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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.

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.

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 | 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.

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.

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