Mohamed Rabeh Hajlaoui

Biological control of grey mould in strawberry fruits by halophilic bacteria

Aims:  Grey mould caused by Botrytis cinerea is an economically important disease of strawberries in Tunisia and worldwide. The aim of this study was to select effective halophilic bacteria from hypersaline ecosystems and evaluate the abilities of antifungal bacteria to secrete extracellular hydrolytic enzymes, anti‐Botrytis metabolites and volatiles.

Pathway of deoxynivalenol-induced apoptosis in human colon carcinoma cells.

The mycotoxin, deoxynivalenol (DON), is generally detected in cereal grains and grain-based food products worldwide. Therefore, DON has numerous toxicological effects on animals and humans. The present investigation was conducted to determine the molecular aspects of DON toxicity on human colon carcinoma cells (HT 29). To this aim, we have monitored the effects of DON on (i) cell viability, (ii) Heat shock protein expressions as a parameter of protective and adaptive response, (iii) oxidative damage and (iv) cell death signalling pathway. Our results clearly showed that DON treatment inhibits cell proliferation, did not induce Hsp 70 protein expression and reactive oxygen species generation. We have also demonstrated that this toxin induced a DNA fragmentation followed by p53 and caspase-3 activations. Finally, our findings suggested that oxidative damage is not the major contributor to DON toxicity. This mycotoxin induces direct DNA lesions and could be considered by this fact as a genotoxic agent inducing cell death via an apoptotic process.

Trichothecene chemotypes of Fusarium culmorum infecting wheat in Tunisia.

Fusarium culmorum is a major pathogen associated with Fusarium head blight (FHB) of wheat in Tunisia. It may cause yield loss or produce mycotoxins in the grain. The objectives of the present study were threefold: to evaluate by PCR assays the type of mycotoxins produced by 100 F. culmorum isolates recovered from different regions in Northern Tunisia, to determine the amount of mycotoxin production by HPLC analysis, and to analyse for correlations between the amount of mycotoxin produced and the aggressiveness of isolates. PCR assays of Tri5, Tri7, Tri13, and Tri3 were used to predict whether these isolates could produce nivalenol, 3-acetyl-deoxynivalenol, or 15-acetyl-deoxynivalenol. Two of the isolates were predicted to produce NIV, whereas the others were predicted to produce 3-AcDON. Trichothecene production was confirmed and quantified by high pressure liquid chromatography (HPLC) in 28 isolates, after growth on wheat grains, and in a liquid Mycotoxin Synthetic medium (MS). All strains produced DON/3-AcDON at detectable levels ranging from 21 microg/g to 11.000 microg/g of dry biomass on MS medium and from 10 microg/g to 610 microg/g on wheat grain. The evaluation of the relationship between 3-AcDON production and aggressiveness of 17 strains revealed a significant difference in aggressiveness among the isolates. Moreover, only a significant correlation was revealed between aggressiveness and the amount of 3-AcDON produced on MS medium (r=0.36). Chemotyping of F. culmorum isolates is reported for the first time for isolates from Tunisia, and highlights the important potential of F. culmorum to contaminate wheat with 3-AcDON trichothecenes.

Involvement of mitochondria-mediated apoptosis in deoxynivalenol cytotoxicity

Deoxynivalenol (DON) is a widespread trichothecene mycotoxin which contaminates cereal crops and harmfully affects the gastrointestinal tract. Since it is well known that mitochondria play a central role in apoptosis triggered by many stimuli, an effort was made to examine whether DON-induced cytotoxicity occurs through mitochondria-mediated apoptotic pathway. The intestinal system being one of the primary targets of mycotoxins, the human colon carcinoma cell line HCT116 was used in this study. Using flow cytometric analyses and immunofluorescence, we showed that DON at 100 μM induced a mitochondria-dependent apoptotic pathway associated with opening of the mitochondrial permeability transition pore (PTP), loss of the mitochondrial transmembrane potential (ΔΨm), downstream generation of O₂·⁻ and cytochrome c release. The DON-induced apoptosis was accompanied by an activation of caspase 9 and 3, as demonstrated by Western blot and caspase activity assay. In addition, by taking advantage of HCT116 cells invalidated for Bax, we showed that this pro-apoptotic protein favored mitochondrial alterations induced by the mycotoxin. Besides, incubation of purified mitochondria with DON indicated that this mycotoxin does not directly target mitochondria to induce PTP-dependent permeabilization of mitochondrial membranes. Altogether, our results indicate that mitochondria-related caspase-dependent apoptotic pathway is involved in this in vitro model of DON induced-cytotoxicity.

Cell death induced by the Alternaria mycotoxin Alternariol.

Mycotoxins are unavoidable contaminants of most foods and feeds, and some are known to be detrimental to human health. It is thus worthwhile to understand how cells of the intestinal system, one of the primary targets of these toxins, respond to their toxic effects. In this study, human colon carcinoma cells were used to elucidate the cell death mode and the pathways triggered by Alternariol (AOH), the most important mycotoxin produced by Alternaria species, which are the most common mycoflora infecting small grain cereals worldwide. Treatment of cells with AOH resulted in a loss of cell viability by inducing apoptosis. AOH-induced apoptosis was mediated through a mitochondria-dependent pathway, characterized by a p53 activation, an opening of the mitochondrial permeability transition pore (PTP), a loss of mitochondrial transmembrane potential (ΔΨm), a downstream generation of O(2)(*-) and caspase 9 and 3 activation. Besides, deficiency of the pro-apoptotic protein Bax partially protected cells against AOH-induced mitochondrial alterations. In addition, experiments performed on purified mitochondria indicated that AOH does not directly target this organelle to induce cell death. Our results demonstrate for the first time that AOH-induced cytotoxicity is mediated by activation of the mitochondrial pathway of apoptosis in human colon carcinoma cells.

Genetic variability and population structure of the wheat foot rot fungus, Fusarium culmorum, in Tunisia

The random amplified polymorphic DNA (RAPD) method was used to investigate the genetic variability and population structure of Fusarium culmorum isolated from wheat stem bases. A total of 108 isolates, representing seven geographically distinct populations, was collected from five climatic regions in Tunisia. Pseudo-allelic frequencies were estimated at each of the 25 putative RAPD loci analyzed by scoring for the presence or absence of amplified fragments; 92 haplotypes were found among the 108 strains. The analysis of the population structure did not reveal any trend with regard to geographic origin. Total gene diversity (HT* = 0.318) was mostly attributable to diversity within populations (HS* = 0.308). Analysis of molecular variance confirmed that most of the genetic variability was within populations. Genetic differentiation among populations was low to moderate (GST* ranged from 0 to 0.190 and averaged 0.041 over all loci). Cluster analysis with UPGMA using genetic distances did not reveal any spatial clustering of the isolates collected from the different geographic regions. Based on these results, we conclude that the F. culmorum isolates recovered from different regions in Tunisia might be part of a single population pool.

In vitro investigation of toxicological interactions between the fusariotoxins deoxynivalenol and zearalenone

It is expected that humans are exposed to combined mycotoxins, which occur simultaneously in the food items, than to individual compounds and that can increase their potential toxicity. Considering this coincident production, deoxynivalenol (DON) and zearalenone (ZEN) as they are produced by several Fusarium species, can interfere at a cellular level. Therefore, these two toxins were chosen to study their interactive effects on human colon carcinoma cells (HCT116), using the endpoints including cell viability, cell cycle analysis, mitochondrial transmembrane potential (ΔΨm) determination and permeability transition pore (PTP) opening. Our results showed that DON and ZEN caused a marked decrease of cell viability in a dose-dependent manner, mediated by an activation of the mitochondrial apoptotic process; characterized by PTP opening and the loss of ΔΨm. Nevertheless, combined DON and ZEN reduced all the toxicities observed with the mycotoxins separately. Therefore, the combination of the two mycotoxins appears as a sub-additive response.

First report ofAlternaria species associated with black point of wheat in Tunisia

Durum wheat (Triticum turgidum) is one of the most important cereal crops in the world and the staple food for the Tunisian population. The main growing areas of durum wheat are located in the Northern part of the country, which is characterised by a sub humid climate favourable for fungal spoilage of grain such asAlternaria species. These species are able to produce plant pathogenic as well as toxic metabolites. For mycological analysis, the agar test was used. After mycelial growth, theAlternaria species were inoculated on potato carrot agar media. A PCR-based assay followed by a sequencing step allowed the identification ofAlternaria alternata; Alternaria tenuissima and Alternaria japonica from grains.

Mechanism of Alternariol monomethyl ether-induced mitochondrial apoptosis in human colon carcinoma cells.

Alternariol monomethyl ether (AME) is a major mycotoxin produced by fungi of the genus Alternaria and a common contaminant of food products such as fruits and cereals worldwide. AME can cause serious health problems for animals as well as for humans. In this study, human colon carcinoma cells (HCT116) were used to explore the mechanisms of cell death induced by AME. Exposure of HCT116 cells to AME resulted in significant cytotoxicity manifested by a loss in cell viability mainly mediated by activation of apoptotic process. AME activated the mitochondrial apoptotic pathway evidenced by the opening of the mitochondrial permeability transition pore (PTP), loss of the mitochondrial transmembrane potential (ΔΨm) downstream generation of O(2)(-), cytochrome c release and caspase 9 and 3 activation. Experiments conducted on isolated organelles indicated that AME does not directly target mitochondria to induce PTP-dependent permeabilization of mitochondrial membranes. Moreover, no difference was observed in Bax-KO cells in comparison to parental cells, suggesting that the pro-apoptotic protein Bax is not involved in AME-induced mitochondrial apoptosis. Our findings demonstrate for the first time that AME induces cell death in human colon carcinoma cells by activating the mitochondrial pathway of apoptosis.

Characterization and selection of Bacillus sp. strains, effective biocontrol agents against Fusarium oxysporum f. sp. radicis-lycopersici, the causal agent of fusarium crown and root rot in tomato.

The antagonistic activities of 20Bacillus isolates were tested with dual culture and greenhouse conditions againstFusarium oxysporum f. sp.Pseudomonas (FORL) race 0, the causal agent of Fusarium crown and root rot of tomato. Under dual culture, 10 isolates inhibited mycelial growth >38% and the most effective inhibited fungal growth >50%. The 20Bacillus isolates were tested for production of volatiles, cyanide, antibiotics, and phosphorus solubilisation; 15 isolates produced volatiles that inhibited growth of pathogens, 9 isolates produced cyanide, 10 produced antibiotics, and five solubilised phosphorus. Greenhouse experiments with the same 20 isolates revealed the effectiveness of 12 strains, which increased the percentage of healthy plants in the tested cultivar from 66 to 96%. The best disease control was achieved by isolates B11, B5, B17, and B18. However, B11 and B17 were the only isolates that produced cyanide, antibiotics, solubilised phosphate and showed 44% inhibition of fungal growth. The selected strains could be considered in plant growth promotion and biological disease control.