Biostimulation of grapevine and wheat : mode of action and possible agronomic uses

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Biostimulation of grapevine and wheat : mode of action and possible agronomic uses

Yuko Krzyżaniak, Franck Paris, Christian Gauvrit, G. Lecollinet, Vincent Ferrières, Marielle Adrian, Laurent Legentil, Xavier Daire, Sophie Trouvelot

To cite this version:

Yuko Krzyżaniak, Franck Paris, Christian Gauvrit, G. Lecollinet, Vincent Ferrières, et al.. Biostim- ulation of grapevine and wheat : mode of action and possible agronomic uses. 11. International Conference on Plant Diseases, Dec 2015, Tours, France. 2015. �hal-02739417�

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Evidence for foliar uptake of sulfated laminarin into grapevine depending on surfactant use and leaf surface.

Yuko Krzyżaniak

¹

, F. Paris

^1,2,3

, C. Gauvrit

¹

, G. Lecollinet

⁴

, V. Ferrières

^2,3

, M. Adrian

⁵

, L. Legentil

^2,3

, X. Daire

¹

and S. Trouvelot

⁵

1INRA, UMR 1347 Agroécologie, BP 86510, F-21000 Dijon, France; ²Ecole Nationale Supérieure de Chimie de Rennes, CNRS, UMR 6226, 11 Allée de Beaulieu, CS 50837, 35708 Rennes, France; ³Université européenne de Bretagne, France ; ⁴Laboratoires Goëmar S.A.S.-Parc technopolitain Atalante, 35435 Saint-Malo, France; ⁵Université de Bourgogne, UMR 1347 Agroécologie BP 86510, F-21000 Dijon, France.

→ Etudiés et développés comme stratégie de protection des cultures dans l’objectif de diminuer l’utilisation de pesticides (Delaunois et al., 2014).

MAIS …

L’efficacité de l’éliciteur est variable au champ.

THE PATHOSYSTEM

GRAPEVINE : Perennial crop with major economic and cultural value, but susceptible to many cryptogamic diseases such as downy mildew.

DOWNY MILDEW : Disease caused by the biotrophic oomycete Plasmopara viticola (Pv), responsible for considerable damages in worldwide vineyards : loads of fungicides are required.

ELICITORS : Compounds able to trigger plant defences, resulting in some cases, in an enhanced resistance to bioagressors. Some oligosaccharides (OS) like ß-glucans are elicitors (Delaunois et al., 2014).

SULFATED LAMINARIN (PS3) : Obtained by chemical sulfation of laminarin, a ß-(1,3)glucan from the brown algae Laminaria digitata. PS3 was shown to elicit plant defences and to induce resistance against Pv in controlled conditions (Trouvelot et al., 2008).

Disease control by OS is often inconsistent. Elicitors’ efficacy depend on many factors (Walters, 2013), including their bioavailiability.

One hypothesis is that hydrophilic elicitors with high MW (>1000Da), such as PS3, have to cross the hydrophobic cuticular barrier, to reach internal receptors.

Obligatory parasitism

Defence mechanisms

Induction

Vitis vinifera (Vv) Plasmapara viticola (Pv)

Sulfated laminarin (PS3)

PS3 = hydrophilic

Cuticle = hydrophobic

AIMS

Understanding the uptake of PS3 in grapevine leaves, to improve its protection rate against Pv by:

(1) Developping a method to vizualize its penetration in planta

(2) Studying the effect of an ethoxylated surfactant use and

influence of leaf surface.

…UNFORTUNATELY ENCOUNTERING MANY BARRIERS.

ELICIORS : A PROMISING CONTROL STRATEGY ....

Native laminarin

HO

OH

OH HO

HO O

O OH O

OH

O HO

OH O HO O

OH O OH O

O N

N⁺

S S

O O

HN

O O O^- HN O

HOHO OH OH

O HOHO

OH OH

n

At ENSCR Rennes by F. Paris:

Chemical labeling of native laminarin with Lissamine Rhodamine B ethylenediamine (LRB)

NaO3SO

OSO3Na

OSO3Na NaO3SO

O O O

OSO3Na O OSO3Na

NaO3SO O

O O

OSO3Na O OSO3Na O

O N

N⁺

S S

O O

HN

O O O^- HN O

NaO3SO NaO3SO

O OSO3Na

NaO3SO O NaO3SO

OSO₃Na OSO3Na

n SO3Na

SO3Na

SO3Na SO3Na

 With or without 0,5% Dehscofix CO125® (DE) as the surfactant

 On the adaxial or abaxial surface

 Different duration of contact: 1, 2, 3 days

HO

OH

OH HO

HO O O

OH O OH

HO O

OH O HO O

OH O OH O

O N

N⁺

S S

O O

HN

O O O^- HN O

HOHO OH OH

O HOHO

OH OH

n

 Removing the unabsorbed PS3-LRB by washing the droplet deposit area with 1 mL of 0,1% Tween20.

 Mounting in distilled water Labeling of laminarin

1 2 Sulfation of labeled laminarin 3 PS3-LRB application

1 µL droplet deposit

detached grapevine leaves (n >3)

Sample preparation

4 Two-Photon Laser Scanning Laser

Microscopy (TPLSM) vizualisations

5

 Laser excitation : 840 nm

 Collecting the fluorescence emission : on orange [563-588] nm and red channels [601-657] nm.

 Cross-sections : 50+/- 10 µm depth, every 1 µm of Z-axis

 5 acquisitions per droplet deposit

Nikon A1-MP x60 objective, DimaCell Platform

Laminarin - LRB PS3 - LRB

- Observation 1 -

Better uptake of PS3-LRB

when formulated in an adequate surfactant than in water.

Preferential uptake of PS3-LRB along the anticlinal cell walls

and especially through stomata.

Better uptake of PS3-LRB with DE into the abaxial foliar tissue

than into the adaxial one.

In bioassays, PS3 in DE reduces the sporulation of Pv by 20 to 30%,

compared to PS3 in water.

DE = Dehscofix CO 125 ® DAT = Days after treatment Cu = Cuticle

St = Stomata

St = Substomatal chamber Ep = Epidermal cells

V = Vein

G= Intercellular gaps Acw =Anticlinal cell wall Bar = 20µm

A

Cu

1 ,2 and 3 DAT

+ water

Cu St

B

^{1 DAT}

C

Ep

St 2 DAT

D

St

Cu

StCh

3 DAT

St

G

+ 0,5% DE

Diff ere nt random areas Horizon tal sec tion Cross sec tion

Adaxial Abaxial

A ^B

St Ep

Acw

St

Acw 5 droplets per leaf

1. The development of this microscopic observation method enabled us to show that an OS elicitor such as PS3 does not readily enter the grapevine leaf without an ad hoc formulation.

2. Spray application of OS elicitors preferentially targeting the abaxial surface of the grapevine leaves may improve the effectiveness of such treatments.

* Want to know more? See our online publication: Paris et al. (2015) An ethoxylated surfactant enhances the penetration of the sulfated laminarin through leaf cuticle and stomata, leading to increased induced resistance against grapevine downy mildew. Physiologia plantarum. doi: 10/1111/ppl.12394

In the literature, surfactants were shown :

1. to promote the uptake of a range of hydrophilic compounds such as glucose derivatives (Forster, 2005).

2. to increase the water content of cuticle, and consequently to improve leaf permeability to water-soluble compounds (Ramsey, 2005).

This could be due:

1. to the fact that grapevine possesses stomata on the abaxial surface only

2. to the difference in waxes and cuticular structure and composition between its leaf surfaces

 Therefore, further studies should focus in

analyzing differences in the thickness and composition between the two leaf surfaces*.

Hydrophilic compounds can enter the leaf via polar pathways in the cuticle. They consist of polysaccharide fibers embedded in the cuticle.

These polar zones were shown to occur preferentially in the cuticle above anticlinal cell walls, around the stomata and at the bases of trichomes (Schönherr 2006).

The beneficial effect of DE on PS3 uptake into grapevine leaf is backed by increased PS3-induced resistance in these biological tests.

 This supports our hypothesis that insufficient

bioavailability of OS elicitors can limit their efficacy.

Delaunois B, Farace G, Jeandet P, et al (2014) Elicitors as alternative strategy to pesticides in grapevine? Current knowledge on their mode of action from controlled conditions to vineyard. Environmental Science & Pollution Research 21:4837.

Forster WA, Zabkiewicz JA, Liu Z (2006) Cuticular uptake of xenobiotics into living plants.part 2: influence of the xenobiotic dose on the uptake of bentazone, epoxiconazole and pyraclostrobin, applied in the presence of various surfactants, into Chenopodium album, Sinapis alba and Triticum aestivum leaves. Pest management science 62, 7 Liu, Z. (2004). Confocal laser scanning microscopy - an attractive tool for studying the uptake of xenobiotics into plant foliage. J. Microsc. 213:87–93 Ramsey R j. l., Stephenson G r., Hall J c. (2005) A review of the effects of humidity, humectants, and surfactant composition on the absorption and efficacy of highly water-soluble herbicides. Pesticide Biochemistry and Physiology 82:162–175 Schönherr J (2006) Characterization of aqueous pores in plant cuticles and permeation of ionic solutes. Journal of Experimental Botany 57:2471–2491 Trouvelot S, Varnier AL, Allegre M, et al (2008) β-1,3 Glucan Sulfate Induces Resistance in Grapevine against Plasmopara viticola Through Priming of Defense Responses, Including HR-like Cell Death. Molecular plant-microbe interactions 21, 2 Walters DR, Ratsep J, Havis ND (2013) Controlling crop diseases using induced resistance: challenges for the future. Journal of Experimental Botany 64:1263–1280

Biostimulation of grapevine and wheat : mode of action and possible agronomic uses

Evidence for foliar uptake of sulfated laminarin into grapevine depending on surfactant use and leaf surface.

Yuko Krzyżaniak

, F. Paris

, C. Gauvrit

, G. Lecollinet

, V. Ferrières

, M. Adrian

, L. Legentil

, X. Daire

and S. Trouvelot

Better uptake of PS3-LRB

when formulated in an adequate surfactant than in water.

Preferential uptake of PS3-LRB along the anticlinal cell walls

and especially through stomata.

Better uptake of PS3-LRB with DE into the abaxial foliar tissue

than into the adaxial one.

In bioassays, PS3 in DE reduces the sporulation of Pv by 20 to 30%,

compared to PS3 in water.

A

+ water

B

C

D

+ 0,5% DE

Diff ere nt random areas Horizon tal sec tion Cross sec tion

Adaxial Abaxial

A B

A ^B