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Recent Doctorate Theses in Plant Protection (2023/24)

Jenfaoui, Houda. 2023. Hypericum triquetrifolium TURRA (Wavy-leaf St John's wort) in Northern of Tunisia: Morphological and genetic characterization, geo-localization and integrated management in cereal cropping systems. Doctorate Thesis in Agronomic Sciences (Phytiatry), INAT, University of Carthage, Tunis, Tunisia, 170 pp. (Public Defense: 25 Novembre 2023).

St John’s wort (Hypericum spp.), a perennial weed known in Tunisia as El Hamra, is a widespread and troublesome weed in cereal crops in northern of Tunisian. The weed causes not only crop yield reduction but also a decrease in the quality and market value of agricultural products and a toxic effect on livestock. Once established in an area, it spreads vegetatively and becomes difficult to control without damaging other plant species. The extensive and deep perennial root system of this weed makes its chemical control difficult and inefficient. This study was undertaken to develop an integrated and sustainable weed management strategy that will decrease the wide spreading and the infestations in cereal areas in northern Tunisia. More specifically, the study was aimed to: (i) identify the weed species genetically and morphologically and better understand its phenological growth stages, (ii) delineate weed infested areas and determine the factors that contributed to the spread of the infestation, and (iii) evaluate the allelopathic activity of selected crops in bioassays and in the field to be used as components of an integrated weed management strategy against this perennial weed. Populations from six locations in northern Tunisian (Zaghouan, El Aroussa, Le Krib, Tastour, Mjez El Bab et Touiref) were studied for their genetic and morphological diversity using 10 Inter Simple Sequence Repeats (ISSR) markers and 10 vegetative morphological characteristics. The phylogenetic analysis, using 308 bp of sequenced ITS1 region, revealed the occurrence of the species Hypericum triquetrifolium TURRA. The structure analysis showed three genetic subpopulations. Morphological data showed a higher diversity than ISSR data. However, there was no evidence of correlation between genetic and morphological traits that could be suggested in this study. Zaghouan, northeastern location in the lower semi-arid, with the highest genetic (I= 0.370) and morphological (I= 0.631) Shannon’s information indexes and regrouping 2 out of the 3 genetic subpopulations, is the most probable zone of the origin of H. triquetrifolium. The results of the study of the life cycle of H. triquetrifolium for three consecutive years showed that the vegetative zero of this species is at 10°C. The highest percentage of weed germination was recorded at the temperature of 20°C for a photoperiod of 16/8 light and darkness. However, no significant difference was found between the final percentages of weed germination in light and in darkness. The predicted base temperature for germination was 8°C. The latter was used to adjust a linear model to predict H. triquetrifolium phenology based on the growing degree-days (GDD). The weed has a linear phenological cycle and requires on average of 7500 GDD to complete its cycle. Field prospections carried out in 146 infested sites in six governorates in northern Tunisia (Beja, Manouba, Bizerte, Zaghouan, Siliana and Kef) showed a variability in the rates of weed coverage which is correlated to the date of the first appearance of the weed. Results of the surveys conducted with seventy farmers showed that simplification of tillage and the absence of summer stubble ploughing, combined with the inefficacy of weed chemical control, were among the principal factors that have contributed to the spread of the weed and to the increase of areas infested by this weed. An initial screening in laboratory bioassays to determine the response of H. triquetrifolium to aqueous extracts of selected allelopathic crops (Alfalfa (Medicago sativa), Sorghum (Sorghum bicolor), Sunflower (Helianthus annuus), Rape (Brassica napus), Rye (Secale cereal), Barley (Hordeum vulgare) and Oat (Avena sativa)) showed that barley and rape had the highest phytotoxic activity on H. triquetrifolium (-74.51% and -72.29% respectively). Both crops were tested in pot experiments at three different concentrations and different growth stages of H. triquetrifolium (S1: H. triquetrifolium pre-germinated seeds, S2: four H. triquetrifolium leaves developed and S3: secondary H. triquetrifolium ramification developed). The effects of both crops’ residues on H. triquetrifolium root growth were dose dependent causing at the highest rape residue concentration an inhibition of 67% compared to the controls. The phytotoxic effect of crop residue was significant in the earliest stage of H. triquetrifolium development with up to 97% reduction caused by the rape residue compared to controls. The GC-MS analysis of rape residue revealed the presence of several allelopathic compounds, with the main compound is Tetracontane (48.25%), followed by 12-Trieosanone (CAS) Lauron (17.74%). The results of a field experiment conducted to assess the effect of the two allelopathic crops (rape and barley) combined with two tillage systems (conventional tillage (CT) and minimum tillage (MT)) and three herbicide treatments with non-treated control for three consecutive growing seasons, showed that the adoption of a conventional tillage for three consecutive years contributed to a 71% decrease in H. triquetrifolium densities. Our results revealed that H. triquetrifolium densities, the weed coverage and the total biomass accumulation, were more affected in barley crop. The lowest densities and percentages of weed coverage were observed by associating the allelopathic effect of barley with conventional tillage as compared to those obtained in barley with minimum tillage and without herbicide treatment. The allelopathic potential of barley alone was not able to reduce H. triquetrifolium infestations. Therefore, the practice of a long-term rotation integrating barley and the adoption of conventional tillage with a summer herbicide treatment may be suggested as an integrated management strategy against this perennial weed and may contribute to reducing a long-term infestation in cereal cropping systems.

Mechichi, Ghaya. Characterization of agricultural and phytosanitary practices adopted in Tunisian viticulture and study of the quality of grapes and their by-products in terms of pesticide residues. Doctorate Thesis in Agronomic Sciences (Phytiatry), INAT, University of Carthage, Tunis, Tunisia, and Faculty of Veterinary Medicine, University of Liège, Belgium, 245 pp. (Public Defense: 26 April 2024)

Viticulture is prone to a wide range of phytosanitary issues, and pesticide application is one of the most effective methods to manage them, despite the adverse effects of these chemicals on both human health and the environment. No previous research has been conducted to highlight the agricultural and phytosanitary practices used in Tunisian vineyards to produce wine and dried grapes. Thus, this study aims to characterize the phytosanitary practices used in Tunisian table and wine vineyards, to describe the vinification and drying processes used and evaluate the factors contributing to the presence of pesticide residues in fresh and processed products. To achieve these goals, field surveys and sampling were conducted during the 2018/19, 2019/20 and 2020/21 grape growing seasons, and samples of wine grapes, must, wine, table grapes before and after treatment, and dried grapes were collected and analyzed using three accredited multi-residue analysis methods in the laboratories of Sciensano. On the other hand, the treatment frequency indices (TFI), and the risk indicators of Quebec pesticides (health (HRI) and environment (ERI)) were also assessed in Tunisian wine vineyards. The results showed a high diversity of grape varieties in wine grape vineyards, while only one grape variety was used for drying. The study also revealed different vinification techniques, particularly for the clarification and filtration steps, as reported by the winemakers. The drying process also showed a high diversity of techniques, especially in the pretreatment step. The most often mentioned phytosanitary problem in both wine and table grape vineyards was fungal diseases, leading to the frequent use of fungicides (79%) in wine grape vineyards, with average TFI values of 14.9 and 18.7 during the 2018/19 and 2019/20 agricultural campaigns, respectively. The TFI values could not be calculated for table grape vineyards due to the lack of information provided by the grape growers. The evaluation of the average risk using the Quebec pesticide risk indicator (QPRI) revealed a high risk for the fungicides thiophanate-methyl (HRI = 295.5), metiram (HRI = 380), and penconazole (HRI = 276.4), as well as for the insecticides chlorpyriphos-ethyl (HRI = 274.4) and lambda-cyhalothrin (HRI = 285) for human health. In addition, insecticides presented a higher risk than fungicides for the environment due to their impact particularly on bees. Regarding the risk for consumers, it has been observed that 15% of the active substances applied by grape growers have also been detected in the wine grape samples such as: thiophanate-methyl, fenhexamid, iprovalicarb, and imidacloprid. These substances have also been detected in the must and wine samples. However, 40% of the molecules detected in the wine grape samples were not mentioned in the survey, such as boscalid and iprodione, but were detected in the samples of must and wines. Furthermore, among all the analyzed samples, exceeding the maximum residue limits set by European and/or international regulations (Codex Alimentarius) was observed in 36 out of 108 fresh wine grape samples, 3 out of 8 table grape samples, 3 out of 15 wine samples and 2 out of 8 dried grape samples. This non-compliance was due either to the use of non-approved active substances in the European Union or to non-compliance with recommended doses (as in the case of carbendazim resulting from the degradation of thiophanate-methyl used at 2.8 times the recommended dose). Therefore, it is recommended that Tunisian authorities train the grape growers in good agricultural practices and establish a monitoring program that includes the use of pesticides and the analysis of wine and table grapes to improve the quality of Tunisian wine and dried grapes in terms of pesticide residue content.


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	- Recent Doctorates - *Recent Doctorate Theses in Plant Protection (2023/24)* Jenfaoui, Houda. 2023. Hypericum triquetrifolium TURRA (Wavy-leaf St John's wort) in Northern of Tunisia: Morphological and genetic characterization, geo-localization and integrated management in cereal cropping systems. Doctorate Thesis in Agronomic Sciences (Phytiatry), INAT, University of Carthage, Tunis, Tunisia, 170 pp. (Public Defense: 25 Novembre 2023). St John’s wort (Hypericum spp.), a perennial weed known in Tunisia as El Hamra, is a widespread and troublesome weed in cereal crops in northern of Tunisian. The weed causes not only crop yield reduction but also a decrease in the quality and market value of agricultural products and a toxic effect on livestock. Once established in an area, it spreads vegetatively and becomes difficult to control without damaging other plant species. The extensive and deep perennial root system of this weed makes its chemical control difficult and inefficient. This study was undertaken to develop an integrated and sustainable weed management strategy that will decrease the wide spreading and the infestations in cereal areas in northern Tunisia. More specifically, the study was aimed to: (i) identify the weed species genetically and morphologically and better understand its phenological growth stages, (ii) delineate weed infested areas and determine the factors that contributed to the spread of the infestation, and (iii) evaluate the allelopathic activity of selected crops in bioassays and in the field to be used as components of an integrated weed management strategy against this perennial weed. Populations from six locations in northern Tunisian (Zaghouan, El Aroussa, Le Krib, Tastour, Mjez El Bab et Touiref) were studied for their genetic and morphological diversity using 10 Inter Simple Sequence Repeats (ISSR) markers and 10 vegetative morphological characteristics. The phylogenetic analysis, using 308 bp of sequenced ITS1 region, revealed the occurrence of the species Hypericum triquetrifolium TURRA. The structure analysis showed three genetic subpopulations. Morphological data showed a higher diversity than ISSR data. However, there was no evidence of correlation between genetic and morphological traits that could be suggested in this study. Zaghouan, northeastern location in the lower semi-arid, with the highest genetic (I= 0.370) and morphological (I= 0.631) Shannon’s information indexes and regrouping 2 out of the 3 genetic subpopulations, is the most probable zone of the origin of H. triquetrifolium. The results of the study of the life cycle of H. triquetrifolium for three consecutive years showed that the vegetative zero of this species is at 10°C. The highest percentage of weed germination was recorded at the temperature of 20°C for a photoperiod of 16/8 light and darkness. However, no significant difference was found between the final percentages of weed germination in light and in darkness. The predicted base temperature for germination was 8°C. The latter was used to adjust a linear model to predict H. triquetrifolium phenology based on the growing degree-days (GDD). The weed has a linear phenological cycle and requires on average of 7500 GDD to complete its cycle. Field prospections carried out in 146 infested sites in six governorates in northern Tunisia (Beja, Manouba, Bizerte, Zaghouan, Siliana and Kef) showed a variability in the rates of weed coverage which is correlated to the date of the first appearance of the weed. Results of the surveys conducted with seventy farmers showed that simplification of tillage and the absence of summer stubble ploughing, combined with the inefficacy of weed chemical control, were among the principal factors that have contributed to the spread of the weed and to the increase of areas infested by this weed. An initial screening in laboratory bioassays to determine the response of H. triquetrifolium to aqueous extracts of selected allelopathic crops (Alfalfa (Medicago sativa), Sorghum (Sorghum bicolor), Sunflower (Helianthus annuus), Rape (Brassica napus), Rye (Secale cereal), Barley (Hordeum vulgare) and Oat (Avena sativa)) showed that barley and rape had the highest phytotoxic activity on H. triquetrifolium (-74.51% and -72.29% respectively). Both crops were tested in pot experiments at three different concentrations and different growth stages of H. triquetrifolium (S1: H. triquetrifolium pre-germinated seeds, S2: four H. triquetrifolium leaves developed and S3: secondary H. triquetrifolium ramification developed). The effects of both crops’ residues on H. triquetrifolium root growth were dose dependent causing at the highest rape residue concentration an inhibition of 67% compared to the controls. The phytotoxic effect of crop residue was significant in the earliest stage of H. triquetrifolium development with up to 97% reduction caused by the rape residue compared to controls. The GC-MS analysis of rape residue revealed the presence of several allelopathic compounds, with the main compound is Tetracontane (48.25%), followed by 12-Trieosanone (CAS) Lauron (17.74%). The results of a field experiment conducted to assess the effect of the two allelopathic crops (rape and barley) combined with two tillage systems (conventional tillage (CT) and minimum tillage (MT)) and three herbicide treatments with non-treated control for three consecutive growing seasons, showed that the adoption of a conventional tillage for three consecutive years contributed to a 71% decrease in H. triquetrifolium densities. Our results revealed that H. triquetrifolium densities, the weed coverage and the total biomass accumulation, were more affected in barley crop. The lowest densities and percentages of weed coverage were observed by associating the allelopathic effect of barley with conventional tillage as compared to those obtained in barley with minimum tillage and without herbicide treatment. The allelopathic potential of barley alone was not able to reduce H. triquetrifolium infestations. Therefore, the practice of a long-term rotation integrating barley and the adoption of conventional tillage with a summer herbicide treatment may be suggested as an integrated management strategy against this perennial weed and may contribute to reducing a long-term infestation in cereal cropping systems. Mechichi, Ghaya. Characterization of agricultural and phytosanitary practices adopted in Tunisian viticulture and study of the quality of grapes and their by-products in terms of pesticide residues. Doctorate Thesis in Agronomic Sciences (Phytiatry), INAT, University of Carthage, Tunis, Tunisia, and Faculty of Veterinary Medicine, University of Liège, Belgium, 245 pp. (Public Defense: 26 April 2024) Viticulture is prone to a wide range of phytosanitary issues, and pesticide application is one of the most effective methods to manage them, despite the adverse effects of these chemicals on both human health and the environment. No previous research has been conducted to highlight the agricultural and phytosanitary practices used in Tunisian vineyards to produce wine and dried grapes. Thus, this study aims to characterize the phytosanitary practices used in Tunisian table and wine vineyards, to describe the vinification and drying processes used and evaluate the factors contributing to the presence of pesticide residues in fresh and processed products. To achieve these goals, field surveys and sampling were conducted during the 2018/19, 2019/20 and 2020/21 grape growing seasons, and samples of wine grapes, must, wine, table grapes before and after treatment, and dried grapes were collected and analyzed using three accredited multi-residue analysis methods in the laboratories of Sciensano. On the other hand, the treatment frequency indices (TFI), and the risk indicators of Quebec pesticides (health (HRI) and environment (ERI)) were also assessed in Tunisian wine vineyards. The results showed a high diversity of grape varieties in wine grape vineyards, while only one grape variety was used for drying. The study also revealed different vinification techniques, particularly for the clarification and filtration steps, as reported by the winemakers. The drying process also showed a high diversity of techniques, especially in the pretreatment step. The most often mentioned phytosanitary problem in both wine and table grape vineyards was fungal diseases, leading to the frequent use of fungicides (79%) in wine grape vineyards, with average TFI values of 14.9 and 18.7 during the 2018/19 and 2019/20 agricultural campaigns, respectively. The TFI values could not be calculated for table grape vineyards due to the lack of information provided by the grape growers. The evaluation of the average risk using the Quebec pesticide risk indicator (QPRI) revealed a high risk for the fungicides thiophanate-methyl (HRI = 295.5), metiram (HRI = 380), and penconazole (HRI = 276.4), as well as for the insecticides chlorpyriphos-ethyl (HRI = 274.4) and lambda-cyhalothrin (HRI = 285) for human health. In addition, insecticides presented a higher risk than fungicides for the environment due to their impact particularly on bees. Regarding the risk for consumers, it has been observed that 15% of the active substances applied by grape growers have also been detected in the wine grape samples such as: thiophanate-methyl, fenhexamid, iprovalicarb, and imidacloprid. These substances have also been detected in the must and wine samples. However, 40% of the molecules detected in the wine grape samples were not mentioned in the survey, such as boscalid and iprodione, but were detected in the samples of must and wines. Furthermore, among all the analyzed samples, exceeding the maximum residue limits set by European and/or international regulations (Codex Alimentarius) was observed in 36 out of 108 fresh wine grape samples, 3 out of 8 table grape samples, 3 out of 15 wine samples and 2 out of 8 dried grape samples. This non-compliance was due either to the use of non-approved active substances in the European Union or to non-compliance with recommended doses (as in the case of carbendazim resulting from the degradation of thiophanate-methyl used at 2.8 times the recommended dose). Therefore, it is recommended that Tunisian authorities train the grape growers in good agricultural practices and establish a monitoring program that includes the use of pesticides and the analysis of wine and table grapes to improve the quality of Tunisian wine and dried grapes in terms of pesticide residue content.
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