9602 that encodes a chitinase (Chi9602) belonging to glycosyl hydrolase family 18 with conserved substrate-binding and substrate-catalytic motifs. We constructed a structural model of a truncated version of Chi9602 (Chi960235-459) containing the substrate-binding domain using the homologous 1ITX protein of Bacillus circulans as the template. We performed molecular docking analysis of Chi960235-459 using di-N-acetyl-D-glucosamine as the ligand. We then selected 10 residues of interest from the docking area for the site-directed mutagenesis experiments and expression in Escherichia coli. Assays of the chitinolytic activity of the purified chitinases revealed that the three mutants exhibited increased chitinolytic activity. The ChiW50A mutant exhibited a greater than 60 % increase in chitinolytic activity, with similar pH, temperature and metal ion requirements, compared to wild-type Chi9602. Furthermore, ChiW50A exhibited pest-controlling activity and antifungal activity. Remarkable synergistic effects of this mutant with B. thuringiensis spore-crystal preparations against Helicoverpa armigera and Caenorhabditis elegans larvae and obvious activity against several plant-pathogenic fungi were observed.

Contribution of S-layer proteins to the mosquitocidal activity of Lysinibacillus sphaericus.

Lysinibacillus sphaericus strains belonging the antigenic group H5a5b produce spores with larvicidal activity against larvae of Culex mosquitoes. C7, a new isolated strain, which presents similar biochemical characteristics and Bin toxins in their spores as the reference strain 2362, was, however, more active against larvae of Culex mosquitoes. The contribution of the surface layer protein (S-layer) to this behaviour was envisaged since this envelope protein has been implicated in the pathogenicity of several bacilli, and we had previously reported its association to spores. Microscopic observation by immunofluorescence detection with anti S-layer antibody in the spores confirms their attachment. S-layers and BinA and BinB toxins formed high molecular weight multimers in spores as shown by SDS-PAGE and western blot detection. Purified S-layer from both L. sphaericus C7 and 2362 strain cultures was by itself toxic against Culex sp larvae, however, that from C7 strain was also toxic against Aedes aegypti. Synergistic effect between purified S-layer and spore-crystal preparations was observed against Culex sp. and Aedes aegypti larvae. This effect was more evident with the C7 strain. In silico analyses of the S-layer sequence suggest the presence of chitin-binding and hemolytic domains. Both biochemical characteristics were detected for both S-layers strains that must justify their contribution to pathogenicity.

Mortality Responses of Spodoptera litura Following Feeding on BT- Sprayed Plants

Bacillus thuringiensis delta-endotoxins are safe biological insecticidal proteins whose usefulness has long been recognized. The first commercialized Bt insecticidal formulations were composed of spore-crystal preparations derived from wild-type strains. These products generally have a limited insecticidal host range and several genetically modified strains have, therefore, been constructed in this study using conjugation procedure. However, addition of a new plasmids to Bt strains already harboring other genes often resulted in broader - spectrum. Bacillus thuringiensis serovar Kurstaki, Bacillus subtilis and four of their transconjugants were used in this study as a biocontral agents against lepidopterous cotton pest. Bacterial transconjugants were evaluated for their hybrid vigor in relation to the mid parents and better parent. This evaluation was related to survival and mortality percentages induced in Spodoptera littoralis larvae. Two groups of bioinsecticides; crystals, crystals + endospores were used to be evaluated in this study. The results appeared that bioinsecticides containing crystals + endospores was more effective than crystals for increasing mortality percentage and reducing survival percentage. This effective was including reduction in the mean number of Spodoptera littoralis larvae feeding on leaves sprayed with crystals + endospores. Increasing mortality percentage of crystals + endospores was due to higher toxicological effects than that of crystals. This recommended bioinsecticide biologists to use crystals + endospores in all bioinsecticides formulations. Higher positive efficiency was appeared at 168 h of treatments. Recombinant Bacillus thuringiensis was more effective as biocontrol agents against lepidopteran pests at the early instars, because susceptibility was decreased with larval development. This indicated that the first instars were more susceptible to Bt sprayed plants than the later instar stages. The combined effects of crystals + endospores produced higher mortality. This factor was important to be considered in designing resistance management strategies.

Diversity of Bacillus thuringiensis strains isolated from citrus orchards in spain and evaluation of their insecticidal activity against Ceratitis capitata.

A survey of Bacillus thuringiensis (Berliner) strains isolated from Spanish citrus orchards has been performed, and the strains were tested for insecticidal activity against the Mediterranean fruit fly Ceratitis capitata (Wiedemann), a key citrus pest in Spain. From a total of 150 environmental samples, 376 isolates were selected, recording a total B. thuringiensis index of 0.52. The collection was characterized by means of phase-contrast microscopy, SDS-PAGE, and PCR analysis with primer pairs detecting toxin genes cry1, cry2, cry3, cry4, cry5, cry7, cry8, cry9, cry10, cry11, cry12, cry14, cry17, cry19, cry21, cry27, cry39, cry44, cyt1, and cyt2. Diverse crystal inclusion morphologies were identified: bipyramidal (45%), round (40%), adhered to the spore (7%), small (5%), and irregular (3%). SDSPAGE of spore-crystal preparations revealed 39 different electrophoresis patterns. All primer pairs used in PCR tests gave positive amplifications in strains of our collection, except for primers for detection of cry3, cry19, cry39, or cry44 genes. Strains containing cry1, cry2, cry4, and cry27 genes were the most abundant (48.7%, 46%, 11.2%, and 8.2% of the strains, respectively). Ten different genetic profiles were found, although a total of 109 strains did not amplify with the set of primers used. Screening for toxicity against C. capitata adults was performed using both spore-crystal and soluble fractions. Mortality levels were less than 30%. We have developed a large and diverse B. thuringiensis strain collection with huge potential to control several agricultural pests; however, further research is needed to find out Bt strains active against C. capitata.

Novel Isolate of Bacillus thuringiensis subsp. thuringiensis That Produces a Quasicuboidal Crystal of Cry1Ab21 Toxic to Larvae of Trichoplusia ni

A new isolate (IS5056) of Bacillus thuringiensis subsp. thuringiensis that produces a novel variant of Cry1Ab, Cry1Ab21, was isolated from soil collected in northeastern Poland. Cry1Ab21 was composed of 1,155 amino acids and had a molecular mass of 130.5 kDa, and a single copy of the gene coding for this endotoxin was located on a approximately 75-kbp plasmid. When synthesized by the wild-type strain, Cry1Ab21 produced a unique, irregular, bipyramidal crystal whose long and short axes were both approximately 1 microm long, which gave it a cuboidal appearance in wet mount preparations. In diet incorporation bioassays, the 50% lethal concentrations of the crystal-spore complex were 16.9 and 29.7 microg ml(-1) for second- and fourth-instar larvae of the cabbage looper, Trichoplusia ni, respectively, but the isolate was essentially nontoxic to larvae of the beet armyworm, Spodoptera exigua. A bioassay of autoclaved spore-crystal preparations showed no evidence of beta-exotoxin activity, indicating that toxicity was due primarily to Cry1Ab21. Studies of the pathogenesis of isolate IS5056 in second-instar larvae of T. ni showed that after larval death the bacterium colonized and subsequently sporulated extensively throughout the cadaver, suggesting that other bacteria inhabiting the midgut lumen played little if any role in mortality. As T. ni is among the most destructive pests of vegetable crops in North America and has developed resistance to B. thuringiensis, this new isolate may have applied value.

Geographical variation in larval susceptibility of the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae) to Bacillus thuringiensis spore–crystal mixtures and purified crystal proteins and associated resistance development in India

The susceptibility of larvae of the diamondback moth, Plutella xylostella Linnaeus to purified crystal proteins and spore-crystal preparations of Bacillus thuringiensis was investigated for 13 populations from seven states in India. The LC50 (microg ml(-1), 48 h) values of Cry proteins for different populations of P. xylostella ranged from 0.14-3.74 (Cry1Aa), 0.007-1.25 (Cry1Ab), 0.18-2.47 (Cry1Ac) and 0.12-3.0 (Cry1C). The LC50 (mg (ai) l(-1), 48 h) of spore-crystal preparations ranged from 0.02-0.98 (HD-1) and 0.06-2.14 (HD-73). Significantly higher LC50 values for all tested toxins and strains were obtained with populations collected from Iruttupallam and Ottanchathiram in the southern state of Tamil Nadu, whereas some of the populations collected from the northern part of India were more susceptible than the susceptible IARI 17-65 population. The high levels of resistance in the Iruttupallam and Ottanchathiram populations to Cry1Ab suggested selection pressure by Cry1Ab, which is the predominant toxin in B. thuringiensis formulations used in India. Cry1Ab was found to be more toxic than the other toxins. The population from Iruttupallam showed increased resistance following selection with Cry1Ab in the laboratory (LC50 from 1.25 to 4.31 microg ml(-1) over two generations) and also showed cross resistance to CrylAa and CrylAc. The resistance to Biobit in the field population from Iruttupallam declined slowly; requiring c. 33 generations for an overall 10-fold decline in LC50 when the insects were reared in the laboratory without exposure to B. thuringensis.

Spore coat protein synergizes bacillus thuringiensis crystal toxicity for the indianmeal moth

Spores from Bacillus thuringiensis serovars kurstaki and entomocidus synergized crystal protein toxicity for larvae of the Indianmeal moth (Plodia interpunctella). Preparations of spore-crystal mixtures of either serovar were more toxic for the larvae than either purified spores or crystals alone (based on dry weight). Spores lost 53% of their toxicity for the Indianmeal moth after 2 h of UV-irradiation, but remained partially toxic (28%) even after 4 h of irradiation. Spore coat protein was toxic for the Indianmeal moth and was synergistic with B. thuringiensis serovar kurstaki HD-1 crystal protein. Enhanced toxicity of the combined spore-crystal preparation was attributed to a combination of crystal and spore coat protein, and included the effects of spore germination and resulting septicemia in the larval hemolymph. Ultraviolet irradiation of spores reduced the toxicity from septicemia but not the synergism caused by spore coat protein. The potencies of spore-crystal preparations must be carefully evaluated on the basis of contributions from all three factors.

Inheritance of Resistance to the Bacillus thuringiensis CrylC Toxin in Spodoptera littoralis (Lepidoptera: Noctuidae)

A strain of Spodoptera littoralis Boisduval, selected for resistance to the Bacillus thuringiensis CryIC toxin, was used to analyze the inheritance of resistance. This strain resulted from a laboratory selection with spore-crystal preparations of B, thttringiensis CryIC toxin. At generation 12, resistance was >;500 times the resistance ratio of the control. Dose-mortality curves of the resistant population, the susceptible strain, the F1 and the backcrosses from reciprocal crosses were compared. The resistance was partially recessive. F1 offspring from resistant males and susceptible females were more resistant than those from susceptible males and resistant females, indicating a clear paternal effect. Segregation of resistance was investigated in the 8 possible backcrosses between F1 and one of the parental strains. Dose-mortality responses significantly differed from those expected under the hypotheses of a major resistance gene that was either autosomal or sex linked. It was concluded that resistance was most probably polyfactorial. These results are discussed in relation to the nature of bacterial toxin resistance genes in various insects.

Spodoptera littoralis (Lepidoptera: Noctuidae) Resistance to CryIC and Cross-Resistance to Other Bacillus thuringiensis Crystal Toxins

Laboratory selection of a strain of Spodoptera littoralis Boisduval with spore-crystal preparations of the Bacillus thuringiensis CryIC toxin for 14 generations resulted in resistance ratios from 10- to >500-fold resistance by using 2 different methods of selection. Partial cross-resistance was exhibited to CryID, CryIE, CryIAb toxins and to the parental strain B. thuringiensis aizawai 7.29. No cross-resistance occurred to CryIF. Resistance declined from >500 times the resistance ratio to >74 times after 1 generation without selection pressure, remained ≈100 times after 3–6 generations and fell to 11 times after 8 generations. Sex ratio remained the same over selection period. Pupal weights of the selected strain varied, but we observed no trend in these differences. Developmental time increased from 5 to 6 d for treated, resistant insects.

Construction of new insecticidal Bacillus thuringiensis recombinant strains by using the sporulation non-dependent expression system of cryIIIA and a site specific recombination vector

Bacillus thuringiensis (Bt) δ-endotoxins are safe biological insecticidal proteins whose usefulness has long been recognized. The first commercialized Bt insecticidal formulations were composed of spore-crystal preparations derived from wild-type strains. These products generally have a limited insecticidal host range and several genetically modified strains have, therefore, been constructed using transformation procedures. However, addition of a new δ-endotoxin gene to strains already harboring other δ -endotoxin genes often resulted in broader-spectrum but less potent products because they produced significantly less of each of the crystal proteins. We report expression of the coding sequence of the sporulation specific cryIC gene from the non-sporulation-dependent cryIIIA promoter. Large amounts of CryIC accumulated in various Bt strains with different genetic backgrounds. Sporulation deficient SpoOA mutants, acrystalliferous derivatives and wild-type Bt strains expressing the engineered cryIII-cryIC gene were obtained. Introduction of the cryIII-cryIC gene whose product is highly active against Spodoptera littoralis into the Kto strain harboring the cryIA(c) gene active against Ostrinia nubilalis resulted in the construction of a new strain with increased potency and broader activity spectrum than the parent strain. Large amounts of each toxin were produced and the expression of the two genes seemed to be summed, presumably because the expression systems of the two genes are different. The plasmid shuttle vector used to introduce the cryIII-cryIC gene into the different Bt hosts utilizes the specific resolution site of transposon Tn 4430 to enable construction of recombinant Bt strains that are free of foreign non- Bt DNA. This should facilitate the approval and acceptance for environmental release of the insecticidal recombinant products.

A new medium for growth and delta-endotoxin production by Bacillus thuringiensis var. kurstaki

The influence of composition of media used for growth and delta-endotoxin production by B. thuringiensis var. kurstaki was studied with the idea of finding a cheap medium for attaining high yields of spore-crystal preparations. A new medium, based on malt sprouts is proposed. Data on growth and bioinsecticidal activity are given. A concentration of 2.1 × 109 spores.ml-1 was attained in a 48 h process. The spore-crystal preparations obtained present a LC50 of 2.18 × 108 spores.g-1 against larvae of Galleria mellonella.