Sameh Sellami

Prays oleae Midgut Putative Receptor of Bacillus thuringiensis Vegetative Insecticidal Protein Vip3LB Differs from that of Cry1Ac Toxin

Vegetative insecticidal protein (Vip) is a class of insecticidal proteins produced by many Bacillus thuringiensis strains during their vegetative growth stage. The vip3LB gene of B. thuringiensis strain BUPM95, which encodes a protein active against the Lepidoptera olive tree pathogenic insect Prays oleae, was cloned into pET-14b vector and overexpressed in Escherichia coli. The expressed Vip3LB protein, found in the E. coli cytoplasmic fraction, was purified and used to produce anti-Vip3LB antibodies. Using the midgut extract of P. oleae, the purified Vip3LB bound to a 65-kDa protein, whereas Cry1Ac toxin bound to a 210-kDa midgut putative receptor. This result justifies the importance of the biological pest control agent Vip3LB that could be used as another alternative particularly in case of resistance to Cry toxins.

Screening and identification of a Bacillus thuringiensis strain S1/4 with large and efficient insecticidal activities.

The bacterium Bacillus thuringiensis was recognized for its entomopathogenic activities related to Cry and Cyt proteins forming the δ‐endotoxins and some extracellular activities like the vegetative insecticidal proteins (VIP) and Cry1I. These activities may act specifically against diverse organisms and some of them typically characterize each strain. Here, we screened a set of 212 B. thuringiensis strains to search the higher insecticidal activities. These strains had bipyramidal and cubic crystal morphologies and 30% of them showed PCR amplification of vip3 internal region, from which five isolates (S1/4, S17, S122, S123, and S144) showed plasmid profile variability. These five strains contained the cry1I, cry1Aa and/or cry1Ac, cry1Ab and cry2 genes, and S1/4 harbored in addition the cry1C, vip1, and vip2 genes. They produced from 25 to 46 µg δ‐endotoxin per 107 spores. Their δ‐endotoxins displayed distinct lethal concentrations 50% against either Spodoptera littoralis or Ephestia kuehniella larvae with the lowest one for S1/4, which was also active against Tuta absoluta. Fortunately, the analysis of the culture supernatants revealed that S1/4 had the higher toxicity towards these lepidopteron but it did not show any toxicity against the Tribolium castaneum coleopteran larvae; additionally, S1/4 displayed an antibacterial activity. S1/4 is a good candidate for agricultural pest control, as it is more efficient than the reference strain HD1.

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.

Antagonist effects of Bacillus spp. strains against Fusarium graminearum for protection of durum wheat (Triticum turgidum L. subsp. durum)

Bacillus species are attractive due to their potential use in the biological control of fungal diseases. Bacillus amyloliquefaciens strain BLB369, Bacillus subtilis strain BLB277, and Paenibacillus polymyxa strain BLB267 were isolated and identified using biochemical and molecular (16S rDNA, gyrA, and rpoB) approaches. They could produce, respectively, (iturin and surfactin), (surfactin and fengycin), and (fusaricidin and polymyxin) exhibiting broad spectrum against several phytopathogenic fungi. In vivo examination of wheat seed germination, plant height, phenolic compounds, chlorophyll, and carotenoid contents proved the efficiency of the bacterial cells and the secreted antagonist activities to protect Tunisian durum wheat (Triticum turgidum L. subsp. durum) cultivar Om Rabiia against F. graminearum fungus. Application of single bacterial culture medium, particularly that of B. amyloliquefaciens, showed better protection than combinations of various culture media. The tertiary combination of B. amyloliquefaciens, B. subtilis, and P. polymyxa bacterial cells led to the highest protection rate which could be due to strains synergistic or complementary effects. Hence, combination of compatible biocontrol agents could be a strategic approach to control plant diseases.

Molecular characterisation of Bacillus thuringiensis strain MEB4 highly toxic to the Mediterranean flour moth Ephestia kuehniella Zeller (Lepidoptera: Pyralidae).

BACKGROUND Cry2 proteins play an essential role in current Bacillus thuringiensis (Bt) applications and in the prevention of insect resistance to Cry1A toxins. This paper reports on the screening and characterisation of novel Bt strains harbouring effective cry2A-type genes and higher insecticidal activity to Ephestia kuehniella. RESULTS A total of 29 native Bt strains were screened to search for the potent strain against E. kuehniella. The plasmid pattern of the selected strains showed interesting variability. PCR-RFLP analysis of two amplified regions showed high sequence identity within the selected cry2A-type genes. SDS-PAGE and western blot analysis revealed the presence of Cry2Aa toxin only in the MEB4 and BLB240 strains. The activation of Cry2Aa protoxins by larval midgut juice, trypsin or chymotrypsin enzymes revealed significant differences in terms of proteolysis profiles. Interestingly, a 49 kDa band was detected in the proteolysis pattern of BLB240, suggesting the presence of a chymotrypsin cleavage site that might have affected its insecticidal activity. Further, bioassays demonstrated that MEB4 (103.08 ± 36 µg g(-1)) was more active than BLB240 (153.77 ± 45.65 µg g(-1)) against E. kuehniella. CONCLUSION Based on its potent insecticidal activity, the MEB4 strain could be considered to be an effective alternative agent for the control of E. kuehniella.

Isolation and characterization of a new Bacillus thuringiensis strain with a promising toxicity against Lepidopteran pests

Insecticides derived from Bacillus thuringiensis are gaining worldwide importance as environmentally desirable alternatives to chemicals for the control of pests in public health and agriculture. Isolation and characterization of new strains with higher and broader spectrum of activity is an ever growing field. In the present work, a novel Tunisian B. thuringiensis isolate named BLB459 was characterized and electrophoresis assay showed that among a collection of 200 B. thuringiensis strains, the plasmid profile of BLB459 was distinctive. SmaI-PFGE typing confirmed the uniqueness of the DNA pattern of this strain, compared with BUPM95 and HD1 reference strains. PCR and sequencing assays revealed that BLB459 harbored three cry genes (cry30, cry40 and cry54) corresponding to the obtained molecular sizes in the protein pattern. Interestingly, PCR-RFLP assay demonstrated the originality of the BLB459 cry30-type gene compared to the other published cry30 genes. Insecticidal bioassays showed that BLB459 spore-crystal suspension was highly toxic to both Ephestia kuehniella and Spodoptera littoralis with LC50 values of about 64 (53-75) and 80 (69-91) μg of toxin cm(-2), respectively, comparing with that of the commercial strain HD1 used as reference. Important histopathological effects of BLB459 δ-endotoxins on the two tested larvae midguts were detected, traduced by the vacuolization of the apical cells, the damage of microvilli, and the disruption of epithelial cells. These results proved that BLB459 strain could be of a great interest for lepidopteran biocontrol.

Effect of adding amino acids residues in N- and C-terminus of Vip3Aa16 (L121I) toxin.

To study the importance of N‐ and C‐terminus of Bacillus thuringiensis Vip3Aa16 (L121I) toxin (88 kDa), a number of mutants were generated. The addition of two (2R: RS) or eleven (11R: RSRPGHHHHHH) amino acid residues at the Vip3Aa16 (L121I) C‐terminus allowed to an unappropriated folding illustrated by the abundant presence of the 62 kDa proteolytic form. The produced Vip3Aa16 (L121I) full length form was less detected when increasing the number of amino acids residues in the C‐terminus. Bioassays demonstrated that the growth of the lepidopteran Ephestia kuehniella was slightly affected by Vip3Aa16 (L121I)‐2R and not affected by Vip3Aa16 (L121I)‐11R. Additionally, the fusion at the Vip3Aa16 (L121I) N‐terminus of 39 amino acids harboring the E. coli OmpA leader peptide and the His‐tag sequence allowed to the increase of protease sensitivity of Vip3Aa16 (L121I) full length form, as only the 62 kDa proteolysis form was detected. Remarkably, this fused protein produced in Escherichia coli (E. coli) was biologically inactive toward Ephestia kuehniella larvae. Thus, the N‐terminus of the protein is required to the accomplishment of the insecticidal activity of Vip3 proteins. This report serves as guideline for the study of Vip3Aa16 (L121I) protein stability and activity.

Efficacy of olive mill wastewater for protecting Bacillus thuringiensis formulation from UV radiations.

The effectiveness of 10 low-cost UV-absorbers in protecting Bacillus thuringiensis subsp. kurstaki BLB1 toxins against inactivation by UV-A and UV-B irradiation was evaluated in this study. Among them, two by-products, molasses and olive mill wastewater (OMW) were selected for further studies. They were tested at different concentrations of 0.05, 0.1, 0.15 and 0.2% using the para-aminobenzoic acid (PABA) as a common UV protectant. Interestingly, addition of PABA and OMW to BLB1 formulations was found to be most effective in protecting BLB1 spores at 90.8 and 76.4% respectively and in preserving delta-endotoxin concentration at a level of 81.7 and 72.2%, respectively when used at a concentration of 0.2%. The lowest preserved spores (46.3%) and delta-endotoxin level (12.4%) was found using molasses. In contrast, spore count and delta-endotoxin concentration were completely reduced after an exposure of unprotected Bt strain BLB1 to UV radiations up to 96h. SDS-PAGE analysis of protected and unprotected samples revealed that delta-endotoxin bands (130, 65-70kDa) were conserved until 96h of UV exposure in presence of PABA or OMW compared with their disappearance in presence of molasses after 72h of exposure and their dramatically decline from 8h of exposure in unprotected mixture. A complete loss of larvicidal toxicity against Ephestia kuehniella was found after 24h of exposure in absence of any UV-absorber. Addition of OMW or PABA offered the highest levels of insecticidal activity with 63.2 and 74.7% of residual toxicity, respectively. Whereas, molasses addition, as UV protectant retained only 26.3% of residual activity after 96h of exposure. Therefore, addition of OMW by-product to Bt formulation may be a suitable alternative to others synthetic chemical compounds. OMW may also provided added value, be environmentally friendly and less hazardous, when used at low concentration.

Influence of Ephestia kuehniella stage larvae on the potency of Bacillus thuringiensis Cry1Aa delta-endotoxin

The economically important crop pest Ephestia kuehniella was tested at two stages of larval development for susceptibility to Bacillus thuringiensis Cry1Aa toxin. Bioassays showed that toxicity decreased during the development of larvae stage. In fact, Cry1Aa toxins from BNS3-Cry- (pHT-cry1Aa) showed low toxicity against the first-instar larvae (L1) with a LC50 value of about 421.02μg/g of diet and was not toxic against the fifth-instar (L5), comparing to the BLB1 toxins used as positive control which represent a LC50 value of about 56.96 and 84.21μg/g of diet against L1 and L5 instars larvae, respectively. Effects of Cry1Aa toxins were reflected in histopathological observations by the weak destruction of midgut epithelium, slight hypertrophy of epithelial cells, and minor alteration of brush border membrane (BBM) detected mainly in L1 larvae stage comparing to the more extensive damage caused by BLB1 toxins. Interestingly, in vitro proteolysis of Cry1Aa toxins was found to correlate with the difference of toxicity during larval stage development. In fact, the weak proteinase activity detected inside the L1 midgut has led to the persistence of the Cry1Aa active forms (65 and 58kDa) during prolonged incubations, causing the alterations described previously. Three subfamilies of aminopeptidase (APN) receptors were detected in both larvae instars with different intensities and molecular weights (150kDa and 55kDa for APN1, and 90kDa for APN2 and APN4). Remarkably, binding assay using Cry1Aa toxin seems to have no direct correlation with larval stages toxicity differences, since same putative receptors were detected. Understanding the reasons for the clear differences in the effectiveness of Cry1Aa toxins during larval development stages of E. kuehniella is very important for the design of future improvement insecticidal approaches and for the accomplishment of resistance prevention strategies.

Combinatorial effect of Photorhabdus luminescens TT01 and Bacillus thuringiensis Vip3Aa16 toxin against Agrotis segetum

&NA; The entomopathogenic Photorhabdus luminescens TT01 is a promoting bacterium that controls effectively many insect pests. Indeed, it exhibited a mortality rate of 32.36% against the first instar larvae of the turnip moth Agrotis segetum, when it was used at a concentration of 5 × 107 cells/ml but no toxicity against the second instar larvae in the same condition. P. luminescens TT01 oral toxicity is associated to septicaemia since cells fraction exhibited the highest mortality rate of 34%. In order to enhance P. luminescens TT01 insecticidal potential, combination with Bacillus thuringiensis Vip3Aa16 toxin was tested. An improvement of insecticidal activity was shown. Indeed, 100% mortality of A. segetum first instar larvae was obtained after 2 days of treatment, when using TT01 cells and Vip3Aa16 toxin at a concentration of 5 × 107 cells/ml and 9.025 ng/cm2, respectively. Moreover, growth inhibition rate of 45% of the second instar larvae was observed, when using the same combination. A. segetum mortality could be the result of several alterations in the midgut epithelium caused by Vip3Aa16 toxin, allowing a rapid invasion of the hemocoel by TT01 cells as demonstrated by histopathological study. Clear symptoms of intoxication were observed for all combinations tested, including swelling, vesicle formation, cytoplasm vacuolization and brush border membrane lysis. Taken together, these results promote the use of P. luminescens TT01 as a potent bioinsecticide to control effectively A. segetum by oral treatment in a mixture with Vip3Aa16 toxin. Graphical abstract Figure. No caption available. HighlightsAt 5 × 107 cells/ml, TT01 cells caused A. segetum mortality of 32.36%.TT01 cells oral toxicity is associated to septicaemia.Combination of TT01 cells and Bt Vip 3 toxin, resulted in 100% A. segetum mortality.