Development of a new TaqMan-based PCR assay for the specific detection and quantification of Simkania negevensis.

Simkania negevensis is an emerging Chlamydia-like bacterium related to human respiratory diseases. An early and accurate detection of this pathogen could be useful to monitor the potential infectious risks and to set suitable outbreak control measures. In Tunisia, distribution and abundance of S. negevensis remain until now largely unknown. In the present work, a qPCR assay, targeting the 16S rRNA gene, for fast detection and quantification of S. negevensis was developed and validated. A high specificity for S. negevensis detection displaying no cross-reaction with the closely related Chlamydia spp. or the other tested microorganisms was noticed. qPCR assay performance was considered very satisfying with detection limits of 5 DNA copies per reaction. qPCR assay validation was performed by screening 37 clinical specimens and 35 water samples. S. negevensis wasn't detected in respiratory samples, but it was found in four cases of water samples. We suggest that the qPCR assay developed in this study could be considered sufficiently characterized to initiate the quantification of S. negevensis in environmental samples.

Comparative analysis of the susceptibility/tolerance of Spodoptera littoralis to Vip3Aa, Vip3Ae, Vip3Ad and Vip3Af toxins of Bacillus thuringiensis.

The cotton leaf worm Spodoptera littoralis is known for causing serious damages to various crops. In this study, the susceptibility/tolerance of this larvae to four Vip3A (Vip3Aa, Vip3Ae, Vip3Ad and Vip3Af) toxins was investigated. UnlikeVip3Ad which showed no activity to S. littoralis, Vip3Aa, Vip3Ae and Vip3Af exhibited high toxicity to this larva with LC50 of 228.42 ng/cm2, 65.71 ng/cm2, and 388.90 ng/cm2, respectively. Activation of the 90 kDa Vip3A proteins by S. littoralis larvae juice generated four major bands of sizes 62, 45, 33 and 22 kDa. Binding experiments between biotinylated Vip3A toxins and the brush border membrane vesicles (BBMV) revealed two binding proteins of 55 and 100 kDa with Vip3Aa. Vip3Ae and Vip3Af recognized one single putative receptor of 65 kDa, whereas Vip3Ad did not bind to S. littoralis BBMV. In histopathological observations, Vip3Aa, Vip3Ae and Vip3Af toxins showed approximately similar damages on S. littoralis midgut including rupture and disintegration of epithelial layer and cellular vacuolization. These findings showed that Vip3Aa, Vip3Ae and Vip3Af might be useful for controlling S. littoralis.

Quantification of Bacillus thuringiensis Vip3Aa16 Entomopathogenic Toxin Using Its Hemolytic Activity.

Vegetative insecticidal proteins produced by some Bacillus thuringiensis strains are specifically toxic to different agricultural pests such as the polyphagous Spodoptera and several other Lepidopteran insects, but one of the major problems found in the use of these biopesticides was the lack of an easy and credible method of quantification of such secreted toxins. Heterologous expression of B. thuringiensis Vip3Aa16 toxin was performed in Escherichia coli then the protein was purified by chromatography. Using blood agar as well as blood agar overlay (zymogram assay), we reported, for the first time, the capacity of Vip3Aa16 to induce hemolysis. The hemolytic activity of this protein was shown to be relatively stable after treatment at 40 °C and at a range of pH between 6.5 and 9. Moreover, a linear relationship was shown between hemolysis levels and Vip3Aa16 concentrations. The model established in the present study could quantify Vip3A toxin as a function of hemolytic activity and the assay proposed showed to be a simple and low-cost method to readily assess Vip3A toxins in liquid cultures and facilitate the use of this kind of bioinsecticides in pest management programs.

Combinatorial effect of Bacillus amyloliquefaciens AG1 biosurfactant and Bacillus thuringiensis Vip3Aa16 toxin on Spodoptera littoralis larvae.

Spodoptera littoralis, one of the most serious and destructive agricultural pests in the world, is very susceptible to Vip3 toxin. In order to develop a new efficient bioinsecticide and to prevent the development of resistance by the target pest, insecticidal activity of biosurfactant produced by Bacillus amyloliquefaciens AG1 was evaluated against S. littoralis. Bioassays revealed the susceptibility of the first instar larvae of this pest to AG1 biosurfactant with an LC50 of 245ng/cm2. Moreover, the histopathology examination of the larval midgut treated by AG1 biosurfactant showed vacuolization, necrosis and disintegration of the basement membrane. Binding experiments revealed that the AG1 biosurfactant recognized three putative receptors located in the brush border membrane vesicles of S. littoralis with sizes of 91, 72 and 64kDa. Competition assays using biotinylated metabolites indicated that AG1 biosurfactant and Vip3Aa16 toxin did not compete for the same S. littoralis receptors. When combined, AG1 biosurfactant and Vip3Aa16 showed an additive effect against S. littoralis larvae. These findings suggested that B. amyloliquefaciens AG1 biosurfactant could be a promising biocontrol agent to eradicate S. littoralis and to prevent resistance development by this pest.

Involvement of the processing step in the susceptibility/tolerance of two lepidopteran larvae to Bacillus thuringiensis Cry1Aa toxin.

Bacillus thuringiensis (Bt) Cry1A toxins are known for their effectiveness against lepidopteran insects. In this study, the entomopathogenic activity of Cry1Aa was investigated against two lepidopteran larvae causing serious threat to various crops, Spodoptera littoralis and Tuta absoluta. Contrarily to S. littoralis, which showed low susceptibility to Cry1Aa (40% mortality with 1µg/cm(2)), T. absoluta was very sensitive to this delta-endotoxin (LC50 of 95.8ng/cm(2)). The different steps in the mode of action of this toxin on the two larvae were studied with the aim to understand the origin of their difference of susceptibility. Activation of the 130kDa Cry1Aa protein by T. absoluta larvae juice generated a 65kDa active toxin, whereas S. littoralis gut juice led to a complete degradation of the protoxin. The study of the interaction of the brush border membrane vesicles (BBMV) with purified biotinylated Cry1Aa toxin revealed six and seven toxin binding proteins in T. absoluta and S. littoralis BBMV, respectively. Midgut histopathology of Cry1Aa fed larvae demonstrated approximately similar damage caused by the toxin in the two larvae midguts. These results suggest that the activation step was strongly involved in the difference of susceptibility of the two larvae to Cry1Aa.

Bacillus amyloliquefaciens AG1 biosurfactant: Putative receptor diversity and histopathological effects on Tuta absoluta midgut.

The use of biosurfactant in pest management has received much attention for the control of plant pathogens, but few studies reported their insecticidal activity. The present study describes the insecticidal activity of biosurfactant extracted from Bacillus amyloliquefaciens strain AG1. This strain produces a lipopeptide biosurfactant exhibiting an LC50 of about 180ng/cm(2) against Tuta absoluta larvae. Accordingly, the histopathologic effect of this biosurfactant on T. absoluta larvae showed serious damages of the midgut tissues including rupture and disintegration of epithelial layer and cellular vacuolization. By PCR, we showed that this biosurfactant could be formed by several lipopeptides and polyketides including iturin, fengycin, surfactin, bacyllomicin, bacillaene, macrolactin and difficidin. Binding experiment revealed that it recognized five putative receptors located in the BBMV of T. absoluta with sizes of 68, 63, 44, 30 and 19kDa. Therefore, biosurfactant AG1 hold potential for use as an environmentally friendly agent to control the tomato leaf miner.

Overproduction of the Bacillus thuringiensis Vip3Aa16 toxin and study of its insecticidal activity against the carob moth Ectomyelois ceratoniae.

The vip3Aa16 gene of Bacillus thuringiensis strain BUPM95 was cloned and expressed in Escherichia coli. Optimization of Vip3A16 protein expression was conducted using Plackett-Burman design and response surface methodology. Accordingly, the optimum Vip3A16 toxin production was 170µg/ml at 18h post-induction time and 39°C post-induction temperature. This corresponds to an improvement of 21times compared to the starting conditions. The insecticidal activity, evaluated against Ectomyelois ceratoniae, displayed an LC50 value of 40ng/cm(2) and the midgut histopathology of Vip3Aa16 fed larvae showed vacuolization of the cytoplasm, brush border membrane destruction, vesicle formation in the apical region and cellular disintegration.

Agrotis segetum midgut putative receptor of Bacillus thuringiensis vegetative insecticidal protein Vip3Aa16 differs from that of Cry1Ac toxin.

Considering the fact that Agrotis segetum is one of the most pathogenic insects to vegetables and cereals in the world, particularly in Africa, the mode of action of Vip3Aa16 of Bacillus thuringiensis BUPM95 and Cry1Ac of the recombinant strain BNS3Cry-(pHTcry1Ac) has been examined in this crop pest. A. segetum proteases activated the Vip3Aa16 protoxin (90kDa) yielding three bands of about 62, 45, 22kDa and the activated form of the toxin was active against this pest with an LC50 of about 86ng/cm(2). To be active against A. segetum, Cry1Ac protoxin was activated to three close bands of about 60-65kDa. Homologous and heterologous competition binding experiments demonstrated that Vip3Aa16 bound specifically to brush border membrane vesicles (BBMV) prepared from A. segetum midgut and that it does not inhibit the binding of Cry1Ac. Moreover, BBMV protein blotting experiments showed that the receptor of Vip3Aa16 toxin in A. segetum midgut differs from that of Cry1Ac. In fact, the latter binds to a 120kDa protein whereas the Vip3Aa16 binds to a 65kDa putative receptor. The midgut histopathology of Vip3Aa16 fed larvae showed vacuolization of the cytoplasm, brush border membrane lysis, vesicle formation in the goblet cells and disintegration of the apical membrane. The distinct binding properties and the unique protein sequence of Vip3Aa16 support its use as a novel insecticidal agent to control the crop pest A. segetum.

The impact of the Bacillus subtilis SPB1 biosurfactant on the midgut histology of Spodoptera littoralis (Lepidoptera: Noctuidae) and determination of its putative receptor.

SPB1 is a Bacillus subtilis strain producing a lipopeptide biosurfactant. The insecticidal activity of this biosurfactant was evaluated against the Egyptian cotton leaf worm (Spodoptera littoralis). It displayed toxicity with an LC(50) of 251 ng/cm(2). The histopathological changes occurred in the larval midgut of S. littoralis treated with B. subtilis SPB1 biosurfactant were vesicle formation in the apical region, cellular vacuolization and destruction of epithelial cells and their boundaries. Ligand-blotting experiments with S. littoralis brush border membrane vesicles showed binding of SPB1 biosurfactant to a protein of 45 kDa corresponding to its putative receptor. The latter differs in molecular size from those recognized by Bacillus thuringiensis Vip3A and Cry1C toxins, commonly known by their activity against S. littoralis. This result wires the application of B. subtilis biosurfactant for effective control of S. littoralis larvae, particularly in the cases where S. littoralis will develop resistance against B. thuringiensis toxins.

Study of the Bacillus thuringiensis Vip3Aa16 histopathological effects and determination of its putative binding proteins in the midgut of Spodoptera littoralis.

The bacterium Bacillus thuringiensis produces, at the vegetative stage of its growth, Vip3A proteins with activity against a broad spectrum of lepidopteran insects. The Egyptian cotton leaf worm (Spodoptera littoralis) is an important agricultural pest that is susceptible to the Vip3Aa16 protein of Bacillus thuringiensis kurstaki strain BUPM95. The midgut histopathology of Vip3Aa fed larvae showed vacuolization of the cytoplasm, brush border membrane destruction, vesicle formation in the apical region and cellular disintegration. Biotinylated Vip3Aa toxin bound proteins of 55- and 100-kDa on blots of S. littoralis brush border membrane preparations. These binding proteins differ in molecular size from those recognized by Cry1C, one of the very few Cry proteins active against the polyphagous S. littoralis. This result supports the use of Vip3Aa16 proteins as insecticidal agent, especially in case of Cry-resistance management.

Investigation of the steps involved in the difference of susceptibility of Ephestia kuehniella and Spodoptera littoralis to the Bacillus thuringiensis Vip3Aa16 toxin.

BUPM95 is a Bacillus thuringiensis subsp. kurstaki strain producing the Vip3Aa16 toxin with an interesting insecticidal activity against the Lepidopteran larvae Ephestia kuehniella. Study of different steps in the mode of action of this Vegetative Insecticidal Protein on the Mediterranean flour moth (E. kuehniella) was carried out in the aim to investigate the origin of the higher susceptibility of this insect to Vip3Aa16 toxin compared to that of the Egyptian cotton leaf worm Spodoptera littoralis. Using E. kuehniella gut juice, protoxin proteolysis generated a major band corresponding to the active toxin and another band of about 22kDa, whereas the activation of Vip3Aa16 by S. littoralis gut juice proteases generated less amount of the 62kDa active form and three other proteolysis products. As demonstrated by zymogram analysis, the difference in proteolysis products was due to the variability of proteases in the two gut juices larvae. The study of the interaction of E. kuehniella BBMV with biotinylated Vip3Aa16 showed that this toxin bound to a putative receptor of 65kDa compared to the 55 and 100kDa receptors recognized in S. littoralis BBMV. The histopathological observations demonstrated similar damage caused by the toxin in the two larvae midguts. These results demonstrate that the step of activation, mainly, is at the origin of the difference of susceptibility of these two larvae towards B. thuringiensis Vip3Aa16 toxin.

Overproduction of Glucose Oxidase by Aspergillus tubingensis CTM 507 Randomly Obtained Mutants and Study of Its Insecticidal Activity against Ephestia kuehniella.

In order to enhance the production of glucose oxidase (GOD), random mutagenesis of Aspergillus tubingensis CTM 507 was performed using the chemical and physical mutagens: nitric acid and UV irradiation, respectively. The majority of the isolated mutants showed good GOD production, but only some mutants presented a significant overproduction, as compared with the parent strain. The selected mutants (19 strains), showing an overproduction larger than 200%, are quite stable after three successive subcultures. Among these, six strains revealed an important improvement in submerged fermentation. The insecticidal activity of GOD produced by the wild and the selected mutant strains was evaluated against the third larval instars of E. kuehniella. Mutant strains U11, U12, U20, and U21, presenting the most important effect, displayed an LC50 value of 89.00, 88.51, 80.00, and 86.00 U/cm2, respectively, which was 1.5-fold more important than the wild strain (61 U/cm2). According to histopathology observations, the GOD enzyme showed approximately similar damage on the E. kuehniella midgut including rupture and disintegration of the epithelial layer and cellular vacuolization. The data supports, for the first time, the use of GOD as a pest control agent against E. kuehniella.

Insecticidal activity, putative binding proteins and histopathological effects of Bacillus thuringiensis Vip3(459) toxin on the lepidopteran pest Ectomyelois ceratoniae.

The carob moth, Ectomyelois ceratoniae, is an important agricultural pest that is susceptible to the Vip3(459) protein. The insecticidal activity, evaluated against this lepidopteran pest, displayed an LC50 value of about 28 ng/cm2. The investigation of the mode of action of this B. thuringiensis protein demonstrated that the active form of this toxin bound to putative receptors in the BBMV of E. ceratoniae. Ligand blotting experiment proved that Vip3(459) specifically bound to two proteins of about 53 and 57 kDa, located on the midgut. This specific binding caused perturbations in midgut tissues. The histopathology of 20 midguts from Vip3(459)-feeding larvae showed cytoplasm vacuolization, brush border membrane destruction, vesicle formation in the apical region and cellular disintegration. These findings suggested that B. thuringiensis Vip3(459) could be a promising biocontrol agent to eradicate E. ceratoniae and to prevent emergence of resistance.

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

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.

Improvement of Vip3Aa16 Toxin Production and Efficiency Through Nitrous Acid and UV Mutagenesis of Bacillus thuringiensis (Bacillales: Bacillaceae).

Bacillus thuringiensis Berliner (Bacillales: Bacillaceae) strain BUPM95 was known by the efficiency of its vegetative insecticidal protein (Vip3Aa16) against different Lepidoptera such as Spodoptera littoralis (Lepidoptera: Noctuidae). To overcome the problem of the low quantities of Vip3 proteins secreted by B. thuringiensis strains in the culture supernatant, classical mutagenesis of vegetative cells of BUPM95 strain was operated using nitrous acid and UV rays. The survivors were screened on the basis of their hemolytic activity and classified in three groups: unaffected, overproducing, and hypo-producing mutants. Using different mutants improved in their hemolytic activity, the supernatants showed an improved toxicity toward S. littoralis larvae (83.33-100% of mortality) compared with the wild-type supernatant (76%). After Vip3 protein quantification in the different supernatants, bioassays against S. littoralis larvae demonstrated that mutants M62, M43, and M76 were improved in the efficiency of their toxin as demonstrated by the lower values of LC50 and LC90 compared with the wild-type Vip3Aa16 protein. However, M26 and M73 mutants were improved in the toxin quantities produced in the supernatant. The improvement of the production and the efficiency of B. thuringiensis Vip3 toxins should contribute to a significant reduction of the production costs of these very interesting B. thuringineis proteins and facilitate the use of these toxins in the pest control management.

Vegetative insecticidal protein of Bacillus thuringiensis BLB459 and its efficiency against Lepidoptera.

Bacillus thuringiensis strain BLB459 supernatant showed a promising activity against Lepidopteran pests with extremely damages in the larvae midgut. Investigations of the genes that encode secreted toxin demonstrated that this strain harbored a vip3-type gene named vip3(459). Based on its original nucleotide and amino acid sequences, this gene was cloned into pET-14b vector and overexpressed in Escherichia coli. The expressed protein was purified and tested against different insects and interestingly the novel toxin demonstrated a remarkable activity against the stored products pest Ephestia kuehniella and the polyphagous insects Spodoptera littoralis and Agrotis segetum. As demonstrated, the acute activity of Vip3(459) protein against A. segetum can be due to its original amino acids sequence and the putative receptors of this toxin in the larvae midgut. These results demonstrated that this Vip3 toxin showed a wide spectrum of activity against Lepidoptera and support its use as a biological control agent.

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.