Bacillus thuringiensis strain QBT220 pBtoxis plasmid structural instability enhances δ-endotoxins synthesis and bioinsecticidal activity.

Bacillus thuringiensis subsp. israelensis (Bti) spherical parasporal crystal contains several insecticidal proteins used as environmentally safe alternative to toxic chemical pesticides. The exploration of a Bti strain isolated from Qatar QBT220 genes encoding the δ-endotoxins responsible of the insecticidal activities revealed the alteration of a 14-kb DNA region including the δ-endotoxins cry10A and cyt1C genes of pBtoxis plasmid. The presence of all the insecticidal genes except cry10A and cyt1C was explained by a structural instability of the plasmid pBtoxis. However, when compared with the Bti reference strains H14 and QBT217 that carry all δ-endotoxins coding genes, it was found that QBT220, has a significantly higher insecticidal activity against the dipteran insect Aedes aegypti larvae despite of the plasmid pBtoxis structural instability due to the alteration of cry10A and cyt1C genes. In addition, QBT220 showed the highest δ-endotoxin synthesis per spore, compared with that of the wildtype strains. These findings confirm that the altered genes cry10A and cyt1C are not mandatory for Bti insecticidal activities and on the other hand show a possible inhibitory effect played by the 2 proteins Cry10A and Cyt1C on the insecticidal activities of the other insecticidal proteins. In addition, the QBT220 increased δ-endotoxins synthesis per cell, makes this strain a good candidate for possible applications in the industrial production of bioinsecticides.

Towards novel Cry toxins with enhanced toxicity/broader: a new chimeric Cry4Ba / Cry1Ac toxin.

Attempts have been made to express or to merge different Cry proteins in order to enhance toxic effects against various insects. Cry1A proteins of Bacillus thuringiensis form a typical bipyramidal parasporal crystal and their protoxins contain a highly conserved C-terminal region. A chimerical gene, called cry(4Ba-1Ac), formed by a fusion of the N-terminus part of cry4Ba and the C-terminus part of cry1Ac, was constructed. Its transformation to an acrystalliferous B. thuringiensis strain showed that it was expressed as a chimerical protein of 116 kDa, assembled in spherical to amorphous parasporal crystals. The chimerical gene cry(4Ba-1Ac) was introduced in a B. thuringiensis kurstaki strain. In the generated crystals of the recombinant strain, the presence of Cry(4Ba-1Ac) was evidenced by MALDI-TOF. The recombinant strain showed an important increase of the toxicity against Culex pipiens larvae (LC50 = 0.84 mg l-1 ± 0.08) compared to the wild type strain through the synergistic activity of Cry2Aa with Cry(4Ba-1Ac). The enhancement of toxicity of B. thuringiensis kurstaki expressing Cry(4Ba-1Ac) compared to that expressing the native toxin Cry4Ba, might be related to its a typical crystallization properties. The developed fusion protein could serve as a potent toxin against different pests of mosquitoes and major crop plants.

Protective effects of ethyl acetate fraction of Lawsonia inermis fruits extract against carbon tetrachloride-induced oxidative damage in rat liver.

This study aimed to investigate the antioxidant properties of different fractions obtained from the fruits of Lawsonia inermis, a widely used medicinal plant, against carbon tetrachloride (CCl4)-induced oxidative stress in rat liver. The results show that several fractions obtained from L. inermis fruits possessed important antioxidant activity. Among them, the ethyl acetate (EA) fraction showed the highest antioxidant activity. Then, EA fraction was selected for the purification of potential antioxidant compounds. The hepatoprotective effects of EA fraction and its most active constituent, gallic acid (GA), were evaluated against CCl4-induced hepatotoxicity in rats. CCl4 induced oxidative stress by a significant rise in serum marker enzymes. However, pretreatment of rats with EA fraction of fruits of L. inermis at a dose of 250 mg kg(-1)body weight and GA significantly lowered some serum biochemical parameters (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and lactate dehydrogenase) in treated rats. A significant reduction in hepatic thiobarbituric acid reactive substances and an increase in antioxidant enzymes namely superoxide dismutase, catalase, and glutathione peroxidase by treatment with plant extract and GA, against CCl4-treated rats, were observed. Histopathological examinations showed extensive liver injuries, characterized by extensive hepatocellular necrosis, vacuolization, and inflammatory cell infiltration. This potential antioxidant activity is comparable to those of the major purified antioxidant compound, GA. Based on these results, it was observed that fruits of L. inermis protect liver from oxidative stress induced by CCl4 and thus help in evaluation of traditional claim on this plant.

Electropolymerized phenol derivatives as permselective polymers for biosensor applications.

Amperometric biosensors are often coated with a polymeric permselective film to avoid electroactive interference by reducing agents present in the target medium. Phenylenediamine and phenol monomers are commonly used to form these permselective films in the design of microsensors and biosensors. This paper aims to evaluate the permselectivity, stability and lifetime of polymers electrosynthesized using either constant potential amperometry (CPA) or cyclic voltammetry (CV) from naturally occurring phenylpropanoids in monomeric and dimeric forms (eugenol, isoeugenol, dehydrodieugenol and magnolol). Sensors were characterized by scanning electron microscopy and permselectivity analysis. Magnolol formed an electro-deposited polymer with a more defined three-dimensional texture in comparison with the other films. The phenol-derived films showed different permselectivity towards H2O2 over ascorbic acid and dopamine, likely to be related to the thickness and compactness of the polymer. The CV-derived films had a better permselectivity compared to the CPA-corresponding polymers. Based on these results, the permselectivity, stability and lifetime of a biosensor for glucose were studied when a magnolol coating was electro-deposited. The structural principles governing the permselectivity of the magnolol-derived film are suggested to be mainly related to the conformational flexibility of this monomer. Newly designed biosensors, coated with electropolymerized natural phenol derivatives, may represent promising analytical devices for different application fields.

Characterisation of novel Bacillus thuringiensis isolates against Aedes aegypti (Diptera: Culicidae) and Ceratitis capitata (Diptera: Tephridae).

Bacillus thuringiensis is successfully used in pest management strategies as an eco-friendly bioinsecticide. Isolation and identification of new strains with a wide variety of target pests is an ever growing field. In this paper, new B. thuringiensis isolates were investigated to search for original strains active against diptera and able to produce novel toxins that could be used as an alternative for the commercial H14 strain. Biochemical and molecular characterization revealed a remarkable diversity among the studied strains. Using the PCR method, cry4C/Da1, cry30Ea, cry39A, cry40 and cry54 genes were detected in four isolates. Three strains, BLB355, BLB196 and BUPM109, showed feeble activities against Aedes aegypti larvae. Interestingly, spore-crystal mixtures of BLB361, BLB30 and BLB237 were found to be active against Ceratitis capitata with an LC50 value of about 65.375, 51.735 and 42.972 µg cm(-2), respectively. All the studied strains exhibited important mortality levels using culture supernatants against C. capitata larvae. This suggests that these strains produce a wide range of soluble factors active against C. capitata larvae.

Susceptibility of Agrotis segetum (noctuidae) to Bacillus thuringiensis and analysis of midgut proteinases.

Seventy-eight Bacillus thuringiensis isolates were selected for a screening against the Lepidoptera species Agrotis segetum to search the higher insecticidal activity. In a preliminary bioassay, the spore-crystal mixture of 78 B. thuringiensis isolates was tested against L1 larvae of A. segetum. Fifty-two isolates had more than 60% corrected mortality after 3 days. Seven isolates caused a corrected mortality of 100% on A. segetum. Twelve isolates were selected for a second bioassay investigating the effect of the vegetative insecticidal protein (Vip) against third-instar larvae. After 7 days, the weight gain and the larval stage of each larva were recorded. This bioassay showed an aberration in larval growth increases, morphology, and weight gain. After plasmid pattern analysis, the most active strains are most likely B. thuringiensis kurstaki strains expressing the Vip3A toxin. The absence of two proteinase activities observed in the case of Cry1Ac would be the consequence of the difference in susceptibility of A. segetum to the toxins used.

Combinatorial effect of Bacillus thuringiensis kurstaki and Photorhabdus luminescens against Spodoptera littoralis (Lepidoptera: Noctuidae).

Spodoptera littoralis, one of the major pests of many important crop plants, is more susceptible to Bacillus thuringiensis aizawai delta-endotoxins than to those of Bacillus thuringiensis kurstaki. Within the framework of the development of efficient bioinsecticides and the prevention against insect resistance, we tested the effect of mixing B. thuringiensis kurstaki delta-endotoxins and Photorhabdus luminescens cells on S. littoralis growth. The obtained results showed that the growth inhibition of this insect was more effective when B. thuringiensis kurstaki spore-crystal mixture and Photorhabdus luminescens cells were used in combination. Furthermore, this synergism is mainly due to the presence of Cry1Ac, which is one of the three delta-endotoxins that form the crystal of B. thuringiensis kurstaki strain BNS3 in addition to Cry1Aa and Cry2Aa. This work shows a possibility to use B. thuringiensis as a delivery means for Photorhabdus bacteria in order to infect the insect hemocoel and to reduce the risk of developing resistance in the target organism.

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.

Heterologous expression and secretion of an antifungal Bacillus subtilis chitosanase (CSNV26) in Escherichia coli.

The aims of the study were the production improvement, the purification, the characterization and the activity investigation of chitosanase CSNV26 of Bacillus subtilis (V26). The gene csnV26 encoding for this protein was amplified and cloned in the pBAD vector then expressed in Escherichia coli (Top10). The SDS-PAGE and zymogram analysis of the recombinant protein showed that it has two active forms sized 27 and 31 kDa, corresponding to the protein with and without signal peptide. This protein has the particularity of being secreted by Top10-pBAD-csnV26 with a high yield of 6.2 g/l. The HPLC purification of CSNV26 from supernatant confirmed the presence of the two sizes. The investigation of the CSNV26 thermostability showed that the pure protein is highly stable keeping 68 % of its activity after 30-min treatment at 100 °C, contrarily to the protein present within the supernatant of E. coli and B. subtilis (V26). The molecular dynamics study of the predicted structure of protein in both forms showed that the presence of the peptide signal in the form of 31 kDa gave it a remarkable thermal stability. The antifungal activity of CSNV26 was evidenced on Rhizopus nigricans and Rhizopus oryzae. Indeed, it has provoked an alteration and embrittlement of their hyphae with onset of protoplast.

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 10(7) 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.

Molecular identification and biocontrol of ochratoxigenic fungi and ochratoxin A in animal feed marketed in the state of Qatar.

Ochratoxin A (OTA) is a toxic fungal metabolite produced by some Aspergillus and Penicillium species. This work was designed to explore the presence of OTA and ochratoxigenic fungi in feed grains marketed in Qatar and their biological control by a bacterium (Burkholderia cepacia). Significantly higher levels of OTA were detected in mixed grains samples (144.59 ± 6.63 µg/kg), compared to the maize (25.27 ± 1.89 µg/kg) and wheat (3.37 ± 0.11 µg/kg). OTA-producing fungi (A. niger, A. ochraceus, A. westerdijkiae, A. carbonarius and P. verrucosum) were identified on the basis of their morphological features as well as through polymerase chain reaction (PCR). Putative ochratoxigenic polyketide genes in these isolates were evidenced by using primers AoOTA-L/AoOTA-R (in A. ochraceus and A. westerdijkiae), AoPks1/AoPks2 (in A. niger and A. ochraceus) and PenPks1/Penpks2 (in P. verrucosum). On synthetic media, A. westerdijkiae showed the highest OTA synthesis (5913 ± 576 µg/kg) than the closely related A. ochraceus (3520 ± 303 µg/kg), A. carbonarius (3064 ± 289 µg/kg) and P. verrucosum (3030 ± 710 µg/kg). Burkholderia cepacia cells and culture extract showed promising biological control potentials against OTA producing fungi. On the basis of these findings, it can be concluded that animal feed samples are generally contaminated with OTA-producing fungi as well as OTA, and Burkholderia cepacia CS5 exhibits promising antifungal activities.

Perfume Guns: Potential of Yeast Volatile Organic Compounds in the Biological Control of Mycotoxin-Producing Fungi.

Pathogenic fungi in the genera Alternaria, Aspergillus, Botrytis, Fusarium, Geotrichum, Gloeosporium, Monilinia, Mucor, Penicillium, and Rhizopus are the most common cause of pre- and postharvest diseases of fruit, vegetable, root and grain commodities. Some species are also able to produce mycotoxins, secondary metabolites having toxic effects on human and non-human animals upon ingestion of contaminated food and feed. Synthetic fungicides still represent the most common tool to control these pathogens. However, long-term application of fungicides has led to unacceptable pollution and may favour the selection of fungicide-resistant mutants. Microbial biocontrol agents may reduce the incidence of toxigenic fungi through a wide array of mechanisms, including competition for the ecological niche, antibiosis, mycoparasitism, and the induction of resistance in the host plant tissues. In recent years, the emission of volatile organic compounds (VOCs) has been proposed as a key mechanism of biocontrol. Their bioactivity and the absence of residues make the use of microbial VOCs a sustainable and effective alternative to synthetic fungicides in the management of postharvest pathogens, particularly in airtight environments. In this review, we will focus on the possibility of applying yeast VOCs in the biocontrol of mycotoxigenic fungi affecting stored food and feed.

Improvement of Photorhabdus temperata strain K122 bioinsecticide production by batch and fed-batch fermentations optimization.

Optimization of a fermentation process for bioinsecticides production by Photorhabdus temperata strain K122 was investigated into fully controlled 3-L fermenter using an optimized medium (OM). Development of large-scale inocula showed that the composition of the growth medium greatly influenced the physiological state of P. temperata cells. The effect of pH, agitation and dissolved oxygen concentration (DO) on the growth, culturability and oral toxicity of P. temperata cells were also investigated. Indeed, maintaining the pH at 7 and controlling DO concentration at 50 % saturation throughout the fermentation process, improved biomass production, CFU counts and oral toxicity by 41.1, 35 and 32.1 %, respectively, as compared to cultures carried out in 500 mL shake flasks. At such conditions, 8 g/L glucose fed-batch fermentation, enhanced cell lysis and variants small colony (Vsm) polymorphism appearance. To overcome such limitations, glucose concentration should be maintained at 4 g/L. In this case, P. temperata cells were produced at high cell density and culturability reaching 4.5 and 1.2 × 10(9) cells/mL, respectively. In addition, the stability of the primary form was maintained for a long period in the stationary growth phase and Vsm polymorphism was completely avoided that can be crucial for scale-up the bioprocess of P. temperata bioinsecticide.

Differential oxidative stress responses to methanol in intraperitoneally exposed rats: ameliorative effects of Opuntia vulgaris fruit extract.

Methanol is primarily metabolized by oxidation to formaldehyde and then to formate. These processes are accompanied by formation of superoxide anion and hydrogen peroxide. This article reports data on the effect of methanol-induced oxidative damage in experimental rats and the role of aqueous extract of Opuntia vulgaris fruit extract (OE) to counteract the toxicity induced by methanol. The animals were exposed to methanol at a dose of 2.37 g/kg body weight intraperitoneally for 30 days. OE was found to contain large amounts of polyphenols and carotenoids and significant antioxidant capacities highlighted by scavenging activities for 2,2-diphenyl-l-picrylhydrazyl. The treatment with methanol exhibited a significant increase in serum hepatic and renal biochemical parameters (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase, bilirubin, urea, and creatinine). Methanol intoxication significantly increased hepatic and renal lipid peroxidation evaluated by thiobarbituric acid reactive substances in treated rats as compared to controls. However, hepatic and renal antioxidant enzymes namely superoxide dismutase, catalase, and glutathione peroxidase were significantly decreased in methanol-treated animals as compared to controls. The results concluded that treatment with OE prior to methanol intoxication has significant role in protecting animals from methanol-induced hepatic and renal histopathological and oxidative damage.

Correlation between synthesis variation of 2-alkylquinolones and the antifungal activity of a Burkholderia cepacia strain collection.

Twenty epiphytic and rhizospheric bacterial strains harbouring strong antifungal activities were isolated from the Tunisian environment. This group of bacteria was identified as Burkholderia cepacia genomovar I using 16S rDNA and recA fragment gene sequence analyses for two selected strains and RFLP technique for the eighteen other ones. This identification did not show variability between isolates despite the significant differences in the antifungal activities of their culture supernatant and the organic crude extract against Aspergillus niger and other phytopathogenic fungi. Chromatographic and mass spectrometric analyses of these extracts allowed us to confirm the difference between strains of the group. Their metabolic production showed differences in term of contents and quantities of secreted molecules, particularly those which were identified to be involved in the antifungal activities. Two metabolites, named Bc-255 and Bc-257 secreted by the entire group at different amounts, have been purified and tested separately against A. niger. Bc-255 showed an activity twice as high as those shown by Bc-257. The structural characterization of these two compounds by mass spectrometry and nuclear magnetic resonance spectroscopy allowed their identification as two analogous 2-alkylquinolones with only one difference at the alkyl chain.

Occurrence of Mycotoxins and Toxigenic Fungi in Cereals and Application of Yeast Volatiles for Their Biological Control.

Fungal infections in cereals lead to huge economic losses in the food and agriculture industries. This study was designed to investigate the occurrence of toxigenic fungi and their mycotoxins in marketed cereals and explore the effect of the antagonistic yeast Cyberlindnera jadinii volatiles against key toxigenic fungal strains. Aspergillus spp. were the most frequent contaminating fungi in the cereals, with an isolation frequency (Fr) of 100% in maize, followed by wheat (88.23%), rice (78.57%) and oats (14.28%). Morphological and molecular identification confirmed the presence of key toxigenic fungal strains in cereal samples, including A. carbonarius, A. flavus, A. niger, A. ochraceus and A. parasiticus. Aflatoxins (AFs) were detected in all types of tested cereal samples, with a significantly higher level in maize compared to wheat, rice, oats and breakfast cereals. Ochratoxin A (OTA) was only detected in wheat, rice and maize samples. Levels of mycotoxins in cereals were within EU permissible limits. The volatiles of Cyberlindnera jadinii significantly inhibited the growth of A. parasiticus, A. niger and P. verrucosum. The findings of this study confirm the presence of toxigenic fungi and mycotoxins in cereals within the EU permissible limits and the significant biocontrol ability of Cyberlindnera jadinii against these toxigenic fungi.

Detection of multimycotoxins in camel feed and milk samples and their comparison with the levels in cow milk.

Camel milk has been considered as an important source of nutrients and is commercialized in many countries of the world including the Middle East. This study aimed to investigate the presence of mycotoxins in camel feed and milk samples in comparison with the cow milk. Fumonisins (FUM), ochratoxin A (OTA), and zearalenone (ZEN) were detected in 14%, 39%, and 39% of the tested camel feed samples, respectively. Among the tested camel feed samples, 8.3% and 5.6% were co-contaminated with OTA+FUM and FUM+ZEN, respectively. In the case of milk samples, 46.15% of camel and 63.63% of cow were found contaminated with aflatoxin M1 (AFM1). In total, 16.2% and 8.1% of the milk samples were simultaneously contaminated with two and three mycotoxins, respectively. Although the levels of individual mycotoxins in the camel feed and milk samples were within the European Union (EU) permissible limits, their co-occurrence may pose severe risk to human and animal health due to possible additive and/or synergistic toxicities.

Biological control of Botrytis cinerea using the antagonistic and endophytic Burkholderia cepacia Cs5 for vine plantlet protection.

Antifungal activity of the Burkholderia cepacia Cs5 was tested in vitro and in vivo for the control of Botrytis cinerea . Bacterial biomass was significantly improved by the amendment of ZnSO(4), Mo(7)(NH(4))(6)O(24), and mannitol to the NBY medium; consequently, the amount of the secreted fungicides was increased. The quantification of B. cinerea inhibition, in liquid and solid conditions, showed an important sensitivity of this fungus to the strain Cs5 fungicides. Microscopic monitoring impact of these fungicides on mycelium structure showed an important increase in their diameter and ramifications in the presence of 0.75% supernatant. For the in vivo application of the strain Cs5, Vitis vinifera plantlets were inoculated with a Cs5 bacterial suspension, then with B. cinerea spores. The plantlets protection was total and durable when these two inoculations were made 3 weeks apart, which is the time for the endophytic bacterium to colonize the plantlets up to the top leaves. This protection is due to Cs5 antagonism and the elicitation of the plantlets self-defense via the root overgrowth.

Improvement of bioinsecticides production by sporeless Bacillus thuringiensis strains in response to various stresses in low cost medium.

The use of bioinsecticides, particularly those produced by sporeless Bacillus thuringiensis strains, has been shown to be a good alternative in pest management. Two types of sporeless mutants were distinguished. The asporogenic mutants which completely lack spores produce a regular bipyramidal crystal inclusion. The oligosporogenic mutants kept the ability to produce insecticidal crystal proteins. However, sporulation in such mutants was not totally blocked and very few of them could still produce spores. In order to improve bioinsecticides production, adaptation of sporeless strains to heat shock and osmotic stress was investigated. Delta-endotoxin production by 78% of sporeless mutants was significantly improved by osmotic stress with an overproduction of about 17%, compared to the wild strain BNS3. However, toxin production was improved by only 21% of mutants after heat shock, in low cost medium. The statistical analysis proved that delta-endotoxin production, cell growth, and spore formation of asporogenic and oligosporogenic mutants depended on the type of applied stress. Each strain has an important potential when applying the adequate stress. Moreover, adaptation of sporeless mutants to NaCl may allow the substitution of all minerals of the medium by diluted sea water which appeared to be a good alternative for the economic production of bioinsecticides at industrial scale which is of great importance from the practical point of view.