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Recent Doctorate Theses in Plant Protection (2022/23)

Hlaiem, Sawssen. 2023. Investigation and characterization of pathogenic fungi associated with forest species in Northern coastal dunes of Tunisia. Doctorate Thesis in Agronomic Sciences (Phytiatry), INAT, University of Carthage, Tunis, Tunisia, 195 pp. (Public Defense: 09 January 2023).

Pathogenic fungi are amongst the main causes of forest trees and shrubs diseases. This study aims to investigate the dieback disease in two forests in the north-east (Henchir Kort) and north (Rimel) of Tunisia. The identification of the fungi was carried out by means of morphological and molecular features; then the pathogenicity of the isolates was evaluated and the fungal growth was evaluated at temperatures ranging from 5 to 40 °C on PDA medium. Antagonism tests were performed using direct and indirect confrontations between pathogenic fungi and the antagonist strain of Trichoderma obtained in this study. A collection of 115 fungal isolates was obtained from symptomatic branches of Pinus trees (Pinus halepensis and P. pinea) and shrubs species (e.g. Juniperus oxycedrus, Tetraclinis articulata, Pistacia lentiscus, Olea europeae, Erica arborea, Retama raetam, Quercus coccifera). Five different genera have been characterized: Botyosphaeria spp., Pestalotiopsis spp., Heterotruncatella spp. Alternaria spp. and Fusarium spp. The results showed that Botryosphaeria spp. were the most frequently isolated fungi, followed by Pestalotiopsis spp. Their isolation frequency has been noticed to be significantly correlated to the dendrometric parameters and ecological factors (p < 0.000). Morphological identification and genetic analysis of internal transcribed spacer region (ITS) of rDNA, and partial sequencing of the translational elongation factor 1-alpha (Tef-1 α) and β-tubulin (TUB) genes identified 12 fungal species: Diplodia pseudoseriata, D. seriata, D. scrobiculata, D. africana, Neofusicoccum luteum, Pestalotiopsis biciliata, P. chamaeropis, Heterotruncatella spartii, Alternaria alternata, A. infectoria, A. tenuissima and Fusarium oxysporum. The evaluation of the pathogenicity of the isolates confirmed their aggressiveness towards their host plants. Diplodia scrobiculata isolate was the most aggressive species. The results of tested temperatures suggested that the optimum growth temperature of the tested isolated was 25°C. The temperatures of 5°C and 40°C inhibited the mycelial growth of all the examined fungi except D. seriata, which could grow at 40°C. The genus Trichoderma is the most fungal used as biocontrol agent. Morphological and molecular characteristics of the ITS region allow to identify 15 isolates as Trichoderma harzianum isolated from asymptomatic branches of P. pinea trees. Direct (on PDA medium) or indirect (remote) confrontation tests between fungal isolates and T. harzianum showed that the antagonist has inhibited mycelial growth of the pathogenic fungus compared to the untreated control. In conclusion, this study indicated that the majority of the investigated forest types are threatened by dieback, and the forest in northern Tunisia (Rimel) appears to be the most affected. This decline in forests could be mainly linked to the impacts of climate change.

Hanafi, Marwa. 2023. Detection and identification of viruses infecting Musa spp. using polymerase chain reaction and high throughput sequencing technologies. Doctorate Thesis in Agronomic Sciences and Biological Engineering, Gembloux Agro-Bio Tech, University of Liege, Belgium, 212 pp. (Public Defense: 03 July 2023).

Banana mild mosaic virus (BanMMV) (Betaflexiviridae, Quinivirinae, unassigned species) is a filamentous virus infecting Musa spp. with a very wide geographic distribution. It is defined as a significant risk for the banana sector in particular in the various DROMs, and seems to fulfil the criteria of a regulated non-quarantine pest. Therefore, it is important to establish appropriate control measures based on the setting-up of specific and sensitive diagnostic techniques. BanMMV displays a very high genome variability which makes its molecular detection by specific primers particularly challenging, and requires the development of diagnostic tests with high inclusivity. BanMMV detection and/or sanitation remains labor- and time-consuming. The current BanMMV indexing process for an accession requires the testing of no less than four plants cultivated in a greenhouse for at least 6 months and causes a significant delay for the distribution of the germplasm. The main objective of this thesis was to improve the diagnostic of BanMMV from Musa accessions. This study aimed to design new diagnostic primers in order to improve detection of BanMMV, following the identification of novel isolates of the virus. It also aimed to test the use of banana in vitro plants in order to accelerate the testing process and evaluate virus therapy success. In this context, a discrepancy in test result was observed between electron microscopy and immunocapture (IC) reverse transcription (RT) polymerase chain reaction (PCR) test results for one asymptomatic banana accession. The absence of molecular detection and the presence of filamentous particles suggested the presence of a new variants or new virus. The accession underwent high throughput sequencing which allowed the identification of two complete genomes of BanMMV with high nucleotide identity that constitute the two novel isolates of BanMMV found in this accession. These findings triggered the development of a new diagnostic primer based on these two new sequences and the BanMMV CP sequences already published in GenBank. A retrospective analysis of 110 different germplasm accessions from diverse origins was performed in order to compare BanMMV CP9 and BanMMCP2 primers. Five accessions showed contrasting results. The new primer missed the detection of BanMMV infection from three accessions. Similarly, BanMMCP2 failed to detect BanMMV infection from two banana accessions. Interestingly, the analytical sensitivity was better with BanMMV CP9 comparing to BanMMCP2. Through this study, we recommended the use of the two primers successively to improve the inclusiveness of the protocol. HTS technologies are one of the most important advances that have revolutionized molecular diagnostics. Their adoption in plant pest diagnostics, in particular plant virus diagnostics, has been growing steadily in the last decades. In same line, this study showed the improved performance of these technologies in BanMMV detection in comparison to the conventional molecular techniques from the same plants (in absence of therapy). These without a priori technologies were much more sensitive than RT-PCR from the same in vitro plants with 100% of diagnostic sensitivity (DSE) for HTS comparing to a DSE of 65% for RT-PCR. Interestingly, HTS technologies allowed the identification of a new species from these samples, with a genome of 7,364 bp, presenting a typical genome organisation of Betaflexiviridae members after annotation of its new contig. Thus, it could be interesting to consider the in-depth biological characterization of this new species. This would help to know more about the symptomatology, transmission, host range of the new species, and its potential impact on either banana industry and/or the environment. In summary, this study suggested two tests for the detection of BanMMV from in vitro plants. HTS technologies could be performed from RNA extracts of pooled leaves or bases. If these technologies are not available or too expensive, RT-PCR could be applied instead on individual RNA extracted from at least four plants. This proposed methodology helps to avoid the greenhouse cultivation and thus can save time and space.


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	- Recent Doctorates - *Recent Doctorate Theses in Plant Protection (2022/23)* Hlaiem, Sawssen. 2023. Investigation and characterization of pathogenic fungi associated with forest species in Northern coastal dunes of Tunisia. Doctorate Thesis in Agronomic Sciences (Phytiatry), INAT, University of Carthage, Tunis, Tunisia, 195 pp. (Public Defense: 09 January 2023). Pathogenic fungi are amongst the main causes of forest trees and shrubs diseases. This study aims to investigate the dieback disease in two forests in the north-east (Henchir Kort) and north (Rimel) of Tunisia. The identification of the fungi was carried out by means of morphological and molecular features; then the pathogenicity of the isolates was evaluated and the fungal growth was evaluated at temperatures ranging from 5 to 40 °C on PDA medium. Antagonism tests were performed using direct and indirect confrontations between pathogenic fungi and the antagonist strain of Trichoderma obtained in this study. A collection of 115 fungal isolates was obtained from symptomatic branches of Pinus trees (Pinus halepensis and P. pinea) and shrubs species (e.g. Juniperus oxycedrus, Tetraclinis articulata, Pistacia lentiscus, Olea europeae, Erica arborea, Retama raetam, Quercus coccifera). Five different genera have been characterized: Botyosphaeria spp., Pestalotiopsis spp., Heterotruncatella spp. Alternaria spp. and Fusarium spp. The results showed that Botryosphaeria spp. were the most frequently isolated fungi, followed by Pestalotiopsis spp. Their isolation frequency has been noticed to be significantly correlated to the dendrometric parameters and ecological factors (p < 0.000). Morphological identification and genetic analysis of internal transcribed spacer region (ITS) of rDNA, and partial sequencing of the translational elongation factor 1-alpha (Tef-1 α) and β-tubulin (TUB) genes identified 12 fungal species: Diplodia pseudoseriata, D. seriata, D. scrobiculata, D. africana, Neofusicoccum luteum, Pestalotiopsis biciliata, P. chamaeropis, Heterotruncatella spartii, Alternaria alternata, A. infectoria, A. tenuissima and Fusarium oxysporum. The evaluation of the pathogenicity of the isolates confirmed their aggressiveness towards their host plants. Diplodia scrobiculata isolate was the most aggressive species. The results of tested temperatures suggested that the optimum growth temperature of the tested isolated was 25°C. The temperatures of 5°C and 40°C inhibited the mycelial growth of all the examined fungi except D. seriata, which could grow at 40°C. The genus Trichoderma is the most fungal used as biocontrol agent. Morphological and molecular characteristics of the ITS region allow to identify 15 isolates as Trichoderma harzianum isolated from asymptomatic branches of P. pinea trees. Direct (on PDA medium) or indirect (remote) confrontation tests between fungal isolates and T. harzianum showed that the antagonist has inhibited mycelial growth of the pathogenic fungus compared to the untreated control. In conclusion, this study indicated that the majority of the investigated forest types are threatened by dieback, and the forest in northern Tunisia (Rimel) appears to be the most affected. This decline in forests could be mainly linked to the impacts of climate change. Hanafi, Marwa. 2023. Detection and identification of viruses infecting Musa spp. using polymerase chain reaction and high throughput sequencing technologies. Doctorate Thesis in Agronomic Sciences and Biological Engineering, Gembloux Agro-Bio Tech, University of Liege, Belgium, 212 pp. (Public Defense: 03 July 2023). Banana mild mosaic virus (BanMMV) (Betaflexiviridae, Quinivirinae, unassigned species) is a filamentous virus infecting Musa spp. with a very wide geographic distribution. It is defined as a significant risk for the banana sector in particular in the various DROMs, and seems to fulfil the criteria of a regulated non-quarantine pest. Therefore, it is important to establish appropriate control measures based on the setting-up of specific and sensitive diagnostic techniques. BanMMV displays a very high genome variability which makes its molecular detection by specific primers particularly challenging, and requires the development of diagnostic tests with high inclusivity. BanMMV detection and/or sanitation remains labor- and time-consuming. The current BanMMV indexing process for an accession requires the testing of no less than four plants cultivated in a greenhouse for at least 6 months and causes a significant delay for the distribution of the germplasm. The main objective of this thesis was to improve the diagnostic of BanMMV from Musa accessions. This study aimed to design new diagnostic primers in order to improve detection of BanMMV, following the identification of novel isolates of the virus. It also aimed to test the use of banana in vitro plants in order to accelerate the testing process and evaluate virus therapy success. In this context, a discrepancy in test result was observed between electron microscopy and immunocapture (IC) reverse transcription (RT) polymerase chain reaction (PCR) test results for one asymptomatic banana accession. The absence of molecular detection and the presence of filamentous particles suggested the presence of a new variants or new virus. The accession underwent high throughput sequencing which allowed the identification of two complete genomes of BanMMV with high nucleotide identity that constitute the two novel isolates of BanMMV found in this accession. These findings triggered the development of a new diagnostic primer based on these two new sequences and the BanMMV CP sequences already published in GenBank. A retrospective analysis of 110 different germplasm accessions from diverse origins was performed in order to compare BanMMV CP9 and BanMMCP2 primers. Five accessions showed contrasting results. The new primer missed the detection of BanMMV infection from three accessions. Similarly, BanMMCP2 failed to detect BanMMV infection from two banana accessions. Interestingly, the analytical sensitivity was better with BanMMV CP9 comparing to BanMMCP2. Through this study, we recommended the use of the two primers successively to improve the inclusiveness of the protocol. HTS technologies are one of the most important advances that have revolutionized molecular diagnostics. Their adoption in plant pest diagnostics, in particular plant virus diagnostics, has been growing steadily in the last decades. In same line, this study showed the improved performance of these technologies in BanMMV detection in comparison to the conventional molecular techniques from the same plants (in absence of therapy). These without a priori technologies were much more sensitive than RT-PCR from the same in vitro plants with 100% of diagnostic sensitivity (DSE) for HTS comparing to a DSE of 65% for RT-PCR. Interestingly, HTS technologies allowed the identification of a new species from these samples, with a genome of 7,364 bp, presenting a typical genome organisation of Betaflexiviridae members after annotation of its new contig. Thus, it could be interesting to consider the in-depth biological characterization of this new species. This would help to know more about the symptomatology, transmission, host range of the new species, and its potential impact on either banana industry and/or the environment. In summary, this study suggested two tests for the detection of BanMMV from in vitro plants. HTS technologies could be performed from RNA extracts of pooled leaves or bases. If these technologies are not available or too expensive, RT-PCR could be applied instead on individual RNA extracted from at least four plants. This proposed methodology helps to avoid the greenhouse cultivation and thus can save time and space.
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