Tunisian Regions Research Articles

Phenotypic and genetic characterization of Pseudomonas syringae strains associated with the recent citrus bacterial blast and bacterial black pit epidemics in Tunisia

In the spring of 2012, symptoms of a disease resembling citrus blast and citrus black pit were observed in some orchards in Tunisia. The epidemic spread rapidly in the following years. Twenty‐four commercial citrus orchards from four Tunisian regions showing characteristic symptoms of bacterial diseases were surveyed during a 3‐year study. Eighty‐eight Pseudomonas‐like bacterial isolates were successfully obtained from the northeast and west of Tunisia. No isolates were recovered from the central region. Overall, 46 isolates were identified as Pseudomonas syringae pv. syringae and most of them showed similar phenotypic and genetic profiles. The virulence of three selected isolates differed from one plant cultivar to another as well as from the type of plant organ used for the inoculation. In a bioassay test, all isolates produced syringomycin, which was confirmed by molecular detection based on the syrB and syrD genes. Only EC122 possessed syrD but not syrB. DNA fingerprints, based on repetitive sequence‐based polymerase chain reaction (rep‐PCR) and PCR melting profile (PCR MP), were used to determine the potential genetic diversity among strains. Clustering of PCR MP fingerprinting data matched with rep‐PCR fingerprinting data. The generated distribution tree showed that Tunisian isolates were closely related to the citrus reference strain LMG5496. In contrast, EC112, isolated from citrus, and the almond isolate EC122 were distantly related to the type strain LMG1247T isolated from lilac. Such studies have not been reported until now for P. syringae from citrus.

Comparative study of phenolic composition and antioxidant activity of leaf extracts from three wild Rosa species grown in different Tunisia regions: Rosa canina L., Rosa moschata Herrm. and Rosa sempervirens L.

Aim of the present study was to characterize and to compare phenolic composition and antioxidant properties of methanol (MeOH) and ethyl acetate (EtOAc) extracts from leaves of Rosa canina, Rosa sempervirens and Rosa moschata collected from different Tunisian regions. Fifteen phenolic compounds were identified by means of HPLC-DAD and HPLC-DAD-ESI–MS analyses. It was shown that phenolic composition of Rosa canina extracts includes two kaempferol derivatives (Kaempferol 3-O-glucoside and kaempferol 7-O-glucoside) which are not present in profiles of the two Synstylae species. Rosa moschata and Rosa sempervirens extracts are rich in quercetin derivatives. As regarding the total phenolic content (evaluated by the Folin–Ciocalteu assay), R. sempervirens and R. moschata showed the highest values with their EtOAc and MeOH extracts, respectively. The highest mean values for DPPH, TEAC and FRAP tests were recorded for the EtOAc extracts of R. sempervirens and for the MeOH extracts of R. canina. In the ORAC assay, R. moschata and R. sempervirens showed the highest effectiveness for EtOAc and MeOH extracts, respectively. Taken together, these data suggest that leaf extracts of different Tunisian Rosa species should be used as a potential source of antioxidant phenolics, and R. sempervirens and R. moschata are a good alternative to R. canina especially in those regions where these species can be easily found.

Growth, photosynthesis and water relations as affected by different drought regimes and subsequent recovery in Medicago laciniata (L.) populations

The aims of this study were to investigate the effects of water deficit and recovery on growth, photosynthesis and water relations in four Medicago laciniata populations from saharian (Ml-90), inferior arid (Ml-204), superior arid (Ml-306) and semi-arid (Ml-376) Tunisian regions. After 28 d of sowing with ample irrigation, the plants were subjected to 4 water regimes: optimal irrigation (100% of field capacity, FC), moderate drought (75% FC), severe drought (35% FC) and rewatering (plants submitted to 35% FC during 7 d, afterwards the plants were rewatered to 100% FC). Harvest was carried out after 28 d of treatments. The drought tolerance in M. laciniata populations was found to be increased particularly with increasing temperatures of collection site of the population. The Ml-204 and Ml-90 populations used mainly physiological strategies for survival under moderate water shortage. Higher severe drought tolerance in both signaled populations would be related to their lower photosynthesis metabolic impairment, relatively higher leaf RWC and greater osmotic potential decrease. The results suggest that plants with low values of leaf features are likely to maintain higher leaf RWC under sever drought. The largest decrease of osmotic potential was found associated with the solute accumulations such as proline and K+.

Characterization and Classification of Different Tunisian Geographical Olive Oils using Voltammetric Electronic Tongue

In this work, we describe a sensor based on glassy carbon electrode, employed to discriminate between olive oils from different Tunisian regions. The characterization was made using three electrochemical techniques, cyclic voltammetry (CV), differential pulse voltammetry (DPV) and square wave voltammetry (SWV). Each type of oil provides a diversity of characteristic signals that can be used as an input variable of different statistics analysis, like principle component analysis (PCA), cluster analysis (CA) and discriminate factorial analysis (DFA).The results resulting from the electrochemical methods are compared. The obtained results show the reliability of the used methods on the discrimination between olive oil qualities obtained from different regions of Tunisia.

Comparison of Leishmania killicki (syn. L. tropica) and Leishmania tropica Population Structure in Maghreb by Microsatellite Typing.

Leishmania (L.) killicki (syn. L. tropica), which causes cutaneous leishmaniasis in Maghreb, was recently described in this region and identified as a subpopulation of L. tropica. The present genetic analysis was conducted to explore the spatio-temporal distribution of L. killicki (syn. L. tropica) and its transmission dynamics. To better understand the evolution of this parasite, its population structure was then compared with that of L. tropica populations from Morocco. In total 198 samples including 85 L. killicki (syn. L. tropica) (from Tunisia, Algeria and Libya) and 113 L. tropica specimens (all from Morocco) were tested. Theses samples were composed of 168 Leishmania strains isolated from human skin lesions, 27 DNA samples from human skin lesion biopsies, two DNA samples from Ctenodactylus gundi bone marrow and one DNA sample from a Phlebotomus sergenti female. The sample was analyzed by using MultiLocus Enzyme Electrophoresis (MLEE) and MultiLocus Microsatellite Typing (MLMT) approaches. Analysis of the MLMT data support the hypothesis that L. killicki (syn. L. tropica) belongs to the L. tropica complex, despite its strong genetic differentiation, and that it emerged from this taxon by a founder effect. Moreover, it revealed a strong structuring in L. killicki (syn. L. tropica) between Tunisia and Algeria and within the different Tunisian regions, suggesting low dispersion of L. killicki (syn. L. tropica) in space and time. Comparison of the L. tropica (exclusively from Morocco) and L. killicki (syn. L. tropica) population structures revealed distinct genetic organizations, reflecting different epidemiological cycles.

Laticifers identification and natural rubber characterization from the latex of Periploca angustifolia Labill. (Apocynaceae)

Periploca angustifolia (Apocynaceae) is a multipurpose shrub widely distributed in arid and semi-arid Tunisian regions. Local people often use this laticiferous shrub us a fodder for their livestock and consume the dried latex as a pleasant taste chewing. This study aims to identify laticifers distribution in vegetative and reproductive organs of Periploca angustifolia and investigate latex composition in order to valorize this constituent for later use. The histological study conducted on this species has confirmed the presence of laticifers in leaf (spongy and palisade mesophyll), stem (cortical parenchyma), root (periderm), floral bud (petal–carpel) and follicle (pericarp). Laticifers in the studied species are non-articulated. The purified rubber fraction from latex was subjected to different chemical analyses, proving that the rubber of P. angustifolia is composed of cis-1,4-polyisoprene as the main constituent. When the molecular weight distribution of rubber components in the exudate was measured using SEC analysis, the number-average molecular weight (Mn), the weight-average molecular weight (Mw), and polydispersity (Mw/Mn) were estimated to be Mn=2.3×105, Mw=4.1×105 and Mw/Mn=1.78, respectively. Thus, the obtained results show that the latex of P. angustifolia contains a natural rubber.

Palynological reconstruction of climate and paleoenvironment during the lower to middle Miocene in northeast Tunisia

The pollen analysis of the sedimentary series of marine origin outlines the environmental and climatic context of the Northeastern Tunisia during the Lower to Middle Miocene. The analysis of the pollen sequence reveals a changing climate: (1) a subtropical climate during Burdigalian (importance of mega-mesothermal and mesothermal elements as Taxodium, Oleaceae, and Fagaceae) and (2) a tropical climate during the upper Langhian-lower Serravallian (megathermes elements (Avicennia and Acacia). This phase corresponds to the Miocene climatic optimum (MCO). The identified pollen associations helped organize the vegetation of the period in altitudinal levels (high areas, plain littoral environment, and river frontiers). As far as the inferior to middle Miocene is concerned, we noticed the existence of a diversified forest, which developed an average altitude with conifer trees of high reliefs. The plain which was dominated by Poaceae and Asteraceae was relatively small. The coastal environment is defined by the presence of Amaranthaceae-Chenopodiaceae and Ephedra. A correlation with known results for other Tunisian and Mediterranean regions, on the one hand, and a comparison with the current climate of Tunisia, on the other hand, have been completed.

Chemical Composition and Antioxidant and Antimicrobial Activities of Wormwood (Artemisia absinthiumL.) Essential Oils and Phenolics

The aim of this study was to determine the chemical variability of wormwood extracts as affected by the growing region. Antioxidant and antimicrobial activities were also investigated. The essential oil composition variability ofA. absinthiumL. aerial parts collected from four different Tunisian regions was assessed by gas chromatography (GC/FID) and by gas chromatography mass spectrometry (GC/MS). In addition, total polyphenols, flavonoids, and condensed tannins as well as antioxidant, antibacterial, and antifungal activities of methanolic extract and essential oils were undertaken. Chromatographic analysis of wormwood essential oils showed the predominance of monoterpene hydrocarbons represented mainly by chamazulene. RP-HPLC analysis of wormwood methanolic extract revealed the predominance of phenolic acids. Antiradical activity was region-dependant and the methanolic extract of Bou Salem region has the strongest activity (CI50=9.38±0.82 µg/mL). Concerning the reducing power, the methanolic extract of Bou Salem, Jérissa, and Boukornine regions was more active than the positive control. Obtained results of antimicrobial activities showed that wormwood essential oil is endowed with important antibacterial activity which was strongly related to the organoleptic quality of oil which appeared strongly region-dependant.A. absinthiumL. EOs investigated are quite interesting from a pharmaceutical standpoint because of their biological activities.

Variations in Tunisian borage essential oil profiles and their antioxidant activities during flowering

This study was conducted to examine the chemical composition and antioxidant activity of the essential oils (EOs) of Borago officinalis stem during its flowering stage. The EO composition was characterised by high proportions of E,E-decadienal, the main compound of monoterpene hydrocarbon class, with values varying from 47.08% to 55.28% in two Tunisian regions. The region of Korba exhibited the highest scavenging activity (2.05 mg/mL) by comparison with Beja region. In all tests, the fructification stage showed the best antioxidant activity of all flowering stages.

Variation in protein and oil content and fatty acid composition of Rhus tripartitum fruits collected at different maturity stages in different locations

The goal of this study is to evaluate the effects of ripening (immature: yellow; intermediate: mahogany brown; mature: dark brown) on the protein and oil content and fatty acid composition of Rhus tripartitum fruits collected from two Tunisian regions. The high protein and oil content were detected in the intermediate stage (ca. 9.5% and ca. 6%, respectively). Fatty acid profiles did not vary among the growing regions; and palmitic (16:0), oleic (18:1), linoleic (18:2), and linolenic (18:3) acids were the predominant fatty acids. Linoleic acid showed the highest level in the intermediate stage (ca. 21%). In the mature stages palmitic and oleic acids were the major fatty acids (more than 31% and 34%, respectively). Our findings showed that R. tripartitum fruits, especially at the intermediate stage, could therefore represent an attractive source of protein, oil and fatty acid for both food, cosmetic, and pharmaceutical industries.

OCCURRENCE AND WIDESPREAD DISTRIBUTION OF GRAPEVINE VIRUS D IN TUNISIAN GRAPEVINES

Rugose wood is a complex disease affecting grapevine worldwide. At present, at least six different viruses belonging to the family Betaflexiviridae are known to be associated with the disease (Saldarelli, 2014). Previous studies had shown that Grapevine virus A (GVA), Grapevine virus B (GVB) and Grapevine rupestris stem pitting associated virus (GRSPaV) are common in Tunisian grapevines (Martelli, 2014), whereas no information is available on the presence of Grapevine virus D (GVD). Therefore, a total of 140 samples from several cultivars, were collected from major Tunisian viticultural regions and tested for the presence of this virus by RT-PCR using the GVD-specific primers CP471C and CP7V (Abou-Ghanem et al., 1997). A 474 bp product corresponding to a fragment of the coat protein gene was amplified from 58 samples, accounting for an infection rate of 41.42%. The virus was present in all the surveyed areas and grape cultivars tested. The infection rate ranged from 11.7% (cv. Red Globe) to 88.8% (cv. Muscat RafRaf). To confirm the identity of the target virus, three RT-PCR products were cloned and sequenced. The Tunisian isolates shared 86-91% common nucleotides with the other GVD isolates from GenBank, and 72-73%, 60-62% and 53-55% nucleotide identity with GVA (JF754577), GVB (NC003602.1) and GVE (JX402759.1), respectively. To our knowledge, this is the first report on the occurrence of GVD in Tunisian grapevines. The infection rate of GVD (41.42%), which is close to that of GVA (47.2%) in the same samples, suggests the presence of common vectors for both viruses in Tunisian vineyards.

Natural occurrence of begomovirus recombinants associated with tomato yellow leaf curl disease co-existing with parental viruses in tomato crops and weeds in Tunisia

Tomato yellow leaf curl virus disease (TYLCVD) is the main limiting factor for tomato production in the Mediterranean Basin, and particularly in Tunisia where climatic and crop production conditions, as well as the occurrence of many weeds, provide suitable conditions for the presence and spread of TYLCVD all year round. In Tunisia, epidemics in tomato-growing regions have been associated with two begomoviruses: Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) but the presence of recombinants between them has never been investigated. A large-scale survey was conducted on leaves sampled from late-season tomato crops exhibiting severe curling symptoms, from beans, and from nine nearby weed species in the South and Sahel regions of Tunisia to investigate: (i) the natural occurrence of the TYLCV and TYLCSV species; (ii) the presence of the two recombinant types, RecA and RecB, derived from these species. Identification of TYLCV and TYLCSV was based on a multiplex PCR with primers generating a typical pattern on gels (750 bp and 366 bp fragments, respectively). Recombinants were identified by simplex PCR, which amplified the intergenic region, the most common recombination site described for begomoviruses, and sequencing. The occurrence of TYLCSV and TYLCV was confirmed in both Tunisian regions. Their co- existence was shown on tomato and in new weed species. This is the first time that recombinants between TYLCV and TYLCSV species showing 93% to 95% nucleotide identity with tomato yellow leaf curl recombinant viruses, are reported in Tunisia in tomato and black nightshade.

Genotypic characterization by spoligotyping and VNTR typing of Mycobacterium bovis and Mycobacterium caprae isolates from cattle of Tunisia

This work is an approach to the molecular epidemiology of Mycobacterium tuberculosis complex (MTBC) bovine infections in Tunisia. A total of 35 MTBC isolates from both lateral retropharyngeal lymph node samples of cattle slaughtered in different Tunisian regions were genotyped by spoligotyping and variable number tandem repeat typing (VNTR)-typing. Spoligotyping allowed to identify two profiles not previously registered, namely SB2024, a Mycobacterium caprae isolate from Nabeul Region (North East Tunisia), the first description of this species in the country, and SB2025 (Mycobacterium bovis) from Sfax Region (Southern Tunisia). A second M. caprae isolate with a spoligotyping profile previously described in Europe mainland, SB0418, was also isolated from a bovine of Sfax region. Both isolates suggest the possibility of a widespread distribution of this species in the country. The predominant spoligotype was SB0120, present in all Tunisian regions selected for the study but Nabeul. Molecular typing also allowed to describe a mixed infection caused by two different M. bovis isolates (SB0120 and SB0848) in the same animal. VNTR typing was highly discriminant by testing a panel of six loci. Loci QUB3232 and QUB11b were the most discriminant, whereas ETR-D and QUB11a had the lower diversity index. The value of allelic diversity can significantly vary among countries; thus, it is important to standardize a panel of loci for future inter-laboratory comparisons. Although VNTR typing proved to be useful for an efficient discrimination among MTBC isolates, especially in combination with spoligotyping, further studies are needed in order to assess the genetic diversity of the MTBC in Tunisia.

English

Polyploidy plays an important role in plant evolution and constitutes an important mechanism of diversification and creation of genetic variability. Artificial polyploidy can be induced using the colchicine. The aim of this study was to determine the Trigonella foenum-greacum (2n= 16) population which resists better to colchicine treatment and which has the highest rate of polyploid formation among 38 populations collected from different Tunisian regions. The comparative study between treated and control plants showed significant increases (p < 0.05) for the parameters survival rate, rate of malformed leaves appearance, stem height and seeds number by pod. The 38 treated populations showed significant variations (p < 0.05) between them for the parameters survival rate, branches number, stem height and seeds number by pod. Results also showed that treatment of the shoot meristem by colchicine allowed to obtain diploid (2n= 16) and mixoploid plants having mixoploid branches (4n = 32 and 2n= 16) and branches entirely tetraploid (4n = 32). The obtained rate of mixoploidy was 65.79% of treated plants. Among the 38 analyzed fenugreek populations, the population 15 of Menzel Temime presented the highest rate of mixoploids formation and survival rate to colchicine treatment.

INFLUENCE OF TRANSPORT PHENOMENA ON POLLUTANTS CONCENTRATION IN MEDITERRANEAN TUNISIAN REGIONS

The objective of this study is to investigate the origin of the polluted air masses in the Tunisian Mediterranean regions and to characterize the atmospheric conditions and the transport and dispersion mechanisms that give rise to pollution events. For this end, we conducted continuous measurements campaigns of surface ozone concentration and meteorological parameters during summer 2011 using mobile and fixed stations in the Tunisian regions. The results indicated that the seasonal variability of ozone concentrations was related to European and Saharan transport events. Long range transport of pollutants from Europe resulted in O3 loading and frequent surpassing of the European standard limits in the Mediterranean Tunisian sites. During these events, the weather conditions support photochemical phenomena. The Saharan events provided the importance of chemical reactions and radiative impact involving dust particles which may reduce the photochemical production of ozone.

Genetic Susceptibility to Type 2 Diabetes: A Global Meta-Analysis Studying the Genetic Differences in Tunisian Populations

The present study is the first meta-analysis to evaluate type 2 diabetes (T2D)- associated polymorphisms in cohorts originated from several Tunisian regions. In fact, we evaluated the effect of seven polymorphisms in the following genes—PPARg (Pro12Ala), TNFα (-308A/G), ENPP1(K121Q), TCF7L2(rs7903146°C/T), MTHFR(C677T), ACE(I/D), and CAPN10(3R/2R)—on T2D risk, through a meta-analysis combining data of previous studies performed on Tunisian populations originating from the north, center, or south of the country. R statistics version 2.12.1 software was used to estimate the heterogeneity between studies. Pooled odds ratios were computed by the fixed-effects method of Mantel-Haenszel if no heterogeneity between studies exists. Despite the similarities founded in a number of loci, the Woolf test reported that the contributions of ENPP1 and ACE loci in T2D risk are dependent on the geographic origin of concerned groups, and this heterogeneity could be attributed not only to the variable contribution of the variant in T2D risk but also to diversities of genetic background between tested groups. Interestingly, observed heterogeneity highlighted founding concerning Y chromosome and the mitochondrial DNA about the genetic structure of Tunisian population and proves once again that Tunisians, like the north-Africans, are a mosaic of subpopulations, with significant differences in genetic structure. In homogeneous groups, we replicated the association of single-nucleotide polymorphisms of TCF7L2, MTHFR, CAPN 10, TNFα, and ACE genes with a T2D risk in the Tunisian population with OR ranging from 1.43 to 6.72. However, we reported an absence of the association of PPARg with T2D in the Tunisian population.

Genetic Susceptibility to Type 2 Diabetes: A Global Meta-Analysis Studying the Genetic Differences in Tunisian Populations

The present study is the first meta-analysis to evaluate type 2 diabetes (T2D)- associated polymorphisms in cohorts originated from several Tunisian regions. In fact, we evaluated the effect of seven polymorphisms in the following genes-PPARg (Pro12Ala), TNFα (-308A/G), ENPP1(K121Q), TCF7L2(rs7903146°C/T), MTHFR(C677T), ACE(I/D), and CAPN10(3R/2R)-on T2D risk, through a meta-analysis combining data of previous studies performed on Tunisian populations originating from the north, center, or south of the country. R statistics version 2.12.1 software was used to estimate the heterogeneity between studies. Pooled odds ratios were computed by the fixed-effects method of Mantel-Haenszel if no heterogeneity between studies exists. Despite the similarities founded in a number of loci, the Woolf test reported that the contributions of ENPP1 and ACE loci in T2D risk are dependent on the geographic origin of concerned groups, and this heterogeneity could be attributed not only to the variable contribution of the variant in T2D risk but also to diversities of genetic background between tested groups. Interestingly, observed heterogeneity highlighted founding concerning Y chromosome and the mitochondrial DNA about the genetic structure of Tunisian population and proves once again that Tunisians, like the north-Africans, are a mosaic of subpopulations, with significant differences in genetic structure. In homogeneous groups, we replicated the association of single-nucleotide polymorphisms of TCF7L2, MTHFR, CAPN 10, TNFα, and ACE genes with a T2D risk in the Tunisian population with OR ranging from 1.43 to 6.72. However, we reported an absence of the association of PPARg with T2D in the Tunisian population.

A Hybrid ARIMA and Artificial Neural Networks Model to Forecast Air Quality in Urban Areas: Case of Tunisia

Forecasting air quality time series represents a very difficult task since air quality contains autoregressive, linear and nonlinear patterns. Autoregressive Integrated Moving Average (ARIMA) models have been widely used in air quality time series forecasting. However, they fail to detect extreme events because of their presumed linear form of data. Artificial Neural Networks (ANN) models have proved to be promising nonlinear tools for air quality forecasting. A hybrid model combining ARIMA and ANN improved forecasting more than either of the models used independently. Experimental results with meteorological and Particulate Matter data indicated that the combined model can be used as an efficient forecasting and early warning system for providing air quality information towards the citizen, not only in Sfax Southern Suburbs but in other Tunisian regions that suffer from poor air quality conditions.

Isolation and molecular characterisation of &lt;i&gt;Mycobacterium bovis&lt;/i&gt; from raw milk in Tunisia

Consumption of raw milk and unpasteurized dairy products is common in Tunisia where bovine tuberculosis remains enzootic. We herein investigated the frequency of M. bovis isolation from raw milk. Three hundred and six milk samples collected from 102 infected cows in different Tunisian regions were analysed. M. bovis isolates were further characterized by spoligotyping and variable number tandem repeat typing. A total of five (4.9 %) M. bovis strains exhibiting three different genotypes were isolated. This study demonstrates that consumers of raw milk or derivatives in Tunisia are at high risk of zoonotic infection with M. bovis.

Morquio A disease: clinical and molecular study of Tunisian patients

Type IVA mucopolysaccharidosis or Morquio A disease is a lysosomal storage disease, autosomal recessive, caused by deficiency of the N-acetylgalactosamine-6-sulfate sulfatase or GALNS. The severe phenotype is characterized by a severe skeletal dysplasia without any mental retardation. The aim of this study was to propose a strategy of molecular and prenatal diagnosis of this pathology. A molecular study was carried out on 7 patients MPS IVA issued from 5 unrelated families recruited from different Tunisian regions. All the patients were offspring of consanguineous marriages. The clinical and biologic study confirmed the diagnosis of MPS IVA within the 7 studied patients. Three GALNS mutations were identified by molecular analysis: IVS1+1G>A, G66R and A85T. The unions between Tunisian relatives are important and increase the Morquio A incidence in Tunisia. The identification of GALNS mutations in the Tunisian population permits better understanding of the Morquio A phenotype, a reliable genetic counselling and a molecular prenatal diagnosis to Tunisian at-risk relatives.