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.

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.

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.

Prevalence of toxigenic fungi and mycotoxins in Arabic coffee (Coffea arabica): Protective role of traditional coffee roasting, brewing and bacterial volatiles.

Fungal infection and synthesis of mycotoxins in coffee leads to significant economic losses. This study aimed to investigate the prevalence of toxigenic fungi, their metabolites, and the effect of traditional roasting and brewing on ochratoxin A (OTA) and aflatoxins (AFs) contents of naturally contaminated coffee samples. In addition, in vivo biocontrol assays were performed to explore the antagonistic activities of Bacillus simplex 350-3 (BS350-3) on the growth and mycotoxins synthesis of Aspergillus ochraceus and A. flavus. The relative density of A. niger, A. flavus, Penicillium verrucosum and A. carbonarius on green coffee bean was 60.82%, 7.21%, 3.09% and 1.03%, respectively. OTA contents were lowest in green coffee beans (2.15 µg/kg), followed by roasted (2.76 µg/kg) and soluble coffee (8.95 µg/kg). Likewise, AFs levels were highest in soluble coffee (90.58 µg/kg) followed by roasted (33.61 µg/kg) and green coffee (9.07 µg/kg). Roasting naturally contaminated coffee beans at three traditional methods; low, medium and high, followed by brewing resulted in reduction of 58.74% (3.50 µg/kg), 60.88% (3.72 µg/kg) and 64.70% (4.11 µg/kg) in OTA and 40.18% (34.65 µg/kg), 47.86% (41.17 µg/kg) and 62.38% (53.73 µg/kg) AFs contents, respectively. Significant inhibitions of AFs and OTA synthesis by A. flavus and A. carbonarius, respectively, on infected coffee beans were observed in presence of Bacillus simplex BS350-3 volatiles. Gas chromatography mass spectrochemistry (GC-MS/MS) analysis of head-space BS350-3 volatiles showed quinoline, benzenemethanamine and 1-Octadecene as bioactive antifungal molecules. These findings suggest that marketed coffee samples are generally contaminated with OTA and AFs, with a significant level of roasted and soluble coffee contaminated above EU permissible limits for OTA. Further, along with coffee roasting and brewing; microbial volatiles can be optimized to minimize the dietary exposure to mycotoxins.

Biocontrol Activity of Bacillus megaterium BM344-1 against Toxigenic Fungi.

Mycotoxins are secondary metabolites of some fungal species and represent important contaminants of food and feed. This study aimed to explore the biological control activity of Bacillus megaterium BM344-1 volatile organic compounds (VOCs) on the growth and mycotoxin production of single representatives of the toxigenic species Aspergillus flavus, Aspergillus carbonarius, Penicillium verrucosum, and Fusarium verticillioides. In vitro co-incubation experiments indicated the P. verrucosum isolate as the most sensitive one, with a growth inhibition ratio of 66.7%, followed by A. flavus (29.4%) and F. verticillioides (18.2%). Exposure of A. flavus, P. verrucosum, and F. verticillioides to BM344-1 VOCs resulted in complete inhibition of aflatoxins (AFB1, AFG1, and AFG2), ochratoxin A, and fumonisin B1 (FB1) synthesis on artificial media, respectively. In vivo experiments on maize kernels showed 51% inhibition of fungal growth on ears simultaneously infected with A. flavus spores and exposed to BM344-1 volatiles. Likewise, AF synthesis by A. flavus was significantly (p < 0.05) inhibited (25.34 ± 6.72 µg/kg) by bacterial volatiles as compared to that in control maize ears (91.81 ± 29.10 µg/kg). Gas chromatography-tandem mass spectrometry-based analysis of headspace volatiles revealed hexadecanoic acid methyl ester (palmitic acid) and tetracosane as bioactive compounds in the BM344-1 volatilome. Bacterial volatiles have promising potential to control the growth and mycotoxin synthesis of toxigenic fungi and may present valuable aid in the efforts to warrant food and feed safety.

Bacillus thuringiensis strains isolated from Qatari soil, synthesizing δ-endotoxins highly active against the disease vector insect Aedes aegypti Bora Bora.

Bacillus thuringiensis (Bt) is a Gram-positive soil bacterium that has been recognized as an effective bioinsecticide active against plant, animal and human pathogenic and disease vector insects. During its sporulation phase, Bt produces crystals consisting of δ-endotoxins, which upon ingestion kill specifically insect larvae. Bt subsp. israelensis (Bti) is very active against dipteran insects. Bti based bioinsecticides are considered as a sustainable solution to control the Dipteran insects responsible of plant, animal and human diseases. In this study, Bti strains isolated from Qatar soil were analyzed for their insecticidal activities against the dipteran insect Aedes aegypti Bora Bora (Culicidae, Diptera) and for their δ-endotoxins yields per cell. Among the local Bti strains, four exceptional strains producing spherical crystals, were found to be more insecticidal than the reference strain Bti H14. When tested for their δ-endotoxin yield, the Bti QBT217 strain, producing typical spherical crystals and having the best insecticidal activity, was recognized as the best candidate strain for potential bioinsecticide production and biological control of dipteran insects, particularly the disease vector insect A. aegypti.

Potential for native hydrocarbon-degrading bacteria to remediate highly weathered oil-polluted soils in Qatar through self-purification and bioaugmentation in biopiles.

Petroleum-derived contamination events constitute a unique environmental issue in the arid areas because of the weathering processes, accentuated due to harsh conditions and representing the main origin of failure of bioremediation applications. The industrial area of Dukhan and the AlZubara coast represent wethered oily-sites as an appropriate model due to the extreme weather, which characterizes Qatar. Indigenous bacteria would be highly adapted and when re-introduced or stimulated would conduct to the remediation of these sites. An appropriate approach to bioremediating-weathered oil was investigated in these two areas. Systematic soil sampling was performed, and the soil samples were analyzed. The results clearly showed the harsh chemical compositions (high salinity and contents of total petroleum components contents in the range of the oil organics). By implementation of a procedure of isolation of highly adapted bacteria, few hydrocarbon-degrading bacteria were isolated in the objective of selecting those for further application. Their identification by ribotyping showed dominance of Bacillus and Virgibacillus in AlZubara site, and Bacillus and Pseudomonas in Dukhan site. All bacterial strains were highly tolerant to 10 % diesel and showed potential of removal of 20 %-85 % of C 10 to C 36 hydrocarbons. This potential was confirmed by determination of the n-heptadecane/pristane and n-octadecane/phytane ratios, indicating in turn their potential to bioremediate oil. This was demonstrated by comparison of the self-remediation to bioaugmentation using two selected Bacillus and Pseudomonas strains using Dukhan soil in biopiles. These strains when biostimulted in such a soil increased the removal of total petroleum components to 53 % compared to 30 % with self-purification, after 90 d. The results indicated that highly adapted endogenous bacteria might be used to bioremediate highly weathered oil-contaminated soil under harsh conditions.

Isolation of a Novel Kluyveromyces marxianus Strain QKM-4 and Evidence of Its Volatilome Production and Binding Potentialities in the Biocontrol of Toxigenic Fungi and Their Mycotoxins.

To overcome the economic losses associated with fungi and their toxic metabolites, environmentally safe and efficient approaches are needed. To this end, biological control using yeasts and safe bacterial strains and their products are being explored to replace synthetic fungicides. In the present study, the biocontrol effect of a yeast strain of Kluyveromyces marxianus, QKM-4, against the growth and mycotoxin synthesis potential of key toxigenic fungi was evaluated. In vitro assays were performed to find the application of yeast volatile organic compounds (VOCs) against fungal contamination on important agricultural commodities. The removal of ochratoxin A (OTA) and deoxynivalenol (DON) by living and heat-inactivated yeast cells was also explored. VOCs produced by strain QKM-4 were able to significantly limit the fungal growth of 17 fungal species belonging to genera Aspergillus, Penicillium, and Fusarium. Yeast VOCs were able to reduce OTA biosynthesis potential of Penicillium verrucosum and Aspergillus carbonarius by 99.6 and 98.7%, respectively. In vivo application of QKM-4 VOCs against Fusarium oxysporum and A. carbonarius infection on tomatoes and grapes, respectively, determined a complete inhibition of fungal spore germination. GC/MS-based analysis of yeast VOCs identified long-chain alkanes, including nonadecane, eicosane, docosane, heptacosane, hexatriacontane, and tetracosane. In vitro testing of the mycotoxin-binding potential of the living and heat-inactivated QKM-4 cells showed a reduction of OTA and DON up to 58 and 49%, respectively, from artificially contaminated buffers. Our findings clearly demonstrate the strong antifungal potential of K. marxianus QKM-4 and propose this strain as a strong candidate for application in agriculture to safeguard food and feed products.

Influence of temperature, salinity and Mg2+:Ca2+ ratio on microbially-mediated formation of Mg-rich carbonates by Virgibacillus strains isolated from a sabkha environment.

Studies have demonstrated that microbes facilitate the incorporation of Mg2+ into carbonate minerals, leading to the formation of potential dolomite precursors. Most microbes that are capable of mediating Mg-rich carbonates have been isolated from evaporitic environments in which temperature and salinity are higher than those of average marine environments. However, how such physicochemical factors affect and concur with microbial activity influencing mineral precipitation remains poorly constrained. Here, we report the results of laboratory precipitation experiments using two mineral-forming Virgibacillus strains and one non-mineral-forming strain of Bacillus licheniformis, all isolated from the Dohat Faishakh sabkha in Qatar. They were grown under different combinations of temperature (20°, 30°, 40 °C), salinity (3.5, 7.5, 10 NaCl %w/v), and Mg2+:Ca2+ ratios (1:1, 6:1 and 12:1). Our results show that the incorporation of Mg2+ into the carbonate minerals is significantly affected by all of the three tested factors. With a Mg2+:Ca2+ ratio of 1, no Mg-rich carbonates formed during the experiments. With a Mg2+:Ca2+ ratios of 6 and 12, multivariate analysis indicates that temperature has the highest impact followed by salinity and Mg2+:Ca2+ ratio. The outcome of this study suggests that warm and saline environments are particularly favourable for microbially mediated formation of Mg-rich carbonates and provides new insight for interpreting ancient dolomite formations.

In-vitro Application of a Qatari Burkholderia cepacia strain (QBC03) in the Biocontrol of Mycotoxigenic Fungi and in the Reduction of Ochratoxin A biosynthesis by Aspergillus carbonarius.

Mycotoxins are secondary metabolites produced by certain filamentous fungi, causing human and animal health issues upon the ingestion of contaminated food and feed. Among the safest approaches to the control of mycotoxigenic fungi and mycotoxin detoxification is the application of microbial biocontrol agents. Burkholderiacepacia is known for producing metabolites active against a broad number of pathogenic fungi. In this study, the antifungal potential of a Qatari strain of Burkholderia cepacia (QBC03) was explored. QBC03 exhibited antifungal activity against a wide range of mycotoxigenic, as well as phytopathogenic, fungal genera and species. The QBC03 culture supernatant significantly inhibited the growth of Aspergillus carbonarius, Fusarium culmorum and Penicillium verrucosum in PDA medium, as well as A. carbonarius and P. verrucosum biomass in PDB medium. The QBC03 culture supernatant was found to dramatically reduce the synthesis of ochratoxin A (OTA) by A. carbonarius, in addition to inducing mycelia malformation. The antifungal activity of QBC03's culture extract was retained following thermal treatment at 100 °C for 30 min. The findings of the present study advocate that QBC03 is a suitable biocontrol agent against toxigenic fungi, due to the inhibitory activity of its thermostable metabolites.

Investigation and Application of Bacillus licheniformis Volatile Compounds for the Biological Control of Toxigenic Aspergillus and Penicillium spp.

The present study was designed to investigate the antagonistic activity of Bacillus licheniformis BL350-2 against mycotoxigenic strains of Aspergillus and Penicillium. In vitro coincubation for 5 days indicated Aspergillus westerdijkiae BA1 as the most sensitive strain, with a growth inhibition of 62%, followed by A. carbonarius MG7 (60%), Penicillium verrucosum MC12 (53%), A. niger MC05 (50%), A. flavus CM5 (49%), A. parasiticus SB01 (47%), and A. ochraceus MD1 (44%). Likewise, the majority of the tested strains on exposure to bacterial volatiles showed complete inhibition of mycotoxin synthesis. In vivo assays on maize ears resulted in 88% reduction in A. flavus CM5 growth and complete inhibition of fungal sporulation and aflatoxin accumulation. The GC-MS-based volatile profile showed 3-methyl-1-butanol as the most abundant compound. The findings of the present study advocate that B. licheniformis BL350-2 is suitable as a biocontrol agent against mycotoxigenic fungi, at least during storage of cereal grains.

A proteomic investigation of Aspergillus carbonarius exposed to yeast volatilome or to its major component 2-phenylethanol reveals major shifts in fungal metabolism.

The use of yeast-derived volatile organic compounds (VOCs) represents a promising strategy for the biological control of various plant pathogens, including mycotoxin-producing fungi. Previous studies demonstrated the efficacy of the low-fermenting yeast Candida intermedia isolate 253 in reducing growth, sporulation, and ochratoxin A biosynthesis by Aspergillus carbonarius MPVA566. This study aimed to investigate whether the inhibitory effect of the yeast volatilome is solely attributable to 2-phenylethanol, its major component, or if a synergistic effect of all volatilome components is required to achieve an effective control of the fungal growth and metabolism. Microbiological methods, HPLC measurements and a UPLC-MS/MS approach were used to investigate the metabolic profile of A. carbonarius MPVA566 at different growing conditions: standard incubation (control), exposed to C. intermedia 253 volatilome, and incubation in the presence of 2-phenylethanol. Both yeast volatilome and 2-phenylethanol succeeded in the macroscopic inhibition of the radial mycelial growth, along with a significant reduction of ochratoxin A production. Functional classification of the fungal proteome identified in the diverse growing conditions revealed a different impact of both yeast VOCs and 2-phenylethanol exposure on the fungal proteome. Yeast VOCs target an array of metabolic routes of fungal system biology, including a marked reduction in protein biosynthesis, proliferative activity, mitochondrial metabolism, and particularly in detoxification of toxic substances. Exposure to 2-phenylethanol only partially mimicked the metabolic effects observed by the whole yeast volatilome, with protein biosynthesis and proliferative activity being reduced when compared with the control samples, but still far from the VOCs-exposed condition. This study represents the first investigation on the effects of yeast-derived volatilome and 2-phenylethanol on the metabolism of a mycotoxigenic fungus by means of proteomics analysis. CHEMICAL COMPOUNDS STUDIED OR USED IN THIS ARTICLE: 2-Phenylethanol (PubChem CID: 6054); ochratoxin-A (PubChem CID: 442530); sodium dodecyl sulfate (PubChem CID: 3423265); dithiothreitol (PubChem CID: 446094); phenylmethylsulfonyl fluoride (PubChem CID: 4784); iodoacetamide (PubChem CID: 3727); ammonium bicarbonate (PubChem CID: 14013); acetic acid (PubChem CID: 176); and acetonitrile (PubChem CID: 6342).

The combinatory effect of Cyt1Aa flexibility and specificity against dipteran larvae improves the toxicity of Bacillus thuringensis kurstaki toxins.

Cyt1A98 is a novel cytolytic protein, from BUPM98 Bacillus thuringiensis strain, characterized by its synergistic activity with B. thuringiensis kurstaki toxins against lepidopteran larvae. In this study, we evidenced that Cyt1A98 improves the toxicity of B. thuringiensis kurstaki toxins against Aedes aegypti larvae. In fact, the strain BNS3pHTcyt1A98 exhibited a larvicidal activity of about 849-fold of that of BNS3pHTBlue against A. aegypti. The molecular and biochemical characterizations, of cyt1A98 gene and its product, were achieved. Cyt1A98 had an LC50 value of about 126.56 mg l-1 against A. aegypti larvae. Compared to Cyt1Aa of B. thuringiensis israelensis, Cyt1A98 amino acid sequence harbours three substitutions of three conserved amino acids among Cyt1Aa family members (Ser42Pro, Pro82Ala, Met188Thr). The Cyt1A98 protein structural analysis evidenced more flexibility than Cyt1Aa. According to the high fluctuation observed for the residue Pro42, the amino acid at position 42 is implicated in the flexibility property of Cyt1Aa especially for the αC and αD helices, involved in the penetration into the cell membrane. The toxicity improvement could be probably due to the higher flexibility combined with the specific affinity toward dipteran larvae. The Cyt1A/B. thuringiensis kurstaki Cry toxins model provides a potential molecular genetic strategy for an efficient bioinsecticide.

Effect of yeast volatile organic compounds on ochratoxin A-producing Aspergillus carbonarius and A. ochraceus.

Many foods and beverages in temperate and tropical regions are prone to contamination by ochratoxin A (OTA), one of the most harmful mycotoxins for human and animal health. Aspergillus ochraceus and Aspergillus carbonarius are considered among the main responsible for OTA contamination. We have previously demonstrated that four low or non- fermenting yeasts are able to control the growth and sporulation of OTA-producing Aspergilli both in vitro and on detached grape berries: the biocontrol effect was partly due to the release of volatile organic compounds (VOCs). Aiming to further characterise the effect of VOCs produced by biocontrol yeast strains, we observed that, beside vegetative growth and sporulation, the volatile compounds significantly reduced the production of OTA by two A. carbonarius and A. ochraceus isolates. Exposure to yeast VOCs also affected gene expression in both species, as confirmed by downregulation of polyketide synthase, non-ribosomal peptide synthase, monooxygenase, and the regulatory genes laeA and veA. The main compound of yeast VOCs was 2-phenylethanol, as detected by Headspace-Solid Phase Microextraction-Gas Chromatography-Tandem Mass Spectrometry (HS-SPME-GC-MS) analysis. Yeast VOCs represent a promising tool for the containment of growth and development of mycotoxigenic fungi, and a valuable aid to guarantee food safety and quality.

Application of Low-Fermenting Yeast Lachancea thermotolerans for the Control of Toxigenic Fungi Aspergillus parasiticus, Penicillium verrucosum and Fusarium graminearum and Their Mycotoxins.

Mycotoxins are important contaminants of food and feed. In this study, low fermenting yeast (Lachancea thermotolerans) and its derivatives were applied against toxigenic fungi and their mycotoxins. A. parasiticus, P. verrucosum and F. graminearum and their mycotoxins were exposed to yeast volatile organic compounds (VOCs) and cells, respectively. VOCs reduced significantly the fungal growth (up to 48%) and the sporulation and mycotoxin synthesis (up to 96%). Very interestingly, it was shown that even 7 yeast colonies reduced Fusarium’s growth and the synthesis of its mycotoxin, deoxynivalenol (DON). Moreover, decreasing yeast nutrient concentrations did not affect the inhibition of fungal growth, but reduced DON synthesis. In addition, inactivated yeast cells were able to remove up to 82% of the ochratoxin A (OTA). As an application of these findings, the potentialities of the VOCs to protect tomatoes inoculated with F. oxysporum was explored and showed that while in the presence of VOCs, no growth was observed of F. oxysporum on the inoculated surface areas of tomatoes, in the absence of VOCs, F. oxysporum infection reached up to 76% of the tomatoes’ surface areas. These results demonstrate that the application of yeasts and their derivatives in the agriculture and food industry might be considered as a very promising and safe biocontrol approach for food contamination.

Use of ß-cyclodextrin as enhancer of ascorbic acid rejection in permselective films for amperometric biosensor applications.

Interference rejection in amperometric biosensors can be more effective introducing some modifiers during electro-deposition of permselective film. Addition of ß-cyclodextrin (ßCD), a cyclic oligosaccharide composed of seven glucose units, to the ortho-phenylendiamine (oPD) monomer were already demonstrated to provide an enhancement in ascorbic acid (AA) rejection. Here we evaluated the improvement in permselectivity of poly-eugenol and poly-magnolol films electro-polymerized in presence of different amounts of ßCD or eugenol-ßCD inclusion complex for amperometric biosensor application. Starting from Pt-Ir wire as transducer several microsensors were covered with polymeric films doped with ßCD-based modifiers through constant potential amperometry. Characterization of modified polymers was achieved by scanning electron microscopy and permselectivity analysis. Poly-magnolol film in combination with ßCD showed a worsening in permselectivity compared to poly-magnolol alone. In contrast, the introduction of ßCD-based modifier enhanced the interference rejection toward the archetypal interferent AA, while slightly affecting permeability toward H2O2 compared to the poly-eugenol without modifier. The AA rejection seems to be influenced by the availability of ßCD cavity as well as film performance due to concentration of ßCD-Eugenol inclusion complex. A poly-eugenol film co-polymerized with 2 mM ßCD-eugenol inclusion complex showed a permselectivity equal to poly-orthophenylendiamine film (PPD), with a lower permeability to AA, likely to be related with a self-blocking mechanism. Based on these results, a biosensor for glutamate was constructed with a poly-eugenol doped with ßCD-eugenol as permselective layer and its permselectivity, stability and lifetime were determined.

Diversity of Bacillus thuringiensis Strains From Qatar as Shown by Crystal Morphology, δ-Endotoxins and Cry Gene Content.

Bacillus thuringiensis (Bt) based insecticidal formulations have been recognized as one of the most successful, environmentally safe and sustainable method of controlling insect pests. Research teams worldwide are in search of Bt diversity giving more choices of bio-insecticides and alternatives to address insect resistance. In fact, there are many unexplored ecologies that could harbor novel Bt strains. This study is the first initiative to explore Bt strain diversity in Qatar. A collection of 700 Bt isolates was constructed. Scanning electron microscopy of Bt crystals showed different crystal forms, with a high abundance of spherical crystals compared to the bipyramidal ones. Among the spherical crystals, four different morphologies were observed. The δ-endotoxin content of parasporal crystals from each Bt isolate revealed that there are 16 different protein profiles among the isolates of the collection. On the other hand, plasmid pattern analysis showed seven different plasmid profiles. Their insecticidal activity was predicted by exploring the δ-endotoxin coding genes and conducting qualitative insecticidal bioassays. 19 smooth spherical crystal producing isolates have been identified that could be possible candidates for endotoxin production targeting Dipteran insects. Another group of 259 isolates producing bipyramidal and cuboidal crystals could target Lepidopteran and Coleopteran insects. The remaining 422 isolates have novel profiles. In conclusion, Qatari soil ecology provides a good collection and diversity of Bt isolates. In addition to strains harboring genes encoding common endotoxins, the majority are different and very promising for the search of novel insecticidal endotoxins.

The Replacement of five Consecutive Amino Acids in the Cyt1A Protein of Bacillus thuringiensis Enhances its Cytotoxic Activity against Lung Epithelial Cancer Cells.

Cyt1A protein is a cytolytic protein encoded by the cyt gene of Bacillus thuringiensis subsp. israelensis (Bti) as part of the parasporal crystal proteins produced during the sporulation. Cyt1A protein is unique compared to the other endotoxins present in these parasporal crystals. Unlike δ-endotoxins, Cyt1A protein does not require receptors to bind to the target cell and activate the toxicity. It has the ability to affect a broad range of cell types and organisms, due to this characteristic. Cyt1A has been recognized to not only target the insect cells directly, but also recruit other endotoxins by acting as receptors. Due to these mode of actions, Cyt1A has been studied for its cytolytic activity against human cancer cell lines, although not extensively. In this study, we report a novel Cyt1A protein produced by a Bti strain QBT229 isolated from Qatar. When tested for its cytotoxicity against lung cancer cells, this local strain showed considerably higher activity compared to that of the reference Bti and other strains tested. The possible reasons for such enhanced activity were explored at the gene and protein levels. It was evidenced that five consecutive amino acid replacements in the ß8 sheet of the Cyt1A protein enhanced the cytotoxicity against the lung epithelial cancer cells. Such novel Cyt1A protein with high cytotoxicity against lung cancer cells has been characterized and reported through this study.