Oligosaccharide induced resistance in grapevine as affected by cuticle permeability

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Oligosaccharide induced resistance in grapevine as affected by cuticle permeability

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

To cite this version:

Franck Paris, Xavier Daire, Christian Gauvrit, G. Lecollinet, Vincent Ferrières, et al.. Oligosaccharide induced resistance in grapevine as affected by cuticle permeability. 4. Journée des Doctorants, Mar 2015, Dijon, France. 2015. �hal-02743003�

Oligosaccharide induced resistance in grapevine as affected by cuticle permeability

F. Paris ^a *, X. Daire ^a , C.Gauvrit ^a , G.Lecollinet ^b , V.Ferrières ^c , M. Adrian ^a , S. Trouvelot ^a

a- UMR 1347 Agroécologie AgroSup-INRA-uB, ERL CNRS 6300, BP 86510, 21000 Dijon, France b- Laboratoires Goëmar S.A.S. - Parc technopolitain Atalante - CS 41908 - 35435 Saint-Malo, France c- Ecole Nationale Supérieure de Chimie de Rennes, UMR CNRS 6226, CS 50837, 35708 Rennes, France

* Franck.Paris@dijon.inra.fr

I. Context

Grapevine & downy mildew

• Grapevine is susceptible to many diseases such as downy mildew, caused by the oomycete Plasmopara viticola (Pv).

• Elicitor induced resistance is an interesting strategy to help control this disease.

Penetration & resistance induction

• OS in general suffer inconsistent disease control levels.

• A hypothesis is that hydrophilic elicitors of high MW (> 1000 Da) do not easily cross the hydrophobic cuticular barrier to reach internal receptors.

Aim

• To understand the penetration of OS in the leaf and to correlate it with the

resistance induced against Pv.

• For this purpose, we studied the uptake of different labeled molecules in grapevine

leaf, the effect of different adjuvants on penetration and PS3 efficacy in Pv

resistance tests.

II. Grapevine leaf is nearly impermeable to sucrose unless an appropriate adjuvant is used

V. Take home message Literature cited

.

IV. PS3 induced resistance is enhanced by the use of an adjuvant when applied on the abaxial surface

0 20 40 60 80 100

0 20 40 60 80

% penetration

t (h)

Penetration rates of ¹⁴C labeled sucrose in grapevine leaf : comparison of abaxial and adaxial side. Effect of De.

Sucr. AB Sucr. AD Sucr. + De (0,5%) AB Sucr. + De (0,5%) AD

III. Comparison of PS3 penetration into arabidopsis and grapevine leaf

Sulfated ß-1,3-glucan

• Sulfated laminarin PS3 is obtained by chemical sulfation of laminarin, a storage oligosaccharide (OS) from the brown algae Laminaria digitata.

• PS3 has been shown to elicit plant defense reactions and to induce resistance against Pv in grapevine (1).

Adjuvant HLB¹ EO² 6h 24h 72h

None 5 5 8

SLP4 (0,1%) 9,7 4 8 10 14

SilwetL77 (0,1%) 8 5 10 22

Tween 20 (0,1%) 16,7 20 5 42 68

DehscofixCO125 (0,1%) 13 35 10,5 43,3 83,1

This difference of penetration between both sides of grapevine leaf could be explained by a difference in thickness, structure and composition of cuticular waxes and by the presence of stomata on the abaxial surface.

The adjuvant probably enhances leaf penetration by solubilisation of these cuticular waxes.

These studies were realized with ¹⁴C labeled sucrose, 2,4-D-acid and ester. Measure of penetration used a wash-off and tissue combustion method after application of these radioactive compounds to the adaxial (AD) or abaxial (AB) leaf surface (2).

2,4-D-ester (hydrophobic) penetrates the leaf through both sides while the

penetration of 2,4-D-acid (less hydrophobic) is

severely reduced in the adaxial surface.

In contrast, only a very small amount of sucrose (hydrophilic) can get

through the cuticle.

With Dehscofix

CO125, sucrose is

effectively able to cross the abaxial cuticle and in lesser amount, the adaxial one.

The hydrophobic cuticular barrier seems to prevent the penetration of hydrophilic compounds whereas hydrophobic ones of the same size easily get through.

Is it possible to increase their penetration, and possibly their efficacy, with an appropriate adjuvant ? % penetration of sucrose in AB side

Some adjuvants are

actually able to enhance the penetration of sucrose through the abaxial side of grapevine leaf. Adjuvants with the highest

ethoxylation increase it further.

Dehscofix CO125 (De) increases tenfold the penetration rate of an hydrophilic

compound through the abaxial side of the leaf. This adjuvant will be used for further experiments.

While ¹⁴C labeled sucrose is commercially available, ¹⁴C PS3 is not. Also, its synthesis would lead to too many radioactive wastes and was not considered . The following

experiments make use of fluoresceine labeled PS3. Treatments are deposited on the leaf, washed off and quantified via spectrofluorimetric measures (3).

0 20 40 60 80 100

PS3 PS3 + De

%EMI

Penetration of PS3 in arabidopsis (Col0).

Effect of De

T0 T24 T48

*

Addition of De (0,1%)

significantly enhances the penetration of PS3 through arabidopsis leaf 48 h post treatment.

However, addition of De (0,5%) seems to have no detectable impact on the penetration of PS3 into grapevine leaf.

While significant penetration rates can be observed after the addition of De in

arabidopsis leaves, this is not the case with grapevine. The spectrofluorimetry method with fluoresceine labeled PS3 does not seem sensitive enough to reveal low

penetration rates. However, a certain amount of PS3 should penetrate into grape leaf since it is effective as resistance inducer.

The efficacy of PS3 against Pv is significantly increased when applied with De on the abaxial side of

grapevine leaves rather than on the adaxial side.

On the adaxial side, treatments provide similar results (low efficacy). On the other hand, on the abaxial side, PS3 significantly induces resistance while the addition of De enhances it even more. This suggests that penetration of PS3 into the leaf is similar to that of sucrose.

• Penetration rate of hydrophilic compounds is higher through the abaxial surface than through the adaxial one, leading to practical consequences for treatment

application.

• The addition of appropriate adjuvants increases the efficacy of oligosaccharide elicitors when sprayed onto the leaf.

• Biodisponibility and formulation are major factors to consider when developing such a treatment.

(1) Trouvelot S, Varnier A, Allegre M, Mercier L, Baillieul F, Arnould C, Gianinazzi-Pearson V, Klarzynski O, Joubert J, Pugin A, Daire X (2008) A beta-1,3 glucan sulfate induces resistance in grapevine against Plasmopara viticola through priming of defense

responses, including HRlike cell death. Mol Plant Microbe Interact 21:232–243

(2) Grangeot M, Chauvel B, Gauvrit C (2006) Spray retention, foliar uptake and translocation of glufosinate and glyphosate in Ambrosia artemisiifolia. Weed Research 46, 152–162.

(3) Liu Z (2004) Effects of surfactants on foliar uptake of herbicides – a complex scenario. Colloids and Surfaces B: Biointerfaces 35 149–153

0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00

Water PS3 2,5 g/l De 0,5% PS3 2,5 g/l + De 0,5%

% sporulation

PS3 efficacy against Pv : abaxial side

a

b

b

c

0,00 0,50 1,00 1,50 2,00 2,50

Water PS3 2,5 g/l De 0,5% PS3 2,5 g/l + De 0,5%

% sporulation

PS3 efficacy against Pv : adaxial side

abc

bc

a

c

(1) HLB = Hydrophilic Lipophilic Balance of the adjuvant determining if it is rather hydrophobic (low HLB) or hydrophilic (high HLB) on a scale ranging from 0 to 20.

(2) EO = Ethylene Oxide residues in a molecule (degree of ethoxylation).

0 10 20 30 40 50 60 70 80 90 100

PS3 AB PS3 + De AB PS3 AD PS3 + De AD

%EMI

Penetration of PS3 in grapevine. Effect of De

T0 T24 T48

28.7%

90.1%

98.9%

31.4%

% protection % protection

0 20 40 60 80 100

0 12 24 36 48

% penetration

t (h)

Penetration rates of compounds in grapevine leaf

2,4D ester AB 2,4D ester AD 2,4D acid AB 2,4D acid AD Sucrose AB Sucrose AD

%EMI = % of fluorescence obtained after wash off.