# Wounding nectarine fruit disrupts Monilinia laxa infection: deciphering fruit gene pathway involved and the role of phenolic and volatile compounds

Bénédicte Quilot-Turion, Marie-Noëlle Corre, Guy Costagliola, Laure Heurtevin, Véronique Signoret, Maria Do Carmo Raseira, Maximiliano Dini Vinoly

<figure><img src="image_1.png" /></figure>

## ► To cite this version:

Bénédicte Quilot-Turion, Marie-Noëlle Corre, Guy Costagliola, Laure Heurtevin, Véronique Signoret, et al.. Wounding nectarine fruit disrupts Monilinia laxa infection: deciphering fruit gene pathway involved and the role of phenolic and volatile compounds. 10th Rosaceae Genomics Conference, Centre for research in agricultural genomics, Dec 2020, Barcelone, Spain. [hal-03267785](https://hal.inrae.fr/hal-03267785)

HAL Id: hal-03267785

https://hal.inrae.fr/hal-03267785

Submitted on 22 Jun 2021

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

INRAe

<figure><img src="image_5.png" /></figure>

WOUNDING NECTARINE FRUIT DISRUPTS *MONILINIA LAXA* INFECTION: DECIPHERING FRUIT GENE PATHWAY INVOLVED AND THE ROLE OF PHENOLIC AND VOLATILE COMPOUNDS

DINI VINOLY Maximiliano, CORRE Marie-Noëlle, COSTAGLIA Guy, HEURTEVIN Laure, SIGNORET Véronique, RASEIRA Maria Do Carmo, QUILOT-TURION Bénédicte

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

Plantes et Systèmes de culture Horticoles

<figure><img src="image_8.png" /></figure>
INRAO

> CONTEXT
## Brown rot

* a very common disease

* main species: *Monilinia laxa*, *M. fructicola* and *M. fructigena*

* flower and stem desiccation
  and fruit rot

* can provoke 30 to 40% of harvest losses

One of the most damaging diseases of peach and Prunus species

<figure><img src="image_9.png" /><figcaption>M. laxa</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>M. fructicola</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>M. fructigena</figcaption></figure>
Prophylaxy is not effective enough

The use of chemicals is the rule both
on flowers and fruit

• environmental impacts

• public health trouble

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

'The' solution → grow resistant cultivars

No resistant cultivar is available for farmers

No major resistance factor has been identified

<figure><img src="image_14.png" /></figure>
The fungus penetrates using 'open doors' or directly through the skin (cutinases)

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

Some resistance factors to brown rot relie on fruit skin

<figure><img src="image_17.png" /></figure>
Infection immediately after wounding and 7 hours after wounding

<figure><img src="image_18.png" /></figure>

The reaction depends on fruit stage

Infection is limited by compounds synthesized
by the fruit following the injury

<figure><img src="image_19.png" /></figure>
> DOES WOUNDING NECTARINE FRUIT DISRUPT MONILINIA LAXA INFECTION ?
Experimental protocol

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

Cultivars : Zephyr and C216
End of June: immature fruit

<figure><img src="image_22.png" /></figure>
Experimental protocol

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

Cultivars : Zephyr and C216
End of June: immature fruit

<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>
Experimental protocol

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /></figure>

<figure><img src="image_29.png" /><figcaption>Cultivars : Zephyr and C216
End of June: immature fruit</figcaption></figure>

<figure><img src="image_30.png" /></figure>

28 samples 2 genotypes

20 samples 2 genotypes

16 libraries Zephyr

<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /></figure>
Infections after wounding

240h after infection

Infection immediately after wounding

<figure><img src="image_33.png" /></figure>

Infection 7h after wounding

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>

The infection 7 hours after wounding resulted in slowed and reduced brown rot infection
Red reactions

immediately
after wounding

<figure><img src="image_36.png" /></figure>

7h after wounding

<figure><img src="image_37.png" /></figure>

<figure><img src="image_38.png" /></figure>
GC-MS analyses : volatile compounds

70 were detected

The treatment effect was significant for
32 volatile compounds

2-Butanone

<figure><img src="image_39.png" /></figure>

11 were present **only** in case of
wounded samples, for both genotypes

Good candidates to explain
wounding effect on infection

7h after wounded

<figure><img src="image_40.png" /></figure>

<table><thead><tr><th>Volatile compound</th><th>Treatment</th><th>Volatile compound</th><th>Treatment</th></tr></thead><tbody><tr><td>Aac,methyl_ester</td><td>4.06<sup>-3</sup> **</td><td>1-Pentene</td><td>8.17<sup>-8</sup> ***</td></tr><tr><td><strong>2-Butanone</strong></td><td>1.35<sup>-6</sup> ***</td><td>Terpinolene_B</td><td>6.40<sup>-9</sup> ***</td></tr><tr><td>Ethyl_Acetate</td><td>0.017<sup>ns</sup></td><td>P-Cymenene</td><td>0.015<sup>ns</sup></td></tr><tr><td>Tropilidene_Toluene</td><td>0.617<sup>ns</sup></td><td>2-tert_Butyltoluene</td><td>0.457<sup>ns</sup></td></tr><tr><td>Hexanal</td><td>0.558<sup>ns</sup></td><td><strong>Cyclohexane_A</strong></td><td>4.35<sup>-7</sup> ***</td></tr><tr><td>Unknown1</td><td>0.053<sup>ns</sup></td><td>Undecane</td><td>1.38<sup>-8</sup> ***</td></tr><tr><td>Unknown2</td><td>3.57<sup>-3</sup> **</td><td>β-Linalool</td><td>1.95<sup>-3</sup> **</td></tr><tr><td>Ethylbenzene</td><td>0.899<sup>ns</sup></td><td>Nonanal</td><td>0.837<sup>ns</sup></td></tr><tr><td><strong>1-Met_2prop_acet</strong></td><td>0.333<sup>ns</sup></td><td><strong>Cyclohexane_B</strong></td><td>6.52<sup>-9</sup> ***</td></tr><tr><td>P-Xylene_A</td><td>0.538<sup>ns</sup></td><td>neo_allo_Ocimene</td><td>5.78<sup>-10</sup> ***</td></tr><tr><td><strong>3-Met_3but1ol,acet</strong></td><td>7.22<sup>-3</sup> **</td><td>2,6-Dimethyl</td><td>0.012<sup>ns</sup></td></tr><tr><td>P-Xylene_B</td><td>0.211<sup>ns</sup></td><td>allo_Ocimene</td><td>2.11<sup>-4</sup> ***</td></tr><tr><td>N-Amyl_acetate</td><td>0.049<sup>ns</sup></td><td>Unknown3</td><td>0.017<sup>ns</sup></td></tr><tr><td>α-Pinene_A</td><td>3.65<sup>-3</sup> **</td><td>Diisopropyl_xanth</td><td>4.83<sup>-3</sup> **</td></tr><tr><td>Benzaldehyde</td><td>0.513<sup>ns</sup></td><td>Dodecane</td><td>0.307<sup>ns</sup></td></tr><tr><td>5-Hep_2one,6met</td><td>0.491<sup>ns</sup></td><td>Decanal</td><td>0.759<sup>ns</sup></td></tr><tr><td>β-Myrcene</td><td>4.11<sup>-8</sup> ***</td><td>Benzothiazole</td><td>0.018<sup>ns</sup></td></tr><tr><td>Furan,2pentyl</td><td>0.021<sup>ns</sup></td><td>cis_3Hexenyl_isoval</td><td>6.98<sup>-12</sup> ***</td></tr><tr><td>Heptane</td><td>0.064<sup>ns</sup></td><td><strong>n-Hexyl_iso_val</strong></td><td>5.02<sup>-12</sup> ***</td></tr><tr><td>Decane</td><td>7.83<sup>-8</sup> ***</td><td><strong>trans-2Hexenyl_val</strong></td><td>2.60<sup>-6</sup> ***</td></tr><tr><td>Octanal</td><td>8.31<sup>-4</sup> ***</td><td>Unknown4</td><td>0.018<sup>ns</sup></td></tr><tr><td>3-Hexen_1ol,acet</td><td>9.58<sup>-6</sup> ***</td><td>Tridecane</td><td>0.997<sup>ns</sup></td></tr><tr><td>3-Heptene</td><td>0.661<sup>ns</sup></td><td>Undecanal</td><td>0.674<sup>ns</sup></td></tr><tr><td><strong>α-Phellandrene</strong></td><td>1.57<sup>-4</sup> ***</td><td>Unknown5</td><td>0.067<sup>ns</sup></td></tr><tr><td>α-Pinene_B</td><td>0.015<sup>ns</sup></td><td>Unknown6</td><td>0.543<sup>ns</sup></td></tr><tr><td>Aac,hexyl_es</td><td>3.63<sup>-6</sup> ***</td><td>Unknown7</td><td>0.012<sup>ns</sup></td></tr><tr><td><strong>2-Hexen_1ol,ac</strong></td><td>3.98<sup>-3</sup> **</td><td>cis-Jasmone</td><td>1.18<sup>-8</sup> ***</td></tr><tr><td>Terpinolene_A</td><td>0.018<sup>ns</sup></td><td>Tetradecane</td><td>0.812<sup>ns</sup></td></tr><tr><td>omp-cymene</td><td>3.80<sup>-4</sup> ***</td><td>Trans-α-Bergamo</td><td>7.38<sup>-4</sup> ***</td></tr><tr><td>Limonene</td><td>4.27<sup>-4</sup> ***</td><td>Trans-Geranylacet</td><td>0.818<sup>ns</sup></td></tr><tr><td>O-cymene</td><td>6.90<sup>-11</sup> ***</td><td>Cis-β-Farnesene</td><td>6.50<sup>-3</sup> **</td></tr><tr><td><strong>β-Ocimene</strong></td><td>1.27<sup>-7</sup> ***</td><td>Pentadecane</td><td>0.683<sup>ns</sup></td></tr><tr><td>Y-Caprolactone</td><td>0.238<sup>ns</sup></td><td>Butylated_Hydroxy</td><td>0.021<sup>ns</sup></td></tr><tr><td>Y-Terpinene</td><td>3.38<sup>-4</sup> ***</td><td>Heneicosane</td><td>0.195<sup>ns</sup></td></tr><tr><td>Acetophenone</td><td>0.201<sup>ns</sup></td><td>Di_n_octyl_ether</td><td>0.808<sup>ns</sup></td></tr></tbody></table>

<figure><img src="image_41.png" /></figure>
HPLC analyses : phenolic and terpenoid compounds

30 phenolic
and terpenoid
compounds

Samples from
fruit with wounds
presented lower
contents

<figure><img src="image_42.png" /></figure>

<figure><img src="image_43.png" /></figure>
HPLC analyses : phenolic and terpenoid compounds in red zones

<table><thead><tr><th>Compound Family</th><th>Compound</th><th>p-value</th></tr></thead><tbody><tr><td rowspan="6">Triterpenoid</td><td>trihydroxy-urs-12-en-28-oic acid</td><td>0.030 ns</td></tr><tr><td>trihydroxy-urs-12-en-28-oic acid</td><td>0.049 ns</td></tr><tr><td>Oleanolic acid</td><td>0.496 ns</td></tr><tr><td>Ursolic acid</td><td>0.676 ns</td></tr><tr><td>Flavan-3-ol</td><td>1.54<sup>-4</sup> ***</td></tr><tr><td>Procyanidin B1</td><td>0.254 ns</td></tr><tr><td rowspan="4">Flavan-3-ol</td><td>Flavan-3-ol</td><td>5.16<sup>-3</sup> **</td></tr><tr><td>Flavan-3-ol</td><td>9.08<sup>-3</sup> **</td></tr><tr><td>Catechin</td><td>8.95<sup>-4</sup> ***</td></tr><tr><td>Flavanone</td><td>only in red reaction</td></tr><tr><td rowspan="7">Flavanone</td><td>Flavanone</td><td>only in red reaction</td></tr><tr><td>Eriodictyol-7-glucoside</td><td>only in red reaction</td></tr><tr><td>Flavanone</td><td>only in red reaction</td></tr><tr><td>Naringenine-7-glucoside</td><td>only in red reaction</td></tr><tr><td>Flavanone</td><td>only in red reaction</td></tr><tr><td>cis-Neochlorogenic acid</td><td>9.19<sup>-3</sup> **</td></tr><tr><td>Neochlorogenic acid</td><td>0.012 ns</td></tr><tr><td rowspan="6">Hydroxycinnamic derivatives</td><td>Hydroxycinnamic derivative</td><td>only in red reaction</td></tr><tr><td>Chlorogenic acid</td><td>0.739 ns</td></tr><tr><td>cis-Chlorogenic acid</td><td>0.733 ns</td></tr><tr><td>Hydroxycinnamic derivative</td><td>0.437 ns</td></tr><tr><td>5-p-Coumaroylquinic acid</td><td>9.90<sup>-3</sup> **</td></tr><tr><td>3,5-Dicaffeoylquinic acid</td><td>0.334 ns</td></tr></tbody></table>

6 compounds present in greater proportion in red zones

<figure><img src="image_44.png" /></figure>

<figure><img src="image_45.png" /></figure>

The two major phenolic compounds

- Eriodictyol-7-glucoside = prunine

- Naringenin-7-glucoside
## HPLC analyses : phenolic and terpenoid compounds in red zones

<table><thead><tr><th>Compound Family</th><th>Compound</th><th>p-value</th></tr></thead><tbody><tr><td rowspan="6">Triterpenoid</td><td>trihydroxy-urs-12-en-28-oic acid</td><td>0.030 ns</td></tr><tr><td>trihydroxy-urs-12-en-28-oic acid</td><td>0.049 ns</td></tr><tr><td>Oleanolic acid</td><td>0.496 ns</td></tr><tr><td>Ursolic acid</td><td>0.676 ns</td></tr><tr><td>Flavan-3-ol</td><td>1.54<sup>-4</sup> ***</td></tr><tr><td>Procyanidin B1</td><td>0.254 ns</td></tr><tr><td rowspan="4">Flavan-3-ol</td><td>Flavan-3-ol</td><td>5.16<sup>-3</sup> **</td></tr><tr><td>Flavan-3-ol</td><td>9.08<sup>-3</sup> **</td></tr><tr><td>Catechin</td><td>8.95<sup>-4</sup> ***</td></tr><tr><td>Flavanone</td><td>only in red reaction</td></tr><tr><td rowspan="7">Flavanone</td><td>Flavanone</td><td>only in red reaction</td></tr><tr><td>Eriodictyol-7-glucoside</td><td>only in red reaction</td></tr><tr><td>Flavanone</td><td>only in red reaction</td></tr><tr><td>Naringenine-7-glucoside</td><td>only in red reaction</td></tr><tr><td>Flavanone</td><td>only in red reaction</td></tr><tr><td>cis-Neochlorogenic acid</td><td>9.19<sup>-3</sup> **</td></tr><tr><td>Neochlorogenic acid</td><td>0.012 ns</td></tr><tr><td rowspan="7">Hydroxycinnamic derivatives</td><td>Hydroxycinnamic derivative</td><td>only in red reaction</td></tr><tr><td>Chlorogenic acid</td><td>0.739 ns</td></tr><tr><td>cis-Chlorogenic acid</td><td>0.733 ns</td></tr><tr><td>Hydroxycinnamic derivative</td><td>0.437 ns</td></tr><tr><td>5-p-Coumaroylquinic acid</td><td>9.90<sup>-3</sup> **</td></tr><tr><td>3,5-Dicaffeoylquinic acid</td><td>0.334 ns</td></tr><tr><td>Hydroxycinnamic derivative</td><td>only in red reaction</td></tr><tr><td>Hydroxycinnamic derivative</td><td>only in red reaction</td></tr></tbody></table>

### The two major phenolic compounds

- Eriodictyol-7-glucoside = prunine

- Naringenin-7-glucoside

6 compounds present in greater proportion in red zones

9 compounds only in red zones

<figure><img src="image_46.png" /><figcaption>Eriodictyol-7-glucoside</figcaption></figure>

<figure><img src="image_47.png" /></figure>
RNAseq analyses

26 873 genes

<figure><img src="image_48.png" /></figure>

<figure><img src="image_49.png" /></figure>

Lambert et al. 2020. DiCoExpress: a tool to process multifactorial RNAseq experiments from quality controls to co-expression analysis through differential analysis based on contrasts inside GLM models. *Plant Methods*, 16:68-68
RNAseq analyses

<figure><img src="image_50.png" /><figcaption>Pathways activated after injury</figcaption></figure>

<figure><img src="image_51.png" /></figure>
RNAseq analyses

Co-expression
analysis

<figure><img src="image_52.png" /></figure>
INRAO

> CONCLUSIONS
Take home messages

* The inoculation done 7 hours after wounding resulted in *slowed and reduced brown rot* infection compared to inoculation done immediately after wounding

* 11 volatile compounds were associated with wounded fruits only

* phenolic and terpenoid compounds were less present in fruit with wounds

* We observed red reactions

* 9 phenolic compounds were present only in red zones
  2 major phenolic compounds : Eriodictyol-7-glucoside and Naringenin-7-glucoside

* RNAseq analyses highlighted pathways activated after injury

<figure><img src="image_53.png" /></figure>
Injury induces an activation of metabolic pathways involved in the susceptibility/resistance of peach to *M. laxa*

<figure><img src="image_54.png" /><figcaption>Wounding</figcaption></figure>

1h

<figure><img src="image_55.png" /></figure>

gene pathways
activated after injury

7h

<figure><img src="image_56.png" /><figcaption>cis-Jasmone</figcaption></figure>

Prupe_1G526000
"glutathione S-transferase tau 7"

<figure><img src="image_57.png" /></figure>

<figure><img src="image_58.png" /></figure>
Injury induces an activation of metabolic pathways involved in the susceptibility/resistance of peach to *M. laxa*

<figure><img src="image_59.png" /><figcaption>Wounding</figcaption></figure>

1h

<figure><img src="image_60.png" /></figure>

gene pathways
activated after injury

<figure><img src="image_61.png" /></figure>

7h

<figure><img src="image_62.png" /><figcaption>cis-Jasmone</figcaption></figure>

volatile compounds
gave off

<figure><img src="image_63.png" /><figcaption>Infection</figcaption></figure>

<figure><img src="image_64.png" /></figure>

phenolic
compounds

<figure><img src="image_65.png" /></figure>

Eriodictyol-7-glucoside
Naringenin-7-glucoside

<figure><img src="image_66.png" /></figure>
<figure><img src="image_67.png" /></figure>

<figure><img src="image_68.png" /></figure>

<figure><img src="image_69.png" /></figure>