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Bertrand S.,University of Geneva | Schumpp O.,Swiss Federal Research Station Agroscope Changins Wadenswil | Bohni N.,University of Geneva | Monod M.,University of Lausanne | And 2 more authors.
Journal of Natural Products | Year: 2013

The co-cultivation of fungi has recently been described as a promising strategy to induce the production of novel metabolites through possible gene activation. A large screening of fungal co-cultures in solid media has identified an unusual long-distance growth inhibition between Trichophyton rubrum and Bionectria ochroleuca. To study metabolite induction in this particular fungal interaction, differential LC-MS-based metabolomics was performed on pure strain cultures and on their co-cultures. The comparison of the resulting fingerprints highlighted five de novo induced compounds, which were purified using software-oriented semipreparative HPLC-MS. One metabolite was successfully identified as 4″-hydroxysulfoxy-2,2″- dimethylthielavin P (a substituted trimer of 3,5-dimethylorsellinic acid). The nonsulfated form, as well as three other related compounds, were found in the pure strain culture of B. ochroleuca. © 2013 The American Chemical Society and American Society of Pharmacognosy.


Bertrand S.,University of Geneva | Petit C.,University of Geneva | Marcourt L.,University of Geneva | Ho R.,University of Geneva | And 4 more authors.
Phytochemical Analysis | Year: 2014

Introduction The search for anti-fungal compounds has maintained a scientific interest notably due to existing difficulties in the treatment of mycoses and their increasing occurrence in hospitals. Objective Development of a simple method to rapidly identify anti-fungal compounds in crude plant extracts based on a HPLC microfractionation approach combined with an at-line anti-Candida assay. Methods The scale of the semi-preparative HPLC microfractionation was adapted to fit the sensitivity of the Candida albicans anti-fungal in a 96-well microdilution assay. This format is also compatible for MS and NMR dereplication of the active compounds. Results Based on the screening of 12 crude extracts of plants from French Polynesia, three plants, which displayed various levels of anti-fungal activities, were selected to assess the efficiency of the HPLC anti-fungal profiling and the scale necessary for microfractionation. The same anti-Candida assay was performed on the HPLC microfractions collected using a generic profiling method. Analysis of active microfractions by MS and NMR issued from the most active extract enabled an efficient dereplication of the compounds responsible for the anti-fungal activity. Conclusion A generic HPLC anti-fungal profiling method was developed which revealed that only 50 mg of crude extract were sufficient for a rapid identification of compound(s) responsible for the anti-Candida activity. This approach was illustrated by the study of Alphitonia zizyphoides, a plant traditionally used to treat dermatomycoses. Copyright © 2013 John Wiley & Sons, Ltd.


Bertrand S.,University of Geneva | Schumpp O.,Swiss Federal Research Station Agroscope Changins Wadenswil | Bohni N.,University of Geneva | Bujard A.,University of Geneva | And 4 more authors.
Journal of Chromatography A | Year: 2013

Access to new biological sources is a key element of natural product research. A particularly large number of biologically active molecules have been found to originate from microorganisms. Very recently, the use of fungal co-culture to activate the silent genes involved in metabolite biosynthesis was found to be a successful method for the induction of new compounds. However, the detection and identification of the induced metabolites in the confrontation zone where fungi interact remain very challenging. To tackle this issue, a high-throughput UHPLC-TOF-MS-based metabolomic approach has been developed for the screening of fungal co-cultures in solid media at the petri dish level. The metabolites that were overexpressed because of fungal interactions were highlighted by comparing the LC-MS data obtained from the co-cultures and their corresponding mono-cultures. This comparison was achieved by subjecting automatically generated peak lists to statistical treatments. This strategy has been applied to more than 600 co-culture experiments that mainly involved fungal strains from the Fusarium genera, although experiments were also completed with a selection of several other filamentous fungi. This strategy was found to provide satisfactory repeatability and was used to detect the biomarkers of fungal induction in a large panel of filamentous fungi. This study demonstrates that co-culture results in consistent induction of potentially new metabolites. © 2013 Elsevier B.V.


Schumpp O.,Swiss Federal Research Station Agroscope Changins Wadenswil | Bruderhofer N.,Swiss Federal Research Station Agroscope Changins Wadenswil | Monod M.,University of Lausanne | Wolfender J.-L.,University of Geneva | Gindro K.,Swiss Federal Research Station Agroscope Changins Wadenswil
Mycoses | Year: 2012

Ultraviolet-C irradiation as a method to induce the production of plant compounds with antifungal properties was investigated in the leaves of 18 plant species. A susceptibility assay to determine the antifungal susceptibility of filamentous fungi was developed based on an agar dilution series in microtiter plates. UV irradiation strongly induced antifungal properties in five species against a clinical Fusarium solani strain that was responsible for an onychomycosis case that was resistant to classic pharmacological treatment. The antifungal properties of three additional plant species were either unaffected or reduced by UV-C irradiation. This study demonstrates that UV-C irradiation is an effective means of modulating the antifungal activity of very diverse plants from a screening perspective. © 2012 Blackwell Verlag GmbH.


Gindro K.,Swiss Federal Research Station Agroscope Changins Wadenswil | Alonso-Villaverde V.,Mision Biologica de Galicia CSIC | Voinesco F.,Swiss Federal Research Station Agroscope Changins Wadenswil | Spring J.-L.,Swiss Federal Research Station Agroscope Changins Wadenswil | And 2 more authors.
Plant Physiology and Biochemistry | Year: 2012

Grape clusters of different Vitis genotypes, including Vitis vinifera cvs Chasselas and Merlot, and two interspecific grape varieties, Solaris (cvs. Merzling* x (Saperavi severneyi x Muscat ottonel)) and 2091 (cvs. Gamaret x Bronner), are susceptible or resistant to downy mildew. These cultivars were inoculated with Plasmopara viticola at three developmental stages (BBCH stages 53, 69 and 75). Samples were examined by scanning electron microscopy and the synthesis of stilbenes was measured. Microscopical examinations of pedicels, rachis and calyptras showed important differences in stomatal structures within seasonal development. At BBCH 53, successful infections were observed on all tested cultivars and functional stomata were present, while no infections were observed after this stage. At BBCH 69 and 75, cracks were observed around the stomata and guard cells were unstructured or completely collapsed, leading to closed-like stomata. At BBCH 53, significant stilbene accumulation was quantified in 2091 and Solaris; pterostilbene and δ-viniferin were produced in large amounts. In the susceptible varieties, only piceid and resveratrol were induced. At the other two stages, the concentration of all measured stilbenes was undetectable. The critical roles of seasonal development and stilbenes in the resistance of grape clusters towards downy mildew are discussed. © 2011 Elsevier Masson SAS.


Gindro K.,Swiss Federal Research Station Agroscope Changins Wadenswil | Berger V.,Swiss Federal Research Station Agroscope Changins Wadenswil | Godard S.,Swiss Federal Research Station Agroscope Changins Wadenswil | Voinesco F.,Swiss Federal Research Station Agroscope Changins Wadenswil | And 3 more authors.
Plant Physiology and Biochemistry | Year: 2012

Plasmopara viticola must successfully infect susceptible grapevine cultivars to complete its biological cycle. In resistant grapevine varieties, P. viticola is blocked by the activation of defense mechanisms; these defense mechanisms produce hypersensitive reactions, which are related to programmed cell death. In animals, programmed cell death is dependent on caspase activities. In plants, different caspase-like proteases assume the same functions. To examine the roles of caspase-like proteases in P. viticola-grapevine interactions, three varieties of grapevine with different levels of P. viticola resistance were chosen. These grapevine varieties were treated with either PMSF, a serine protease inhibitor, or E-64, a cysteine protease inhibitor. The development of the pathogen was followed microscopically, and the plant defense reactions were estimated through stilbene quantification. Both protease inhibitor treatments increased the infection rate in the resistant and immune varieties, diminished the production of toxic stilbenes and changed the level of the plants' susceptibility to the pathogen. In particular, after either protease treatment, the cultivar that was originally immune became resistant (hyphae and haustoria were observed), the resistant cultivar reached the level of a susceptible cultivar (sporulation was observed) and the susceptible cultivar became more sensitive (P. viticola colonized the entirety of the leaf mesophyll). © 2012 Elsevier Masson SAS.


Alonso-Villaverde V.,Mision Biologica de Galicia CSIC | Voinesco F.,Swiss Federal Research Station Agroscope Changins Wadenswil | Viret O.,Swiss Federal Research Station Agroscope Changins Wadenswil | Spring J.-L.,Swiss Federal Research Station Agroscope Changins Wadenswil | Gindro K.,Swiss Federal Research Station Agroscope Changins Wadenswil
Plant Physiology and Biochemistry | Year: 2011

Leaves of different Vitis vinifera L. cultivars, susceptible or resistant to downy mildew, Chasselas, Solaris, IRAC 2091 (cvs. Gamaret x Bronner) and Muscadinia rotundifolia were inoculated with Plasmopara viticola. Samples were then examined by scanning and transmission electron microscopy, by light microscopy and for their ability to synthesise stilbenes. These phytoalexins were strictly analysed at infection sites. In the susceptible Chasselas, P. viticola colonises, at 72h post-infection (hpi), all of the spongy mesophyll with functional haustoria and produces mainly the non toxic piceide. No necrotic zone was observed on Chasselas leaves. The ultrastructural response to downy mildew infection is different in each of the other three resistant grape cultivars. In Solaris, where leaf necrosis are rapidly induced, the infection is restricted to the upper part of the loose spongy mesophyll, and associated with a rapid cell wall disruption and the dispersion of cytoplasmic content along with the production of viniferins. In IRAC 2091, leaf necrosis are quite similar to those observed on Solaris but the infected plant cell, as well as the haustoria, show high electron dense cellular particles without any recognisable organelles, probably related to the effect of the toxic compound pterostilbene, which is synthesised in this grape cultivar. In M. rotundifolia leaf necrosis are much more scarce and smaller than in other cultivars, but pathogen and plant cells are both strongly affected, with concomitant expulsion of cytoplasmic materials through the stomata after P. viticola penetration. In this cultivar, the concentration of all identified stilbenes exceeds 1×103μmolmg-1 FW. The critical role of stilbenes in the resistance of Vitis spp. is discussed. © 2011 Elsevier Masson SAS.


Alonso-Villaverde V.,Mision Biologica de Galicia CSIC | Viret O.,Swiss Federal Research Station Agroscope Changins Wadenswil | Gindro K.,Swiss Federal Research Station Agroscope Changins Wadenswil
Vitis - Journal of Grapevine Research | Year: 2011

Leaves of different Vitis vinifera cultivars, susceptible ('Chasselas' and '2185'), less susceptible ('2142') or resistant to downy mildew ('Solaris' and '2091'), were inoculated with four different concentrations of an aqueous sporangia suspension of Plasmopara viticola (5 × 105, 2 × 105, 6 × 104 and 2 × 104 sporangia · ml -1). The infection rate of these samples was then examined by light microscopy and synthesis of stilbenes was analysed at infection sites. Infection rate increased parallel with inoculum concentration, but there was no correlation between the infection rate and resistance to P. viticola. Moreover, at the lowest inoculum concentration, the infection rate is similar for susceptible and resistant grapevine varieties. Quantification of stilbenes at 72 hpi showed that at the lowest inoculum concentration, the most susceptible grape variety synthesized the largest amount of stilbenes, whose level remained however below the ED50 values defined for each of them. Conversely, at the highest inoculum concentration, the most resistant varieties produced the highest amounts of the most toxic stilbenes against P. viticola. The critical role of the inoculum concentration used for artificial inoculation to evaluate grapevine resistance to downy mildew is discussed.


PubMed | Swiss Federal Research Station Agroscope Changins Wadenswil
Type: | Journal: Plant physiology and biochemistry : PPB | Year: 2012

Grape clusters of different Vitis genotypes, including Vitis vinifera cvs Chasselas and Merlot, and two interspecific grape varieties, Solaris (cvs. Merzling* x (Saperavi severneyi x Muscat ottonel)) and 2091 (cvs. Gamaret x Bronner), are susceptible or resistant to downy mildew. These cultivars were inoculated with Plasmopara viticola at three developmental stages (BBCH stages 53, 69 and 75). Samples were examined by scanning electron microscopy and the synthesis of stilbenes was measured. Microscopical examinations of pedicels, rachis and calyptras showed important differences in stomatal structures within seasonal development. At BBCH 53, successful infections were observed on all tested cultivars and functional stomata were present, while no infections were observed after this stage. At BBCH 69 and 75, cracks were observed around the stomata and guard cells were unstructured or completely collapsed, leading to closed-like stomata. At BBCH 53, significant stilbene accumulation was quantified in 2091 and Solaris; pterostilbene and -viniferin were produced in large amounts. In the susceptible varieties, only piceid and resveratrol were induced. At the other two stages, the concentration of all measured stilbenes was undetectable. The critical roles of seasonal development and stilbenes in the resistance of grape clusters towards downy mildew are discussed.


PubMed | Swiss Federal Research Station Agroscope Changins Wadenswil
Type: | Journal: Plant physiology and biochemistry : PPB | Year: 2012

Plasmopara viticola must successfully infect susceptible grapevine cultivars to complete its biological cycle. In resistant grapevine varieties, P.viticola is blocked by the activation of defense mechanisms; these defense mechanisms produce hypersensitive reactions, which are related to programmed cell death. In animals, programmed cell death is dependent on caspase activities. In plants, different caspase-like proteases assume the same functions. To examine the roles of caspase-like proteases in P.viticola-grapevine interactions, three varieties of grapevine with different levels of P.viticola resistance were chosen. These grapevine varieties were treated with either PMSF, a serine protease inhibitor, or E-64, a cysteine protease inhibitor. The development of the pathogen was followed microscopically, and the plant defense reactions were estimated through stilbene quantification. Both protease inhibitor treatments increased the infection rate in the resistant and immune varieties, diminished the production of toxic stilbenes and changed the level of the plants susceptibility to the pathogen. In particular, after either protease treatment, the cultivar that was originally immune became resistant (hyphae and haustoria were observed), the resistant cultivar reached the level of a susceptible cultivar (sporulation was observed) and the susceptible cultivar became more sensitive (P.viticola colonized the entirety of the leaf mesophyll).

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