An ARIMNet project
WP coordinator: UNINA
Other partners involved: CSIC, ENSA
Objectives
- Development of protocols to extract and purify plant and fungal metabolites
- Determination of the structure of bioactive plant and fungi metabolites using advanced spectroscopic and optical methods
- Optimization of chemical reactions to prepare derivative for structures-activity relationships studies
Methodology and study materials
T6.1: Search and exploitation of allelopathy for broomrape control: root exudates will be obtained from hydroponically grown seedlings of nonhosts (oat and rye with known allelopathic effect) and hosts (faba bean, pea, lentil and chickpea) will be extracted. Bioguided-fractionation of crude organic extract will be performed with column and thin layer chromatography in order to isolate and identify the active metabolites responsible for inhibition of weed germination and growth. Structural characterisation will be carried out using advanced spectroscopic methods (IR, UV, but essentially 1D and 2D 1H- and 13C-NMR and EI- and ESI-MS), optical (optical rotation and CD) methods. When needed, the structure of new metabolites will be confirmed through the preparation of their key derivatives
T6.2: Structure-activity relationship study: some suitable derivatives of fungal metabolites as phyllostin and scytolide produced by Phyllosticta cirsii which showed a stimulatory activity on Orobanche in a preliminary investigation, are currently being in preparation in the laboratory of UNINA by modification of their funtionalities. Inhibitory and stimulatory activity of available derivatives will be tested in a range of Orobanche spp.
T6.3. Search and exploitation of allelopathy for fungal disease control: isolation and chemical characterization of metabolites from different legumes, resistant to different pathogens, with putative antifungal activity (phytoalexins). Assessment of the compound activity in the development of fungal pathogens. Extracts of plants showing significant inhibitory effects in various aerial fungi will be extracted with the most suitable organic solvent. The crude extract will be fractionated using bioguide-combined chromatographic methods as in details reported above. The pure bioctive metabolites will be used to test their inhibitory effect on different fungal pathogens, both, in vivo by spraying susceptible plants and in vitro in petri dishes. Chemical structure of the inhibitors will be determined by spectroscopic (IR, UV, but essentially 1D e 2D 1H- and 13C-NMR and EI- and ESI-MS), optical (optical rotation and CD) and chemical methods.
T6.4: Exploitation of phytotoxins isolated from pea and lentil pathogens as tool for crop protection: toxic liquid and/or solid fungal culture filtrates will be obtained from inoculated tissue. The lypophilic bioactive metabolites will be purified by extraction with most suitable organic solvent, followed by classic chromatographic fractionation (using both normal and reverse phases supports); the hydrophilic ones will be purified by fractionated precipitation combined with suitable chromatographic methods. The structure determination of phytotoxins will be performed using spectroscopic (IR, UV, but essentially 1D e 2D 1H- and 13C-NMR and EI- and ESI-MS), and optical (optical rotation and CD) methods. In some cases their structure will be confirmed using chemical methods based on the preparation of key derivatives through their functional group transformation. The purification processes of phytotoxins will be checked with opportune biological assays on host and non-host plant organs. The same assays will be used for the activity characterization of purified phytotoxins. For these last the micotoxic and antimicrobic activity will be also assayed.
T6.5: Search and exploitation of allelopathy for control of broomrape and dodder and plant pathogens: a previous screening made by ENSA partner identified several Mediterranean plants (Inula viscosa in particular) that produced low molecular weight lipophlic metabolites with herbicidal activity against two broomrapes species (P. ramosa, O. crenata), dodder (C. campestris) and several plant pathogens (fungal and bacterial diseases). This will be studied further by identifiying and characterizing the phytotoxic metabolites following the procedures described in T6.4.
Tasks T6.1, T6.2, T6.3 and T6.4 will be performed by UNINA in collaboration with CSIC and T6.5 in collaboration with ENSA. These partners will supply dried plants and root plant exudates, liquid and/or solid lyophilized fungal culture filtrates and will carry out in vitro bioassays and field experiments.
Deliverables
D6.1: Protocols for the extraction and purification of bioactive metabolites (phytotoxins, herbicides, phytoalexins)
D6.2: Structure determination of new bioactive compounds with original carbon skeleton
D6.3: Characterization of phytotoxic, antifungal, mycotoxic and allelopatic activities of the pure metabolites isolated
D6.4: Preparation of key derivatives for structure confirmation and structure-activity relationships studies
Other partners involved: CSIC, ENSA
Objectives
- Development of protocols to extract and purify plant and fungal metabolites
- Determination of the structure of bioactive plant and fungi metabolites using advanced spectroscopic and optical methods
- Optimization of chemical reactions to prepare derivative for structures-activity relationships studies
Methodology and study materials
T6.1: Search and exploitation of allelopathy for broomrape control: root exudates will be obtained from hydroponically grown seedlings of nonhosts (oat and rye with known allelopathic effect) and hosts (faba bean, pea, lentil and chickpea) will be extracted. Bioguided-fractionation of crude organic extract will be performed with column and thin layer chromatography in order to isolate and identify the active metabolites responsible for inhibition of weed germination and growth. Structural characterisation will be carried out using advanced spectroscopic methods (IR, UV, but essentially 1D and 2D 1H- and 13C-NMR and EI- and ESI-MS), optical (optical rotation and CD) methods. When needed, the structure of new metabolites will be confirmed through the preparation of their key derivatives
T6.2: Structure-activity relationship study: some suitable derivatives of fungal metabolites as phyllostin and scytolide produced by Phyllosticta cirsii which showed a stimulatory activity on Orobanche in a preliminary investigation, are currently being in preparation in the laboratory of UNINA by modification of their funtionalities. Inhibitory and stimulatory activity of available derivatives will be tested in a range of Orobanche spp.
T6.3. Search and exploitation of allelopathy for fungal disease control: isolation and chemical characterization of metabolites from different legumes, resistant to different pathogens, with putative antifungal activity (phytoalexins). Assessment of the compound activity in the development of fungal pathogens. Extracts of plants showing significant inhibitory effects in various aerial fungi will be extracted with the most suitable organic solvent. The crude extract will be fractionated using bioguide-combined chromatographic methods as in details reported above. The pure bioctive metabolites will be used to test their inhibitory effect on different fungal pathogens, both, in vivo by spraying susceptible plants and in vitro in petri dishes. Chemical structure of the inhibitors will be determined by spectroscopic (IR, UV, but essentially 1D e 2D 1H- and 13C-NMR and EI- and ESI-MS), optical (optical rotation and CD) and chemical methods.
T6.4: Exploitation of phytotoxins isolated from pea and lentil pathogens as tool for crop protection: toxic liquid and/or solid fungal culture filtrates will be obtained from inoculated tissue. The lypophilic bioactive metabolites will be purified by extraction with most suitable organic solvent, followed by classic chromatographic fractionation (using both normal and reverse phases supports); the hydrophilic ones will be purified by fractionated precipitation combined with suitable chromatographic methods. The structure determination of phytotoxins will be performed using spectroscopic (IR, UV, but essentially 1D e 2D 1H- and 13C-NMR and EI- and ESI-MS), and optical (optical rotation and CD) methods. In some cases their structure will be confirmed using chemical methods based on the preparation of key derivatives through their functional group transformation. The purification processes of phytotoxins will be checked with opportune biological assays on host and non-host plant organs. The same assays will be used for the activity characterization of purified phytotoxins. For these last the micotoxic and antimicrobic activity will be also assayed.
T6.5: Search and exploitation of allelopathy for control of broomrape and dodder and plant pathogens: a previous screening made by ENSA partner identified several Mediterranean plants (Inula viscosa in particular) that produced low molecular weight lipophlic metabolites with herbicidal activity against two broomrapes species (P. ramosa, O. crenata), dodder (C. campestris) and several plant pathogens (fungal and bacterial diseases). This will be studied further by identifiying and characterizing the phytotoxic metabolites following the procedures described in T6.4.
Tasks T6.1, T6.2, T6.3 and T6.4 will be performed by UNINA in collaboration with CSIC and T6.5 in collaboration with ENSA. These partners will supply dried plants and root plant exudates, liquid and/or solid lyophilized fungal culture filtrates and will carry out in vitro bioassays and field experiments.
Deliverables
D6.1: Protocols for the extraction and purification of bioactive metabolites (phytotoxins, herbicides, phytoalexins)
D6.2: Structure determination of new bioactive compounds with original carbon skeleton
D6.3: Characterization of phytotoxic, antifungal, mycotoxic and allelopatic activities of the pure metabolites isolated
D6.4: Preparation of key derivatives for structure confirmation and structure-activity relationships studies
WP 5 - Epidemiological studies