# Strong elicitation of plant defense pathways by foliar and collar inoculations of wheat with the Bacillus pumilus strain JM79

Aline Ballot, Matthieu Gaucher, Marjolaine Rey, Marie-Noelle Brisset, Pierre

Joly, Assia Dreux-Zigha, Ahmed Taïbi, Thierry Langin, Claire

Prigent-Combaret

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Aline Ballot, Matthieu Gaucher, Marjolaine Rey, Marie-Noelle Brisset, Pierre Joly, et al. Strong elicitation of plant defense pathways by foliar and collar inoculations of wheat with the Bacillus pumilus strain JM79. Current Plant Biology, 2025, 43 (100524), 10.1016/j.cpb.2025.100524. hal-05311520

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# Strong elicitation of plant defense pathways by foliar and collar inoculations of wheat with the *Bacillus pumilus* strain JM79

Aline Ballot<sup>a,1</sup>, Matthieu Gaucher<sup>b</sup>, Marjolaine Rey<sup>a</sup>, Marie-Noelle Brisset<sup>b</sup>, Pierre Joly<sup>c,2</sup>, Assia Dreux-Zigha<sup>c</sup>, Ahmed Taïbi<sup>c</sup>, Thierry Langin<sup>d</sup>, Claire Prigent-Combaret<sup>a,* ID</sup>

## ARTICLE INFO

### Keywords:

Plant defense responses

Induced Systemic Resistance

Pumilacidin

*Bacillus pumilus*

*Fusarium graminearum*

Wheat

## ABSTRACT

The ability of the *Bacillus pumilus* JM79 strain to induce systemic resistance in wheat against *Fusarium graminearum* (Fg), a major wheat pathogen, was investigated using the Fusarium crown rot (FCR) pathosystem. The *B. pumilus* strain JM79 exhibited the ability to colonize both root and leaf surfaces while secreting surfactin-like pumilacidin in the root zone of wheat plantlets. Experiments involving foliar inoculation with JM79 revealed its ability to induce a strong local defense response in wheat, characterized by the selective overexpression of genes associated with phenylpropanoid metabolism and cell wall reinforcement pathways. Moreover, pre-inoculation of the wheat collar, at the soil surface interface, with the JM79 strain prior to Fg inoculation led to the over-expression of wheat genes linked to both jasmonic acid/ethylene (JA/ET) and salicylic acid (SA)-dependent defense pathways. This direct induction occurred during the asymptomatic phase of Fg infection, compensating for the lack or absence of an early immune response triggered by Fg infection. Collectively, these findings reveal for the first time that the *B. pumilus* strain JM79 produces a higher proportion of long-chain pumilacidins under *in planta* conditions than under *in vitro* conditions, and is capable of activating both local and systemic resistance in wheat plants, underscoring its potential as a biocontrol agent against major wheat fungal diseases.

## 1. Introduction

Wheat is a host target for various fungal pathogens, including those belonging to the *Fusarium* genus, which cause three of the most important diseases of wheat and other small grains worldwide: Fusarium head blight (FHB), Fusarium crown rot (FCR), and Fusarium root rot (FRR). While FHB primarily affects the wheat head, FCR mainly impacts the stem base and crown, resulting in necrotic lesions in these tissues, and FRR affects the root system, reducing nutrient and water uptake and leading to stunted growth and significant yield losses in affected crops [53,70,77]. Although FHB has been extensively studied due to its global significance, *Fusarium* infections affecting underground plant tissues (FCR and FRR) have garnered comparatively less interest [69]. *Fusarium* diseases in underground organs are typically associated with infections

of seedling stem bases by *F. culmorum*, *F. pseudograminearum*, or *F. graminearum* (Fg). These pathogens, which include a saprophytic phase in their life cycle, are integral members of the rhizosphere community, and their prevalence is rising due to intensive agricultural practices and climate changes [37]. Among these, *F. graminearum* is a hemibiotrophic fungal pathogen that adopts different lifestyles depending on the tissue it colonizes, complicating the study of this host-pathogen interaction.

In conventional agriculture, fungicides have been extensively used to control many fungal plant pathogens. However, given the urgent need to reduce pesticide use—a concern prominently highlighted in global policy agendas and supported by public opinion—alternative strategies for fungal infection control are imperative [21]. Alongside breeding resistant wheat genotypes, an alternative strategy involves reinforcing

<footnote><sup>a</sup> Université Claude Bernard Lyon 1, Laboratoire d'Ecologie Microbienne, UMR CNRS 5557, UMR INRAE 1418, VetAgro Sup, Villeurbanne 69622, France</footnote>

<footnote><sup>b</sup> IRHS-UMR1345, Université d'Angers, INRAE, Institut Agro, SFR 4207 QuaSaV, 42 rue Georges Morel, Beaucouzé cedex F-49071, France</footnote>

<footnote><sup>c</sup> Greencell, Saint-Beauzire, France</footnote>

<footnote><sup>d</sup> Université Clermont Auvergne, INRAE, GDEC, Clermont-Ferrand, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: claire.prigent-combaret@univ-lyon1.fr (C. Prigent-Combaret).</footnote>

<footnote n="1">¹ Present address: Department of Microbiology and Ecosystem Science, Centre for Microbiology and Environmental Systems Science, University of Vienna, Vienna, Austria</footnote>

<footnote n="2">² Vivagro, Technopole Montesquieu, 5 allée Jacques Latrille, 33650 Martillac</footnote>
plant natural resistance through plant-defense stimulators (PDS, also known as exogenous elicitors) [43]. The goal of PDS application is to compensate for the lack of early or complete pest perception in susceptible host plants by inducing temporary activation of their defense mechanisms. Some PDSs directly stimulate plant defense (a direct effect), while others “prime” the plant to activate its defenses more effectively upon subsequent infection [49]. PDSs offer several advantages: unlike pesticides, most PDSs lack a direct biocidal effect on pests or the broader ecosystem (flora and fauna), thereby minimizing adverse impacts on non-target organisms and the environment, as well as reducing the development of pathogen resistance. Instead, they trigger a wide range of plant defenses, making them effective against multiple pests on the same host. This indirect and multi-faceted mode of action also reduces the likelihood of pests developing resistance.

Plant growth-promoting rhizobacteria (PGPR) are a diverse group of microorganisms that reside in the rhizosphere, either by colonizing the root surface or entering within plant tissues, thereby establishing beneficial interactions with plants. PGPRs have been shown to enhance plant growth and health through diverse co-occurring mechanisms, such as promoting nutrient uptake, modulating plant hormonal pathways, producing antimicrobial compounds against pathogens, or stimulating induced systemic resistance (ISR) [4,33,34,58]. In the context of wheat diseases, some PGPR strains have been used as PDS [48] [68]. *Bacillus* species, including *Bacillus pumilus*, are among the most studied PGPRs due to their ability to produce antifungal compounds, induce ISR, and provide biocontrol against a variety of plant pathogens [10,15,32,41,42, 76]. However, the molecular mechanisms by which these strains trigger plant protection remain incompletely understood [34].

Insights into the defense-related events in plant cells following PDS perception often reveal the activation of plant defense genes linked to jasmonic acid (JA) and ethylene (ET) signaling pathways, resulting in ISR. Specific PGPR components responsible for ISR induction include lipopeptides (LPs), lipopolysaccharides, siderophores, polyketides such as 2,4-diacetylphoroglucinol, and volatile compounds like 2,3-butadiol [36,50,57,79]. Recent studies indicate that both salicylate- and jasmonate-dependent defense pathways can be activated simultaneously, suggesting dual activation of the SA-dependent systemic acquired resistance (SAR) pathway and the ISR pathway [75].

One key compound produced by *B. pumilus* strains is pumilacidin, a surfactin-like cyclic lipopeptide. Few studies have reported its antimicrobial properties [13] but its role in direct antagonism against fungal pathogens or in triggering plant defense against fungal infection has been quite poorly investigated [58]. Yet, in a previous study, the *B. pumilus* JM79 strain demonstrated plant protection properties against crown rot (CR) caused by the necrotrophic soil-borne fungal pathogen *F. graminearum* strain *Fg1* when applied to seeds [7]. Interestingly, JM79 did not exhibit a strong direct antagonistic effect on fungal growth under *in vitro* conditions, suggesting that its biocontrol mechanism might be linked to ISR induction in plant tissues [7].

The primary objective of the present study was thus to investigate the ISR-inducing capacity of JM79 against *F. graminearum*, a major cause of FCR, and to elucidate the plant defense pathways triggered by this strain. Additionally, we aimed to determine if this *B. pumilus* strain produces pumilacidins when it colonizes wheat roots, and if their structural forms differ depending on the environmental conditions (i.e. planktonic versus biofilm; *in vitro* versus *in planta*), an aspect that has received little attention to date. To achieve this, comparative genomics combined with chromatography and mass spectrometry was employed to characterize the LPs secreted by JM79 during wheat colonization. Subsequently, qRT-PCR analysis was performed to assess the expression levels of 29 plant defense-related genes in wheat inoculated with JM79 in two different ways: either on wheat leaves (in the absence of the pathogen) or on the collar near the soil surface interface prior to pathogen inoculation during FCR disease assays. This work provides original evidence that JM79 can produce specific pumilacidins with long carbon chains under *in planta* conditions, and activate both local and systemic

resistance in wheat. These findings reveal promising strategies for overcoming *Fusarium* diseases, offering new mechanistic insights into how *Fg* suppresses plant immune response during the asymptomatic phase of its life cycle. This knowledge could facilitate the development of novel PGPR-based biocontrol products to combat this important cereal pathogen.

## 2. Material and methods

### 2.1. Evaluation of ecological and metabolic features of *Bacillus* JM79

#### 2.1.1. Bacterial and fungal strains and growth conditions

The *B. pumilus* JM79 strain, originally isolated from the wheat rhizosphere and selected for its protective effects against *Fusarium graminearum* CR [7], was utilized in this study. The strain was stored at -80°C in 20% glycerol and routinely cultured on Tryptic Soy media (Broth or Agar, i.e., TSB or TSA) (Tryptone Soya, Carl Roth, Karlsruhe, Germany) at 28°C for 48 h (h). The virulent *Fusarium graminearum* *Fg1* strain [2,19] used in the *in planta* experiments was routinely cultured on Potato Dextrose Agar (PDA, Conda Pronadisa, Madrid, Spain) at 20°C in the dark for 8 days and subsequently stored at 4°C until further use.

#### 2.1.2. *gfp* tagging of JM79 by bacterial transformation under nitrogen-limiting conditions

The transformation of the JM79 strain was performed using competent cells prepared following a protocol adapted from Jarmer et al. [28], exploiting the natural competence of *Bacillus* cells under nitrogen-limiting conditions. A single colony of JM79 was cultured in 10 mL of LB medium at 37°C under shaking conditions for 4 h. A 2 mL aliquot of culture was centrifuged at 5000 × g for 5 min (min), washed once with 0.1% peptone water, and recentrifuged. The cells were then suspended to an OD₆₀₀ nm of 0.01 in 1 mL of glucose minimal medium. Subsequently, 1 µg of pHAPII, a pUBC19 plasmid containing the *gfp* gene fused to the *HapII* promoter [11], was added to the competent cells. Following 24 h of growth at 30°C, the bacteria were plated on LB agar supplemented with 20 µg.mL⁻¹ kanamycin and incubated overnight at 28°C. Compared to the WT strain, no phenotypic change was observed in the transformed strain.

#### 2.1.3. Growth of JM79 on wheat plantlets under gnotobiotic conditions

In order to evaluate the ability of JM79 to colonize wheat plantlets, *in vitro* plant assays were conducted using the 'Recital' French bread wheat cultivar. Seeds were first surface-disinfected following a previously described protocol [26] and germinated on plant agar plates (8 g. L⁻¹), with the embryo facing upward. Plates were incubated in the dark at 27°C for 48 h at 75% relative humidity. To verify seed surface-sterility, 250 µL of the final wash was spread on TSA plates, which were subsequently incubated at 28°C. Pre-germinated wheat seeds were aseptically placed, with the embryo facing upward, in 20 cm-high glass tubes (2 cm in diameter) containing 30 mL of 2 g.L⁻¹ plant agar. For root colonization experiments, a JM79-*gfp* inoculum (JM79 strain carrying the pHAPII plasmid conferring kanamycin resistance (KmR)) was prepared from TSB cultures grown with Km for 24 h. Cells were collected by centrifugation, washed gently, and resuspended in sterile MgSO₄ (10 mM) to a final concentration of 10⁸ cells.mL⁻¹. Bacterial inoculum (100 µL; 10⁷ cells per seed) was applied to each pre-germinated seed. A minimum of four plants were inoculated per treatment, sampling time point, and experiment type (microscopy, enumeration of epiphyte cells, enumeration of endophyte cells). Tubes were sealed with sterile cotton and incubated for 7 days in a growth chamber set at 21°C, with a 16/8 h day/night cycle and 60% relative humidity.

#### 2.1.4. Epifluorescence and confocal microscopic for observing epiphyte colonization of wheat tissues by JM79 strain

To analyse the distribution of the JM79 strain on wheat plantlets,
fresh shoots and roots of wheat plants grown under gnotobiotic systems were collected at 3 and 7 days post-inoculation with JM79-gfp. Segments of shoots and roots (1–2 cm) were sampled from at least two independent seedlings from two separate inoculation experiments. For roots, samples were collected along the entire length of the root system, from the seed to the apical root zone, including in the hair root zone. Samples were mounted in Aqua-Poly/Mount (Polysciences, Eppelheim, Germany). Bacterial colonization along the roots was visualized using an Axioskop 40 epifluorescence microscope (Carl Zeiss, Le Pecq, France) with the appropriate filter set for GFP observation (BP 450–490 nm for excitation and LP 515 nm for emission). Confocal microscopic analysis was performed using an LSM 800 confocal microscope (Carl Zeiss) equipped with argon–krypton and He-Ne lasers (excitation at 488 nm, detection between 504 and 555 nm). GFP fluorescent images were overlaid with bright field transmitted light image using LSM software release 3.5 (Carl Zeiss). Ten images per region of interest and per plant were captured and analyzed.

### 2.1.5. Analysis of bacterial epiphyte and endophyte root colonization by CFU quantification

To determine the external and/or internal colonization of wheat roots by *B. pumilus* JM79, cell counting was performed on surface and interior root tissues of wheat cultivar 'Recital' plants grown for 7 days under gnotobiotic conditions. Surface root colonization was quantified by vortexing 2 cm root sections at maximum speed for 5 min in 5 mL of 10 mM MgSO₄ containing two metal beads to detach bacteria from the root surface.

For endophytic colonization, 7 days old root systems were subjected to a surface sterilisation process. Root systems were transferred to a 50 mL-tube containing 40 mL of a disinfecting solution (1 % chloramine T with 3 drops of Tween 20 for 100 mL), and mixed by inversion for 15 min. The disinfecting solution was discarded, and roots were rinsed for 5 min with 40 mL of 0.85 % NaCl. A second sterilization step was performed by dipping roots in 70 % ethanol for 60 s (s), followed by four rinses with 0.85 % NaCl. To confirm the effectiveness of the surface-sterilization process, 150 µL of the last NaCl rinse and a fragment of the surface-sterilized root were plated onto LB medium to check the absence of microbial growth.

The sterilized root systems (from at least 3 seedlings) were crushed in 5 mL of 10 mM MgSO₄ using a sterile mortar and pestle. The resulting suspension was transferred to 15 mL tubes, and root debris were removed by centrifugation at 2000 rpm. Bacterial suspensions from surface washed or crushed roots were centrifuged at 6500 rpm and resuspended in 1 mL of 10 mM MgSO₄, and serially diluted from 10⁻¹ to 10⁻⁷. Aliquots of the dilutions were plated onto TSA medium and incubated at 30°C for 3 days before colony-forming unit (CFU) counting.

### 2.1.6. Metabolites extraction of JM79 and pumilacidins quantification in various media and wheat rhizosphere

To evaluate whether the production of potentially active compounds, like pumilacidins, differs according to growth conditions, bacterial cultures for metabolite extraction were grown in Root Exudates (RE) or TS medium under both planktonic and biofilm colony conditions. The RE medium was prepared as described by Nihorimbere et al. [55]. For solid media, 12 g.L⁻¹ agar (European biological agar, Pronadisa, Conda, Madrid, Spain) was added. Experiments were conducted in 24-well cell culture (Cellstar®, Greiner Bio-One, France) using 1 mL cultures inoculated with 5.10⁶ CFU.mL⁻¹ of JM79 preculture. Plates were incubated at 28°C for 4 days with agitation 120 rpm agitation. Uninoculated media (broth and agar) served as controls. All treatments were performed in four independent replicates. Metabolites were extracted as described by Rieusset et al. [63], yielding 50 µL of crude methanol extract. Twenty microliters of each extract were analyzed via ultra-high-pressure liquid chromatography coupled with mass spectrometry (UHPLC-MS). The extraction efficiency for planktonic and biofilm samples was verified by quantifying a tryptophan internal standard in both RE and TS broth and

agar media, showing no significant differences (as already confirmed by Rieusset et al. [63]).

To evaluate in planta metabolites production by JM79, 20 mL of 2 % agar plant medium, from 7 days old JM79-inoculated wheat plantlets grown under gnotobiotic conditions were subjected to metabolite extraction. After removing the roots, two successive liquid/liquid partitions were performed using 20 mL of ethyl acetate. The agar plant medium was vortexed for 30 s, sonicated for 10 min, allowed to decant for 20 min, and the supernatant was recovered. The final volume of 40 mL of the ethyl acetate extraction organic phase was dried in 50 mL falcon tubes using a SpeedVac (Centrivap Cold Trap Concentrator LABCONCO). Agar samples from uninoculated wheat plantlets served as controls. All treatments consisted of four independent replicates. Dried extracts were also suspended in 50 µL of methanol and transferred to vials for spectrometry analysis. Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) analysis of the various specialized metabolites extracts was conducted as described by Rieusset et al. [63], using positive mode mass analysis.

The antifungal activity of JM79 secondary metabolite extracts was evaluated by performing spore germination and/or mycelium growth assays on *Fg1* spores as previously described in Besset-Manzoni et al. [7]. Methanol extracts containing JM79 secondary metabolites were added to wells of a 96-well microtiter plate, then dried before adding media and fungal inoculum (and using methanol as negative control). The microtiter plate was then incubated for 5 days at 28 °C and analyzed using an Infinite M200 Pro microplate reader (TECAN, Switzerland). The turbidimetry was measured at 492 nm (data not shown).

### 2.1.7. MS/MS analysis for structure elucidation of pumilacidins

To determine the specific structure of *m/z* parent ion masses corresponding to either pumilacidin or surfactin in both RE and wheat cultures of JM79, one sample per condition (equivalent to a 6 µL mixture pooled from the four replicates) was analyzed with tandem mass spectrometry (MS/MS). MS/MS analyses were performed by collision-induced dissociation (CID), with a collision energy of 40 eV. Chromatograms were processed using MassHunter Qualitative Analysis B.07.00 software (Agilent Technologies).

### 2.2. Analysis of the genomic determinants of Bacillus JM79

To explore the genomic features potentially relevant to rhizosphere competence and plant-microbe interactions, including the potential ability of JM79 to produce pumilacidin, the genome of the *B. pumilus* JM79 strain was sequenced. Genomic DNA was extracted from an overnight JM79 culture using the NucleoSpin Microbial DNA kit (Ref - 740235.50, Macherey-Nagel, Hoerdt, France). Sequencing was performed at Mr DNA (Shallowater; USA) using MiSeq Illumina technology to generate a 2 × 300 bp paired-end library. The sequences were trimmed and assembled *de novo* using assembling using the NGen DNA assembly software by DNASTar, Inc. The draft genome was deposited in the European Nucleotide Archive under BioProject ID PRJEB80234 with accession number GCA_964264945. Genome annotation was conducted using the MicroScope platform [74]. The final draft genome consisted of 32 contigs, with a maximum contig length of 0.837 Mbp, a total genome size of 3.82 Mbp, and an average GC content of 41.33 %. Comparative genomic analysis was performed using MicroScope gene families (MICFAM) with parameters of 80 % amino acid identity and 80 % alignment coverage.

Specialized metabolite biosynthetic gene clusters (BGCs) were identified using the **Antibiotics and Secondary Metabolite Analysis Shell** (antiSMASH) software [8]. Predictions were cross-referenced with the MIBIG database to identify known compounds, and cluster completeness was verified manually. SignalP v5.0 software [1] was used to predict secretory proteins containing signal peptides, while Localizer v1.0.4 [71] was employed to predict protein subcellular localization within the plant host.
## 2.3. Determination of defense gene induction in wheat

### 2.3.1. Plant and fungal materials

Experiments were conducted on the FCR-susceptible French winter wheat cv. 'Recital'. Root and leaf inoculation experiments were carried out independently. For both inoculation experiments, seeds were sown in 7 × 7 cm plastic pots (1 plant per pot) filled with seedling soil (Humustar soil, NPK 14–16–18, SARL Activert, Riom, France). Wheat plants were grown under greenhouse semi-controlled conditions with a natural photoperiod, supplemented with artificial light if necessary, and maintained at 20 °C ± 5 depending on sunlight intensity. Plants were watered every three days to maintain soil moisture at 40 % of Water Holding Capacity. The *F. graminearum* strain *Fg1* was used for plant inoculation during root inoculation trials. Spore fungal cultures were prepared as described previously [5].

### 2.3.2. Wheat leaf bacterial inoculation experiment

The bacterial inoculum was prepared from *B. pumilus* JM79 grown in TSB medium for 24 h. The suspension was adjusted to 5.10⁶ cells·mL⁻¹ in 10 mM MgSO₄ and Tween 0.05 %. At the 3–4 leaf stage (15 days post-sowing), the last fully developed leaves were sprayed to runoff using a micro-diffuser sprayer (Ecospray) with either 10 mM MgSO₄ and Tween 0.05 % as negative control, methyl jasmonate (MeJA, 6 mM in 3 % ethanol and Tween 0.05 %) as positive control or JM79 inoculum (about 2.10⁶ cells/plant) (Fig. S1A). No corresponding negative control for the MeJA treatment (i.e., 3 % ethanol and Tween 0.05 %) was included because we observed in a previous experiment that ethanol 3 % had a low impact on the response of defense genes in wheat (Fig. S10). Leaves from four independent plants per condition were harvested 3 days post-inoculation, immediately frozen in liquid nitrogen, and stored at –80 °C for RNA extraction.

### 2.3.3. Wheat collar bacterial inoculation experiment

The bacterial inoculum was prepared as described for leaf inoculation experiment. In this case, JM79 cells adjusted at 2.10⁶ cells·mL⁻¹ were applied to the wheat collar, near the soil surface, at the one-leaf stage (6-day post sowing) (Fig. S1B). Negative controls were treated with 10 mM MgSO₄. After 3 days, *Fg1* spores (5.10⁵ spores/plantlet) were inoculated on the collars. Plants treated only with *Fg1* spore suspension served as positive controls and un-inoculated plants were used as negative controls. The last fully developed leaves were collected at 3, 6, 9 and 13 days post-bacterial inoculation from four independent plantlets per condition (e.g., resulting in a total of 56 samples). They were immediately frozen in liquid nitrogen for further analysis.

### 2.3.4. RNA extraction and quantification of gene expression by quantitative RT-PCR

To prevent RNA degradation, steel balls (3 mm) were treated with acetone for 2 h, dried, and sterilized by heating at 180°C for 1 h. Two RNase-free balls (Tungsten carbide grinding balls, Retsch, Luxembourg) were added to each sample tube, and samples were flash-frozen in liquid nitrogen before storage at –80°C. Total RNA was extracted from 100 mg of plant tissue using NucleoSpin RNA Plant kit (Macherey-Nagel, Düren, Germany) as described by Le Mire et al. [35]. Reverse transcription was performed using the M-MLV system (Promega Corp.). The absence of genomic DNA contamination was confirmed on cDNA by PCR, using elongation factor α primers flanking an intron, as described by Gilliland et al. [24]. Quantitative RT-PCR (qRT-PCR) was performed to analyze 29 genes involved in pathogenesis-related (PR) proteins, oxidative stress, secondary metabolism, cell-wall reinforcement and defense signaling pathways (e.g., salicylic acid, jasmonic acid, and ethylene). The methods described by Brisset & Bernonville [9] and Gaucher et al. [23] were followed, using n = 4 independent samples per condition per time point. Gene expression changes (log₂ ratios) were calculated using the 2⁻⁰⁻⁰ΔCt method [65] with three internal reference genes (e.g., Tubulin B, GAPDH, and actin) for normalization.

Changes in gene expression were calculated relative to the mean values of control samples at each time point. More precisely, the mean log₂ expression level for each gene in the control replicates was calculated and subtracted from the log₂-transformed value for each replicate across all conditions, including the control replicates themselves. This transformation emphasizes deviations from the control baseline, allowing for a more direct comparison of effects of treatments. Because 3 % ethanol and 0.05 % Tween (co-applied with MeJA) had minimal effects on defense gene expression in wheat cv. 'Sacramento' in a preliminary experiment (i.e., -1 < Δ log2 ratio < 1 for 21 out of 27 genes, with the exception of PAL, FNS, GST, CSL, PR4, and PR5) (Fig. S10), all data were calibrated with the same control: MgSO₄ + 0.05 % Tween. A log₂ fold-change threshold of 1 was selected, as all statistically significant differences in gene expression between the 'Recital' wheat cultivar treatments and the control condition were found to be equal to or greater than this value. While this calibration approach may introduce a slight bias for genes responsive to ethanol and Tween, the magnitude of this effect was assessed beforehand and is taken into account in the interpretation of the results. ANOVA followed by Tukey's Honest Significant Difference (HSD) test or the non-parametric Kruskal Wallis test (when needed) were used to compare more than two treatments.

## 2.4. Statistical methods

Data were analyzed using R Studio (v.4.2.1). The normality of data distribution and homogeneity of variance were tested using Shapiro-Wilk and Bartlett tests, respectively. For data meeting these assumptions, differences between groups were assessed using Student's t-test (for two groups) or ANOVA followed by Tukey post-hoc (*P* < 0.05) test (for more than two groups). When the assumptions of normality and variance homogeneity were not met, the non-parametric Kruskal-Wallis test with Bonferroni correction method was applied (*P* < 0.05).

# 3. Results

## 3.1. JM79 is a *Bacillus pumilus* strain that colonize wheat roots as an epiphyte and that partially shows endophyte colonization

The *B. pumilus* strain JM79 was selected for this study due to its previously demonstrated ability to protect wheat against FCR [7]. When inoculated onto seeds at a concentration of 10⁷ cells per plant, JM79 significantly improved various growth and yield parameters in *F. graminearum*-infected wheat plants. These included chlorophyll content, shoot dry weight, ear number, ear weight, strand length, and reduced symptoms severity [7].

To better understand the characteristics of this biocontrol agent, its genome was sequenced and analyzed (Fig. S2). The assembled genome of JM79 was compared to the publicly available *B. pumilus* genome in the MaGe genome database. A phylogenetic analysis of *B. pumilus* genome sequences was conducted, confirming that the JM79 strain belongs to the *B. pumilus* species (Fig. S3).

To evaluate the ability of JM79 to colonize wheat roots and form biofilms, we analyzed its interaction with the wheat roots system 7 days post-inoculation. Seeds of the wheat cv, 'Recital' were inoculated with JM79 at a concentration of 1.10⁷ cells per seed. Observations using epifluorescence and confocal laser scanning microscopy revealed that JM79 successfully colonized the wheat root system, extending from the seed zone to the apex. Colonization was also observed on the leaf surface (Fig. 1A-B). JM79 formed small aggregates on lateral root surfaces, and thicker aggregates in the seed and apical root zones, indicating robust colonization of the entire wheat root system. The presence of JM79 cells in foliar parts suggested both maintenance and/or colonization of this ecological niche after seed inoculation. CFU counting of JM79 on the root surface confirmed the microscopic observations, with colonization levels reaching up to 7.70 × 10⁸ CFU per g of fresh root (corresponding to 1.23 × 10⁸ CFU per root system, with an inoculum of 10⁷ cells per
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Laser confocal microscopy and epifluorescence images (A-B), and quantitative analysis (C) of root and shoot colonization by *B. pumilus* JM79 on wheat genotype 'Recital', 7 days post bacterial inoculation (7 dpi). (A) Photographs of seed, lateral and apical root zones were taken using a confocal laser scanning microscope with an excitation of 488 nm and an emission filter between 504 and 555 nm. (B) Photographs of leaves were done using epifluorescence (left) or confocal (right) microscopy. Cells expressing *gfp* are green and grey backgrounds correspond to root or shoot views observed with transmitted light. Scale bars measure 100 µm for epifluorescence pictures (B-left) or 20 µm for confocal laser scanning pictures (A and B-right). (C) Quantification of the number of JM79 cells (in log10 CFU·g⁻¹ fresh tissue) recovered from the surface of root and shoot tissues [mean ± SE, n = 10] such as inside root tissues [mean ± SE, n = 13].</figcaption></figure>

plant) (Fig. 1C). JM79 cells enumeration after surface disinfection of wheat root tissues revealed an endophytic population reaching a density of approximately 10³ CFU per g fresh roots, indicating that JM79 is also able to colonize internal wheat root tissues, albeit at a lower level (Fig. 1C).

## 3.2. *In silico* prediction of secretome, extracellular enzymes and metabolic activities of JM79 reveals enzymatic activities that are common to plant endophytes

A general view of the genomic structure of the *B. pumilus* JM79 chromosome is depicted in Fig. S2. The genome of 3825,136 pb is predicted to encode 4251 coding sequences (CDSs), including 12 fragmented coding sequences (fCDSs). Putative biological functions were assigned to 4100 CDS after manual curation, and 241 (7.15 %) of these were conserved hypothetical proteins. The functional classification assigned 3413 CDS (82.20 %) in different COG (cluster of orthologs groups) functional groups (Fig. S4). This analysis revealed that, apart from essential housekeeping gene functions, the genome appears to be biased towards five major functional CDS subclasses: amino acid metabolism (E), carbohydrate metabolism (G), inorganic ion (P) transport

and metabolism, along with CDS involved in defense mechanisms (V) and cell motility (N), representing 20 % of total CDS. The high proportion of these genes in the genome is indicative of the inherent potential of JM79 to be a rhizosphere-competent strain.

In the *B. pumilus* JM79 genome, 251 out of 4100 genes were predicted to encode proteins from the secretory pathways (Table S1, Fig. 2). Among these 251 genes, 140 (56 %) had Sec-dependent signal peptide cleaved by a type I signal peptidase, 106 (42 %) were identified as lipoproteins consisting of SPII/Lsp, 2 contained a TAT motif (twin-arginine translocation), and 3 a PILIN motif for pilin proteins secretion. Eighty-eight genes (35 % of the potential secreted proteins) were found to encode enzymes (15 hydrolases, 27 proteases, 5 nucleases, and 48 with other activities) (Fig. 2). COG classification revealed that 50 % of the predicted secreted proteins were not classified, 28 (11.2 %) were involved in cell wall/membrane/envelope biogenesis, and 22 (8.8 %) in inorganic ion transport and metabolism. In accordance with its ecological niche, the secretome of JM79 comprises various enzymes assigned to carbohydrate transport and metabolism (19 CDS, 7.6 %) and to amino acid transport and metabolism (23 CDS, 9.2 %) of polysaccharides, proteins and other compounds available in the rhizosphere. Part of this enzymatic repertoire may be used by JM79 to colonize the rhizoplane
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** *In silico* functional classification of JM79 genes encoding proteins from its predicted secretome. Distribution of the 251 JM79 genes encoding proteins predicted to be secreted, according to COG functional classes. Genes that did not have any inferred COG annotation were assigned to category S.</figcaption></figure>

and potentially the internal root tissues. Half of the genes with SPI (Signal Peptidase I) peptide signals encode enzymes (Fig. 2). Some are involved in polysaccharide degradation, including *xynA* and *xynC* encoding endoxylanases (GH11 and GH5, respectively), *xynD* encoding an arabinofuranohydrolase (glycoside hydrolase family GH43), *bglS* encoding a lichenase (GH16), and *eglS* encoding an endo-glucanase/cellulase (GH5). Members of the polysaccharide lyase family were also identified including a pectate lyase (*pelA*), a putative pectin lyase (PBJM79_v1_40256) and an endo-beta-1,4-galactanase (BPJM79_v1_40140) which may be involved in the degradation of plant material. Further investigation by sequence feature analysis of CDSs predicted to encode secreted proteins revealed that endo-β-1,4-xylanase encoded by *xynA* possesses a predicted chloroplast targeting signal peptide (i.e., with the sequence AYA-KT in position 48) [67]. The *yfkN* gene encoding a 2',3'-cyclic-nucleotide 2'-phosphodiesterase (CNPase) (BPJM79_v1_210018) possesses a predicted plant-nucleus targeting signal (KKPSVVDRKKNR, Table S1).

**3.3. Specialised metabolites, siderophores and antibiotics biosynthesis pathways prediction**

JM79 genome harbors 12 biosynthetic gene clusters (BGC) predicted to be involved in the biosynthesis of several specialised metabolites (Table. S2). Among them, clusters 5, 7 and 9 show high similarity score with well-characterized BGCs of *B. velezensis* FZB42 involved in the production of bacillibactin iron-siderophore (Fig. S5C), bacilysin-dipeptide antibiotic (Fig. S5B) and lichenysin surfactin-like cyclo-lipopetide (Fig. S5A), respectively.

The *srfA* operon of JM79 (cluster 9, Fig. S5A) includes the three genes *srfAA*, *srfAB*, and *srfAC*, encoding the surfactin synthase subunits, and *srfAD*, encoding a surfactin synthase thioesterase subunit. Similar to other *B. pumilus* or *B. safensis* pumilacidin-producing strains [18,64], JM79 *srf* cluster contains two still-uncharacterized genes, *orfX* and *orfY* which are absent in surfactin- or lychenisin-producing strains (Fig. S5A). In JM79, as in other lychenysin- or pumilacidin-producing bacteria, the *yxc* operon comprises only two genes, *ycxC* and *ycxD* while homologs of

*ycxAB* genes from *B. subtilis* and *B. amyloliquefaciens* are absent. Additionally, an insertion element is present between the *srfAD* and *ycxC* genes in JM79. This insertion element is absent in the *B. pumilus* ATCC 7061 reference strain while it contains five genes that were previously described in some *B. safensis* [18] and *B. pumilus* strains [64]. This cluster analysis suggests that JM79 may produce pumilacidin rather than lichenysin, as predicted by antiSMASH. The production of such lipopeptide is known to facilitate adherence to roots and swarming [29, 57]. Pumilacidins are also known to be highly effective ISR inducers in plants [20]. Further analyses were then performed to confirm that JM79 can produce pumilacidins.

**3.4. JM79 pumilacidin lipopeptide production pattern differs depending on growth media and life style**

To assess the ability of JM79 to produce pumilacidin and confirm that pumilacidin is produced when JM79 interacts with wheat, a metabolomic analysis was performed via ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-qTOF MS). In addition, secondary metabolite profiles were compared between different growing conditions: growth in liquid media in a planktonic state (TSB and RE) *versus* in biofilm-colony conditions (TSA and REA), and in complex rich TSB/TSA medium *versus* in an exudate-mimicking medium RE medium (RE and REA) [3].

Based on the accurate mass determined via UPLC-qTOF MS, the presence of surfactin-like molecules was identified in JM79 supernatants from all four conditions. By comparing the *m/z* values with the mass numbers reported for the pumilacidins commonly produced by *B. pumilus* strains, signals at *m/z* 1008.6, 1022.6, 1036.6, 1050.7, 1064.7 and 1078.7 were detected in all four different extracts and were hypothetically attributed to the hydrogen adducts of six pumilacidin isoforms whose structural differences and ion MS profiles are indicated in Fig. 3A, and Fig. S6, respectively. Pumilacidins are lipopeptide-type biosurfactants, mainly produced by *B. pumilus* species, having a cyclic heptapeptide moiety of the sequence Gln-Leu-Leu-Leu-Asp-Leu-(Leu/Ile/Val) and a beta-hydroxy fatty acid chain length of 13–18 carbons,
<table><thead><tr><th rowspan="2">Pumilacidin lipopeptide</th><th rowspan="2">[M+H]+<br>(m/z)</th><th rowspan="2">[M+Na]+<br>(m/z)</th><th colspan="6">Peak RT (min)</th></tr><tr><th>9.10</th><th>9.16</th><th>9.26</th><th>-</th><th>-</th><th>-</th></tr></thead><tbody><tr><td>C13 [Val7]</td><td>1008.67</td><td>1030.6</td><td>9.33</td><td>9.42</td><td>9.49</td><td>9.78</td><td>-</td><td>-</td></tr><tr><td>C14 [Val7], C13 [Leu/Ile7]</td><td>1022.68</td><td>1044.7</td><td>9.38</td><td>9.44</td><td>9.54</td><td>9.58</td><td>9.70</td><td>9.81</td></tr><tr><td>C15 [Val7] (pumilacidin B)<sup>a</sup>, C14 [Leu/Ile7]</td><td>1036.67</td><td>1058.7</td><td>9.64</td><td>9.88</td><td>9.97</td><td>10.13</td><td>10.21</td><td>10.57</td></tr><tr><td>C16 [Val7] (pumilacidin F/G)<sup>a</sup>, C15<br>[Leu/Ile7] (pumilacidin A)</td><td>1050.67</td><td>1072.67</td><td>10.25</td><td>10.35</td><td>10.53</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C17 [Val7] (pumilacidin D)<sup>a</sup>, C16 [Leu/Ile7]<br>(pumilacidin E)</td><td>1064.7</td><td>1086.7</td><td>10.66</td><td>10.89</td><td>11.06</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C18 [Val7], C17 [Leu/Ile7] (pumilacidin C)<sup>a</sup></td><td>1078.7</td><td>1100.7</td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Pumilacidin ions identified in JM79 cultures under *in vitro* conditions (A, B) and *in planta* conditions (C). (A) Lipopeptide ion isoform parameters of retention time (RT, min) and masses (m/z) identified by QTOF mass spectrometry (EIC+) (according to the Pumilacidin nomenclatureª referenced in Naruse et al. [54]). (B) Peak area means of identified pumilacidin ions within TSB and RE (liquid media) and TSA and REA (solid media) [mean + SE, n = 4]. (C) Peak area means of identified pumilacidin ions within *in planta* condition [mean + SE, n = 4]. Percentages shown are calculated from the total peak areas per growth condition. Different letters indicate significant differences (Tukey post-hoc test, *P* < 0.05).</figcaption></figure>

as previously characterized by [54]. The pumilacidins identified in JM79 extracts shared these characteristics and differ in the fatty acid chain length and amino acid sequence, resulting in the presence of six pumilacidin variants and homologs, whose structural differences are described in Fig. 3A. However, to each *m/z* mass of pumilacidin corresponds a [Val2] surfactin isomer, and only MS/MS analysis could confirm whether the masses detected in JM79 extracts were [Leu2] pumilacidin isomers.

Consequently, an LC tandem-mass MS/MS spectrometry analysis was performed, enabling the fragmentation of both 1050.9 and 1064.9 *m/z* [M+H]<sup>+</sup> ions in supernatants from all synthetic media (Retention Time (RT) = 9.88 and 10.35 min, respectively, Fig. 3A). The MS/MS mass fragmentation of those two ions in RE medium extract is shown in Fig. S8. In the fragmentation profile of *m/z* 1050.9, pumilacidin A ion was detected, b+ fragments ranging from (*m/z*) 370.2–937.6, and in the

fragmentation profile of *m/z* 1064.9, pumilacidin E ion was detected, b+ fragments ranging from (*m/z*) 470.3–951.6 (Fig. S8). These fragmentation profiles were consistent with specific [Leu2] amino-acid sequence, i.e., C15 [Leu/Ile7] (pumilacidin A) and C16 [Leu/Ile7] (pumilacidin E) pumilacidin structures, respectively. In addition, a previous study reported a correlation between the specific gene cluster structure described in the JM79 strain genome (Fig. S5) and the specific production of these pumilacidin isoforms, whose structures were also verified by an MS/MS experiment [64]. Moreover, specific fragmentation of the surfactin C14 standard (Sigma Aldrich, France) was compared with fragmentation profiles in JM79, revealing that none of the fragmented molecules of extracted masses in EIC could match (data not shown). Several peaks at different retention times were detected for the same *m/z* mass, indicating potential isomeric forms of pumilacidins (Fig. 3A). However, their structures could not be further characterized
since the developed MS/MS method and limited extracted material did not permit their fragmentation. For some masses, the number of detected peaks from the chromatograms was higher than the number of isoforms deduced from the known variability in amino acid sequences and fatty acid chain length for pumilacidins [54]. This suggests that some peaks may not correspond to surfactin-like isomeric molecules or that there is some divergence in -iso or -anteiso stereochemical configurations of the fatty acid chain.

Variability in surfactin-like lipopeptide structure led to variability in biological activity [27], and such variability is partly influenced by the composition of the available resources in the environment [81]. However, little is known about the influence of both biofilm versus planktonic growth mode and of root exudate-like medium versus rich medium on the type and proportion of produced pumilacidin isoforms. Here, it was found that the six pumilacidin isoforms were produced under all conditions; furthermore, the profile of pumilacidin production by JM79 was overall weakly affected by the medium composition when comparing liquid conditions (TSB rich vs RE mineral media) and by growth modes in the rich medium (i.e., biofilm colony on agar vs planktonic state). The most important variations concerned the global abundance of C15/C14 (m/z of 1036.7), C16/C15 (m/z of 1050.7) and C18/C17 (m/z of 1078.7) that are significantly lower in the REA medium than in the three other conditions (m/z of [M+H]⁺ ions; *P* < 0.05) (Fig. 3B). In addition, it could be noticed that the proportion of the most abundant C13[Val7] isomer (m/z of 1008.7; RT = 9.16 min) is significantly higher in the biofilm colony mode (i.e., ranging 7.5 and 10 % in the TSA and REA agar media, respectively) than in the planktonic state (i.e., ranging around 6.5 % in the liquid TSB and RE conditions) (*P* < 0.05). Regarding the proportions of the C14/C13 (m/z of 1022.7) isoform (4 isomers), an influence of the substrate composition on the production of this isoform was observed with a significant higher proportion ranging about 20 % in the RE and REA conditions compared to about only 10 % in TSB and TSA media.

To validate the *in planta* production of pumilacidins, JM79 was inoculated into pregerminated wheat seeds grown under gnotobiotic conditions and the metabolites present near the wheat roots were extracted after 7 days of growth (Fig. 3C). Here, we confirmed the production of C13 [Val7] (m/z of 1008.7) and C14 [Val7], C13 [Leu/Ile7] (m/z of 1022.6) pumilacidin isoforms, showing similar RT in the EIC chromatograms from *in vitro* and *in planta* conditions (Fig. 3). In contrast, the pattern of LPs production is altered from those observed in the RE and TSB media for the four greatest masses, with EIC peaks exhibiting a slight drift in their RT in comparison to those observed in the RE and TS media (Fig. S7). MS/MS analysis was then required to confirm that the detected peaks correspond to pumilacidin LPs. As shown in Fig. S8, the MS/MS fragmentation pattern of both m/z 1050.7 and 1064.7 ions (pumilacidins A and E, respectively) from different retention times in the two JM79 culture conditions, i.e., RE medium and *in planta*, were exactly the same. These results indicate that the pumilacidins share the same peptide sequence and fatty acid mass, but differ in the stereochemistry of the fatty acid chain, with iso- or anteisobranching. Interestingly, a notable different proportion of C₁₃/C₁₄/C₁₅/C₁₆/C₁₇/C₁₈ pumilacidin isoforms was observed compared to JM79 grown under *in vitro* conditions. Indeed, the *in planta* pumilacidin production profile showed that the C₁₃/C₁₄ isoforms represent only 5.35 % of the total isoforms and isomers (Fig. 3A), whereas in artificial media, they represent between 17.12 % (TSB medium), and 34.8 % (REA medium) of the total isoforms and isomers (Fig. 3B). As negative controls, extracts prepared from plants inoculated with pumilacidin non-producing rhizobacteria in the same gnotobiotic system showed no traces of any pumilacidin (N = 16, data not shown). We concluded from these results that the JM79 strain is therefore able to produce pumilacidin under *in planta* condition.

### 3.5. Local defense-related genes induction profile in wheat in response to leaf inoculation of JM79

The expression pattern of 29 wheat defense-related genes was analyzed three days after JM79 direct inoculation (dpi) on wheat leaf tissues by spraying to runoff 2.10⁶ cells/plant. This expression pattern was compared to that elicited by spraying to runoff 6 mM methyl jasmonate (MeJA, a defense elicitor).

Gene expression changes were observed in response to JM79, with 10 and 2 out of 29 genes (38 %) significantly over- and down-regulated, respectively, compared to the negative control condition of the bacterial treatment (MgSO₄ 10 mM) (Fig. 4, Fig. S9).

Calibrating the data using 10 mM MgSO₄ instead of 3 % ethanol for the MeJA treatment may introduce potential biases for certain genes, specifically *PAL*, *FNS*, *GST*, *CSL*, *PR4*, and *PR5*. For the other genes, these biases are likely negligible (Fig. S10). Given this, the plant response to JM79 showed some similarities to the response induced by MeJA. Except for the phenylpropanoid pathway gene *CHS* (chalcone synthase), all genes induced by the inoculation of JM79 cells were similarly induced by MeJA. The latter led to the significant overexpression of 16 genes (constituting 55 % of total genes screened). MeJA up-regulation levels reached up to 8.5 log2 (observed in *Lox*: 13-Lipoxygenase), whereas the JM79 condition showed a maximum up-regulation of 4 log2 (observed in *CalS*: callose synthase) (Fig. 4; Fig. S9). Unlike the MeJA condition, the jasmonic acid biosynthesis-related gene *Lox* did not exhibit up-regulation in response to inoculation of JM79 cells (Fig. 4). No oxidative stress-related genes (i.e., *Apox*, *GST*, *POX*) were significantly induced by JM79 at 3 dpi, while the *POX* gene is specifically induced in presence of MeJA. Among the other genes up-regulated by both JM79 and MeJA were genes related to the ethylene pathway, *ACCS* (1-aminocyclopropane-1-carboxylate synthase) and *EIN3*, (Fig. 4; Fig. S9). The two PR encoding genes *PR2* and *PR8* were positively and significantly up-regulated by both JM79 and MeJA. Other screened PR genes were not induced by JM79. Thus, MeJA but not JM79 cells significantly induced the expression of *PR14* and potentially of *PR5* (Fig. 4). However, since ethanol alone induced *PR5* expression (Fig. S10), the significant overexpression of this gene in the MeJA treatment (Fig. 4) may be partly attributed to the solvent. For the *PAL* and *FNS* genes, ethanol and MeJA treatments appear to exert opposing effects (Fig. S10), suggesting that MeJA-induced expression of *PAL* and *FNS* genes (Fig. 4) might be higher than shown results. The representative gene of the cysteine sulfoxide pathway, *CSL*, was also overexpressed in JM79 and MeJA treatments (Fig. 4 and Fig. S9), suggesting that signals from both treatments can activate sulphur metabolism pathways, leading to sulphur-based defense mechanisms in wheat. All genes encoding for parietal reinforcement exhibited up-regulation in both JM79 and MeJA conditions: *CalS* (callose synthase), *CesA* (cellulose synthase), and *CAD* (cinnamyl-alcohol dehydrogenase). JM79 specifically enhanced the expression *CHS* involved in the phenylpropanoid pathway.

Thus, the leaf inoculation of JM79 cells induced defense responses in wheat with the over-expression of genes related to changes in cell wall composition, synthesis of defensive specialized metabolites (i.e., from the phenylpropanoid pathway and the sulphur metabolism pathway), induction of the ethylene pathway, but only a few PR proteins genes.

### 3.6. Wheat defense-related gene expression in response to collar inoculation of JM79

A second greenhouse experiment was conducted to assess the defense-related gene expression responses of the FCR susceptible wheat cultivar 'Recital' to JM79 collar inoculation and *Fg1* infection 3 days later. Expression of the 29 selected wheat genes was measured at 3, 6, 9, and 13 dpi (corresponding to 1 day before and 2, 5, and 9 days post-*Fg1* inoculation). These analyses were thus focused on the biotrophic stage of *F. graminearum* during FCR, as previously shown in a study of *F. graminearum* life stage dynamics, which outlined the necrotrophic
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Heatmap profiling and hierarchical clustering of 29 defense-related genes differentially expressed in wheat in response to foliar inoculation of JM79 applied at 2.10⁶ UCF per plant, foliar treatment with MeJA (6 mM, positive control) and MgSO₄ (10 mM, negative control). Apox, ascorbate peroxidase; CalS, callose synthase; CesA, cellulose synthase; CHS, chalcone synthase; CAD, cinnamyl-alcohol dehydrogenase; CSL, cysteine sulfoxide; EIN3, EIN3-binding F box protein; EDS1, enhanced disease susceptibility 1; Far, (E,E)-α-farnesene synthase (i.e., TPS, terpene synthase); FNS, flavone synthase; FPPS, farnesyl pyrophosphate synthase; GST, glutathione S-transferase; HMGR, hydroxymethyl glutarate-CoA reductase; JAR, jasmonate resistant 1; Lox2, 13-lipoxygenase 2; PAL, phenylalanine ammonia-lyase; PR, pathogenesis-related protein; PPO, polyphenol oxidase; POX, peroxidase; WRKY, WRKY transcription factor 30. At 15-day post-sowing, each gene expression replicate (expressed in log₂) was subtracted with the averaged defense gene expression from the negative control (MgSO₄ 10 mM). For the MeJA treatment (indicated by a gray dotted line), gene expression was normalized against the MgSO₄ negative control instead of the 3 % ethanol control but the bias introduced by this normalization was considered during results interpretation. For each gene, results of each of the 4 replicates per condition are shown. Asterisks indicate significant differences compared with the negative control according to the Student's t-test (n = 4; \*: P < 0.05, \*: P < 0.01, \*: P < 0.001 \*: P < 0.0001).</figcaption></figure>

switch at 28 dpi [72]. Consistently, no lesions were observed in roots or collars before 13 dpi in our experiment (data not shown).

Hierarchical clustering of genes based on their expression levels across the four conditions revealed four gene clusters with similar dynamics over time, grouping genes from different pathways (Fig. 5).

First, regarding the *Fg1*-infected positive control condition, the induction of defense genes mostly began nine days after *Fg1* inoculation (13 dpi), although the differences were not statistically significant compared to the un-inoculated control condition. The induced genes primarily belonged to cluster 2, with the *WRKY* (*P* < 0.05, *t*-test) and *CSL* genes (*P* < 0.1, *t*-test) being significantly induced (Fig. 5, Fig. S11). Interestingly, at 5-days post *Fg1* inoculation (9 dpi), a significant lower

expression levels (*P* < 0.05, *t*-test) of *PAL* (phenylalanine ammonia lyase) and *Lox2* was observed as compared to the negative control. *PR2*, *EIN3* (*P* < 0.05, Kruskal Wallis; Fig. S11), *ACCS*, and *PR8* also tended to be less expressed (*P* < 0.1, *t*-test, Fig. 5).

Second, regarding the JM79-elicited expression profile, no plant response was observed before 9 dpi except the significant down-regulation of *PAL* at 6 dpi (Fig. 5, and Fig. S12). At 9 dpi, 18 out of 29 genes (62 %) from clusters 1–3 showed an induction profile; among them, 14 genes showed a significant up-regulation (*P* < 0.05, *t*-test) and 2 genes showed a trend (*P* < 0.1, *t*-test) (Fig. 5). These genes represent all key plant defense pathways, from signalization to the establishment of effective physiological plant defense modifications. An intricate
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Heatmap profiling and hierarchical clustering of 29 defense-related genes differentially expressed in wheat in response to collar inoculation of the strain JM79, in presence or absence of the *Fg1* pathogen applied 3-days later, at several days post-bacterial inoculation. *Apox*, ascorbate peroxidase; *CalS*, callose synthase; *CesA*, cellulose synthase; *CHS*, chalcone synthase; *CAD*, cinnamyl-alcohol dehydrogenase; *CSL*, cysteine sulfoxide; *EIN3*, EIN3-binding F box protein; *EDS1*, enhanced disease susceptibility 1; *Far*, (*E*,*E*)-α-farnesene synthase; *FNS*, flavone synthase; *FPPS*, farnesyl pyrophosphate synthase; *GST*, glutathione S-transferase; *HMGR*, hydroxymethyl glutarate-CoA reductase; *JAR*, jasmonate resistant 1; *Lox2*, 13-lipoxygenase 2; *PAL*, phenylalanine ammonia-lyase; *PR*, pathogenesis-related protein; *PPO*, polyphenol oxidase; *POX*, peroxidase; *WRKY*, WRKY transcription factor 30. For each time-points and studied gene, the averaged defense gene expression level expressed in log<sub>2</sub> was subtracted to that of the negative control (MgSO<sub>4</sub> 10 mM). Gene expression levels were averaged over 4 independent biological replicates. The JM79 strain was applied at 10<sup>6</sup> UCF per plant, at 7-day post sowing, and 4 days before *Fg1* inoculation in the JM79+Fg1 condition. Asterisks indicate significant differences compared to the negative control (MgSO<sub>4</sub>) according to the Student's *t*-test (n = 4; *: P* < 0.1, **: P* < 0.05, **:** P* < 0.01, ***: P* < 0.001 ****: P* < 0.0001).</figcaption></figure>

network of defense signaling pathways was revealed with significant up-
regulation of genes encoding the Apox protein (ascorbate peroxidase)
involved in the oxidative stress response, two genes from the ethylene
hormone pathway (EIN3, ACCS), one gene from the acid salicylic hor-
mone pathway (EDS1: enhanced disease susceptibility 1), one from the
jasmonic acid pathway (JAR: jasmonate resistant 1), and genes from the
phenylpropanoid and isopropanoid pathways (FNS: flavone synthase,
PPO: polyphenol oxidase, FPPS: farnesyl pyrophosphate synthase) such
as from the cysteine pathway (CSL: cysteine sulfoxide) (Fig. S11). The
PR14 gene was also significantly upregulated as well as genes coding for
effective cell wall reinforcement proteins (CAD: cinnamyl-alcohol de-
hydrogenase, CalS: callose synthase). Contrariwise, two genes from
cluster 4, Lox2 and HMGR, were significantly repressed compared to the
negative control (Fig. 5). Taken together, these results indicate a direct
systemic elicitation of key plant defense genes after 9 dpi, by inoculation
with the B. pumilus JM79 strain (Fig. 5; Fig. S11).

Third, comparison of JM79 and *Fg1* single inoculation conditions
with JM79-*Fg1* double inoculation on wheat gene expression allowed to
assess the potential ability of JM79 to induce defense responses in the
presence of the pathogen (Fig. 5). The gene expression profile in
response to the JM79-*Fg1* double inoculation was highly similar to the
one elicited by single JM79 inoculation at 9 dpi, with similar genes being
induced or repressed, but generally to a lesser extend in the case of
JM79-*Fg1* treatment (Fig. 5; Fig. S11). Given the overall repression of
plant defense gene expression by *Fg1* at 9 dpi, this suggests a potential
averaging or additive effect of the two consecutive inoculations. At 13
dpi, compared to the *Fg1* single inoculation condition, the plant defense
profile of the *Fg1*-JM79 treatment showed minimal changes, with a
slightly stronger induction of defense gene expression (Fig. 5). Accord-
ingly, six genes from different defense pathways (*EDS1*, *CSL*, *Far*, *PPO*,
*CAD*, *PR14*) were more highly expressed in the double JM79-*Fg1* inoc-
ulation than in single *Fg1* inoculation (*P* < 0.05, Kruskal Wallis;
Fig. S11).

A specific focus on the transcriptional profile of the *PAL* gene over time (Fig. S12) reveals a significant regulation of this gene. We observe a notable and sustained repression of *PAL* after JM79 and *Fg1* single inoculation (between 6 and 9 dpi for JM79 and 9 and 13 dpi for *Fg1*), followed by an up- regulation, at 13 dpi, in all conditions where JM79 was inoculated.

## 4. Discussion

This study aimed to elucidate the plant-defense mechanisms activated in wheat by the inoculation of the PGPR *B. pumilus* JM79, in both the presence and absence of the soil-borne pathogen *F. graminearum* *Fg1*. More precisely, we aimed to determine whether the pumilacidin-producing JM79 strain was able to activate plant immunity and/or to have direct antifungal activity against *F. graminearum*. Combined antagonistic assays, wheat protection experiments against *F. graminearum* crown rot [7], and root and leaf colonization analyses indicate that *B. pumilus* JM79, isolated from the wheat rhizosphere, does not exhibit direct antifungal activity against the pathogen. Instead, this suggests that molecules secreted by the PGPR JM79 may act indirectly by inducing resistance mechanisms in wheat, rather than functioning as biofungicides. Genomic analyses of JM79 revealed traits essential for plant colonization, such as genes associated with cell motility. These observations align with the JM79's demonstrated ability to colonize roots and leaves when inoculated onto pre-germinated seeds. Furthermore, the production of surfactin-like compounds appears to play a critical role in bacterial motility and biofilm formation [73], potentially contributing to JM79's colonization capacity. In accordance with the fact that *B. pumilus* species are often reported as plant endophytes [46] [47,80], cell counts after root surface disinfection, together with the presence in the JM79 genome of genes associated with sugar catabolism and encoding secreted enzymes targeting plant cell structures, suggest that JM79 may function as an endophyte, interacting intimately with its host. This is consistent with findings that endemic endophytes exhibit stronger interactions with plants compared to epiphytes [61].

Surfactins, often referred to as natural elicitors for inducing plant resistance, have been widely studied for their efficacy in controlling various plant diseases [27,29,57]. *Bacillus* isolates producing surfactins and stimulating ISR [12] have emerged as promising biocontrol tools. Studies have documented the efficacy of surfactins in reducing wheat diseases such as Septoria [51], powdery mildew [31] and fusariosis [39]. In this study, it was crucial to confirm that JM79 produces surfactin-like pumilacidin and to assess its production levels under both *in vitro* and *in planta* conditions, as well as under planktonic and biofilm lifestyles, as these comparisons have rarely been explored in the literature. However, such exploration is relevant as *Bacillus* rhizobacteria are known to produce biofilms on plant roots, as well as on soil particles or fungal mycelium [62].

The profiling of JM79's secondary metabolites revealed that surfactin-like molecules constitute the major component of its metabolome, both in terms of abundance and diversity. In biofilm-like growth conditions (REA medium), JM79 produced pumilacidins in amounts and with a diversity comparable to those found in nutrient rich media, suggesting robust surfactant production under conditions mimicking the rhizosphere [3]. Furthermore, we observed changes in the pumilacidin isoform profiles depending on JM79 growth conditions, with a higher proportion of long-chain (C14 to C18) pumilacidin isoforms under *in planta* conditions, underscoring their relevance in the interaction of JM79 with plants. These results are consistent with a previous study that demonstrated an increase in the proportion of C15 among co-produced C12/C13/C14/C15 surfactins, secreted by a *Bacillus* strain growing on tomato roots, as compared to artificial medium [55]. These longer-chain variants are known to exhibit stronger ISR-inducing activity due to their enhanced ability to integrate more effectively into plant cell membranes, while the C12 and C13 variants showed almost no activity [27,

29].

Upon local spraying of the pumilacidin-producing JM79 strain onto wheat leaves, a rapid and strong plant defense response was observed, characterized by the up-regulation of secondary metabolite production genes (e.g., *CHS* and *CSL* genes) and enhanced cell wall reinforcement (e.g., *CAD* and *CalS* genes). This suggests that JM79 could be an effective biocontrol agent against wheat foliar pathogens by reinforcing the cell wall through lignification and callose deposition, and by limiting pathogen proliferation in leaf tissues, which is crucial given the dependency of vascular pathogen on the apoplast environment of leaf cells [6].

In the present study, we investigated the dynamics of defense-related gene transcriptional responses in wheat following infection with *Fg1*. By analyzing these responses from 2 to 9 days post-infection (6-13 dpi), we gained valuable insights into the asymptomatic biotrophic phase of this dual soil-borne and aerial wheat pathogen. Notably, no visible root or crown rot symptoms were observed before 17 dpi, underscoring the latent progression of the pathogen during its early biotrophic stage. Our results demonstrated that *Fg1* treatment repressed plant-defense genes expression at 2 and 5 dpi, consistent with the hemi-biotrophic nature of *F. graminearum*. This observation aligns with previous findings that a biotrophic progression of *F. graminearum* in wheat roots precedes the necrotrophic phase in the collar tissue [78]. It is plausible that this repression of defense gene expression is mediated by *Fg1* effectors, deployed as part of a strategic mechanism to evade the host plant's immune responses. This finding is particularly important given the limited understanding of *F. graminearum* interaction with wheat during the crown rot disease process, especially during the biotrophic phase. Interestingly, the induction of certain defense-related genes, including *WRKY*, *CSL* and, to a lesser extent, several PR genes (*PR2*, *PR1*, *PR4*, *PR8* and *PR15*), was observed after 9 days post *Fg1* infection (i.e., 13 dpi). This suggests the onset of a systemic acquired resistance (SAR)-mediated defense response. These results are consistent with SAR being a major defense mechanism against biotrophic and hemi-biotrophic pathogens [25,66]. For instance, it has been reported that SA production is enhanced in the roots of *Brachypodium distachyon* as early as 5 dpi following inoculation with *F. graminearum*, compared to uninoculated roots [16]. Accordingly, deficiencies in SA accumulation in wheat and *B. distachyon* have been shown to increase susceptibility to FHB caused by *F. graminearum* and FCR caused by *F. pseudograminearum* [45,59].

The genes expression profile induced by JM79 in the FCR experiment (i.e., collar inoculation) demonstrates a simultaneous activation of both ISR and SAR pathways. This activation involves genes associated with the jasmonic-acid (JA) /ethylene (ET) and acid-salicylic (SA) hormonal signaling pathways. The signaling molecules SA and JA are known to regulate distinct but interactive pathways, each modulating a specific set of defense-related genes [30]. While beneficial microbes typically activate ISR, our findings align with previous studies that showed the co-elicitation of SA and JA pathways by biocontrol *Pseudomonas* or *Bacillus* in various plants species [22,56,75]. In wheat, the responses triggered by JM79 in combination with *Fg1* at 9 dpi — a period corresponding to the asymptomatic phase before plant defense activation against *Fg1* —, suggest additive effects of both *Fg1* and JM79 on gene expression. This co-inoculation appears to counteract the *Fg1* induced suppression of wheat defense genes. Notably, our experimental setup involved inoculating both *Fg1* and JM79 in the same plant compartment. Interestingly, *Fg1* did not seem to impair JM79's ability to induce wheat defenses. For specific genes such as *Far* (at 9 dpi), *WRKY* transcription factor (at 13 dpi), and *CSL* (at 9 and 13 dpi), higher levels of expression were observed under JM79-*Fg1* co-inoculation than with either treatment alone. This observation suggests a priming effect induced by JM79, consistent with the general idea that beneficial microbes can elicit a 'priming' state in plants, allowing faster and stronger defense responses when confronted with a pathogen. However, the observed repression of *PAL* by both JM79 and *Fg1* suggests an active strategy to bypass this key defense gene. *PAL*, the first and rate-limiting enzyme in the
phenylpropanoid pathway, is widely associated with plant resistance. While studies on *PAL* in wheat remain limited, recent findings indicate that *F. graminearum* infection upregulates 31 copies of the *PAL* gene, with only one being down-regulated [82]. In this context, the up-regulation of *PAL* in the JM79-*Fg1* condition at 13 dpi could be a critical factor in the biocontrol efficacy of JM79.

Based on this study, we propose that the JM79 strain could be developed as a biocontrol product to protect both below- and above-ground tissues. In our experiments, inoculating wheat with JM79 four days before *Fg1* inoculation elicited strong defense responses, particularly during the phase when *Fg1* effectors typically suppress wheat defenses. This interference in host-pathogen interaction may underpin JM79's protective mechanism against *Fg1*. While it is not known whether JM79 defense responses remain elicited at later stages of infection-beyond 9 or 13 days post-inoculation- this strain was shown in a previous study to be able to reduce symptoms of FCR after 45 days post-inoculation [7], suggesting that the defense responses induced during the *Fg* asymptomatic phase might be still effective. Additionally, while our study focused on crown rot, it is worth considering whether similar ISR-based protection might extend to Fusarium head blight (FHB), which is also caused by *F. graminearum* but affects above-ground floral tissues. Traditionally, SA and JA /ET phytohormones have been associated with defense against biotrophic and necrotrophic pathogens, respectively. However, defense against hemi-biotrophic pathogens like *F. graminearum* likely requires the coordinated activation of both pathways at different stages to confer tolerance or resistance. JA signaling has been shown to play a critical role in the rapid induction of defense against FHB in resistant wheat cultivars [16,40,60], and in seedling resistance to FCR caused by *F. pseudograminearum* [59]. Furthermore, JA treatments in cereals have been reported to delay FCR symptoms caused by *F. pseudograminearum* [14] and *Fusarium culmorum* [52] even while suppressing SA-related defenses [44,45]. These results suggest that SA-signaling likely contributes to resistance by limiting *Fusarium* infection during the initial biotrophic phase, whereas JA signaling is proposed to have a dichotomous role by attenuating SA-signaling during initial infection but promoting resistance during the necrotrophic phase of *Fusarium* infection [14,45,52]. Supporting this, studies have demonstrated the synergistic effects of combining a PGPR with a SAR-chemical inducer, the benzothiadiazole (BTH), to enhance resistance capacity of pepper against bacterial spot diseases caused by *Xanthomonas axonopodis* pv. vesicatoria, in field conditions [80]. Interestingly, while some studies have highlighted cross-talk and synergy between SA and JA/ET pathways in wheat, others have reported trade-offs between these hormonal pathways [38]. Wheat appears capable of precisely adjusting its defense responses based on the specific pathogen, with SA and JA signaling acting either synergistically or antagonistically to regulate wheat defense gene expression [17].

## 5. Conclusion

In conclusion, our findings provide valuable mechanistic insights into the biocontrol potential of *B. pumilus* strain JM79 as a wheat defense elicitor, particularly against the relatively understudied wheat crown rot (FCR) disease caused by *F. graminearum*. The secretion of various isoforms of surfactine-like pumilacidins by JM79, especially those with long carbon chains, was confirmed to occur in planta, suggesting their potential role in mediating the observed biocontrol effects. Our investigation of the early biotrophic / asymptomatic infection stage of FCR revealed that *F. graminearum* first suppresses wheat defense responses during this phase. However, soil pre-inoculation with JM79 was found to induce a direct, earlier, and more robust activation of wheat defense mechanisms, involving a complementary interplay of SAR- and ISR-like pathways, to counteract *F. graminearum*. Additionally, the strong and direct elicitation of defense response observed following JM79 leaf-inoculation further highlights the potential of this strain as a versatile biocontrol agent against crown rot and foliar pathogens. These findings

offer promising insights into the use of bacterial defense elicitors like JM79 as biocontrol agents, emphasizing their potential to mitigate *F. graminearum* induced FCR disease. Optimization of JM79-based biocontrol strategies could significantly enhance sustainable disease management in wheat.

## Author contribution

Authors contributions are as follow

1) conceived and designed the experiments: AB, MG, AT, TL, CP-C.

2) performed the experiments: AB, MG, MR.

3) analyzed and interpreted the data: AB, MG, MR, M-NB, PJ, AD-Z, AT, TL, CP-C.

4) contributed reagents, materials, analysis tools or data: AB, MG, AD-Z, AT, TL, CP-C.

5) wrote the paper: AB, AT, TL, CP-C.

## Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

Aline Ballot's work was supported by a Ph.D. grant from the Auvergne Rhône Alpes Region. The authors also thank the Auvergne Rhône Alpes Region for funding the DOPELARA project (Pack Ambition Recherche 2018). The authors would like to thank the iBio and the CESN platforms and the quarantine laboratory platform P2 from the Microbial Ecology laboratory (UMR CNRS 5557) for their technical support, such as the Centre Technique des Microstructures from the FR BioEEnVis, and the technical staff of the Greencell SME and of the Rhizosphere team from the Microbial Ecology laboratory. We also thank the experimental infrastructure VégéPole for their help in plant cultivation, as well as Florian Rocher, Olivier Soudière, Michael Denefle, Mélanie Sicard, Mélissa Battache and Benoit Keraval for their help in sampling. Finally, the LABGeM (CEA/Genoscope & CNRS UMR8030), the France Génomique and French Bioinformatics Institute national infrastructures (funded as part of Investissement d'Avenir program managed by Agence Nationale pour la Recherche, contracts ANR-10-INBS-09 and ANR-11-INBS-0013) are acknowledged for support within the MicroScope annotation platform.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.cpb.2025.100524.

## Data Availability

Data will be made available on request.

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