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Recent Doctorate Theses in Plant Protection (2016/17)


Chattaoui, Mayssa. 2016. Foliar fungal diseases of Olive Tree in Tunisia: Characterization of causal agents and proposal of control strategies. Doctorate Thesis in Biological Sciences, Faculty of Sciences of Tunis, University El-Manar, Tunis, Tunisia, 161 pp. (Public Defense: 12 November 2016)

The introduction of new cultivation systems based on high-density plantings, combined to the current climate change, have led to a proliferation of already known diseases, as well as an emergence of new symptoms caused by foliar pathogens. One of the main objectives of this work was to characterize these causal agents. Several samples were collected, mostly from fields situated in the North, and served for isolations. Based on morphological (macroscopic, microscopic ,and cultural characters), physiological (optimal temperature growth, Benomyl susceptibility, protease activity), pathogenic, and molecular (ITS, TUB2, ISSR-PCR) studies, 3 new emergent foliar pathogens were identified and characterized for the first time in Tunisia: Colletotrichum acutatums.s, Botryosphaeria dothidea, and B. obtusa, responsible for olive anthracnose, dalmatian disease (olive fruit rot) and branch dieback, respectively.

Secondly, in order to develop “eco-friendly” and sustainable control strategies of major foliar mycosis of olive tree, two aspects were studied: variety resistance and biological control. In the first case, forty descendants from a crossing between olive cultivar Picholine and the local cultivar Meski were evaluated for their susceptibility to olive leaf spot (Fusicladium oleagineum) in field and laboratory studies. Disease incidence varied greatly among the descendants and was correlated with the severity of leaf symptoms. Based on field observations and laboratory tests, the hybrids were classified into five groups: 1) very susceptible to the disease: Meski and 17H; 2) susceptible to the disease: 20J, 8I, 11I, 21I, 14H, 16H, 23H, 6J, 8J and 23J; 3) moderately susceptible to the disease: 15I, 16I, 22I, 23I, 8K, 13H, 18H, 22H, 9J, 10J, 11J, 12J, 14J, 16J, 18J, 4) moderately tolerant to the disease: 21J, 12I, 13I, 18I, 19I, 9K, 10K, 12H, 15H, 19H, 15J and 22J; 5) resistant to the disease: cv. Picholine and hybrids 6I and 19I. In order to know the factors involved in the resistance of these latter, different parameters were studied. Analysis revealed that the polyphenol content in the leaves as well as its composition were not correlated with the disease incidence, while the anatomical study of the leaves showed that cuticle thickness and trichome density were proven to be very important physical parameters that could determine the degree of susceptibility of hybrids to the disease. On the other hand, we also studied, under laboratory conditions, the susceptibility of a collection of Tunisian and introduced cultivars to C. acutatums.s and B. dothidea. Results revealed, for both tested pathogens, a high tolerance of Tunisian cultivars: Chetoui and Rkhami and a high susceptibility of cv. Chemlali.

For biological control, 3 different bacteria (Bacillus sp.) were considered. Their antagonistic activity was evaluated in vitro (dual culture, double layer method, effect on conidial germination) then in vivo (against C. acutautms.s). Among the considered bacteria, B. amyloliquefaciens isolate IOT M2 was significantly the most effective.

Khammassi, Messaad. 2016. Herbicide resistance of ryegrass (Lolium rigidum Gaud.) and integrated resistance management strategies in the production systems dominated with cereal. Doctorate Thesis in Agronomic Sciences (Plant Production), INAT, University of Carthage, Tunis, Tunisia, 258 pp. (Public Defense: 19 November 2016)

In Tunisia, rigid ryegrass is infesting several crops including cereal crops. Over the last years and since the first reports of herbicide resistant populations of ryegrass, there have been few studies of the prevalence of the weed, crop yield damages as well as factors contributing to its development. This thesis was undertaken to design effective ryegrass management strategies in cereal crops given weed distribution, its susceptibility to commercial herbicides and the extent of herbicide resistance in ryegrass populations. Field surveys, farmers interviewers as well as laboratory and field trials were conducted in order to meet the objective of this study.

Surveys conducted in 312 fields showed that the frequency of infested fields by ryegrass represents 32%. Weeds infestation of prospected sites are highly correlated with annual (R˛ = 0.99) and seasonal rainfall [Autumn (R˛ = 0.81), Winter (R ˛= 0.97) and Spring (R˛ = 0.99)]. The highest ryegrass densities (> 300 plants/m˛) occurred in the sub humid (Bizerte and North of Beja) while medium to low densities were found in the semi-arid (Jendouba, Kef, Siliana, and Zaghouan). The yield losses of wheat were highly correlated (R˛ = 0.93 and P = 0.0001) with ryegrass infestations. Crops yields decrease as weed densities increase (Y = 109.6 e-0.001*X).

The study of Bioassays, using the RISQ Test (Resistance In Season Quik Test) were conducted to detect herbicide resistance of 171 ryegrass populations randomly collected from cereal areas, to the commonly used herbicides: Topik® (clodinafop-propargil, ACCases inhibitor), Amilcar WG® (iodosulfuron+mesosulfuron, inhibitor ALS) and Axial® (pinoxaden, inhibitor ACCases), showed that resistance to the ACCase inhibitors and to the ALS inhibitors was detected in 58 and 52% of the tested populations, respectively. Furthermore, ryegrass collected from Bizerte exhibited multiple (23%) and cross (28%) resistance. Results of the RISQ Test are highly correlated (R˛ = 0.99 for ACCase and R˛ = 0.89 for ALS) with those of pot experiments.

The exam of the cultural practices revealed with the interviewed farmers showed the lack of stubble (67%), early sowing (70%), use of self-produced seeds (46%), no-till (26.4%), mono-cropping (59%) and lack of rotating herbicide groups (56%). These factors that may contribute to the evolution of herbicide resistance in ryegrass. The correlation analysis showed that the evolution of resistance in ryegrass is highly correlated with the frequency of use of the ACCase (R˛ = 0.98 and P = 0.009) and ALS (R˛ = 0.99 and P = 0.0004) inhibiting herbicides.

Field trials conducted in an integrated weed management (IWM) approach revealed that ryegrass infestation may be increased by early sowing and mono-cropping of wheat as well as by minimum tillage. In contrast, the false seeding (45%), soil tillage (44%), late sowing (71%), crop rotation and alternating herbicide groups have significantly reduced ryegrass infestations. Results showed that the use of herbicides of the group (N), recently introduced in Tunisia, combined with late sowing of wheat may reduce ryegrass populations by an average of 80%. Rotating herbicide groups (A/no herbicide/ K1+C2+B) in combination with the practice of a triennial rotation (wheat/oat for silage/small faba bean) and soil tillage resulted in 99.7% reduction in ryegrass densities and in a significant decrease in weed seedbank.

Mejdoub-Trabelsi, Boutheina. 2016. Potato Fusarium diseases: Interactions between Fusarium species, their influence on disease severity and cultivar response and biocontrol using nonpathogenic potato-associated fungi. Doctorate Thesis in Agronomic Sciences (Plant Protection and Environment), ISA Chott-Mariem, University of Sousse, Chott-Mariem, Tunisia, 215 pp. (Public Defense: 14 December 2016)

Potato Fusarium diseases occur worldwide and are responsible for considerable plant and yield losses. Fusarium dry rot during storage and Fusarium wilt of plants are both caused by a complex of Fusarium species of variable aggressiveness. The aim of this study is to elucidate the interactions occurring between the different Fusarium species associated to potato and their influences on disease severity and cultivar response. Some fungi naturally associated to potato were isolated and tested for their in vitro and in vivo efficacy against Fusarium spp.

Tested as a mixture of four isolates, Fusarium sambucinum, F. oxysporum, F. solani, and F. graminearum species, known to be involved in potato dry rot disease, were also found able to induce a severe plant wilting. The assessment of the response of 11 potato cultivars to these four Fusarium species showed that cultivar tolerance to a given species depended on the targeted plant part (whole plant or just tuber). Based on Fusarium dry rot severity, cv. ‘Oceania’ was found to be the most tolerant to F. solani, F. oxysporum and F. graminearum. However, based on Fusarium wilt severity, cv. ‘Bellini’ was found to be tolerant to F. solani and F. sambucinum. Overall, the tolerance of the tested cultivars was more expressed toward dry rot than toward Fusarium wilt.

Based on single and mixed inoculations using these Fusarium species, tolerance of five potato cultivars toward the tested species mixtures was less frequent as compared to mono-specific inoculations. The mixed infection based on F. sambucinum and F. solani was found to be the most aggressive on tubers stored at at 20 and 30°C. The most aggressive species mixtures on tubers, leading to the most severe dry rot, were composed of F. sambucinum among others and none of the cultivars tested was resistant to the tested single and mixed inocula. However, based on Fusarium wilt severity, the mixed inocula composed of F. oxysporum, F. sambucinum and F. graminearum was found to be the most aggressive on plants.

For the biological control of the predefined most aggressive Fusarium spp. complexes, 20 fungal isolates, naturally associated to potato, were selected among 30 collected based on their ability to reduce by 30 to 60% the mycelial growth of Fusarium spp. Tested on tubers, 10 isolates had decreased by 50% dry rot severity induced by F. sambucinum and F. solani. Assessed on plants, seven isolates had reduced by 29 to 47% Fusarium wilt severity caused by F. oxysporum, F. sambucinum and F. graminearum compared to the inoculated and untreated control.

Four associated fungi that inhibited by 34- 60% the in vitro growth of Fusarium spp. and that limited by 41- 60% and 45-50% Fusarium dry rot and Fusarium wilt severity, respectively, were selected for the assessment of the antifungal activity of their extracellular metabolites. The study showed that the culture filtrates of Aspergillus flavus (E.25.11) and Penicillium chrysogenum (E.36.11) were the most active and had reduced the mycelial growth of Fusarium spp. by 66.34- 68.78%, respectively, and had suppressed by 50% Fusarium dry rot severity. The chloroform extracts of these fungi had inhibited the mycelial growth of Fusarium spp. by more than 76% and the mean penetration of dry rot by 46% compared to the untreated control.

Nada, Ouhaibi-Ben Abdeljalil. 2016. Characterization of tomato-associated rhizobacteria and assessment of their suppressive potential against root rots caused by Rhizoctonia solani and Sclerotinia sclerotiorum and their plant growth-promoting effect. Thesis in Agronomic Sciences (Plant Protection and Environment), ISA Chott-Mariem, University of Sousse, Chott-Mariem, Tunisia, 260 pp. (Public Defense: 20 December 2016)

Some rhizobacteria associated to tomato were isolated from different growing areas, under greenhouse and open field, characterized and assessed for their antagonistic potential against Rhizoctonia solani and Sclerotinia sclerotiorum and their growth-promoting effect on plants. The 25 selected isolates, among 200 collected, showed morphological, biochemical and metabolic diversity. The 16S rRNA and rpoB gene sequencing allowed their affiliation to Bacillus subtilis, B. amyloliquefaciens, B. megaterium, B. thuringiensis, Enterobacter cloacae, Chryseobacterium jejuense, and Klebsiella pneumoniae species. Genes encoding for fengycin A and bacillomycin D biosynthesis were detected in 18 and 16 isolates, respectively. Concerning main traits involved in plant growth promotion, all isolates had produced indole-3-acetic acid, 20 synthesized siderophores and 18 had solubilized phosphate. These rhizobacteria degraded 31 carbon sources. As for their effects on both pathogens, their diffusible and volatile metabolites had inhibited R. solani growth by 34- 59% and 18- 45% and that of S. sclerotiorum by 37- 57% and 24- 54%, respectively. The selected isolates had enhanced plant height, relative to control; they doubled the height of the plants. These isolates had reduced up to 100% the disease severity caused by R. solani and S. sclerotiorum and enhanced growth of inoculated plants. In fact, plants inoculated with both pathogens, respectively, showed over to 62 and 52% greatest height, up to 67 and 74% increase in their fresh weight and over to 53 and 66% enhancement in their root fresh weight as compared to the inoculated and untreated control. The most effective rhizobacteria in inhibiting both pathogens and promoting plant growth (B. subtilis str. B10, B. thuringiensis str. B2, and E. cloacae str. B16), were re-evaluated, singly or in combination, on two tomato cultivars tested during two year trials. This study showed that combined treatment had reduced by over to 74% disease severity caused by R. solani and by over to 80% that due to S. sclerotiorum compared to a reduction by over to 60 and 70% noted using these isolates singly. As for their effects on treated and inoculated plants' growth with R. solani and S. sclerotiorum, the study showed that the combination had enhanced height by more than 49% and by more than 38%, respectively, compared to more than 42 and 32% recorded using isolates singly. Similar effects were observed on the aerial part fresh weight where the respective improvements in this parameter exceeded 19.79 and 21.48% using a mixture of isolates and 1.47 and 9.22%, respectively, using single bacterial treatments. Similar results were observed for the root fresh weight that increased respectively by more than 59 and 53% using consortium and by more than 51.50 and 42.01% using single isolates.The structure of microbial populations in the rhizosphere was analyzed using the technique Single Strand Conformation Polymorphism (SSCP) for root DNA of both tomato cultivars inoculated with R. solani and S. sclerotiorum and treated with rhizobacteria either singly or in consortium. The study showed that the shifts recorded in the structure of bacterial and fungal community in the rhizosphere were more affected by cultivars grown (Rio Grande or Marmande) than by the other studied factors (fungal inoculation or bacterial treatment).

El Khaldi, Rabeb 2016. Evaluation of the suppressive potential of two oases composts and their associated microorganisms against stem canker and black scurf on potato (Solanum tuberosum L.). Doctorate Thesis in Agronomic Sciences (Plant Production), INAT, University of Carthage, Tunis, Tunisia, 165 pp. (Public Defense: 29 December 2016)

Two types of compost prepared from date palm wastes and sheep or cattle manure and their associated microorganisms were evaluated for their suppressive potential against Rhizoctonia solani and their biofertilizing effect on potato. Their physico-chemical and biological parameters were monitored throughout the composting process. After 190 days of composting, stability recorded in the various parameters noted indicated maturity achievement for prepared composts where their C/N ratio ranged between 18.59 and 20.49 and their texture was fine and homogeneous.

Sterilized and unsterilized compost extracts were assessed for their antifungal activity against R. solani. Sterilization by heating or microfiltration had induced a loss in their antifungal activity while the unsterilized extracts had inhibited the mycelial growth of the pathogen by 53.05 to 100% compared to the untreated control.

Fungal and bacterial isolates associated with the studied composts were isolated and screened for their suppressive potential against R. solani. All tested isolates had decreased the mycelial growth of the pathogen by 30 to 100%. They were affiliated with three fungal genera namely Penicillium, Aspergillus and Talaromyces and one bacterial isolate was identified as Serratia marcescens. The antifungal activity of extracellular metabolites of the selected isolates varied depending on tested isolate and the highest pathogen inhibition (by 91.6%) was obtained using the extracellular filtrate of Penicillium chrysogenum.

The incidence and the severity of black scurf and stem canker were significantly reduced in potato plants cv. Nicola grown in a culture substrate amended with tested composts compared to peat-sand mixture only. The highest decreases in disease incidence and severity ranged from 97.1 to 100% on tubers, and from 64 to 74.9% on collars, respectively. For plants treated using fungal and bacterial isolates, the highest reductions in black scurf incidence and severity on tubers, of about 51.4 and 81.5% respectively, were noted on plants treated with Aspergillus nidulans. Composts issued from palm wastes and sheep manure, incorporated into the culture substrate, had increased the tuber fresh weight and the aerial part dry weight by 30.1 and 90.3%, respectively, compared to the control substrate. This biofertilizing effect was not noted using composts composed of date palm wastes and cattle manure nor using fungal and bacterial isolates based treatments.

The selected fungal and bacterial isolates were able to produce chitinolytic enzymes. The bacterial isolate S. marcescens exhibited the highest specific N-acethyl ß- D-glucosaminidase activity (232.25 U / mg protein). The activity of ß-1,3-glucanase seems to be not involved in the degradation of the pathogen cell wall in this study. The molecular detection of genes encoding chitinase from S. marcescens indicated the presence of two chitinase genes of 400 bp and 500 bp belonging to family 18 group A and a chitinase gene of 750 bp belonging to family 19.

Ouerghi, Fethi. 2017. Morpho-histological, biochemical and molecular characterization of the resistance of the olive tree to leaf spot disease caused by Venturia oleaginea and proposition of some alternative methods of control. Doctorate Thesis in Agronomic Sciences (Plant Production), INAT, University of Carthage, Tunis, Tunisia, 168 pp. (Public Defense: 06 January 2017)

This thesis was carried out in order to study the histological, biochemical and molecular characterization of the resistance of the olive tree to leaf spot disease caused by Venturia oleaginea and the proposition of some alternative methods of control.

The first part conducted in the field concerned some factors influencing the response of Meski cultivars (susceptible) and Chetoui (moderately resistant). The incidence of the disease varies greatly among cultivars submitted to similar conditions. The results revealed strong correlations between the incidence of the disease on the first hand and the leaf height, the orientation, the age of the leaf and the relative humidity on the other hand.

In the second part, a histological characterization was conducted on varieties Meski, Arbequina, Picholine and Chetoui. Analysis of the results showed a difference between the varieties in the thickness of the cuticle, the density and the diameter of the trichomes. Cv. Meski, the most susceptible variety to the leaf spot disease, is characterized by the thinnest cuticle and the lowest density and diameter of the trichomes. In contrast cultivars Picholine, Chetoui and Arbequina showed a low susceptibility to the disease associated with thicker cuticles and higher densities and diameters of trichomes.

A third part was carried out in laboratory in order to study the role of total phenolic compounds in the defense of the host plant against leaf spot disease. Quantitative and qualitative analyzes of total phenolic compounds were carried out through the high performance liquid chromatography (HPLC). Although there is no correlation between the incidence of the disease and the total quantity of the phenols, there was a tendency of a slightly higher production of some types of phenols in cv. Picholine resistant than cv. Meski sensitive. These phenols are chlorogenic acid, versbacoside oleuropein and its derivatives and apigenin.

To evaluate the resistance of some hybrid olive to leaf spot disease, a fourth part of the study was carried out on cv. Meski sensitive, 3 low sensitive or resistant varieties and 9 hybrids. Molecular techniques using microsatellite markers were used to analyze the genetic relationships of cultivars and hybrids. Genetic analyzes showed that hybrids and cultivars differ in their resistance to this disease and the data revealed that hybrid obtained from the cross between cv. Picholine and cv. Meski showed a significant resistance to the leaf spot disease.

In a fifth and final part, experiments were conducted in greenhouse to study the effects of the adding of nitrogen and potassium as well as the treatment with two antiperspirants (linseed oil and Vapor Gard) on the disease incidence in the susceptible variety Meski. The antitranspirant Vapor Gard 3%, in pre-inoculation treatment, has reduced the incidence of disease. In another hand, the post-inoculation addition of potassium reduced the incidence of the disease, contrarily to the addition of the nitrogen which increased the sensitivity of the plant to the pathogen.

Aydi Ben Abdallah, Rania. 2017. Biological control of Fusarium wilt of tomato using endophytic bacteria naturally associated with wild and cultivated Solanaceae. Doctorate Thesis in Agronomic Sciences (Phytiatry), INAT, University of Carthage, Tunis, Tunisia, 212 pp. (Public Defense: 06 January 2017)

One hundred sixty eight (168) bacterial isolates were selected as endophytes among 258 recovered from cultivated and wild Solanaceae. They were screened in vivo against Fusarium oxysporum f. sp. lycopersici (FOL), the causal agent of Fusarium wilt in tomato, and for their effects on plant growth. The most effective isolates (40) leading to 76- 100% decrease in disease severity and to an improvement of growth parameters by 35-74% in plants inoculated with FOL and by 18-70% in those uninoculated, compared to controls, were subjected to macro-morphological and biochemical characterization. Sequencing of the gene 16S rDNA allowed the affiliation of the selected isolates to the genera Bacillus, Stenotrophomonas, Alcaligenes, Pseudomonas, Azotobacter, and Serratia.

The antifungal activity of the 40 selected isolates was screened in vitro using two confrontation methods. Twenty six (26) isolates were selected and re-evaluated in vivo, as whole bacterial cells or cell-free culture filtrates, for their ability to limit Fusarium wilt and to stimulate the growth of tomato plants cv. Rio Grande. These isolates had reduced disease severity by 35-100% and improved the growth of tomato cv. Rio Grande plants inoculated with FOL by 18-72% compared to the inoculated and untreated controls. Eight isolates were selected, among the most bioactive in suppressing disease by about 69-100%, and were subjected for further investigations on activity and nature of their extracellular metabolites.

The antifungal activity of the cell-free culture filtrates of these isolates was screened depending on the duration of incubation of bacterial cultures and on different treatments (heating to 50 or 100°C, pH modification or addition of proteinase K). These investigations revealed, among others, the protein nature of their extracellular metabolites. In addition, n-butanol and chloroform extracts from culture filtrates of these isolates, tested at 2.5 and 5% (v/v), had limited the in vitro FOL growth by 17- 90% compared to controls.

Tested under greenhouse conditions, B. subtilis SV41 and B. amyloliquefaciens subsp. plantarum SV65 had decreased Fusarium wilt severity by 82-88% and had improved the growth of tomato plants cv. Sahel by 10-16% and their fruit yield by 8-12% compared to the untreated controls.

The mechanisms of action involved in the promotion of plant growth and/or in the colonization of the host plant have been elucidated. The 40 tested isolates were able to produce indole-3-acetic acid (AIA) and showed pectinolytic activity. Twenty five (25) isolates were shown able to solubilize phosphate. Two isolates (B. subtilis SV41 and B. amyloliquefaciens subsp. plantarum SV65) were able to produce oxalic and malic acids.

As for the mechanisms of action involved in pathogen suppression, the 40 tested isolates were able to produce pectinases, 35 had the capacity to produce proteases and chitinases. The detection of chitinase gene (chiA) was confirmed by PCR and sequencing in some isolates. Seven isolates of Bacillus spp. were shown to be able to synthesize surfactin, fengycin D and bacillomycin D and eight isolates were capable to produce hydrogen cyanide (HCN). Eight selected isolates were able to produce siderophores. Moreover, phthalic acids were identified by GC-MS in three most bio-active organic extracts of the isolates against FOL.

Induction of tomato resistance system through the activation of the expression of proteins related-pathogenesis (PRs) genes and lipoxygenase gene (LOXD) was assessed using quantitative RT-PCR in tomato seedlings inoculated or not with FOL and treated with B. subtilis SV41 and B. amyloliquefaciens subsp. Plantarum SV65. The results revealed over expression of the gene LOXD in tomato seedlings treated with B. amyloliquefaciens subsp. Plantarum SV65 compared to FOL-uninoculated and untreated ones. This isolate had also induced activation of defense genes PR1a and PR3.

This study clearly demonstrated that wild and cultivated Solanaceae can be naturally colonized by endophytic bacteria with antifungal activity against FOL and bio-fertilizing effects on plants.

Mejri Manel. 2017. Study of wood boring insects and nematodes that they convey in Tunisian pinewoods. Doctorate thesis in Agronomic Sciences (Phytiatry), INAT, University of Carthage, Tunis, Tunisia, 196 pp. (Public Defense: 11 February 2017)

This thesis considers taxonomic and bio-ecological aspects of bark and wood boring insects threatening the main Pinus halepensis, P. pinea and P. pinaster stands in Tunisia and their associated phoretic nematodes of Bursaphelenchus genus. The overalls objectives were to obtain a knowledge about the frequent xylophagous species in the Tunisian pine forests and to assess the presence of the quarantine nematode pest Bursaphelenchus xylophilus (Aphelenchida: Parasitaphelenchidae), causal agent of Pine Wilt Disease and its potentials vectors of Monochamus genus (Coleoptera: Cerambycidae).

This study was carried out in sixteen representative forests belonging to different bioclimatic area and altitudinal range. Twenty xylophagous species were identified. The species Orthotomicus erosus, Hylurgus ligniperda, Pityogenes calcaratus, Carphoborus pini, Cryptergus mediterraneus, Chrysobothris solieri, Phaenops cyanae, Arhopalus syriacus, Pogonocherus perroudi and Monochamus galloprovincialis are the most abundant. Their geographical distributions, their relationship with their host-plants and their main bio-ecological traits were reported. M. galloprovincialis was the only species of the genus Monochamus found in Tunisia. This species is mainly associated with P. halepensis, but was also reported for the first time on P. pinea and P. pinaster. Its bio-ecological and phylogenetic characteristics were documented.

The assess of the presence of Bursaphelenchus species, associated with P. halepensis, P. pinea and P. pinaster wood and the main bark and wood boring species (M. galloprovincialis, O. erosus, H. ligniperda, P. perroudi, A. syriacus and C. solieri) was carried out using the tray method. Nematode identification was based on molecular characterization performed using PCR on the internal transcribed spacer regions (ITS1 & ITS2) and the large Sub- Unit (LSU) regions of the rDNA. Molecular characterization allowed us to confirm the identity of two nematode species belonging to the Aphelenchida order: namely Bursaphelenchus tusciae from H. ligniperda extracts and Cryptaphelenchus sp. from the three pine wood species extracts. Both nematode species and the association B. tusciae-H. ligniperda are documented for the first time in Tunisia. The phylogenetic analysis of both nematodes species sequences revealed that the Tunisian B. tusciae cluster together with an Italian and a Spanish isolates of B. tusciae registered on the NCBI database and all three isolates are forming a distinct phylogenetic group close to B. hildegardae and B. eggersi groups. As for the Tunisian Cryptaphelenchus sp. isolate, it iss grouped with a Japanese, an Australian and an Iranian Cryptaphelenchus sp. isolates, forming a separate phylogenetic group from the other analyzed Bursaphelenchus, Ektaphelenchus, Devibursaphelenchus genera. All Tunisian rDNA sequences of M. galloprovincialis, B. tusciae and Cryptaphelenchus sp. were registered in the NCBI database.

Despite the fact that we could not confirm the absence of B. xylophilus from the prospected pine woods, this study form an important contribution to the knowledge of xylophagous fauna associated Tunisian pine stands and the Bursaphelenchus species associated with those insects. Moreover, this study demonstrates the value of the molecular methodologies as helpful tools for the Tunisian pine stands identification of Bursaphelenchus species. However, further studies are needed to assess the presence of B. xylophilus and other Bursaphelenchus species.

Bouri, Meriam. 2016. Ecological specificity of Agrobacterium in Tunisian soils. Doctorate Thesis in Biological Sciences. Faculty of Sciences of Tunis, University El-Manar, Tunis, Tunisia, 168 pp. (Public Defense: 18 Mars 2017)

Agrobacteria are a group of bacteria taxonomically very much diversified, composed of many species. They are common in most soils and closely interact with plants. Rhizospheric interactions are always known to be of an anodyne type but in most instances they are likely beneficial to plants. Nevertheless, once they carry a Ti plasmid they can induce crown gall disease, a genetic disease very dreaded in Tunisia. Ti plasmids, responsible for the disease are conjugative and can easily spread in indigenous soil agrobacteria. Therefore, an epidemiological survey of the crown gall requires an ecological knowledge of these bacteria in the soil and an expert determination for the diversity of agrobacteria. Thus, the aim of this work was to assess the presence of Agrobacterium spp. populations and Ti plasmids in several soils of Tunisia, which had or had not a history of crown gall. The disease showed an emergence in fields after being for longtime limited to nurseries. The presence of agrobacteria in soil was especially correlated with the presence of stone fruit and grapevine plantations. Meanwhile, Ti plasmids were detected in fields with high evidences of the disease. Tunisian agrobacterial population was marked by the dominance of genomic species G4 and G7 of Agrobacterium spp. and two types of Ti plasmids. Populations associated to galled grapevines were distinct from those associated to galled stone fruit trees, particularly based on Ti plasmid type and L-tartrate utilization. MLST analyses revealed strong genetic relationships between the pathogenic population from galls and the pathogenic one from soils underneath corresponding galled plants, as a proof for the role of infected plants in soil contamination by pathogenic agrobacteria. There was no evidence in genetic profiles, obtained by BOX-PCR, for a clear clonal spread of Agrobacterium spp. G4 and G7 in Tunisian soils. According to competition tests and intrinsic trophic proprerties of strains from genomic species G1, G4, G7 and G8, these species can be sympatric or in competition once they are together in the soil. The degree of competition seems to be in relation with their capacity of adaptation to biotic and non-biotic soil conditions. Supported by these observations, this work concluded that the dominance of G4 and G7 in Tunisian soils is probably due to the selective effect of the soil on agrobacterial populations introduced by plants.

Haouel Hamdi, Soumaya. 2017. Insecticidal potentialities of essential oils from the genus Eucalyptus: Identification of bioactive compounds and applications for the control of the cowpea beetle Callosobruchus maculatus (Coleoptera: Chrysomelidae). Doctorate Thesis in Biological Sciences, Faculty of Mathematical, Physical and Natural Sciences of Tunis, FST, University of Tunis El-Manar, Tunisia, 209 pp. (Public Defense: 08 April 2017)

The present work aims to investigate the biology and population dynamic of Callosobruchus maculatus, to estimate quantitative and qualitative damage on three food legume hosts (chickpea, lentils, cowpea) and assess the insecticidal activity (adulticide and ovicidal) of pure and formulated aromatized powder and solid formulation of six Eucalyptus species essential oils and their major compound 1,8-cineol. Moreover, yield, chemical composition and three extraction procedures of these oils were investigated.

The results showed that C. maculatus populations were characterized by an adaptive dynamic and coevolution in relation with host substrates. The net reproduction rate (Ro), the intrinsic rate of increase (rm), the generation time (Tg) and the sex-ratio (SR) were significantly correlated with host substrates. Thus, according to our results, the preferential trophic order of C. maculatus was ranged as follow: chickpea (Amdoun 1 > Béja1) > lentils > cowpea. Besides, regarding the enormous multiplication ability and the rapidity of development time of the insect, our study confirms that C. maculatus is one of the most damaging pests of food legume stored grains in Tunisia. Otherwise, the results indicated that C. maculatus cause both quantitative and qualitative damage on different substrates. C. maculatus attacks induced decrease in grains germination, weight loss and alteration of physical-chemical and functional proprieties.

Additionally, the present study revealed the richness of the six Eucalyptus oils on bioactive compounds with insecticidal virtue (1,8-cineol, a-pinene, o and ß-cymene, aromadendrene, globulol, cryptone, a and ß-thujene). These oils were efficient in the protection of stored food legume grains against C. maculatus attacks. The superior potential of E. leucoxylon, E. lehmani and E. camaldulensis essential oils against C. maculatus could be attributed to high quantities of the majors compounds 1,8-cineol and a-pinene. In addition, interesting adulticide and ovicidal activities of aromatized powder were obtained. The aromatized powder and solid formulations improved the efficacy and remanence compared to pure oils.

This work pointed out the interest of modern extraction procedure ultrasound assisted extraction and Microwave assisted extraction in comparison to traditional procedure hydrodistillation in terms of yield improvement and enhancement of insecticidal activities. Moreover, an inter-season variability was illustrated between winter and spring harvest. Furthermore, variability in terms of numbers and proportions of identified bioactive compounds was revealed. Indeed, the winter harvest permitted to identify 6 (total of identified compounds 99.99%) compounds against 17 (total of identified compounds 97.67%) for the spring harvest.

Zammouri, Semia. 2017. Molecular identification and characterization of viral species belonging to the genus «Begomovirus» infecting vegetable crops and weeds in Tunisia. Doctorate Thesis in Agronomic Sciences (Plant Protection and Environment), ISA Chott-Mariem, University of Sousse, Chott-Mariem, Tunisia, 160 pp. (Public defense: 11 February 2017)

Plant viruses belonging to the genus Begomovirus and transmitted by whiteflies were the main cause of various emerging diseases and major economic losses that affect several crops. The Yellow Leaf Curl Disease associated with complex TYLCV (Czosnek, 2007) is one of the most damaging and emerging plant viruses in the world. In Tunisia, the consecutive emergence of two TYLCV species (Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus) gave us the opportunity to study the spatial and temporal evolution of this complex of virus.

Our results showed a partial substitution of the resident species TYLCSV by the arrival Israelite species (TYLCV) and an interaction between the two species through the coinfection and the rising of recombinant virus.

Both species associated with this virus (TYLCV and TYLCSV) were reported on a known invasive plant (Solanum elaeagnifolium) in Tunisia. Our analyzes have also shown the presence of this virus complex on watermelon showing hard watermelon syndrome. Polymorphic analysis using the Maximum Likelihood Method of 16 Tunisian strains of TYLCV reveal high mutation rate within the core region of the capsid protein. Phylogenetic Tree has shown that Tunisian isolates are part of the group of severe strain of monopartite begomovirus. Further, the ability of begomovirus to exchange genetic material through recombination, due to the presence of two species within the same host, has been demonstrated through the dissemination of Rec A and the identification of a new recombinant type B. This report was the singular in Tunisia and in the world. Thereby, these viruses are able to survive and colonize new hosts.

We have also reported the presence of a new virus causing severe disease on melon, zucchini and cucumber cultivated under geothermal plastic tunnels in regions of Gabes and Kebili. This disease was induced by a new Begomovirus; Tomato Leaf Curl Virus New Delhi (ToLCNDV) confirming, thus, the high capacity of global and local emergence and dissemination of begomoviruses.

Farhat, Imen. 2017. Identification of the export problems of Maltaise demi-sanguine orange and prospects for post-harvest citrus quality improvement. Doctorate Thesis in Agronomic Sciences (Phytiatry), INAT, University of Carthage, Tunis, Tunisia, 226 pp. (Public defense: 28 June 2017)

In the present work, the quality criteria of the orange Maltaise demi-sanguine cultivated in Cap Bon and in the new airs of extension of the citrus cultivation were studied in a first part. It consists on a diagnosis of the Maltaise demi-sanguine export sector following the 4S approach of Mainguy (health, flavor, security and service) which were assessed through the study of nutritional, organoleptic, sanitary and techno-functional quality.

The comparative study of pomological and physico-chemical attributes was carried out over three successive campaigns 2010/11, 2011/12 and 2012/13 and revealed that the orange retains its internal characteristics better than those external when grown outside Cap Bon but no region effect related to maturity index, color or other was perceived. In the case of the study of the nutritional quality of the fruit carried out during the third campaign through the evaluation of the antioxidant capacity, total phenols, phenolic compounds, vitamin C as well as organic acids and sugars, we found that the nutritional quality of the fruits is not affected by the region effect too. Nevertheless, the study of the organoleptic parameters showed significant differences in the composition of essential oils of oranges grown in different bioclimatic stages. Similarly, the presence of markers for each region leads us to conclude that the Maltaise demi-sanguine orange is affected by the environment in which it is grown and that the Cap Bon region is better for the cultivation of this variety.

The study of the sanitary quality through the analysis of the residues of 142 samples of Maltaise demi-sanguine orange taken from packing houses during the two campaigns 2012/13 and 2013/14 showed that malathion, dimethoate and methidation were the only active ingredients exceeding the MRL set by the European Union. The frequency of exceeding the MRL varied with the seasons, and methidathion and malathion decreased from 85.71 to 80% and from 54.55 to 36.36%, respectively, while dimethoate decreased from 62.5 to 75%.

In addition to the residues, a study of the epiphytic population of fungi present in the atmosphere and on fruit surfaces in nine packing houses revealed a variation in population density of Penicillium spp. between the unloading and packing compartments. However, Penicillium remains the most dominant genus with a prevalence of P. digitatum species. To control Penicillium, three active ingredients are currently used in Tunisia: benomyl, thiabendazol and imazalil. A study of the resistance of Penicillium strains to these fungicides was carried out in the context of techno-functional quality. The latter shows resistance problems with the benzimidazoles (benomyl and thiabendazol) and no resistance problems with imazalil. Hence, the orange Maltaise demi-sanguine which is characterized by exceptional flavor and nutritional quality, faces health and service problems that hinder its export.

In the second part of the work which aims to find ways to improve the post-harvest quality of citrus fruits, the 4S approach enabled the establishment of a trial to conserve two varieties of blood oranges, Sanguinelli and Tarocco, in order to optimize the nutritional, organoleptic and techno-functional quality of fruits and a trial of salt and hot water treatments of Valencia Late oranges to minimize the use of fungicides. In the first trial, the results showed that fruit flavor was not impaired by fruit storage and that the storage period of 15, 30 and 45 days did not affect the quality of the fruit. However, fruit conservation at 1°C is much better than 5 and 9°C to limit the weight loss and firmness of the two studied varieties and to maintain their nutritional quality. For the second test, the in vitro results revealed that the use of hot water alone or in the presence of salts at 52°C would inhibit the germination of P. digitatum and P. italicum conidia up to 100%, while in vivo, hot saline solutions of Na2CO3 and CaCl2 have been shown to be more effective as a curative than a preventive alternative control method. Dipping with hot water as a curative method also, reduced the diameter of P. italicum lesions.



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