alexins as new antifungal compounds - iobc-wprs · département fédéral de l'économie dfe...

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.


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

OO

12

4a4

2-Me

56

78

8a

5-OMe

3


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)


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


Stilbenes

• Natural phenolic compounds• Identical basic chemical stucture (biphenyl )• Family of molecules

benzene biphenyl Phenol

HO


Stilbenes ?

• The first stilbene was identified in 1939 by Prof. Takaoka (Japan) from Veratrum grandiflorum


Stilbenes ?

• named R E S V E R A T R O L

Class of the moleculeResorcinol

PlantVeratrum

Alcohol function-OH


H H

OH

R E S V E R A T R O L


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


Stilbenes in grapevine

Resveratrol Pterostilbene Piceid

• Initial works: Agrosocope Changins in 1985

ε- α- δ-viniferin

• Works on new antifungal stilbenes


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.


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.


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


Localization

• Induced (newly synthetized after stresses)Green parts: leaves, young berries…

• Constitutive (pre-existing):In wood and lignified canes


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.


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


Localization• Induced (newly synthetized after stresses)

Green parts: leaves, young berries…

• Constitutive (pre-existing):In wood and lignified canes


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)


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


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


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


Where to find mycoalexins?

Specific fungal communities are living in all living lignifiedplants in a confined environment (e.g. grapevine trunks)


Where to find mycoalexins?• Each fungal species fight against the others to keep its

own territory

• Development of competition zone lines


At the chemical and enzymatic level: clash of the Titans

Where to find mycoalexins?


In vitro confrontationfour type of interactive behaviours


Chemical induction ?

Eutypa lata

Botryosphaeria obtusa


Chemical war between fungi

New source of induced antifungal compounds


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.


Examples• Botryosphaeria obtusa against Eutypa lata

• More than15 induced compounds

O

O

128a

4a/b 3 3-Me

87

65

5a

8-OMeO

O

OO

12

4a4

2-Me

56

78

8a

5-OMe

3

New myco-phenolic compound exhibitinghigh antifungal and phytotoxic activities


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


Merci de votre attention

Thanks foryour attention

alexins as new antifungal compounds - iobc-wprs · département fédéral de l'économie dfe...

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