# Preservation of Seed Microbiota: a challenge to maintain viability and function

Céline Mirguet, Isa Hollopp, Clara Maire, Géraldine Taghouti, Cécile Dutrieux, Perrine Portier

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Céline Mirguet, Isa Hollopp, Clara Maire, Géraldine Taghouti, Cécile Dutrieux, et al. Preservation of Seed Microbiota: a challenge to maintain viability and function. 17 th Plants-Bacteria Meeting, Jan 2025, Aussois (FR), France. 2025. [hal-05140475](https://hal.inrae.fr/hal-05140475v1)

HAL Id: hal-05140475

https://hal.inrae.fr/hal-05140475v1

Submitted on 2 Jul 2025

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# Preservation of Seed Microbiota : a challenge to maintain viability and function

Céline MIRGUET, Isa HOLLOPP, Clara MAIRE, Géraldine TAGHOUTI, Cécile DUTRIEUX, Perrine PORTIER

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

Univ Angers, Institut Agro, INRAE, IRHS, SFR QUASAV, CIRM-CFBP, F-49000 Angers, France

celine.mirguet@inrae.fr

## Heterogeneous Community

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

Plant-associated microbiota are complex heterogeneous communities which preservation will lead to heterogeneous survival, altering the taxonomic composition which in turn can alter the functionalities of the microbiota. The preservation processes mastered by Biological Resource Centers (BRC) are adapted to isolated strains but :

## Which procedures to minimize the preservation effects ?

To overcome these limitations, the MICROBE project (EU) aims at developing protocols and a framework to preserve viable microbiota. At CIRM-CFBP, we focus on the Seed Microbiota Preservation.

## Viabilities results

<figure><img src="image_3.png" /></figure>

The cultivable fraction is observed at T0, T+7 days and T+3 months.

We can conclude that after 3 months of preservation we can cultivate a great part of the microbiota diversity but the result depends of the condition.

Metabarcoding results will be necessary to indicate the real extend of the diversity we can culture after preservation.

## Metabolic profiling results

Biolog Ecoplate results - All conditions and all time for each metabolite, the fold change between before and after preservation is displayed.

We can observe metabolic functions which are not impacted (near 0 or positive fold change) or repressed (negative fold change).

## Mean of each conservation modality

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

### Lost capacities :

- α-Cyclodextrin

- M-Erythritol

- L-Threonine

- β-Phenylethylamine

### Variable loss of capacities :

- Glycogen

- α-D-Lactose

- D-Malic-Acid

- L-Phenylalanine

We can conclude that the condition C3: Trehalose 13,7 % (pink) seems to be the condition with which the less capacities are lost from the reference measures.

<table><thead><tr><th rowspan="3">Times</th><th colspan="2">T+7 days</th><th colspan="2">T+3 months</th></tr><tr><th>C3</th><th>C4</th><th>C3</th><th>C4</th></tr><tr><th>-80°C directly</th><th>Slow freezing</th><th>-80°C directly</th><th>Slow freezing</th></tr></thead><tbody><tr><td>Condition</td><td></td><td></td><td></td><td></td></tr><tr><td>Freezing</td><td></td><td></td><td></td><td></td></tr><tr><td>Methyl_Pyruvate</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Tween_40</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Tween_80</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>a-Cyclodextrin</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Glycogen</td><td>-</td><td>-</td><td>+/-</td><td>+/-</td></tr><tr><td>D-Cellobiose</td><td>-</td><td>-</td><td>+/-</td><td>+/-</td></tr><tr><td>a-D-Lactose</td><td>-</td><td>-</td><td>+/-</td><td>+/-</td></tr><tr><td>b-Methyl-D-Glucoside</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>D-Xylose</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>m-Erythritol</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>D-Mannitol</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>N-Acetyl-D-Glucosamine</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>D-Glucosaminic_Acid</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>a-D-Glucose-1-Phosphate</td><td>+</td><td>+</td><td>+/-</td><td>+/-</td></tr><tr><td>D,L-a-Glycerol-Phosphate</td><td>-</td><td>-</td><td>+/-</td><td>-</td></tr><tr><td>D-Galactonic_Acid-g-Lactone</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>D-Galacturonic_Acid</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>2-Hydroxy-Benzoic_Acid</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>4-Hydroxy-Benzoic_Acid</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>g-Hydroxy-Butyric_Acid</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Itaconic_Acid</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>a-Keto-Butyric_Acid</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>D-Malic_Acid</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>L-Arginine</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>L-Asparagine</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>L-Phenylalanine</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>L-Serine</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>L-Threonine</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Gly-Glu</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>b-Phenylethylamine</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Putrescine</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Total of active functions</td><td>19</td><td>19</td><td>24</td><td>23</td></tr></tbody></table>

Biolog Ecoplate results - Condition Trehalose 13,7% of 5 replicates, comparison between fast (C3) and slow (C4) freezing at 7 days and 3 months after preservation in liquid nitrogen.

<table><tr><td>+++</td><td>+</td></tr><tr><td>++-</td><td>+</td></tr><tr><td>+-</td><td>-</td></tr><tr><td>---</td><td>-</td></tr></table>

We can conclude that the preservation of metabolic functions with the Trehalose 13,7 % is not influenced by the method of freezing.

We can observe a variability of metabolic functions between the preservation at 7 days and 3 months. Some capacities appear at 3 months. This may be due to the disappearance of taxonomic diversity, activating capacities that were initially inhibited.

## Conclusion and Perspective

Based on the first results obtained, we can already guide the preservation of the seed microbiota so that it remains alive and functional. The use of Trehalose as a cryoprotectant, preserved in liquid nitrogen at -195°C, currently allows at least the preservation of a part of the taxonomic composition and most of the functional diversity for 3 months.

We need to analyse all the results at 1 year to confirm the first findings and conclude about the variability in the metabolic functions and if there is a relation between the taxonomic diversity and metabolic functions.

We need to obtain all metabarcoding's results to study all different correlation.

Next, we plan to investigate how to preserve and propagate SynComs for use by scientists for their experiments.

In this mixed SynCom, we will associate several of Bacteria, Fungi and Yeast in the same solution.

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

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

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

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

<figure><img src="image_9.png" /></figure>