Département fédéral de l'économie DFE
Station de recherche Agroscope Changins-Wädenswil A CW
16 october 2013
Alexins as new antifungal compoundsin plant and human pathology
Katia GindroOlivier Viret
2IOBC-WPRS Meeting | Integrated protection and produc tion in viticultureKatia Gindro and Olivier Viret, Ascona, 14-17 October 2013
Alexins ?
• Low molecular-weight secondary metabolites synthetized after biotic or abiotic stresses - INDUCED metabolites
• Acting as general antimicrobials of interest for Agriculture and medicine
• Observed and named by Harry Marschall Ward in 1891
Infection with a pathogen depends on
the presence of enzymes, toxins and
antitoxins in the parasite and host.
3IOBC-WPRS Meeting | Integrated protection and produc tion in viticultureKatia Gindro and Olivier Viret, Ascona, 14-17 October 2013
Alexins: main sources
• Plants : officially named by Müller & Börger en 1940
PHYTOALEXINS
On internet: > 1’000’000 references
• Fungi : officially named by Katia Gindro et al. en 2010
MYCOALEXINS
On internet: 31 referencesO
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4IOBC-WPRS Meeting | Integrated protection and produc tion in viticultureKatia Gindro and Olivier Viret, Ascona, 14-17 October 2013
Phytoalexins
• Several chemical classes, several different pathways
• Related to defence reactions of the plants against aggression: happens only in living cells.
• Molecules related to the death of the cells of the host (collateral damage)
5IOBC-WPRS Meeting | Integrated protection and produc tion in viticultureKatia Gindro and Olivier Viret, Ascona, 14-17 October 2013
Phytoalexins
Coniferol (Gymno-Angiospermes)Alcohol
Capsidiol (tobacco, pepper)Terpenoid
Avenanthramide A (oat)Polyphenol
Brassinine (cabbage)Indole
Pisatine (pea)Pterocarpane
Camalexine (Arabidopsis)Indole
6-methoxymelleine (carrot)Phenolic compounds
STILBENES
6IOBC-WPRS Meeting | Integrated protection and produc tion in viticultureKatia Gindro and Olivier Viret, Ascona, 14-17 October 2013
Stilbenes
• Natural phenolic compounds• Identical basic chemical stucture (biphenyl )• Family of molecules
benzene biphenyl Phenol
HO
7IOBC-WPRS Meeting | Integrated protection and produc tion in viticultureKatia Gindro and Olivier Viret, Ascona, 14-17 October 2013
Stilbenes ?
• The first stilbene was identified in 1939 by Prof. Takaoka (Japan) from Veratrum grandiflorum
8IOBC-WPRS Meeting | Integrated protection and produc tion in viticultureKatia Gindro and Olivier Viret, Ascona, 14-17 October 2013
Stilbenes ?
• named R E S V E R A T R O L
Class of the moleculeResorcinol
PlantVeratrum
Alcohol function-OH
9IOBC-WPRS Meeting | Integrated protection and produc tion in viticultureKatia Gindro and Olivier Viret, Ascona, 14-17 October 2013
H H
OH
R E S V E R A T R O L
10IOBC-WPRS Meeting | Integrated protection and produc tion in viticultureKatia Gindro and Olivier Viret, Ascona, 14-17 October 2013
Roles of stilbenes in plants?
• Molecules with antifungal activities• Linked to defence reactions against fungal pathogens, and in
some cases against insects
White Pine
Pinosylvin
Spruce
Piceatannol
Grapevine
Resveratrol + others
11IOBC-WPRS Meeting | Integrated protection and produc tion in viticultureKatia Gindro and Olivier Viret, Ascona, 14-17 October 2013
Stilbenes in grapevine
Resveratrol Pterostilbene Piceid
• Initial works: Agrosocope Changins in 1985
ε- α- δ-viniferin
• Works on new antifungal stilbenes
12IOBC-WPRS Meeting | Integrated protection and produc tion in viticultureKatia Gindro and Olivier Viret, Ascona, 14-17 October 2013
Pterostilbene
Role of stilbenes in grapevine
• Natural defence mechanism
δδδδ-viniferin
E vitisine BReviewGindro, K. et al. 2012. Stilbenes: biomarkers of grapevine resistance to disease of high relevance for agronomy, enology, and human health. In Plant defence: biological control (J-M. Mérillon and K.G. Ramawat, Eds.), Editions Springer, Dordrecht, Heidelberg, London, New York, 412 p: 25-54.
13IOBC-WPRS Meeting | Integrated protection and produc tion in viticultureKatia Gindro and Olivier Viret, Ascona, 14-17 October 2013
Role of stilbenes in grapevine• Quantification of stilbenes for characterization of resistant
grapevine varieties• Fundamental tool for breeding programs at Agroscope to select
resistant seedlings
ReviewGindro, K. et al. 2012. Stilbenes: biomarkers of grapevine resistance to disease of high relevance for agronomy, enology, and human health. In Plant defence: biological control (J-M. Mérillon and K.G. Ramawat, Eds.), Editions Springer, Dordrecht, Heidelberg, London, New York, 412 p: 25-54.
14IOBC-WPRS Meeting | Integrated protection and produc tion in viticultureKatia Gindro and Olivier Viret, Ascona, 14-17 October 2013
Stilbenes for human health• Strong antioxidants (human health and beauty)• Important role in the decrease of oxidative stress
(protein oxidation, DNA/RNA damage): arteriosclerosis, Parkinson disease, cancer, cardiovascular disease…anti skin-aging
15IOBC-WPRS Meeting | Integrated protection and produc tion in viticultureKatia Gindro and Olivier Viret, Ascona, 14-17 October 2013
Localization
• Induced (newly synthetized after stresses)Green parts: leaves, young berries…
• Constitutive (pre-existing):In wood and lignified canes
16IOBC-WPRS Meeting | Integrated protection and produc tion in viticultureKatia Gindro and Olivier Viret, Ascona, 14-17 October 2013
New biomarkers for resistance
• To overcome artificial infections• Experimental design: UVc Stress
UVc-light (280 to 10 nm)
Not existing on earth
Used as sterilizing agent (280 to 10 nm), extremely harmful
Other way to induce antifungal compounds by triggering the production of phytoalexins without pathogens
Schumpp, O., Gindro, K. et al. 2012. Ultraviolet induction of antifungal activity in plants. Mycosis 55:507-513.
17IOBC-WPRS Meeting | Integrated protection and produc tion in viticultureKatia Gindro and Olivier Viret, Ascona, 14-17 October 2013
New biomarkers for resistance• 3 kg UVc leaves and 3 kg downy mildew-infected leaves• UHPLC-TOFMS - metabolomic approach• Identification of induced metabolites (by principal component analysis)
Control
UVc
Divico (new Agroscope resistant cultivarGamaret x Bronner)
Downy mildew
Control 48h
VitisinolViniferetherFerulic acidSyringineAmurensine…
Marti, G., Gindro, K . et al. 2013. J. Nat. Prod. submitted
18IOBC-WPRS Meeting | Integrated protection and produc tion in viticultureKatia Gindro and Olivier Viret, Ascona, 14-17 October 2013
Localization• Induced (newly synthetized after stresses)
Green parts: leaves, young berries…
• Constitutive (pre-existing):In wood and lignified canes
19IOBC-WPRS Meeting | Integrated protection and produc tion in viticultureKatia Gindro and Olivier Viret, Ascona, 14-17 October 2013
New antifungal compounds
• The project runs from early 2010 (find new natural fungicides compounds from vine and other plants)
• Private funding (groupement d’intérêt économique des 9)
20IOBC-WPRS Meeting | Integrated protection and produc tion in viticultureKatia Gindro and Olivier Viret, Ascona, 14-17 October 2013
New fungicides • Valorisation of grapevine by-products (e.g. lignified canes) • Development of a formulated active extract against the three
major fungal diseases of grape• Side Project: chemistry of wood (different varieties), chemical
identification and bioactivity
• S. Schnee, K. Gindro et al. 2013 . Vitis vinifera canes, a new source of antifungal compounds against Plasmopara viticola, Erysiphe necator, and Botrytis cinerea. J. Agric. Food Chem. 2013. 61(23): 5459-5467.
• Halabalaki, Gindro et al. 2013 . J. of chromatography, in press
21IOBC-WPRS Meeting | Integrated protection and produc tion in viticultureKatia Gindro and Olivier Viret, Ascona, 14-17 October 2013
Results• Development of miniaturized bioassays • Identification of 10 active molecules among the 90 remaining
extracted molecules• Characterization and determination of their ED50
• One patent on going (methods of extraction and separation)• Pre-formulation (photo-stabilisator, sticker, adjuvants…)• Promising field trials including ecotoxicology
22IOBC-WPRS Meeting | Integrated protection and produc tion in viticultureKatia Gindro and Olivier Viret, Ascona, 14-17 October 2013
Mycoalexins• Induced in fungi after different stresses• Not to be confused with mycotoxins:
secondary metabolites produced during fungal infection in crops(e.g. ergotamin, aflatoxin, trichothecen)
ergotamin aflatoxin trichothecen
23IOBC-WPRS Meeting | Integrated protection and produc tion in viticultureKatia Gindro and Olivier Viret, Ascona, 14-17 October 2013
Where to find mycoalexins?
Specific fungal communities are living in all living lignifiedplants in a confined environment (e.g. grapevine trunks)
24IOBC-WPRS Meeting | Integrated protection and produc tion in viticultureKatia Gindro and Olivier Viret, Ascona, 14-17 October 2013
Where to find mycoalexins?• Each fungal species fight against the others to keep its
own territory
• Development of competition zone lines
25IOBC-WPRS Meeting | Integrated protection and produc tion in viticultureKatia Gindro and Olivier Viret, Ascona, 14-17 October 2013
At the chemical and enzymatic level: clash of the Titans
Where to find mycoalexins?
26IOBC-WPRS Meeting | Integrated protection and produc tion in viticultureKatia Gindro and Olivier Viret, Ascona, 14-17 October 2013
In vitro confrontationfour type of interactive behaviours
27IOBC-WPRS Meeting | Integrated protection and produc tion in viticultureKatia Gindro and Olivier Viret, Ascona, 14-17 October 2013
Chemical induction ?
Eutypa lata
Botryosphaeria obtusa
28IOBC-WPRS Meeting | Integrated protection and produc tion in viticultureKatia Gindro and Olivier Viret, Ascona, 14-17 October 2013
Chemical war between fungi
New source of induced antifungal compounds
29IOBC-WPRS Meeting | Integrated protection and produc tion in viticultureKatia Gindro and Olivier Viret, Ascona, 14-17 October 2013
Mycoalexins
• In the chemical zone lines: 80-90% of induced compoundsare unknown after dereplication (NMR compared to dictionaryof natural products - 250’000 molecules)
• Different bioactivities• Chemical identification by NMR (nuclear magnetic resonance)
• Bertrand, Schumpp, O., Gindro, K. 2013. Detection of metabolite induction in fungal co-cultures on solid media by high-throughput differential ultra-high pressure liquid chromatography-time-of-flight mass spectrometry. J. Chromatography, 1292: 219-228.
• Glauser, G., Gindro, K. et al. 2009 . Differential analysis of mycoalexins in confrontation zones of grapevine fungal pathogens by ultrahigh pressure liquid chromatography /time-of-flight mass spectrometry and capillary nuclear magnetic resonance. J. Agr. Food. Chem. 57: 1127-1134.
30IOBC-WPRS Meeting | Integrated protection and produc tion in viticultureKatia Gindro and Olivier Viret, Ascona, 14-17 October 2013
Examples• Botryosphaeria obtusa against Eutypa lata
• More than15 induced compounds
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New myco-phenolic compound exhibitinghigh antifungal and phytotoxic activities
31IOBC-WPRS Meeting | Integrated protection and produc tion in viticultureKatia Gindro and Olivier Viret, Ascona, 14-17 October 2013
Conclusion
� In natural systems, defence mechanismsinduce powerful biochemical pathways, leading to the synthesis of new chemicals with a high potential for agriculture and human health
� Phytoalexins: highly preferment fungal killers active in grapevine against the major fungal diseases (breeding resistant cultivars)
� Mycoalexins: source of extremely toxic biocide and unknown active compounds
32IOBC-WPRS Meeting | Integrated protection and produc tion in viticultureKatia Gindro and Olivier Viret, Ascona, 14-17 October 2013
Merci de votre attention
Thanks foryour attention