# Deciphering ecological interactions between grapevine leaf microbiome and pathogens

**Corinne VACHER**, Paola FOURNIER, Didac BARROSO-BERGADA, Charlie PAUVERT, Lucile PELLAN

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RÉPUBLIQUE
FRANÇAISE

Liberté
Égalité
Fraternité

INRAO

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# Reducing the use of chemical pesticides is a major goal in Europe

## 2030 Targets for sustainable food production

### PESTICIDES

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Reduce the overall use and risk of chemical and hazardous pesticides

### NUTRIENT LOSSES

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Reduce nutrient losses by 50% whilst retaining soil fertility, resulting in 20% less fertilisers

### ANTIMICROBIALS

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Reduce sales of antimicrobials for farmed animals and aquaculture

### ORGANIC FARMING

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Increase the percentage of organically farmed land in the EU

\#EUFarm2Fork

\#EUGreenDeal

The European
Green Deal

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# Developping a pesticide-free viticulture is a key priority

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Traitement préventif des vignes contre le mildiou, le 9 juillet 2019 à Epernay (Marne). (ALEXANDRE MARCHI / MAXPPP)

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France Info
Radio France
# Developping a pesticide-free viticulture is a key priority

<figure><img src="image_10.png" /><figcaption>A levées et enseignants s'étaient trouvés très mal après un épandage de produits pour la vigne près de leur école le Crédit photo : archives Jérôme Jamot Par sudouest.fr avec AFP</figcaption></figure>

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Traitement préventif des vignes contre le mildiou, le 9 juillet 2019 à Epernay (Marne). (ALEXANDRE MARCHI / MAXPPP)

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France Info
Radio France
# Grapevine is among the most treated crops in France

<figure><img src="image_14.png" /><figcaption>Treatment Frequency Index (TFI)</figcaption></figure>
# The majority of treatments are fungicides targeting mildews

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**Downy mildew**
*Plasmopara viticola*
(oomycete)

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**Powdery mildew**
*Erysiphe necator*
(fungi)

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Two pathogens of North-American origin introduced in Europe in the 19th century
# Harnessing the grapevine microbiome could be part of the solution to reduce fungicide use

## Functional groups

* Plant pathogen

* Pathogen antagonist

* Plant growth promoter

* Wine-related microorganism

## Ecological interactions

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positive effect

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negative effect

## Microbial fluxes

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Fournier, Pellan *et al*. The functional microbiome of grapevine. *Advances in Ecological Research*, 2022

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# Our goals

* Elucidate the **ecological interactions** within the grapevine leaf microbiome

* Discover novel **microbial antagonists** targeting downy and powdery mildews

* Investigate **environmental reservoirs** and **viticultural practices** selecting for protective microbes to develop **conservation biocontrol**

* Engineer robust **microbial consortia** with biocontrol activity to develop inoculation-based biocontrol strategies
# Our approach to identify the protective microbiome

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# Our approach to identify the protective microbiome

Culture
collection
&WGS

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SynComs
assembly

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Confrontation
tests & qPCR

Microbial consortia with
biocontrol activity

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Yeasts, fil. fungi, bacteria

Dynamics
modelling from
time series

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Leaf samples

Tissue scale

Plot scale

Species scale

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Network inference
from eDNA
# Experiment #1 – Interactions between leaf microbiome and powdery mildew

## STUDY SITE

* Experimental vineyard with no fungicide application

* Planted with Merlot

* Two soil management systems: Cover crop (CC) vs Bare Soil (BS)

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## EXPERIMENTAL DESIGN

* Epidemic triggered by inoculation of *Erysiphe necator*

* Sampling of ~20-day old leaves

* 192 leaves = 2 soil management systems (CC vs BS) × 3 dates × 2 leaf types (asymptomatic vs symptomatic) × 4 sampling zones × 4 leaf replicates

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# Experiment #1 – Interactions between leaf microbiome and powdery mildew

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## MICROBIOME ANALYSIS

* *Myco*biome composition analyzed with a metabarcoding approach targeting fungal ITS1

* *Myco*biome abundance quantified by ddPCR

## ENVIRONMENTAL COVARIATES

* Spatial position of the vine

* Spatial position of the leaf within the canopy

* Weather conditions experienced by the leaf since flush

* Leaf ecophysiological traits (leaf water content, specific leaf area)

## ENVIRONMENTAL RESERVOIRS

* Old leaves

* Bark

* Upper soil
The pathogen *Erysiphe necator* dominates the mycobiome of disease lesions

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# Mycobiome changes induced by infection remain confined to the disease lesion

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**PERMANOVA**

AA vs AS: R²= 1.3%, p-value=0.006

AS vs DL: R²= 17.3%, p-value<0.001
The mycobiome in asymptomatic tissues exhibits greater richness, diversity, and lower abundance

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AS

DL

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<figure><img src="image_38.png" /><figcaption># Network inference reveals associations between *E. necator* and basidiomycete yeasts</figcaption></figure>

Network for
asymptomatic tissues

Inference performed
using PLN models with
environmental covariates
# Some interactions between *E. necator* and basidiomycete yeasts were validated *in vitro*

Confrontation tests to assess the effect of yeasts on *E. necator* growth 16 foliar disks per test (8 treated, 8 control-) incubated for 12 days at 22°C

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<table><thead><tr><th rowspan="3">Yeast strain</th><th colspan="4">PREVENTIVE TREATMENT</th><th colspan="4">CURATIVE TREATMENT</th></tr><tr><th colspan="2">PELLET</th><th colspan="2">SUPERNATANT</th><th colspan="2">PELLET</th><th colspan="2">SUPERNATANT</th></tr><tr><th>En growth (in %)</th><th>p-value</th><th>En growth (in %)</th><th>p-value</th><th>En growth (in %)</th><th>p-value</th><th>En growth (in %)</th><th>p-value</th></tr></thead><tbody><tr><td>Buckleyzyma aurantiaca</td><td>-1.87</td><td>0.33</td><td>2.25</td><td>0.99</td><td>-19.63</td><td>0.18</td><td>-31.25</td><td>&lt;0.001</td></tr><tr><td>Filobasium wieringae</td><td>0.5</td><td>0.98</td><td>6.38</td><td>0.8</td><td>-10.63</td><td>0.75</td><td>-1.87</td><td>1</td></tr><tr><td>Udeniomyces pyricola</td><td>-1</td><td>0.85</td><td>6</td><td>0.83</td><td>-32.5</td><td>&lt;0.001</td><td>-3.75</td><td>0.97</td></tr><tr><td>Vishniacozyma victoriae</td><td>-0.37</td><td>0.99</td><td>7.63</td><td>0.67</td><td>-10</td><td>0.79</td><td>3.88</td><td>0.97</td></tr><tr><td>Growth inhibitor (control+, BTH)</td><td>-98.62</td><td>&lt;0.001</td><td>-90.62</td><td>&lt;0.001</td><td>-85.62</td><td>&lt;0.001</td><td>-86.25</td><td>&lt;0.001</td></tr></tbody></table>
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The yeasts of interest are relatively rare in environmental reservoirs

Species more abundant in asymptomatic tissues (DESeq2 analysis)

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# Soil management does not drive the grapevine leaf mycobiome

<table><thead><tr><th>Most explanatory variables</th><th>Adj. R2 (%)</th><th>Adj. p-value</th></tr></thead><tbody><tr><td>Sampling date</td><td>13.9</td><td>&lt;0.001</td></tr><tr><td>+ Distance to cordons</td><td>16.0</td><td>&lt;0.001</td></tr><tr><td>+ Distance to inoculated leaves</td><td>16.7</td><td>0.42</td></tr><tr><td>+ Mean VPD since flush</td><td>17.4</td><td>0.48</td></tr><tr><td>+ Spatial position (PCNM4)</td><td>18.0</td><td>0.49</td></tr><tr><td>+ All variables</td><td>21.3</td><td></td></tr></tbody></table>

June 1

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June 23

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July 5

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CAP analysis with forward selection of variables
# Experiment #2 – Interactions between leaf microbiome and downy mildew

## STUDY SITE

* 3 French regions

* 3 vineyards per region

* Untreated grapevine rows

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## EXPERIMENTAL DESIGN

* Sampling performed at the peak of infection by *Plasmopara viticola*

* 270 leaves = 3 regions × 3 plots × 30 leaf replicates

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# Experiment #2 – Interactions between leaf microbiome and downy mildew

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## MICROBIOME ANALYSIS

* Composition analyzed with a metabarcoding approach targeting fungal ITS1 and bacterial 16S

* Abundance quantified by ddPCR

* *P. viticola* quantified by qPCR

### Fungi

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### Bacteria

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Leaf microbiome composition is mainly
driven by geography

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Leaf microbiome composition is mainly
driven by geography

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# Infection by downy mildew has variable effects on leaf microbiome composition

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## Fungi - Plot A

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PERMANOVA: $R^2$= 4.8%, p-value=0.001

## Fungi - Plot C

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PERMANOVA: $R^2$= 1.5%, p-value=0.76
A few microbial taxa are consistently more abundant in asymptomatic leaf tissues

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MaAsLin2
differential
abundance
analysis
# Network learning suggests ecological interactions involving *P. viticola* occur on leaves

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## Fungi - Plot B

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InfIntE
package

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Barroso-Bergadà D. Automatic learning of interaction networks.
PhD thesis, Univ. Bourgogne, 2022
Conclusions

* Our statistical analyses generated new hypotheses regarding interactions between powdery mildew, downy mildew, and the grapevine leaf microbiome.

* Certain interactions, involving basidiomycete yeasts, were validated *in vitro*.

* These yeasts were detected in environmental reservoirs such as bark and upper soil, although they were found infrequently.

* Soil management has minimal effect on the leaf microbiome and cannot be used to harness the leaf microbiome.
# Perspectives: use the culture collection to test network models

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Cultiver la vigne sans pesticides

# Perspectives: from tissue scale to biogeographic scale

Culture
collection
&WGS

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Leaf samples

Tissue scale

Plot scale

Species scale

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Network inference
from eDNA

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Native range of:

V. acerifolia

V. arizonica

V. aestivalis

V. berlandieri

V. cinerea

V. labrusca

V. palmata

V. riparia

V. rotundifolia

V. rupestris

V. vulpina

Native range of :

V. amurensis

V. piasezkii

V. romanettii

Locations where the microbiome of American and Asian *Vitis* species has been described (references below)

① Brum et al. (2012)

③ Kernaghan et al. (2017)

⑤ Fan et al. (2020)

⑦ Aleynova et al. (2022)

② Felber et al. (2016)

④ Cureau et al. (2021)

⑥ Sun et al. (2021)

Native range of American and Asian *Vitis* species with known genetic resistance to North American pathogens

Fournier, Pellan *et al.*
The functional
microbiome of
grapevine. *Advances
in Ecological
Research, 2022

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INRAO

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Many thanks to....

**UMR SAVE**: François Delmotte, Jessica Vallance, Paola Fournier, Lucile Pellan, Aarti Jaswa, Marie-Cécile Dufour, Isabelle Demeaux, Laurent Delière, Agnès Calonnec

**UMR BIOGECO/PGTB**: Charlie Pauvert, Simon Labarthe, Tania Fort, Erwan Guichoux, Emilie Chancerel, Marie Massot, Julie Faivre d'Arcier

**UMR Agroécologie**: David Bohan, Didac Barroso-Bergadà

**MIA**: Julien Chiquet, Stéphane Robin, Julie Aubert

**UMR AGAP**: Patrice This

**Cornell University**: Lance Cadle-Davidson, Erin Galarneau

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RÉPUBLIQUE
FRANÇAISE

Liberté
Égalité
Fraternité

anr

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agence nationale
de la recherche

AU SERVICE DE LA SCIENCE

CONSORTIUM

biocontrôle

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BORDEAUX
SCIENCES
AGRO

INRAO

Université
de BORDEAUX

MoëtHennessy
Thanks for your attention!

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Corinne VACHER (**corinne.vacher@inrae.fr**)

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RÉPUBLIQUE
FRANÇAISE

Liberté
Égalité
Fraternité

INRAE