EFFECT OF TEMPERATURE ON THE PATHOGENICITY OF MEDITERRANEANNATIVE ENTOMOPATHOGENIC NEMATODES (STEINERNEMATIDAEAND HETERORHABDITIDAE) FROM NATURAL ECOSYSTEMS

Seven strains of entomopathogenic nematodes (EPNs) belonging to three species (Steinernema feltiae, S. ichnusaeandHeterorhabditis bacteriophora) naturally isolated from Mediterranean countries (Southern Italy and Lebanon) wereevaluated for their potential to infest greater wax moth (Galleria mellonella) larvae at different temperatures underlaboratory conditions. The laboratory bioassay was conducted at six different temperatures ranging from 10°C to 35°C.Nematode Infective Juvenile (IJs) were put in contact with G. mellonellalarvae in Petri dishes and mortality rates wererecorded after 72 hours. The purpose of the study was to evaluate the temperature range in which the EPNs caused larvalmortality; higher mortalities were recorded at 15°C and 20°C. All species failed at lower temperatures except for S.ichnusaeItS-SAR4, which caused 7% mortality. At 35°C S. ichnusaemaintained its infectious activity (24%) along withH. bacteriophora ItH-LU1(38%); both were isolated from Italy and were more efficient at high temperatures than theremaining Lebanese isolates

PHYLOGENETIC RELATIONSHIPS OF ENTOMOPATHOGENIC NEMATODES AND THEIR BACTERIAL SYMBIONTS FROM COASTAL AREAS IN LEBANON

Entomopathogenic nematodes (EPNs) are parasites of soil-dwelling insects that occur in natural and agricultural soils around the world. The current study focuses on the unexplored coastal zone of Lebanon where soil samples were taken in different sites chosen randomly along the coast like beaches, agricultural and herbaceous fields. In total, 350 soil samples were collected, mainly from the southern part of the country. An integrated approach, combining both traditional (morphological) and molecular methods, was used to characterize entomopathogenic nematode species encountered. Two named-species are added to the EPNs catalog in Lebanon from 4 samples out of the total 350 samples isolated: Heterorhabditis indica, reported for the first time in the country (samples AYAB6 and BRA20) and Steinernema feltiae (samples ANFA5 and EDA1). Furthermore, one undescribed potential entomopathogenic nematode belonging to Oscheius genus was recovered. The symbiotic bacteria from S. feltiae and H. indica were also molecularly identified through the use of five gene fragments recA, gyrB, dnaN, gltX and infB. Phylogenetic relationships of entomopathogenic nematodes and their symbiotic bacteria were inferred by using maximum-likelihood analysis. Soil studies were subsequently carried out in order to assess a possible relationship between soil parameters and their effects on EPNs. Results indicate that sandy texture and moisture are key factors for the presence and survival of EPNs in the soil in Lebanon

Biodiversity and biogeography of Entomopathogenic nematodes in Lebanon : phylogenetic study and valorisation in biological control

Les Nématodes Entomopathogènes (NEP) de la famille des Steinernematidae et Heterorhabditidae sont des parasites pour les insectes. Ils hébergent dans leur intestin un symbiote bactérien (genres Xenorhabdus et Photorhabdus) essentiel au succès parasitaire. Les NEP sont présents dans les sols des cinq continents de la planète. Leur diversité génétique et leurs propriétés biologiques constituent une intéressante ressource biologique. Grâce à leur entomotoxicité, les NEP sont de bons outils de lutte biologique en agriculture et en culture ornementale un peu partout dans le monde. La diversité et la biogéographie des NEP dans les cinq continents de la Terre (à l'exception de l'Antarctique) ont été étudiées partout dans le monde mais le Liban est parmi les rares pays du moyen orient où aucune prospection de ces nématodes n'a été réalisée alors que des NEP en Turquie, Syrie, Jordanie, Palestine et Egypte ont déjà été trouvés et caractérisés. L'objet de la thèse est de procéder à une étude biogéographique dans le but de connaître la diversité des NEP au Liban. L'enjeu scientifique est donc de combler un « vide » dans la connaissance de la répartition et de la biodiversité mondiale des NEP. Pour cela, un échantillonnage à l'échelle des étages de végétation est mené au Liban. Des échantillons de sol sont ainsi prélevés, mis en contact avec des larves de Galleria mellonella pour isoler les nématodes entomopathogènes et leurs symbiotes. Les nématodes et leurs symbiotes sont par la suite identifiés morphologiquement et moléculairement. Par la suite, une approche à l'échelle de l'habitat fait l'objet de cette thèse également pour étudier les interactions biotiques et abiotiques influençant la présence des nématodes entomopathogènes dans le sol. Les enjeux technologiques, exposés au second volet de la thèse, sont liés aux propriétés biologiques des nématodes et de leurs symbiotes afin de valoriser leur entomotoxicité en lutte biologique. Dans ce cadre, la sensibilité des Cephalcia tannourinensis, ravageur des cédraies au Liban, par rapport aux nématodes entomopathogènes est exploitée in vitro ; différentes espèces de nématodes entomopathogènes sont testées pour suivre leur cycle à l'intérieur des Cephalcia.Entomopathogenic nematodes (EPNs) are parasites of soil-dwelling insects that occur in natural and agricultural soils around the world. Thanks to their entomotoxicity, EPNs are good tools for biological control in agriculture almost everywhere in the world. They are ubiquitous, having been isolated from every inhabited continent (except Antartica) from a wide range of ecologically diverse soil habitats including cultivated fields, forests, grasslands, deserts, and even ocean beaches. Biogeographic assessments of EPNs in the Eastern Mediterranean basin have been conducted in several countries such as Turkey, Syria, Jordan, Israel, Palestine and Egypt. Lebanon is among the few countries of the Middle East for which no survey of EPNs has been done. The scientific stake is thus to fill a gap in our knowledge of EPNs distribution in the Mediterranean basin. Survey of EPNs was conducted in this framework to cover the different vegetation levels defined in Lebanon. Soil samples were removed placed in contact with Galleria mellonella to isolate entomopathogenic nematode and their symbiotic bacteria. EPNs and their bacteria were then identified morphologically and molecularly. On the other hand, despite the different national surveys conducted on EPNs distribution around the world, habitat preferences remain inadequately known for entomopathogenic nematodes. Therefore, a comprehensive understanding of their distribution and the various biotic and abiotic factors influencing their presence is also a second object of our work. Beside a technological approach related to the biological properties of the nematodes and their symbiotics: valorisation of the entomotoxicity in biological control will be part of the third shutter of the thesis. In this framework, the sensibility of cedar pests, Cephalcia tannourinensis against entomopathogenic nematodes is exploited in vitro; different EPNs species were tested to study their life cycle inside Cephalcia larvae

Biodiversity and biogeography of Entomopathogenic nematodes in Lebanon (phylogenetic study and valorisation in biological control)

Les Nématodes Entomopathogènes (NEP) de la famille des Steinernematidae et Heterorhabditidae sont des parasites pour les insectes. Ils hébergent dans leur intestin un symbiote bactérien (genres Xenorhabdus et Photorhabdus) essentiel au succès parasitaire. Les NEP sont présents dans les sols des cinq continents de la planète. Leur diversité génétique et leurs propriétés biologiques constituent une intéressante ressource biologique. Grâce à leur entomotoxicité, les NEP sont de bons outils de lutte biologique en agriculture et en culture ornementale un peu partout dans le monde. La diversité et la biogéographie des NEP dans les cinq continents de la Terre (à l'exception de l'Antarctique) ont été étudiées partout dans le monde mais le Liban est parmi les rares pays du moyen orient où aucune prospection de ces nématodes n'a été réalisée alors que des NEP en Turquie, Syrie, Jordanie, Palestine et Egypte ont déjà été trouvés et caractérisés. L'objet de la thèse est de procéder à une étude biogéographique dans le but de connaître la diversité des NEP au Liban. L'enjeu scientifique est donc de combler un vide dans la connaissance de la répartition et de la biodiversité mondiale des NEP. Pour cela, un échantillonnage à l'échelle des étages de végétation est mené au Liban. Des échantillons de sol sont ainsi prélevés, mis en contact avec des larves de Galleria mellonella pour isoler les nématodes entomopathogènes et leurs symbiotes. Les nématodes et leurs symbiotes sont par la suite identifiés morphologiquement et moléculairement. Par la suite, une approche à l'échelle de l'habitat fait l'objet de cette thèse également pour étudier les interactions biotiques et abiotiques influençant la présence des nématodes entomopathogènes dans le sol. Les enjeux technologiques, exposés au second volet de la thèse, sont liés aux propriétés biologiques des nématodes et de leurs symbiotes afin de valoriser leur entomotoxicité en lutte biologique. Dans ce cadre, la sensibilité des Cephalcia tannourinensis, ravageur des cédraies au Liban, par rapport aux nématodes entomopathogènes est exploitée in vitro ; différentes espèces de nématodes entomopathogènes sont testées pour suivre leur cycle à l'intérieur des Cephalcia.Entomopathogenic nematodes (EPNs) are parasites of soil-dwelling insects that occur in natural and agricultural soils around the world. Thanks to their entomotoxicity, EPNs are good tools for biological control in agriculture almost everywhere in the world. They are ubiquitous, having been isolated from every inhabited continent (except Antartica) from a wide range of ecologically diverse soil habitats including cultivated fields, forests, grasslands, deserts, and even ocean beaches. Biogeographic assessments of EPNs in the Eastern Mediterranean basin have been conducted in several countries such as Turkey, Syria, Jordan, Israel, Palestine and Egypt. Lebanon is among the few countries of the Middle East for which no survey of EPNs has been done. The scientific stake is thus to fill a gap in our knowledge of EPNs distribution in the Mediterranean basin. Survey of EPNs was conducted in this framework to cover the different vegetation levels defined in Lebanon. Soil samples were removed placed in contact with Galleria mellonella to isolate entomopathogenic nematode and their symbiotic bacteria. EPNs and their bacteria were then identified morphologically and molecularly. On the other hand, despite the different national surveys conducted on EPNs distribution around the world, habitat preferences remain inadequately known for entomopathogenic nematodes. Therefore, a comprehensive understanding of their distribution and the various biotic and abiotic factors influencing their presence is also a second object of our work. Beside a technological approach related to the biological properties of the nematodes and their symbiotics: valorisation of the entomotoxicity in biological control will be part of the third shutter of the thesis. In this framework, the sensibility of cedar pests, Cephalcia tannourinensis against entomopathogenic nematodes is exploited in vitro; different EPNs species were tested to study their life cycle inside Cephalcia larvae.MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Efficacy of local strains of entomopathogenic Beauveria bassiana (Bals.) Vuill. and Steinernema feltiae (Filipjev) on the pronymphs and eonymphs of Cephalcia tannourinensis (Chevin) under laboratory conditions.

peer reviewedThe Lebanese web spinning cedar sawfly, Cephalcia tannourinensis (Chevin), is the main cedars defoliator in Lebanon. The study is to determine the effectiveness, competition and the host finding ability of two indigenous entomopathogens; a fungi Beauveria bassiana (Balsamo) and a nematode Steinernema feltiae (Filipjev) under laboratory conditions. The study included also the use of a commercial entomopathogenic nematode heterorhabditis bacteriophora (Poinar). The study targeted the two diapauses stages of the cedar sawfly, the pronymphs, and the eonymphs. One dose of the entomopathogenic nematodes and two doses of the entomopathogenic fungi were applied as well as, mixtures of the two. Local strain of B. bassiana caused mortalities of 60 and 53% on the pronymphes when applied at 50 and 500 spores/larvae, respectively. A synergistic effect was proven when the treatment consisted of two mixtures of the entomopathogens, B. bassina and S. feltiae / H. bacteriophora, and caused mortalities of 100% on the pronymphs and 86.6 to 100% on the eonymphs. The host finding ability was higher for the entomopathogenic nematode than for the entomopathogenic fungus. The use of a combination of entomopathogens proved to be more effective when only one is used. The local strain of B. bassiana when used at the dose of 500 spores/larvae was effective

Effect of temperature on the pathogenicity of mediterranean native entomopathogenic nematodes (steinernematidae and heterorhabditidae) from natural ecosystems

Seven strains of entomopathogenic nematodes (EPNs) belonging to three species (Steinernema feltiae, S. ichnusae and Heterorhabditis bacteriophora) naturally isolated from Mediterranean countries (Southern Italy and Lebanon) were evaluated for their potential to infest greater wax moth (Galleria mellonella) larvae at different temperatures under laboratory conditions. The laboratory bioassay was conducted at six different temperatures ranging from 10°C to 35°C. Nematode Infective Juvenile (IJs) were put in contact with G. mellonella larvae in Petri dishes and mortality rates were recorded after 72 hours. The purpose of the study was to evaluate the temperature range in which the EPNs caused larval mortality; higher mortalities were recorded at 15°C and 20°C. All species failed at lower temperatures except for S. ichnusae ItS-SAR4, which caused 7% mortality. At 35°C S. ichnusae maintained its infectious activity (24%) along with H. bacteriophora ItH-LU1 (38%); both were isolated from Italy and were more efficient at high temperatures than the remaining Lebanese isolates

Potential Factors behind the Decline of Pinus pinea Nut Production in Mediterranean Pine Forests

Mediterranean stone pine nut is appreciated for its high economic and nutritional value. Starting in 2012, Pinus pinea nut production declined throughout the Mediterranean area. The dry cone syndrome associated with this decline and the introduction of Leptoglossus occidentalis occurred simultaneously. This study aims to evaluate potential reasons behind the decline in pine nut production in Lebanon, considering climatic factors and the invasion of L. occidentalis. Correlation analysis was used to examine a potential relationship between cone yield and the percentage of damaged seeds per cone. Climatic variables were also tested. Two time periods were considered for analysis: before and after 2012. Cone production and the percentage of damaged seeds were negatively correlated (r = −0.42). From 2012 to 2017, cone production declined by 50% and the percentage of damaged seeds increased on average from 3% in 2012 up to 60% in 2017. Correlations were detected between cone production and the temperature of the hottest three months of the year of harvesting, and between cone production and average temperatures during the year of cone initiation. A conjunction of factors that include L. occidentalis and climatic factors might have affected the pine nut production in Lebanon

PHYLOGENETIC RELATIONSHIPS OF ENTOMOPATHOGENIC NEMATODES AND THEIR BACTERIAL SYMBIONTS FROM COASTAL AREAS IN LEBANON

Entomopathogenic nematodes (EPNs) are parasites of soil-dwelling insects that occur in natural and agricultural soils around the world. The current study focuses on the unexplored coastal zone of Lebanon where soil samples were taken in different sites chosen randomly along the coast like beaches, agricultural and herbaceous fields. In total, 350 soil samples were collected, mainly from the southern part of the country. An integrated approach, combining both traditional (morphological) and molecular methods, was used to characterize entomopathogenic nematode species encountered. Two named-species are added to the EPNs catalog in Lebanon from 4 samples out of the total 350 samples isolated: Heterorhabditis indica, reported for the first time in the country (samples AYAB6 and BRA20) and Steinernema feltiae (samples ANFA5 and EDA1). Furthermore, one undescribed potential entomopathogenic nematode belonging to Oscheius genus was recovered. The symbiotic bacteria from S. feltiae and H. indica were also molecularly identified through the use of five gene fragments recA, gyrB, dnaN, gltX and infB. Phylogenetic relationships of entomopathogenic nematodes and their symbiotic bacteria were inferred by using maximum-likelihood analysis. Soil studies were subsequently carried out in order to assess a possible relationship between soil parameters and their effects on EPNs. Results indicate that sandy texture and moisture are key factors for the presence and survival of EPNs in the soil in Lebanon

New insights in biocontrol strategy against Cephalcia tannourinensis, the principal insect defoliator of lebanese cedars

editorial reviewedTannourine cedars forest, dominated by cedars (Cedrus libani, Richard), suffers from native sawfly attacks, Cephalcia tannourinensis (Chevin). The current study assesses the presence of entomopathogenic nematodes (EPNs) endemic to the forest for their potential use as biocontrol agents in an integrated pest management to control C. tannourinensis. A survey was conducted using Galleria mellonella baits in fifteen selected sites taking into consideration the cedars' different habitats. One EPN species, Steinernema feltiae, as determined by morphometric and molecular analyses, was found in one site in the forest and was tested under laboratory conditions against C. tannourinensis prepupae, causing a mortality of 64% at 250 Infective Juveniles (IJs)/prepupa. In light of previous studies, Heterorhabditis bacteriophora (commercial strain) was accordingly considered for the controlled in situ experiments where the pathogenicity of this EPN was evaluated on C. tannourinensis. A concentration of 625,000 IJs/m2 of H. bacteriophora caused a mortality rate of 85% on C. tannourinensis following inundative treatments conducted in jars buried in the forest's soil. While EPN succeeded to emerge from G. mellonella cadavers under the same conditions, no EPN emerged out of C. tannourinensis despite the high mortality percentage; also, opportunistic nematodes emerged unexpectedly out of C. tannourinensis cadavers