Publications of Caroline De Clerck
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See detailLinolenic fatty acid hydroperoxide acts as biocide on plant pathogenic bacteria: biophysical investigation of the mode of action
Deboever, Estelle ULiege; Lins, Laurence ULiege; Ongena, Marc ULiege et al

in Bioorganic Chemistry (2020)

Fatty acid hydroperoxides (HPO) are free phyto-oxylipins known for their crucial role as signalling molecules during plant defense mechanisms. They were also demonstrated to have direct biocidal ... [more ▼]

Fatty acid hydroperoxides (HPO) are free phyto-oxylipins known for their crucial role as signalling molecules during plant defense mechanisms. They were also demonstrated to have direct biocidal activities against plant pathogens including gram negative bacteria. In the present work, the biocidal effect of one linolenic fatty acid hydroperoxide, namely 13-HPOT has been investigated on three plant pathogen gram negative bacteria: Pectobacterium carotovorum, Pseudomonas syringae and Xanthomonas translucens. We showed that 13-HPOT has a strong dose response effect on those phytopathogens. In a second part, the molecular mechanism behind the antibacterial effect of 13-HPOT was investigated at a molecular level using an integrative biophysical approach combining in vitro and in silico methods. Since other antimicrobial amphiphilic molecules have been shown to target the lipid membrane of the organisms they act on, we focused our study on the interaction of 13-HPOT with biomimetic membranes. In a first step, we hypothesized that the inner membrane of the bacteria was the main site of action of 13-HPOT and hence we used lipids representative of this membrane to form our models. Our results indicated that 13-HPOT can interact with the lipid representative of the inner bacterial plasma membrane. A strong membrane insertion is suggested but no major permeabilization of the membrane is observed. Phosphatidylethanolamine (PE) and cardiolipin (CL), present in the bacterial plasma membrane, appear to play important roles in this interaction. We suggest that the mode of action of 13-HPOT should involve either subtle changes in membrane properties, such as its lateral organization and distribution, and/or interactions with membrane proteins. [less ▲]

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See detailMolecular biophysics as a tool to investigate the bioherbicide effect of essential oils related to their interaction with plant plasma membrane
Lins, Laurence ULiege; De Clerck, Caroline ULiege; Fauconnier, Marie-Laure ULiege et al

Conference (2020, February)

Essential oils (EOs) are used in an increasingly number of sectors like medicine, cosmetics, food industry and more recently in agronomy. In agronomy, EOs are used as bio-pesticides for their insecticidal ... [more ▼]

Essential oils (EOs) are used in an increasingly number of sectors like medicine, cosmetics, food industry and more recently in agronomy. In agronomy, EOs are used as bio-pesticides for their insecticidal, antifungal or bactericidal effects but also as bio-herbicides. Owing to the current attraction for natural products, a better understanding of their mode of biological action for new and optimal applications is of importance. It has been shown that EOs antimicrobial activity, quite well described in the literature, is at least partly due to their interaction with the plasma membrane. They notably change the lipid composition, altering fluidity, leading to various effects which can induce cell lysis, apoptosis or necrosis. Citronellol, a major compound of lemongrass EO was notably shown to have antifungal activities by changing the membrane composition and inhibiting cell growth. Cinnamaldehyde (cinnamon EO) has been reported to have a broad spectrum of antibacterial activity, notably by affecting cell morphology, membrane integrity, permeability and composition. We are currently working on the development of a bioherbicide made from Cinnamomum zeylanicum Blume (cinnamon) and Cymbogognon winterianus Jowitt (citronella) EOs. We have shown that the application of the whole EOs and their major individual compounds on the leaves and cotyledons of A. thaliana appears to be promising: when applied on cotyledons or leaves, EOs induce damages that are as important as those observed for commercial herbicides. Since EOs are small amphiphilic molecules, they can cross the mesh of cell wall and interact directly with the plant plasma membrane (PPM). Modifying the lipid organization could lead to crucial cellular effects, notably on protein function. We used a unique and original combination of in silico (molecular dynamics simulations) and in vitro (Langmuir monolayers, isothermal calorimetry, fluorescence and infrared spectroscopies) biophysical approaches, previously developed to study structure-function relationships of molecules of biological interest (pharmacological drugs , proteins, peptides, surfactants…) to investigate the interaction of EOs or their individual compounds with bio-mimetic plant plasma membranes to better understand the structure- activity relationships in the context of their bioherbicide activity. [less ▲]

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See detailIdentification and management of vanilla diseases in Madagascar
Raveloson, Lanto ULiege; El Jaziri, Mondher; De Clerck, Caroline ULiege et al

Poster (2020, January 31)

About 97% of the real vanilla come from Madagascar worldwide. Madagascar’s vanilla is well known for its quality, and its production is very important for the country’s economy. However this culture is ... [more ▼]

About 97% of the real vanilla come from Madagascar worldwide. Madagascar’s vanilla is well known for its quality, and its production is very important for the country’s economy. However this culture is threatened by the emergence of fungal and viral diseases that have been recorded for a decade, causing significant devaluations of the production’s level of quality and an important reduction of the quantities annually exported. In order to be able to scientifically construct a strategy of prevention and control against the main pathogens of vanilla, large sampling campaign was carried out in Madagascar’s SAVA region. Lianas showing symptoms of cryptogamic diseases such as rot, wilt, necrosis or melanosis were harvested. 75 bacteria and 61 fungi were isolated on NA and PDA medium and identified. Morphological and molecular identifications (based on the sequencing of 16S rRNA and ITS regions) of these isolates have shown that the genus Fusarium is the most present (27% of fungal isolates) and is represented by species of F. oxysporum, F. proliferatum, F. concentricum, F. equiseti, F. pseudocircinatum and F. mangiferae. While for bacteria, the genus Bacillus predominates (44% of purified bacterial strains). Many bacteria of the genus Bacillus are known for their biocontrol properties; this is why we have tested the potential antagonistic effect of all vanilla-isolated Bacillus strains against two species of Fusarium. Nine Bacillus strains were identified for reducing the growth of the fungi, and are of great potential for the development of a biocontrol strategy against Fusarium attacking vanilla in Madagascar. [less ▲]

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See detailCan fungal volatile organic compounds be used to develop aflatoxin-specific sensors?
Josselin, Laurie ULiege; De Boevre, Marthe; De Clerck, Caroline ULiege et al

Poster (2020, January 31)

Foodstuff (corn, wheat, rice, etc.) can be contaminated by several filamentous fungal species in pre or post-harvest conditions. Some of these, such as Aspergillus, Fusarium and Penicillium produce ... [more ▼]

Foodstuff (corn, wheat, rice, etc.) can be contaminated by several filamentous fungal species in pre or post-harvest conditions. Some of these, such as Aspergillus, Fusarium and Penicillium produce secondary metabolites, highly toxic at low concentrations to all vertebrates including humans: they can cause severe illnesses upon chronic exposure and can even lead to death after acute exposure. These non-volatile molecules are named mycotoxins and current methods to detect them, involving the use of ELISA tests or HPLC, are quite time consuming and expensive. At present there is no rapid test that does not require extensive sample preparation to detect the presence of mycotoxin directly in a production line (e.g. grain storage companies). Therefore, the aim of this work is to identify volatile organic compounds (VOCs) markers, specific of mycotoxins’ production in foodstuff. Using the SPME technique, we have characterized and compared the VOCs produced in vitro by non-aflatoxigenic (not producing aflatoxins) and aflatoxigenic strains of Aspergillus flavus (producing aflatoxins B1, B2 and G2, three types of mycotoxins). Preliminary analyses have shown similarities and differences between the two strains. Both of them emit VOCs as 1-octen-3-ol, 3-methylbutan-1-ol, octan-3-one, 2-methylbutanal, 3-methylbutanal, known in the literature to be specific of fungi. In particular, we have identified several strain-specific terpenes that are of interest for the development of the future molecular foot-print sensor. The next step is to study the VOCs produced in in vivo conditions, when the fungi are growing on stored cereals; and the correlation between specific VOCs and mycotoxin production. [less ▲]

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See detailCynara cardunculus crude extract as a powerful natural herbicide and insight into the mode of action of its bioactive molecules
Ben Kaab, Sofiène ULiege; Lins, Laurence ULiege; Hanafi, Marwa ULiege et al

in Biomolecules (2020)

The use of chemical herbicides could not only potentially induce negative impacts on the environment, animals, and human health, but also increase the weed resistance to herbicides. In this context, the ... [more ▼]

The use of chemical herbicides could not only potentially induce negative impacts on the environment, animals, and human health, but also increase the weed resistance to herbicides. In this context, the use of plant extracts could be an interesting and natural alternative to chemical products. It is important to understand the mode of action of their bioactive compounds. This is why we have studied the herbicidal effect of Cynara cardunculus crude extract in terms of inhibition of weeds’ seedling growth and its impact on physiological parameters of treated plantlets, like conductivity, dry weight, and fluorescence, and biochemical parameters linked to oxidative stress. We have observed that C. cardunculus crude extract induces oxidative stress in the treated plants and consequently disturbs the physiological and biochemical functions of the plant cells. We have investigated the herbicidal activity of three bioactive compounds, naringenin, myricitrin, and quercetin, from the C. cardunculus crude extract. In both pre- and post-emergence trials, naringenin and myricitrin were significantly more phytotoxic than quercetin. We suggest that their differential initial interaction with the plant’s plasma membrane could be one of the main signals for electrolyte leakage and production of high levels of phenoxyl radicals. [less ▲]

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See detailScreening of Tunisian plant extracts for herbicidal activity and formulation of a bioherbicide based on Cynara cardunculus
Ben Kaab, Sofiène ULiege; Bettaieb, Iness ULiege; Hanafi, Marwa ULiege et al

in South African Journal of Botany (2020)

The need to reduce the use of chemical pesticides currently fosters great interest in eco-friendly biological control agents. In addition, the isolation of plant allelopathic substances and the evaluation ... [more ▼]

The need to reduce the use of chemical pesticides currently fosters great interest in eco-friendly biological control agents. In addition, the isolation of plant allelopathic substances and the evaluation of their phytotoxic effects can lead to the discovery of new natural herbicides. In this context, our study aimed to assess the herbicidal activity of ten crude extracts obtained from aerial parts of Tunisian spontaneous plants against Trifolium incarnatum, Silybum marianum and Phalaris minor. It confirmed that the Cynara cardunculus methanolic extract best inhibited weed germination and seedling growth, and caused necrosis or chlorosis. Following a bioassay-guided fractionation, five main phenolic compounds, (1) syringic acid, (2) p-coumaric acid, (3) myricitrin, (4) quercetin and (5) naringenin were identified in the most active crude methanolic extract. Then, only 3 of the flavonoids contained in the most active fraction were tested on Trifolium incarnatum. The 3 compounds had a significant phytotoxic effect and therefore could be employed in a new composition of botanical herbicides to control crop weeds. Besides, a novel herbicide composition was designed to improve the post-emergence activity of the methanolic extract. The formulation containing the C. cardunculus crude methanolic extract showed the same herbicidal activity as the standard industrial bioherbicide containing pelargonic acid. These results make C. cardunculus a suitable source of natural compounds potentially usable as natural herbicides. [less ▲]

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See detailInsights into the Relationships Between Herbicide Activities, Molecular Structure and Membrane Interaction of Cinnamon and Citronella Essential Oils Components
Lins, Laurence ULiege; Dal Maso, Simon ULiege; Foncoux, Bérénice ULiege et al

in International Journal of Molecular Sciences (2019), 20

Since the 50’s, the massive and “environmental naïve” use of synthetic chemistry has revolutionized the farming community facing the dramatic growth of demography. However, nowadays, the controversy grows ... [more ▼]

Since the 50’s, the massive and “environmental naïve” use of synthetic chemistry has revolutionized the farming community facing the dramatic growth of demography. However, nowadays, the controversy grows regarding the long-term harmful effects of these products on human health and the environment. In this context, the use of essential oils (EOs) could be an alternative to chemical products and a better understanding of theirmode of biological action for new and optimal applications is of importance. Indeed, if the biocidal effects of some EOs or their components have been at least partly elucidated at the molecular level, very little is currently known regarding their mechanism of action as herbicides at themolecular level. Here, we showed that cinnamon and Java citronella essential oils and some of their main components, i.e., cinnamaldehyde (CIN), citronellal (CitA), and citronellol (CitO) could act as efficient herbicides when spread on A. thaliana leaves. The individual EO molecules are small amphiphiles, allowing for them to cross the mesh of cell wall and directly interact with the plant plasma membrane (PPM), which is one of the potential cellular targets of EOs. Hence, we investigated and characterized their interaction with biomimetic PPMwhile using an integrative biophysical approach. If CitO and CitA, maintaining a similar chemical structure, are able to interact with the model membranes without permeabilizing effect, CIN belonging to the phenylpropanoid family, is not. We suggested that different mechanisms of action for the two types of molecules can occur: while the monoterpenes could disturb the lipid organization and/or domain formation, the phenylpropanoid CIN could interact with membrane receptors. [less ▲]

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See detailMolecular biophysics: An integrative approach to investigate the bioherbicide effect of essential oils related to their interaction with plant plasma membrane
Lins, Laurence ULiege; De Clerck, Caroline ULiege; Foncoux, Bérénice et al

Conference (2019, July)

Essential oils (EOs) are used in an increasingly number of sectors like medicine, cosmetics, food industry and more recently in agronomy. In agronomy, EOs are used as bio-pesticides for their insecticidal ... [more ▼]

Essential oils (EOs) are used in an increasingly number of sectors like medicine, cosmetics, food industry and more recently in agronomy. In agronomy, EOs are used as bio-pesticides for their insecticidal, antifungal or bactericidal effects but also as bio-herbicides. Owing to the current attraction for natural products, a better understanding of their mode of biological action for new and optimal applications is of importance. It has been shown that EOs antimicrobial activity, quite well described in the literature, is at least partly due to their interaction with the plasma membrane. They notably change the lipid composition, altering fluidity, leading to various effects which can induce cell lysis, apoptosis or necrosis. Citronellol, a major compound of lemongrass EO was notably shown to have antifungal activities by changing the membrane composition and inhibiting cell growth. Cinnamaldehyde (cinnamon EO) has been reported to have a broad spectrum of antibacterial activity, notably by affecting cell morphology, membrane integrity, permeability and composition. We are currently working on the development of a bioherbicide made from Cinnamomum zeylanicum Blume (cinnamon) and Cymbogognon winterianus Jowitt (lemongrass) EOs. We have shown that the application of the whole EOs and their major individual compounds on the leaves and cotyledons of A. thaliana appears to be promising: when applied on cotyledons or leaves, EOs induce damages that are as important as those observed for commercial herbicides. Since EOs are small amphiphilic molecules, they can cross the mesh of cell wall and interact directly with the plant plasma membrane (PPM). Modifying the lipid organization could lead to crucial cellular effects, notably on protein function. We used a unique and original combination of in silico (molecular dynamics simulations) and in vitro (Langmuir monolayers, isothermal calorimetry, fluorescence and infrared spectroscopies) biophysical approaches, previously developed to study structure-function relationships of molecules of biological interest (pharmacological drugs , proteins, peptides, surfactants…) to investigate the interaction of EOs or their individual compounds with bio-mimetic plant plasma membranes to better understand the structure- activity relationships in the context of their bioherbicide activity. [less ▲]

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See detailScreening of essential oil as potential postharvest biogungicide
Dal Maso, Simon ULiege; De Clerck, Caroline ULiege; Parisi, Olivier ULiege et al

Poster (2019, May)

Plant extracts, such as essential oils (EOs), have been known for centuries for their ability to prevent and/or to cure diseases through their fungicidal and bactericidal effect. In this project we ... [more ▼]

Plant extracts, such as essential oils (EOs), have been known for centuries for their ability to prevent and/or to cure diseases through their fungicidal and bactericidal effect. In this project we evaluated the fungicidal activity of 90 essential oils on several pathogens associated with post-harvest diseases (Botrytis cinerea, Penicillium expensum, Pectobacterium atrosepticum and Pectobacterium carotovorum). The efficacy of the EOs was first tested in vitro using 96 wells ELISA microplates. This step allowed the selection of 9 EOs, sufficiently effective (complete growth inhibition up to 72 hours of contact with pathogen in liquid of medium) against these pathogens to be tested under in-vivo conditions. The phytotoxicity of the selected EOs was then tested on apples, pears and potatoes. While no phytotoxicity was observed when the EOs were applied on intact fruits and tubers, a clear toxicity was observed when EOs were applied on wounded fruits. For the EOs showing a moderate toxicity, the in-vivo tests were carried on by inoculating the pathogens into wounded apples (P. expensum), pears (B. cinerea) and potatoes (P. atrosepticum and P. carotovorum) treated with lower EOs concentration. At these concentration, the EOs showed less phytotoxicity but also a lower efficiency (30% in the best case). [less ▲]

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See detailScreening of essential oil as potential postharvest biogungicide
Dal Maso, Simon ULiege; De Clerck, Caroline ULiege; Parisi, Olivier ULiege et al

Poster (2019, May)

Plant extracts, such as essential oils (EOs), have been known for centuries for their ability to prevent and/or to cure diseases through their fungicidal and bactericidal effect. In this project we ... [more ▼]

Plant extracts, such as essential oils (EOs), have been known for centuries for their ability to prevent and/or to cure diseases through their fungicidal and bactericidal effect. In this project we evaluated the fungicidal activity of 90 essential oils on several pathogens associated with post-harvest diseases (Botrytis cinerea, Penicillium expensum, Pectobacterium atrosepticum and Pectobacterium carotovorum). The efficacy of the EOs was first tested in vitro using 96 wells ELISA microplates. This step allowed the selection of 9 EOs, sufficiently effective (complete growth inhibition up to 72 hours of contact with pathogen in liquid of medium) against these pathogens to be tested under in-vivo conditions. The phytotoxicity of the selected EOs was then tested on apples, pears and potatoes. While no phytotoxicity was observed when the EOs were applied on intact fruits and tubers, a clear toxicity was observed when EOs were applied on wounded fruits. For the EOs showing a moderate toxicity, the in-vivo tests were carried on by inoculating the pathogens into wounded apples (P. expensum), pears (B. cinerea) and potatoes (P. atrosepticum and P. carotovorum) treated with lower EOs concentration. At these concentration, the EOs showed less phytotoxicity but also a lower efficiency (30% in the best case). [less ▲]

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See detailRosmarinus officinalis essential oil as an effective antifungal and herbicidal agent
Ben Kaab, Sofiène ULiege; Bettaieb Rebey, Iness ULiege; Hanafi, Marwa ULiege et al

in Spanish Journal of Agricultural Research (2019), 17(2),

In order to reduce the use of chemical pesticides, great interest has been focused on environment-friendly biological control agents and botanicals that preserve biodiversity. In this context, our study ... [more ▼]

In order to reduce the use of chemical pesticides, great interest has been focused on environment-friendly biological control agents and botanicals that preserve biodiversity. In this context, our study aimed to assess the antifungal and herbicidal activities of Rosmarinus officinalis essential oil (EO) to find an alternative to synthetic pesticides. The chemical composition of R. officinalis essential oil was determined by gaz chromatography-mass spectrometry analysis (GC-MS). Results showed that R. officinallis EO was rich in monoterpenes and the major constituents were 1,8-cineole (54.6%), camphor (12.27%) and α-pinene (7.09%). However, under laboratory condition, two tests were carried out. The first one consisted on the study of EO antifungal activity using ELISA microplates and the second one consisted on evaluating the effect of EO on seedling growth of weeds. It was confirmed that this EO significantly inhibits spore germination of Fusarium oxysporum, Fusarium culmorum, Penicillium italicum and at 6 mM, the percentage of inhibition reached 100% on Fusarium oxysporum. Indeed, EO slows down seedling growth of Trifolium incarnatum, Silybum marianum, and Phalaris minor. In fact, EO at 5 mM completely inhibits seed germination. On the other hand, another experiment was carried out to evaluate the herbicidal activity by spraying EO on weeds. This showed that a novel herbicide formulation was set up for the first time to improve the activity of R. officinalis EO on post-emergence. Overall, R. officinalis EO can be suggested as a potential eco-friendly pesticide and suitable source of natural compounds potentially usable as natural pesticides. [less ▲]

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See detailEtude de composés organiques volatils (COVs) émis lors de l'interaction hôte - pathogène par des souches produisant des mycotoxines.
Josselin, Laurie ULiege; De Boevre, Marthe; De Clerck, Caroline ULiege et al

Poster (2019, April 03)

Method for the qualitative detection (SPME) of VOCs emitted by toxigenic (mycotoxin-producing) and non-toxigenic (non mycotoxin-producing) strains.

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See detailHow could the herbicidal effects of selected essential oil compounds be related to their membrane activity ?
Lins, Laurence ULiege; Bettaieb, Ines; Dal Maso, Simon ULiege et al

Poster (2018, September 25)

The European legislation on plant protection products (PPP) is about to undergo important changes in the coming years. The chemical PPP destined to be removed from the European market are responsible for ... [more ▼]

The European legislation on plant protection products (PPP) is about to undergo important changes in the coming years. The chemical PPP destined to be removed from the European market are responsible for the appearance of resistance phenomena to plants pests. It is therefore necessary to explore new alternatives, one of them being the search for natural herbicides. Essential oils (EO) could provide a welcome alternative due to their proven activity as PPP. Even if these compounds seem to have a bright future as PPP, their activity is mainly approached through empirical observations. EO are also a complex mix of of different molecules that could act individually or in synergy. Particularly, very little is known when it comes to molecular mechanisms of action and the relations between structure and activity of the active compounds. This study aims to investigate the structure/activity relationships of some EO molecules, among which cinnamaldehyde (CIN) from cinnamon and citronellal and citronellol from lemongrass. Due to their lipophilic properties, EO tend to interact with one or more of the layers found in the outer plant tissues, among which the cell membranes. For cinnalmaldehyde (and other EO compounds), it has been shown that they are able to interact with bacterial phospholipids and induce change in lipid organization (fluidity, packing,..) on model lipid monolayers (Nowotarska et al, 2014). Citronnellol was notably shown to displace cholesterol from its phospholipid complexes (Lange, Y et al, 2009). However, nothing is known about any interaction with lipids specific to plant plasma membrane (PPM). In this study, we analyzed the effects of the above three EO compounds on model PPM by complementary in vitro and in silico biophysical approaches. We showed that the three compounds have differential effects on plant lipids and different herbicidal properties on plantae. While part of the herbicidal activity could be related to membrane perturbation, some clues remain to be elucidated. Future studies at a molecular point of view would help to better decipher the herbicidal action involving the membrane, other outer plant tissues such as the cuticule and/or and a potential effect on EO compounds on proteins or genomic DNA, as it was shown for CIN on E. Coli (He, TF et al, 2018). [less ▲]

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