Marginal cross-resistance to mosquitocidal Bacillus thuringiensis strains in Cry11A-resistant larvae: presence of Cry11A-like toxins in these strains

Culex quinquefasciatus mosquito larvae resistant to the Cry11A toxin showed marginal cross-resistance to the multiple toxin crystals from B. thuringiensis subsp. israelensis and also to toxin crystals from three other mosquitocidal strains, i.e. B. thuringiensis subsp. fukuokaensis, subsp. jegathesan, and subsp. kyushuensis. Cross-resistance patterns of the Cry11A-resistant larvae to mosquitocidal strains of B. thuringiensis together with the immunological screening using antisera raised against Cry11A indicated the presence of Cry11A-like toxins in these strains and could be used as a screening tool for the identification of novel toxins. The Cry11A-resistant larvae had significantly less resistance to the Cry11B toxin from B. thuringiensis subsp. jegathesan. The occurrence of cytolytic toxins in all of these mosquitocidal strains partially explains the marginal cross-resistance observed with multiple toxin crystals since each of these crystals also contains cytolytic toxins.     

Specificity of Bacillus thuringiensis Delta-Endotoxin

The insecticidal activity of the delta-endotoxins of 14 Bacillus thuringiensis strains belonging to 12 subspecies was determined against Pieris brassicae, Heliothis virescens, and Spodoptera littoralis. Larvae of P. brassicae were highly susceptible to purified crystals of strains of B. thuringiensis subsp. thuringiensis and B. thuringiensis subsp. morrisoni, whereas H. virescens responded best to B. thuringiensis subsp. kenyae and B. thuringiensis subsp. kurstaki. The crystals of the B. thuringiensis subsp. entomocidus strain were the most potent against S. littoralis. It was shown that the solubility of the crystals within the gut of the three insect species is a first important step in the mode of action. Predissolution of the crystals especially enhanced the insecticidal activity against H. virescens. When in vitro-activated toxins were applied, the relative potency range varied greatly from one insect species to another. It can be concluded that at least three factors influence the potency of B. thuringiensis delta-endotoxins: the strain-related origin of the toxin, the degree of solubility of the crystals in the gut juice, and the intrinsic susceptibility of the insect to the toxin.     

Bifidobacterium animalis subsp. lactis ATCC 27673 Is a Genomically Unique Strain within Its Conserved Subspecies

Many strains of Bifidobacterium animalis subsp. lactis are considered health-promoting probiotic microorganisms and are commonly formulated into fermented dairy foods. Analyses of previously sequenced genomes of B. animalis subsp. lactis have revealed little genetic diversity, suggesting that it is a monomorphic subspecies. However, during a multilocus sequence typing survey of Bifidobacterium, it was revealed that B. animalis subsp. lactis ATCC 27673 gave a profile distinct from that of the other strains of the subspecies. As part of an ongoing study designed to understand the genetic diversity of this subspecies, the genome of this strain was sequenced and compared to other sequenced genomes of B. animalis subsp. lactis and B. animalis subsp. animalis. The complete genome of ATCC 27673 was 1,963,012 bp, contained 1,616 genes and 4 rRNA operons, and had a G+C content of 61.55%. Comparative analyses revealed that the genome of ATCC 27673 contained six distinct genomic islands encoding 83 open reading frames not found in other strains of the same subspecies. In four islands, either phage or mobile genetic elements were identified. In island 6, a novel clustered regularly interspaced short palindromic repeat (CRISPR) locus which contained 81 unique spacers was identified. This type I-E CRISPR-cas system differs from the type I-C systems previously identified in this subspecies, representing the first identification of a different system in B. animalis subsp. lactis. This study revealed that ATCC 27673 is a strain of B. animalis subsp. lactis with novel genetic content and suggests that the lack of genetic variability observed is likely due to the repeated sequencing of a limited number of widely distributed commercial strains.     

Resistance to Toxins from Bacillus thuringiensis subsp. kurstaki Causes Minimal Cross-Resistance to B. thuringiensis subsp. aizawai in the Diamondback Moth (Lepidoptera: Plutellidae)

Repeated exposure in the field followed by laboratory selection produced 1,800- to >6,800-fold resistance to formulations of Bacillus thuringiensis subsp. kurstaki in larvae of the diamondback moth, Plutella xylostella. Four toxins from B. thuringiensis subsp. kurstaki [CryIA(a), CryIA(b), CryIA(c), and CryIIA] caused significantly less mortality in resistant larvae than in susceptible larvae. Resistance to B. thuringiensis subsp. kurstaki formulations and toxins did not affect the response to CryIC toxin from B. thuringiensis subsp. aizawai. Larvae resistant to B. thuringiensis subsp. kurstaki showed threefold cross-resistance to formulations of B. thuringiensis subsp. aizawai containing CryIC and CryIA toxins. This minimal cross-resistance may be caused by resistance to CryIA toxins shared by B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai.     

Enzymatic Ability of Bifidobacterium animalis subsp. lactis To Hydrolyze Milk Proteins: Identification and Characterization of Endopeptidase O

The proteolytic system of Bifidobacterium animalis subsp. lactis was analyzed, and an intracellular endopeptidase (PepO) was identified and characterized. This work reports the first complete cloning, purification, and characterization of a proteolytic enzyme in Bifidobacterium spp. Aminopeptidase activities (general aminopeptidases, proline iminopeptidase, X-prolyl dipeptidylaminopeptidase) found in cell extracts of B. animalis subsp. lactis were higher for cells that had been grown in a milk-based medium than for those grown in MRS. A high specific proline iminopeptidase activity was observed in B. animalis subsp. lactis. Whole cells and cell wall-bound protein fractions showed no caseinolytic activity; however, the combined action of intracellular proteolytic enzymes could hydrolyze casein fractions rapidly. The endopeptidase activity of B. animalis subsp. lactis was examined in more detail, and the gene encoding an endopeptidase O in B. animalis subsp. lactis was cloned and overexpressed in Escherichia coli. The deduced amino acid sequence for B. animalis subsp. lactis PepO indicated that it is a member of the M13 peptidase family of zinc metallopeptidases and displays 67.4% sequence homology with the predicted PepO protein from Bifidobacterium longum. The recombinant enzyme was shown to be a 74-kDa monomer. Activity of B. animalis subsp. lactis PepO was found with oligopeptide substrates of at least 5 amino acid residues, such as met-enkephalin, and with larger substrates, such as the 23-amino-acid peptide α_s1-casein(f1-23). The predominant peptide bond cleaved by B. animalis subsp. lactis PepO was on the N-terminal side of phenylalanine residues. The enzyme also showed a post-proline secondary cleavage site.     

Isolation and Characterization of Coproporphyrin Produced by Four Subspecies of Bacillus thuringiensis

It was found by using spectrophotometric, spectrofluorometric, and high-pressure liquid chromatography that four subspecies of Bacillus thuringiensis produce coproporphyrin. The porphyrin isomer was identified as coproporphyrin I for B. thuringiensis subsp. kurstaki (HD1). The porphyrin was isolated both from spores and from a variety of spent growth media. The quantity of porphyrin released by each Bacillus subspecies differed. The rank order of porphyrin production follows: B. thuringiensis subsp. kurstaki HD1 > B. thuringiensis subsp. thuringiensis HD27 > B. thuringiensis subsp. thuringiensis HD41 > B. thuringiensis subsp. darmstadiensis HD199.     

A new subspecies of <Emphasis Type="Italic">Centaurea cassia</Emphasis> (Asteraceae) from Turkey

A new subspecies in sect. Jacea (Mill.) DC., Centaurea cassia Boiss. subsp. dumanii M. Dinç, A. Duran &; B. Bilgili subsp. nov., collected by the authors from South Anatolia, is described and illustrated. The new subspecies is restricted to Abies cilicica (Ant. &; Kotschy) Carr. subsp. cilicica forest above Göller Yaylas? (C6 Adana-Kozan). Diagnostic morphological characters from C. cassia subsp. cassia are discussed. The ecology, biogeography and conservation status of the new taxon are also presented.     

Arceuthobium (Viscaceae) in Mexico and Guatemala: Additions and range extensions

Three new dwarf mistletoes are described:Arceuthobium globosum subsp.grandicaule (Mexico and Guatemala),A. aureum subsp.aureum (Guatemala) andA. aureum subsp.petersonii (Chiapas, Mexico).Arceuthobium guatemalense is recorded for the first time in Mexico. Significant range extensions are recorded forA. abietisreligiosae, A. divaricatum,A. gillii subsp.nigrum, andA. rubrum. New hosts are reported for several taxa. Nineteen members of the genus are presently known from Mexico, and three (possibly four) from Guatemala. Chromosome counts are reported for the first time for 3 taxa.     

Ribosomal protein profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for phylogenety-based subspecies resolution of Bifidobacterium longum

The taxonomic positions of the subspecies of Bifidobacterium longum (B. longum subsp. longum, subsp. infantis, and subsp. suis) have been controversial. A current proposal is that the former two species “B. infantis” and “B. suis” be unified with B. longum and all three reclassified as three subspecies. To test this proposal, ribosomal protein profiling as observed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was applied to the classification of 17 strains of B. longum, including three subspecies. Among 41 different kinds of ribosomal proteins selected as biomarkers whose masses were calculated from their amino acid sequences, 31-41 ribosomal proteins were observed in sample strains with the same masses as the references. The high matching rate indicates high conservation of ribosomal proteins within the sample strains, and therefore strongly supports the unification of the former species. However, the masses of some ribosomal proteins varied within species. The phylogenetic tree constructed from the profiles of ribosomal proteins matched the references, showing a clear cluster of the subsp. longum and the subsp. infantis strains. This result supports the proposal to reclassify B. longum into subsp. longum and subsp. infantis. The subsp. suis strains formed an individual sub-cluster within the infantis cluster. However, their ribosomal proteins have both characters of longum and infantis types. This result suggests that the taxonomic position of the subsp. suis should be reconsidered.     

Morphological differentiation supports the genetic pattern of the geographic structure of Juniperus thurifera (Cupressaceae)

Juniperus thurifera is an important component of woodland communities of dry sites within the West Mediterranean region and is characterised by a strongly disjunctive geographic range. Two subspecies were recognised, subsp. thurifera in Europe and subsp. africana in Africa. The aim of the study was the comparison of phenetic diversity to the pattern of AFLP geographic differentiation of the species described in the literature. The examination of phenetic diversity was based on the biometrical analysis of 17 populations using 12 morphological characters of cone and seed. The differences among populations were analysed using Student’s t test, analysis of discrimination, UPGMA agglomeration and hierarchical analysis of variance. The majority of morphological characters differentiated at a statistically significant level between populations and between J. thurifera subsp. thurifera and subsp. africana. Three groups of populations were detected using multivariate statistical analyses. The first, well separated, is subsp. africana, while the following two concern subsp. thurifera. The morphological differentiation of populations appeared similar to that described on the AFLP. The Gibraltar Straight appeared to be the most important barrier.     

Allelopathic potential of alkylphenols from Dactylis glomerata subsp. hispanica (Roth) Nyman

Eleven alkylphenols were isolated from the aerial parts of Dactylis glomerata subsp. hispanica, six of them described for the first time. The structural characterization of these compounds has been elucidated by 1D and 2D NMR techniques. The fragmentation patterns of the metabolites obtained by GC–MS analysis allowed the side chain to be elucidated. The allelopathic potential of three alkylphenols, representative of each homologous series of alkylphenols from D. glomerata subsp. hispanica, has been assayed on D. glomerata subsp. hispanica and an herbaceous coexisting species, Phleum subulatum. The bioassay results showed a high auto-stimulation values of germination and root and shoot elongation for D. glomerata subsp. hispanica at high concentrations.     

Recombinant Strain of Bacillus thuringiensis Producing Cyt1A, Cry11B, and the Bacillus sphaericus Binary Toxin

A novel recombinant Bacillus thuringiensis subsp. israelensis strain that produces the B. sphaericus binary toxin, Cyt1Aa, and Cry11Ba is described. The toxicity of this strain (50% lethal concentration [LC₅₀] = 1.7 ng/ml) against fourth-instar Culex quinquefasciatus was higher than that of B. thuringiensis subsp. israelensis IPS-82 (LC₅₀ = 7.9 ng/ml) or B. sphaericus 2362 (LC₅₀ = 12.6 ng/ml).     

New substituted bibenzyls of Frullania brittoniae subsp. Truncatifolia

The two dihydrostilbenes, Brittonin A and Brittonin B, have been isolated from Frullania brittoniae subsp. truncatifolia (F. F. muscicola). They were identified as 3,4,5,3′,4′,5′-hexamethoxybibenzyl and 3,4,5,3′-tetra- methoxy-4′,5′,-methylenedioxybibenzyl respectively.     

Experimental evidence that refuges delay insect adaptation to Bacillus thuringiensis

Theoretical projections suggest that refuges from exposure can delay insect adaptation to environmentally benign insecticides derived from Bacillus thuringiensis, but experimental tests of this approach have been limited. We tested the refuge tactic by selecting two sets of two colonies of diamondback moth (Plutella xylostella) for resistance to B. thuringiensis subsp. aizawai in the laboratory. In each set, one colony was selected with no refuge and the other with a 10 per cent refuge from exposure to B. thuringiensis subsp. aizawai. Bioassays conducted after nine selections were completed show that mortality caused by B. thuringiensis subsp. aizawai was significantly greater in the refuge colonies than in the no-refuge colonies. These results demonstrate that the refuges delayed the evolution of resistance. Relative to a susceptible colony, final resistance ratios were 19 and 8 for the two no-refuge colonies compared to 6 and 5 for the refuge colonies. The mean realized heritability of resistance to B. thuringiensis subsp. aizawai was 0.046 for colonies without refuges, and -0.002 for colonies with refuges. Selection with B. thuringiensis subsp. aizawai decreased susceptibility to B. thuringiensis toxin Cry1Ab, but not to Cry1C or B. thuringiensis subsp. kurstaki. Although the ultimate test of refuges will occur in the field, the experimental evidence reported here confirms modelling results indicating that refuges can slow the evolution of insect resistance to B. thuringiensis.     

Transfer of plasmids pBC 16 and pC 194 into Bacillus thuringiensis subsp. israelensis

A lysozyme sensitive strain of B. thuringiensis (strain O 016) was isolated and shown to be effectively transformed with plasmids pC 194 and pHV 33 using the protoplast transformation technique. The plasmid pC 194 from one successful transformant, strain O 016–194, was subsequently transferred to B. thuringiensis subsp. israelensis by a “conjugation-like” process. The plasmid pBC 16 from B. cereus could also be transferred to B. thuringiensis subsp. israelensis with high frequency using the conjugation-like process. Further, both plasmids, pC 194 and pBC 16, were transferred between strains of B. thuringiensis subsp. israelensis to yield transcipient strains that harbored and expressed properties of both plasmids. This work constitutes effective gene transfer system in B. thuringiensis subsp. israelensis.     

The structure of antibiotic eremomycin B

A new biologically active component, antibiotic eremomycin B, was isolated from the culture liquid of Amycolatopsis orientalis subsp. eremomycini, the producing strain for antibiotic eremomycin. Its structure was established by NMR spectroscopy and mass spectrometry. Eremomycin B was shown to differ from eremomycin by the presence of an N-carboxymethyl substituent in the disaccharide eremosamine fragment.     

Antagonism between Cry1Ac1 and Cyt1A1 Toxins of Bacillus thuringiensis

Most strains of the insecticidal bacterium Bacillus thuringiensis have a combination of different protoxins in their parasporal crystals. Some of the combinations clearly interact synergistically, like the toxins present in B. thuringiensis subsp. israelensis. In this paper we describe a novel joint activity of toxins from different strains of B. thuringiensis. In vitro bioassays in which we used pure, trypsin-activated Cry1Ac1 proteins from B. thuringiensis subsp. kurstaki, Cyt1A1 from B. thuringiensis subsp. israelensis, and Trichoplusia ni BTI-Tn5B1-4 cells revealed contrasting susceptibility characteristics. The 50% lethal concentrations (LC₅₀s) were estimated to be 4,967 of Cry1Ac1 per ml of medium and 11.69 ng of Cyt1A1 per ml of medium. When mixtures of these toxins in different proportions were assayed, eight different LC₅₀s were obtained. All of these LC₅₀s were significantly higher than the expected LC₅₀s of the mixtures. In addition, a series of bioassays were performed with late first-instar larvae of the cabbage looper and pure Cry1Ac1 and Cyt1A1 crystals, as well as two different combinations of the two toxins. The estimated mean LC₅₀ of Cry1Ac1 was 2.46 ng/cm² of diet, while Cyt1A1 crystals exhibited no toxicity, even at very high concentrations. The estimated mean LC₅₀s of Cry1Ac1 crystals were 15.69 and 19.05 ng per cm² of diet when these crystals were mixed with 100 and 1,000 ng of Cyt1A1 crystals per cm² of diet, respectively. These results indicate that there is clear antagonism between the two toxins both in vitro and in vivo. Other joint-action analyses corroborated these results. Although this is the second report of antagonism between B. thuringiensis toxins, our evidence is the first evidence of antagonism between toxins from different subspecies of B. thuringiensis (B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. israelensis) detected both in vivo and in vitro. Some possible explanations for this relationship are discussed.     

Persistence and Recycling of Bioinsecticidal Bacillus thuringiensis subsp. israelensis Spores in Contrasting Environments: Evidence from Field Monitoring and Laboratory Experiments

Sprays of commercial preparations of the bacterium Bacillus thuringiensis subsp. israelensis are widely used for the control of mosquito larvae. Despite an abundant literature on B. thuringiensis subsp. israelensis field efficiency on mosquito control, few studies have evaluated the fate of spores in the environment after treatments. In the present article, two complementary experiments were conducted to study the effect of different parameters on B. thuringiensis subsp. israelensis persistence and recycling, in field conditions and in the laboratory. First, we monitored B. thuringiensis subsp. israelensis persistence in the field in two contrasting regions in France: the Rhône-Alpes region, where mosquito breeding sites are temporary ponds under forest cover with large amounts of decaying leaf matter on the ground and the Mediterranean region characterized by open breeding sites such as brackish marshes. Viable B. thuringiensis subsp. israelensis spores can persist for months after a treatment, and their quantity is explained both by the vegetation type and by the number of local treatments. We found no evidence of B. thuringiensis subsp. israelensis recycling in the field. Then, we tested the effect of water level, substrate type, salinity and presence of mosquito larvae on the persistence/recycling of B. thuringiensis subsp. israelensis spores in controlled laboratory conditions (microcosms). We found no effect of change in water level or salinity on B. thuringiensis subsp. israelensis persistence over time (75 days). B. thuringiensis subsp. israelensis spores tended to persist longer in substrates containing organic matter compared to sand-only substrates. B. thuringiensis subsp. israelensis recycling only occurred in presence of mosquito larvae but was unrelated to the presence of organic matter.     

Chromosome numbers within the genusBolboschoenus in Central Europe

Two chromosome numbers n=54, n=55 were found inBolboschoenus plants studied from Central Europe (Czech Republic, Slovakia, Hungary, Poland) and coastal regions of Europe (the Netherlands, Sweden). The number n=55 is typical forB. maritimus subsp.maritimus with narrow fruits and mostly also forB. maritimus subsp.compactus; the number n=54 characterizesB. planiculmis auct. The morphological type ofB. maritimus subsp.maritimus with wide fruits represents a stable taxon occurring in freshwater habitats throughout Europe. Its variation in chromosome numbers (both n=54, n=55) indicates a possible hybrid origin, probably resulting from hybridization betweenB. maritimus subsp.maritimus with narrow fruits andB. planiculmis auct. Spontaneous hybridization betweenBolboschoenus taxa in the regions with mixed populations may explain the origin of the intermediate morphological and anatomical characters of plants from some localities and the deviations in chromosome numbers.