Najla Sadfi-Zouaoui

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.

Characterization of a novel chitinase from a moderately halophilic bacterium, Virgibacillus marismortui strain M3-23

A new chitinase produced by the moderately halophilic bacterium Virgibacillus marismortui strain M3-23 was identified and characterized. Distinguishable characteristics of high activity and stability at different pH, temperatures and salinity of M3-23 chitinase are reported. Analysis of the catalytic domain sequence from the enzyme highlighted its relationship to glycosyl hydrolase family 18. Comparison of the deduced chitinase sequence from strain M3-23 to known chitinases from Bacillus species showed low similarity (82%), suggested its novelty. This is the first report of the characterization of chitinase from the species V. marismortui. The halo- and thermo-tolerant nature of the chitinolytic enzyme allows its potential use in agricultural and industrial applications.

Antimicrobial Behavior of Intracellular Proteins from Two Moderately Halophilic Bacteria: Strain J31 of Terribacillus halophilus and Strain M3-23 of Virgibacillus marismortui

In the present study, we firstly aimed to determine the ability of halophilic bacteria to improve tomato growth. as well as to detect the antimicrobial activities from two moderately halophilic bacteria strain M3-23 of Virgibacillus marismortui and strain J31 of Terribacillus halophilus exhibited by their intracellular proteins. The results showed that both bacteria were able to improve stem tomato growth by comparison of untreated tomato. The halophilic bacteria were also able to produce intracellular antifungal enzyme: glucanase produced by V. marismortui (1.74U/mg) and chitinase (39.39U/mg) produced by T. halophilus. Both chitinases were halotolerants (active in the presence of (0% to 30% NaCl (w/v)). Chitinase produced by strain J31 was alcaline (pH optimum pH 12), but chitinase from strain M3- 23 was acidic (pH 4 optimum) more than 90% and 80% of activities were retained in the presence of pH value from 4 to 12, respectively for strain J31 and M3-23. Both enzymes were thermotolerants; optimum temperature was 80°C and 90°C respectively for strain J31 and strain M3-23. Both strains have lysozyme activity and value ranging from 6.6 U/ml to 6.8 U/ml respectively for strain J31 and strain M3-23. On the whole, the most potent in vitro antifungal effect was demonstrated by intracellular compound produced by strain J31 compared to strain M3-23. This study, was the first showing the antimicrobial efficiency of moderately halophilic bacteria by means of their intracellular compounds, by means of the spore germination reduction and the destroy of mycelial growth of Botrytis cinerea, in vitro. The distinguishable characteristics of their intracellular halotolerant and thermotolerant chitinases make them as good candidates for biotechnological applications.

First report on the use of moderately halophilic bacteria against stem canker of greenhouse tomatoes caused byBotrytis cinerea

Experiments were conducted to study the effect of moderately halophilic bacteria isolated from different Tunisian Sebkhas (hypersaline soils), on stem canker caused byBotrytis cinerea on tomato plants grown under greenhouse conditions. Treatments performed with moderately halophilic isolates ofBacillus subtilis J9 andHalomonas sp. K2-5 significantly reduced stem lesion expansion byB. cinerea on tomato plants under greenhouse conditions. The use of such bacteria may constitute an important alternative to synthetic fungicides, which failed to suppress the development of the fungal pathogen.

Genetic diversity assessed by SSR markers and chemotyping of Fusarium culmorum causal agent of foot and root rot of wheat collected from two different fields in Tunisia

Fusarium culmorum is a major pathogen able to cause foot and root rot and the incitant of Fusarium head blight in wheat in Tunisia. The aims of the present study were to evaluate by PCR the type of mycotoxins produced, to determine the mating type and to analyse the genetic diversity by microsatellite markers of 82 F. culmorum isolates recovered from two separated Tunisian fields. Specific sequences in the Tri6-Tri5 intergenic region, Tri7 and Tri13 were used to identify 3-AcDON- or 15-AcDON-. All studied F. culmorum isolates, were of the 3-AcDON- type. No 15-AcDON- and NIV types were detected in this research. Both mating types MAT1-1 and MAT1-2 were recovered from the two fields in approximately equal proportions. Five polymorphic microsatellite markers were applied to F. culmorum isolates, to determine the genetic variation in and among populations. Sixty-four haplotypes were identified; the analysis of the population structure did not reveal a strong variation between fields. Total gene diversity (HT = 0.505; HS = 0.497) and analysis of molecular variance confirmed that most of the genetic variability was within populations (ΦST = 0.033; P < 0.0039). Gene flow (Nm = 31.05) indicated little differentiation among populations. Based on these results, the F. culmorum isolates collected from different fields might be part of one large panmictic population and in addition the low linkage disequilibrium values with high genetic variation within populations suggest that the population is recombining sexually.

Prevalence, Etiology, and Risk Factors of Tinea Pedis and Tinea Unguium in Tunisia

Background Foot mycoses are a frequent disease that represents a public health problem worldwide. Objectives This study aims to evaluate the epidemiology of foot mycoses among Tunisian patients, in order to determine the fungal etiological agents and to identify possible risk factors. Patients and Methods A prospective study of three hundred and ninety-two patients was undertaken during one year (2013-2014). All subjects were asked to collect demographic data related to the risk factors of foot mycoses. A complete mycological diagnosis was carried out on all patients. Results A total of 485 samples were collected; tinea pedis and tinea unguium were confirmed in 88.2% of cases. Dermatophytes were isolated in 70.5% and the most frequent pathogen was Trichophyton rubrum (98.1%), followed by yeasts (17.7%) commonly Candida parapsilosis. Non-dermatophyte molds (NDMs) were observed in 8.02% cases and Fusarium sp. was the frequent genus (29.1%). The main predisposing factors of fungal foot infections were practicing ritual washing (56.6%) and frequentation of communal showers (50.5%). Conclusion This is a recent survey of foot mycoses in Tunisia. Epidemiological studies can be useful to eradicate these infections and to provide further measures of hygiene and education.

High salt-tolerant protease from a potential biocontrol agent Bacillus pumilus M3-16

In this paper, we investigate the characterization and evaluation of the antifungal protease activity from a halotolerant strain M3-16 ofBacillus pumilus, earlier isolated from a shallow salt lake in Tunisia. Protease enzyme was highly induced by the pathogen testedin vitro (27.4 U/ml). This is the first report on high salt-tolerant protease fromB. Pumilus, since it was active at high salinity (from 5 to 30% NaCl, w/v) as well as in the absence of salinity. This enzyme showed optimal activity at 60 °C and pH 8. At 80 °C and 30 min, the enzyme retained up to 91% and it showed stability over a wide pH range (from pH 5 to 11). The enzyme was found to be monomer with an estimated molecular mass of 31 kDa. The amino acid sequence showed high similarity (94%) to ATP-dependent protease fromB. Pumilus strain ATCC 7061. Thus, our alkaline thermostable and high salt-tolerant protease induced by a phytopathogenic fungus, could be useful for application in diverse areas such as biotechnology alimentary and agronomy industries.

Analysis of the diversity of Trichoderma spp. in soil horizons using digested ITS regions

We investigated the occurrence ofTrichoderma in the Oueslatia forest located in the centre-west of Tunisia, which represents a good example for studying a distribution ofTrichoderma between different soil horizons of a forest with Mediterranean bioclimate. One hundred-five isolates obtained were identified at the genus level by analysis of morphological characters. The diversity ofTrichoderma between soil horizons was evaluated by analysis of the internal transcribed spacer (ITS) of the rDNA region using restriction fragment length polymorphism (RFLP). The ITS region was first amplified by polymerase chain reaction (PCR) with specific primers and then cleaved with two restriction enzymes. Amplification products showed extensive length polymorphism and RFLP analysis generated bands ranging from 100 to 620 bp. The restriction profiles of the PCR products showed thatHaeIII enzyme has more discriminatory power thanRsaI. A high degree of polymorphism was detected in rhizosphere rather than soil. Thus the RFLP analysis produced different DNA profiles on the gels denoting significant intraspecific genetic variation. Our data confirm the potential of ITS region PCR-RFLP for the study of polymorphism amongTrichoderma population from different soil layers.

Synthesis, crystal structure and potential antimicrobial activities of di (4-sulfamoyl-phenyl-ammonium) sulphate

A new organic-inorganic hybrid SO4[C6H9N2O2S]2, has been synthesized and characterized by X-ray diffraction. This compound crystallizes in the orthorhombic system, spaces group Pbcn. In the title compound, the packing is stabilized by intermolecular N-H⋯O hydrogen bonds and π-π stacking interactions between the phenyl rings, linking the molecules into three-dimensional network. The in vitro antimicrobial activity of di (4-sulfamoyl-phenyl-ammonium) sulphate was determined by the broth dilution method against several strains selected to define their spectrum and potency. Here we show that the synthetic sulfanilamide exhibits promising antibacterial potency. High inhibition was also detected against Candida albicans. In this paper we firstly showed the antifungal activity of the sulfanilamide against two serious phytopathogenic fungi. Interestingly, the new compound was able to suppress mycelial growth as well as the spores germination of tested fungi, values of spore germination vary from 97.6% to 37.5%, respectively for Botrytis cinerea and Fusarium species. The minimal inhibitory concentrations (MIC) ranging from 8 to 100 μg ml(-1) and IC50 values varying from 5.81 to less than 100 μg ml(-1)), showed that the sulfanilamide sulphate had high activity against bacteria, yeast and fungi, compared to others published antifungal compounds.

In vitro biofilms and antifungal susceptibility of dermatophyte and non‐dermatophyte moulds involved in foot mycosis

Tinea pedis and onychomycosis are among the commonest fungal diseases in the world. Dermatophytes and, less frequently, non‐dermatophyte moulds are aetiological agents of foot mycosis and are capable of forming biofilms. Fungal biofilm has demonstrated increasing drug resistance. This work aims to evaluate, in vitro, the ability to form biofilm and the susceptibility to antifungal drugs of sessile dermatophytes and non‐dermatophyte moulds involved in foot mycosis. Thirty‐six dermatophytes and non‐dermatophyte moulds isolated from Tunisian patients with foot mycoses, and identified with MALDI‐TOF have been tested. MICs of fluconazole, econazole, itraconazole, terbinafine and griseofulvin were carried out using CLSI broth microdilution method. The ability to form biofilm and antifungal activities of drugs against fungal biofilm formation has been quantified by Crystal Violet and Safranin Red staining. Biomass quantification revealed that all species studied were able to form biofilms in vitro after 72 hours. Fluconazole, econazole, itraconazole and terbinafine inhibited fungal growth with MIC values ranging from 0.031 to >64 μg mL−1. The best antifungal activity has been obtained with terbinafine against Fusarium solani. Econazole showed the highest activity against fungal biofilm formation. These findings can help clinicians to develop the appropriate therapy of foot mycosis.