# Dark septate endophyte Periconia macrospinosa enhances wheat seedling growth and mitigates Fusarium crown rot by reprogramming the host's immune responses

Mouadh Saadaoui, Théo Morlevat, Olivier Loison, Christelle Blavignac, Mohamed Faize, Lalla Aicha Rifai, Chaimae Nouri, Jamila Dich, Ninon Magliaraschi, Géraldine Philippe, et al.

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Mouadh Saadaoui, Théo Morlevat, Olivier Loison, Christelle Blavignac, Mohamed Faize, et al. Dark septate endophyte Periconia macrospinosa enhances wheat seedling growth and mitigates Fusarium crown rot by reprogramming the host's immune responses. Physiological and Molecular Plant Pathology, 2025, 141, pp.102995. 10.1016/j.pmpp.2025.102995. hal-05473781

HAL Id: hal-05473781

https://hal.inrae.fr/hal-05473781v1

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# Dark septate endophyte *Periconia macrospinosa* enhances wheat seedling growth and mitigates Fusarium crown rot by reprogramming the host's immune responses

Mouadh Saadaoui<sup>a</sup>, Théo Morlevat<sup>a</sup>, Olivier Loison<sup>a</sup>, Christelle Blavignac<sup>b</sup>, Mohamed Faize<sup>c</sup>, Lalla Aicha Rifai<sup>c</sup>, Chaimae Nouri<sup>c</sup>, Jamila Dich<sup>c</sup>, Ninon Magliaraschi<sup>d</sup>, Géraldine Philippe<sup>d</sup>, Ludovic Bonhomme<sup>d</sup>, Jean-Stéphane Venisse<sup>a, *, e</sup>

## ARTICLE INFO

### Keywords:

Biocontrol agent

Dark septate endophytes (DSE)

*Fusarium graminearum*

Plant growth promoting fungi

Plant immunity

## ABSTRACT

Fusarium crown rot (FCR) in wheat, caused by *Fusarium graminearum*, is among the world's major fusarium diseases. This study aimed to assess the potential of the dark septate endophyte *Periconia macrospinosa* PmVS01 in promoting wheat seedlings growth and inducing resistance against *F. graminearum* Fg1 under greenhouse conditions, as well as its ability to inhibit pathogen growth under axenic culture. The results demonstrated that *P. macrospinosa* PmSV01 enhances key plant ecophysiological traits, including photosynthetic activity (18 %), nitrogen nutritional status (18 %), and the accumulation of defensive flavonol compounds (22 %), which collectively result in average increase of 10 %–30 % in both root and shoot growth and biomass. These stimulating effects correlate with its symbiotic nature, as it colonizes the rhizodermis and peripheral parenchyma regions of the roots without causing morbid symptoms. It also significantly protects seedlings against FCR disease. Transcriptomic analyses by qPCR indicate that this protective effect is associated with its ability to enhance host immunity and to reprogram multiple metabolic processes involved in plant defense, including pathways related to oxidative and water stress responses, as well as jasmonic acid and salicylic acid signaling. In addition, confrontation assays revealed an antagonistic activity of *P. macrospinosa* PmSV01 against *F. graminearum* Fg1 through the production of diffusible and volatile compounds, leading to an inhibition rate of the pathogen's mycelia growth that ranges from 28 % to 85 %. This is the first report of the beneficial effect of *P. macrospinosa* PmSV01 on wheat and resistance to FCR, and provides new alternatives for exploiting the potential of DSEs as plant growth-promoting fungi and biocontrol agents for sustainable crop production.

## 1. Introduction

Wheat is one of the world's major crops, contributing to nearly 35 % of the world's traded food grains. In 2020, the total wheat production was around 760 Mt, but it will need to increase by at least 20 % to meet the projected global food demand by 2050 [1,2]. After several decades of steady growth, current wheat production is leveling off because of the limits of genetic breeding and mechanization, as well as environmental fluctuations that adversely affect the overall physiological performance of plants, including growth and immunity [3].

Fusarium crown rot (FCR) is one of the most widespread wheat diseases in the world [4]. FCR is caused by various fungi forming the *Fusarium* species complex, such as *F. graminearum*. Saprophytic in its nature, this fungus uses dead organic matter as a carbon source but can shift to hemibiotrophy, invading the host's living cells in order to extract nutrients and water. The fungus can infect a plant at various developmental stages, but its primary infection sites are the roots and crown of young seedlings, and/or the developing ears of mature plants. The initial symptoms of crown rot in winter cereals include a slowdown in plant development, followed by the emergence of necrotic lesions on the coileptiles, the basal parts of the leaf sheaths, sub-crown internodes, and

<footnote><sup>a</sup> University Clermont Auvergne, INRAE, PIAF, 63000, Clermont-Ferrand, France</footnote>

<footnote><sup>b</sup> Centre Imagerie Cellulaire Santé (CICS), UCA PARTNER, UFR de Médecine, 28 Place Henri Dunant- TSA 50400, 63000, Clermont-Ferrand, France</footnote>

<footnote><sup>c</sup> Laboratory of Plant Biotechnology, Ecology and Ecosystem Valorisation CNRST-URL10, Faculty of Sciences, University Chouaib Doukkali, 24000, El Jadida, Morocco</footnote>

<footnote><sup>d</sup> UMR 1095 Génétique Diversité Ecophysiologie des Céréales, Université Clermont Auvergne, INRAE, Clermont-Ferrand, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: j-stephane.venisse@uca.fr (J.-S. Venisse).</footnote>
List of abbreviations

**BCA** Biocontrol agents

**DSEs** Dark septate endophytes

**FCR** Fusarium crown rot

**NBI** Nitrogen balance index

**PDA** Potato dextrose agar

**PGMs** Plant growth-promoting microorganisms

**PGFs** Plant growth-promoting fungi

**SDW** Sterile distilled water

**SMs** Non-volatile or diffusible Secondary Metabolites

**VOCs** Volatile organic compounds

root emergence points [5]. Depending on the aggressiveness of the pathogen and prevailing environmental conditions, the most susceptible cultivars undergo premature senescence after germination and die within a few weeks [6].

At present, the management of FCR is primarily reliant on the application of fungicides. Nevertheless, these chemical agents are anticipated to face future bans or stringent regulatory restrictions owing to their detrimental impacts on human health and biodiversity, the emergence of fungicide-resistant pathovars, and their limited efficacy beyond the seedling stage -a consequence of natural degradation in the soil and insufficient systemic activity within the host plants [7]. An alternative to agrochemicals is the use of plant growth-promoting and biocontrol microorganisms (PGMs), such as plant growth-promoting fungi (PGFs). Their application in both field crops and greenhouses aligns with the strategic goals of sustainable, environmentally friendly disease management by inducting efficient host immunity and optimizing plant growth potential.

Plant growth-promoting fungi are non-pathogenic, naturally occurring saprophytes. Their ability to establish contact with and/or penetrate plant roots is regarded as the principal mechanism by which they promote plant growth, primarily through the enhanced uptake and assimilation of diverse organic and inorganic nutrients, as well as the reinforcement of plant immunity by inhibiting pathogen infection. The ability to suppress phytopathogens involves a combination of complementary mechanisms, including antibiosis, mycoparasitism, the production of cell-wall degrading enzymes and phytohormone-like compounds, competition for nutrients and ecological niches, as well as the elicitation of a broad spectrum of induced systemic resistance (ISR) responses [8–10]. Paradoxically, although an expanding volume of scientific literature underscores the dual functionality of plant growth promotion (PGP) and biological control activity (BCA), the commercial deployment of these beneficial organisms remains constrained, particularly within the context of wheat cultivation. This restricted deployment is attributed to several factors, among which the most frequently cited are the inconsistent efficacy observed under field or greenhouse conditions and the limited shelf life of existing bioformulations [11]. In addition, the spectrum of identified PGFs is still too narrow, limited to a few major fungal clades such as *Trichoderma*, *Cladosporium*, *Penicillium*, *Phoma*, and some non-pathogenic *Fusarium* species [12]. Nonetheless, the remarkable diversity of fungi suggests that a wide range of novel and interesting PGPF strains remains to be discovered.

In this study, we focused on a fungal species isolated from wheat roots, *Periconia macrospinosa*, which belongs to the Periconiaceae family [13]. The Periconiaceae are part of an artificial group of fungi known as dark septate endophytes (DSEs). DSEs are a common and widely distributed group of fungi that colonize plant roots, and classified as Class 4 non-clavicipitaceous fungal endophytes [14]. These fungi are characterized by their melanized septate hyphae and their ability to form microsclerotia within plant roots. To date, the specific roles of DSEs in ecosystems are not fully understood [15], and information about their

effects on plant growth and health remains limited. DSEs, and specifically *P. macrospinosa*, have been shown to improve plant growth by increasing chlorophyll content and enhancing nitrogen uptake from organic sources, particularly when subjected to abiotic stressors, such as salinity and drought [16–18]. Additionally, *P. macrospinosa* has demonstrated antifungal activity and/or reduced the incidence of plant diseases caused by various *Fusarium* species in cucumber, barley, and red ginger [19–21].

To our knowledge, this study represents the first examination into the application of *P. macrospinosa* in wheat for the dual purpose of enhancing plant growth and conferring disease resistance against FCR caused by *F. graminearum*. A multidisciplinary approach, integrating ecophysiological, morphometric, and transcriptomic methodologies, was designed to evaluate the efficacy of the *P. macrospinosa* strain PmSV01 in promoting wheat seedling development and inducing resistance to *F. graminearum* under controlled greenhouse conditions. Furthermore, the study sought to determine the strain's ability to inhibit *F. graminearum* growth under axenic conditions. Finally, these beneficial effects were assessed in conjunction with the potential of *P. macrospinosa* PmSV01 to establish symbiotic interactions with host plant roots, as demonstrated through scanning electron microscopy (SEM) analyses. The cultivar Recital was specifically chosen due to its marked susceptibility to *F. graminearum* (MDC_Fg1) [22]. This pathosystem allowed us to target gene networks that either induce disease development through the modulation of potential susceptibility mechanisms related to Fg1's pathogenicity, or confer resistance by eliciting a broad range of genes involved in various defense pathways, mediated by *P. macrospinosa* PmSV01. These pathways include those related to phenylpropanoids, sesquiterpenoids, cyanogens, PR-proteins, oxidative and water stresses, and the regulation of phytohormone balances. Our study supports the consideration of *P. macrospinosa* PmSV01 as a plant growth-promoting fungus, presenting it as a suitable and safe alternative for controlling FCR disease in wheat.

## 2. Materials and methods

### 2.1. Fungal strains, and culture conditions

*P. macrospinosa* PmSV01 strain was isolated from healthy wheat roots (*Triticum aestivum* L., var. Florence-Aurore), cultivated in an organic farming system in Tunisia (36.924096° N, 9.699089° E) [23]. The virulent isolate of *F. graminearum* MDC_Fg1 [24], referred to as Fg1 in this study, was kindly provided by the “Cereal diseases” team from the GDEC UMR (Genetics, Diversity and Ecophysiology of Cereals; https://e ng-umr1095.clermont.hub.inrae.fr/). Each strain was routinely grown and maintained on PDA medium in the dark at 25 °C, stored at –20 °C in a 30 % glycerol solution.

### 2.2. Plant material, and seed sterilization

*In planta* experiments were conducted using *Triticum aestivum* L. subsp. *aestivum* cv. Recital (accession number: 6027, GDEC). Seeds were surface sterilized in an aqueous solution of 0.5 % (v/v) sodium hypochlorite for 2 min, washed twice with sterile distilled water (SDW) for 1 min, sterilized with 70 % ethanol for 1 min, and then washed three times with SDW for 1 min each. Subsequently, the seeds were dried on filter paper and maintained on ice in sterile Falcon tubes until the time of sowing.

### 2.3. Priming of wheat seeds with *P. macrospinosa* PmSV01

*P. macrospinosa* PmSV01 has the distinctive feature of producing very few spores. In our study, we adapted a previously described method for inoculum preparation, using plugs of actively growing DSEs on PDA medium for seedling inoculation [25]. Specifically, we applied this principle by crushing actively growing fungal plugs to prepare a
suspension for wheat seed priming. Two plugs (12 mm in diameter) from a 2-week-old culture of *P. macrospinosa* PmSV01 were sampled, coarsely crushed in 20 mL of SDW, and used to inoculate sterilized seeds for 1 h at 25 °C. After treatment, seeds were placed on a 1 % (w/v) water/agar medium in 120 × 120 mm square Petri dishes. To optimize fungal adhesion to the seeds, the same *P. macrospinosa* PmSV01 preparation was delicately sprayed onto the seeds 24 and 48 h after the initial treatment (1 mL per Petri dish). Seeds not primed with *P. macrospinosa* PmSV01 were treated with SDW as controls.

## 2.4. *F. graminearum* Fg1 inoculation, and FCR disease evaluation

*F. graminearum* Fg1 inoculations were initiated on PmSV01-primed plants (denoted as PmSV01+Fg1 in Figs. 6 and 7), and on non-primed plants serving as infected controls (denoted as Fg1). A volume of 125 µL of a spore solution, adjusted to $10^5$ spores mL$^{-1}$ in SDW, was carefully applied to each sown seed with a micropipette, prior to covering the seeds with soil. Seeds not infected with *F. graminearum* Fg1 were covered with potting soil immediately after sowing.

The progression of early events in the development of FCR symptoms was recorded daily and categorized into three symptoms scores: S0, no apparent symptoms; S1, slowing of plant growth with no apparent necrosis, as compared to SDW-control plants; and S2, initial necrosis at the basal part of the leaf sheaths, corresponding to a slowdown of plant growth and progressive necrotic lesions on the crown that extend along the stem.

## 2.5. Germination and growth conditions of PmSV01-primed and control seedlings in both axenic environment and trays

*P. macrospinosa* PmSV01-primed and control seeds were sown either on a new 1 % (w/v) water-agar medium to test germination vigor, with 32 seeds per biological condition (i.e., 8 seeds per 12 × 12 cm square Petri dish, across 4 replicates), or in trays with 60 cells, one seed per cell, to evaluate plant growth promotion (PGP) and biocontrol agent (BCA) abilities. In the trays, each 75 mL cell was filled with a mixture of 2/3 commercial potting soil (Humustar®; vermiculite; N, 105 g m⁻³; P₂O₅, 120 g m⁻³; K₂O, 135 g m⁻³; pH, 6.4), 1/3 sand, and the equivalent of 2 g of solid, coated time-release fertilizer per liter of soil (Nutricote 18-8-18 T140, France). The seeds were sown on the surface of the water-agar medium, or 1 cm deep in the compost, respectively. Petri dishes and trays were placed in a controlled growth chamber with the following illumination conditions: a 14/10 h light/dark photoperiod, with 0 µmol m⁻² s⁻¹ photosynthetic photon flux density from 0:00 to 8:00, 150 µmol m⁻² s⁻¹ from 8:00 to 22:00, and 15 µmol m⁻² s⁻¹ from 22:00 to midnight. The temperature was maintained at 21 °C during the day and 18 °C the night (±0.5 °C), with a continuous relative humidity of 70 ± 5 %. Trays were watered daily with tap water.

## 2.6. Symptomatology, ecophysiological, biochemical, and molecular analyses on seedlings

Non-destructive (symptomatology, morphometry, and ecophysiology) and destructive (transcriptomic) analyses were conducted to characterize physiological abilities of *P. macrospinosa* PmSV01's for plant growth promotion (PGP) and biopesticide activity (BCA). PGP capacity was assessed in both aerial and root tissues by non-destructive methods. The BCA capacity was evaluated based on the potential occurrence of Fusarium symptoms at the whole-plant level. Samples for transcriptomic analysis were collected from the primary sites of Fusarium infection, namely the crown tissue, the basal 2 cm of the stem, and the proximal 2 cm of the root.

Symptomatology, ecophysiological analyses, and measurements of aerial part growth were conducted on all seedlings cultivated in the trays. Fresh and dry weights were measured from twenty randomly selected seedlings. Transcriptomic analyses were performed on 150 mg

of fresh tissue collected from four randomly selected seedlings. Samples were placed in 2 mL Eppendorf tubes with three metal beads (Ø 2 mm), immediately frozen in liquid nitrogen, and stored at -80 °C until use. Molecular analyses were carried out on Fg1-infected plants exhibiting S1 and S2 symptom scores during the 21-days assay period, as well as on 21-days-old PmSV01-primed seedlings (infected or not with Fg1) and control seedlings.

## 2.7. Morphometric measures and sampling

Germination efficiency was measured two days after planting the trays, and again over a period of 6 days. Length of the aerial organs (measured from the base of the crown to the apex of the seedling) and root length were recorded at 7, 14, and 21 days of growth using a ruler. After 21 days, twenty plants were carefully harvested. The roots were removed from the soil and washed with tap water. Fresh weights of leaves and roots were recorded using a digital balance. The fresh biomass was then oven-dried at 60 °C for 7 days, and the dry weights were measured.

## 2.8. Ecophysiological variable analyses

The contents of total chlorophylls (Chl, essential pigments involved in photosynthesis), the epidermal flavonols index (Flav, indicative of plant responses to environmental stressors), and the nitrogen balance index (NBI, a proxy for the nitrogen nutritional status of the plant) were assessed in 21-day-old seedlings using the DUALEX® 4 Scientific Leaf-Clip device (Force-A, Paris, France; https://www.force-a.com/wp-content/uploads/2019/09/BROCHURE-DUALEX-1.pdf). This non-destructive, portable optical sensor allows for in situ and instantaneous measurement of these three physiological proxies. Measurements were performed on all the plants in the trays, specifically on the fully developed adaxial surface of the apical and second leaves that were most exposed to light.

## 2.9. Total RNA isolation, cDNA synthesis, and RT-qPCR

Samples for molecular analysis, corresponding to four biological repetitions per biological conditions, were ground under vigorous agitation for three 30-s cycles with three metal beads (Ø 2 mm). The extraction of total RNA using cetyltrimethylammonium bromide (CTAB) extraction buffer, the elimination of genomic DNA, and the verification of template quantity and quality were performed as previously described in Ref. [26]. First-strand cDNA was synthesized from 1 µg of total RNA, using the SuperScript® III First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA, USA). qPCR was conducted in a 20 µl reaction volume, using TakyonTM Rox SYBR® MasteMix dTTP Blue (Eurogentec, Liege Science Park, Belgium), with 2 µL of cDNA diluted 30-fold with sterile water, and 100 nM primers. PCR reactions were carried out on an Applied Biosystems StepOnePlusTM Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). qPCR reactions were performed according to the manufacturer's instructions, using the following thermal cycling conditions: an initial denaturation at 95 °C for 3 min, followed by 35 cycles of 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 20 s, and a final extension at 72 °C for 15 min. Expression levels of the candidate genes were normalized to those of five wheat internal reference genes, previously validated for their stability in each sample, using the 2⁻$ΔΔCt$ method [27]. A melting curve analysis of amplification products was performed at the end of each PCR, by generating a dissociation curve from 60 °C to 95 °C, with a temperature increment of 0.3 °C every 15 s, to confirm that only a single PCR product was amplified and the absence of primer dimers. Primer pairs are listed in Table S1. PCR efficiencies (E) were determined for each primer pair by comparing threshold values across a dilution series (× 10, × 20, × 40, and × 80) of a cDNA mix synthesized from all reverse transcription products. PCR efficiencies are provided in Table S1.
## 2.10. Molecular identification and bioinformatic analyses of P macrospinosa PmSV01

Mycelia of *P. macrospinosa* PmSV01 (10 days old) was frozen in liquid nitrogen, and ground into a fine powder through vigorous agitation with three metal beads (Ø 5 mm) in three 30-s cycles. The extraction of genomic DNA, concentration and quality controls of nucleic matrices, and PCR amplification of two loci (*nuclear ribosomal internal transcribed spacer* ITS, and partial *nuclear small subunit (18S) ribosomal RNA gene* SSU), were performed according to Ref. [23]. ITS (ITS1, ITS4; [28]) and SSU (NS1, NS4; [29]) primers are detailed in Table S1. Amplicons were sequenced by Genewiz Company, and nucleic sequences were identified using BLASTn from the public NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The newly generated partial ITS and SSU sequences were submitted to the NCBI GenBank database (http://submit.ncbi.nlm.nih.gov). Accession numbers for these sequences, along with those of related *Periconia* strains used in the phylogenetic analyses, are listed in Table 1.

Phylogenetic analyses were performed on the concatenated gene sequence 5'-ITS (470 bp)/SSU (997 bp)-3'. All nucleotide data were weighted equally, and gaps were treated as relevant missing polymorphic characters. Multiple DNA sequence alignments were carried out using the MUSCLE algorithm with default settings. After removing ambiguously aligned regions with Gblocks, the final alignment consisted of 1467 nucleotides. Maximum likelihood analysis was used to construct the phylogenetic tree, as implemented in the PhyML program. The tree topology was evaluated by bootstrap analysis based on 1000 random resamplings. Graphical representation and editing of the phylogenetic tree were done using TreeDyn.

## 2.11. Optical microscopy evaluation of the morphology of P. macrospinosa PmSV01

Mycelia and spores were observed under a microscope at 20X and 40X magnifications. After three weeks of fungal culture on PDA medium at 25 °C, the cell structures were collected using transparent adhesive tape, stained with filtered toluidine blue (0.1 %, w/v) for 10 s, and rinsed with sterile water for 5 s. The preparation was then mounted on a glass slide for light microscopy observation.

<table><caption>**Table 1**

Fungal species and their GenBank accession numbers used in phylogenetic analyses. Percent identity of sequences compared to *P. macrospinosa* PmSV01 is shown in brackets.</caption><thead><tr><th>Organism</th><th>ITS loci</th><th>SSU loci</th><th>Isolates/strains</th></tr></thead><tbody><tr><td><i>Periconia macrospinosa</i></td><td>PQ348100.1</td><td>PQ348541.1</td><td>PmSV01</td></tr><tr><td><i>Periconia macrospinosa</i></td><td>MT446138.1 (100%)</td><td>KP183999.1 (100%)</td><td>ZMXR33</td></tr><tr><td><i>Periconia artemisiae</i></td><td>MW448657.1 (89%)</td><td>MW448658.1 (99%)</td><td>KUMCC 20-0265</td></tr><tr><td><i>Periconia byssoides</i></td><td>OQ981406.1 (89%)</td><td>OQ981398.1 (99%)</td><td>KUMCC 21-0578</td></tr><tr><td><i>Periconia cookei</i></td><td>OP955968.1 (90%)</td><td>OP955968.1 (98%)</td><td>UESTCC 22.0134</td></tr><tr><td><i>Periconia elaeidis</i></td><td>MH109537.1 (90%)</td><td>MH108551.1 (98%)</td><td>MFLUCC 17-0087</td></tr><tr><td><i>Periconia igniaria</i></td><td>MH858969.1 (98%)</td><td>MH858798.1 (99%)</td><td>CBS 298.66</td></tr><tr><td><i>Periconia verrucosa</i></td><td>OP955970.1 (90%)</td><td>NG_070659.1 (98%)</td><td>MFLUCC 17-2158</td></tr><tr><td><i>Arthopyrenia salicis</i></td><td>KM030296.1 (83%)</td><td>AY538333.1 (93%)</td><td>CBS 368.94</td></tr><tr><td><i>Juxtiphoma eupyrena</i></td><td>KJ686363.1 (81%)</td><td>OM618776.1 (94%)</td><td>Isolate P2</td></tr></tbody></table>

## 2.12. Visualization of endophytic colonization of wheat roots by P. macrospinosa PmSV01 using SEM analyses

To visualize *P. macrospinosa* PmSV01 and evaluate its symbiotic nature, we conducted scanning electron microscopy (SEM) analyses on wheat roots colonized by the fungus. Root endophytic colonization by *P. macrospinosa* PmSV01 was investigated on sterilized PmSV01-primed and non-primed seeds under axenic conditions. Three PmSV01-primed seedlings and three water-control seedlings were germinated on Petri dish with PDA at 25 °C. Fifteen days after inoculation, root samples were carefully collected and fixed in a solution of 4 % (v/v) paraformaldehyde and 3 % (v/v) glutaraldehyde in 0.1 M phosphate-buffered saline (PBS). The fixed samples were stored at 4 °C until use. Roots were post-fixed with 1 % (w/v) osmium tetroxide in 0.1 M PBS for 1 h. After rinsing in distilled water for 20 min, dehydration was carried out using a graded ethanol series (25°-100°) as follows: 25° for 10 min, 50° for 10 min, 70° for 3 × 10 min, 95° for 3 × 10 min, and 100° for 3 × 10 min. The samples were then treated with hexamethyldisilazane for 30 min. Two-millimeter-long root segments were cut, mounted on stubs using adhesive carbon tabs, and coated with platinum (Quorum Q150 TES, UK). The samples were examined using a Field Emission Scanning Electron Microscope (Regulus 8230, Hitachi, Japan) at 2 kV with a secondary electron detector.

## 2.13. Growth inhibition effects of P. macrospinosa PmSV01 on F. graminearum Fg1

Three antagonist tests were conducted: a direct confrontation assay between fungal strains, and two indirect confrontation assays to assess the effects of volatile organic compounds (VOCs) and non-volatile or diffusible secondary metabolites (SMs). All experiments were performed on PDA medium. In the direct confrontation assay, a 5 mm-plug of each strain was placed on opposite sides of the Petri dish (Ø 85 mm), 55 mm apart and equidistant from the plate's edge. VOCs effects were evaluated using two-compartment Petri dishes, allowing VOCs to exchange freely. In this setup, a 5 mm plug of each strain as placed in each compartment, ensuring that the colonies remained out of physical contact during the co-culture. The inhibition effect of SMs was determined using the cellophane culture method [30]. PDA plates were covered with a sterile cellophane sheet (325P cellulose Ø 80 mm, AA Packaging, France), and a 5 mm plug of *P. macrospinosa* PmSV01 was placed on the cellophane. When the fungal colony had covered half the surface of the cellophane, both the colony and the cellophane were removed. Then, a 5 mm-plug of *F. graminearum* Fg1 was inoculated in the center of each treated plate. Control plates, inoculated only with *P. macrospinosa* PmSV01 or *F. graminearum* Fg1, were included for each treatment. Plates were carefully sealed with parafilm, and incubated at 25 °C for 7 days for the dual confrontation and VOCs experiments, and for 2 weeks for the SMs experiments. Each treatment was repeated seven times. Mycelial growth was photographed daily, and growth rates were quantified using ImageJ (v. 1.54g/Java 1.8.0_345).

## 2.14. Statistical analysis

The results presented in Figs. 4–6, Fig. S2, and the three tables are expressed as the means of biological replicates from individual experiments. Vertical bars indicate the standard error of the mean (SEM). Data were analyzed using One-way analysis of variance (ANOVA), followed by Tukey's HSD test with Bonferroni-adjusted *p*-values to determine statistically significant differences between the means of treated samples and untreated controls, with a significance threshold of 5 % (*p* ≤ 0.05). Means for fungal-treated seeds that significantly differed from the untreated controls are indicated with significance levels of * *p* ≤ 0.05, ** *p* ≤ 0.01, *** *p* ≤ 0.001, and **** *p* ≤ 0.0001, or by different letters. Statistics for Fig. 3 were calculated based on a Kruskal-Wallis test (*p* < 0.05, rstatix).
# 3. Results and discussion

## 3.1. Molecular identification and morphological features of *P. macrospinosa* PmSV01

### 3.1.1. Identification of *P. macrospinosa* PmSV01

The *Periconia* genus is a well-known group of fungi that belongs to the *Periconiaceae* family [13,31]. *P. macrospinosa* PmsSV01 exhibited distinct morphological features when cultured on PDA medium, consistent with the typical morphology of *Periconia* (Fig. 1A). The colonies appeared circular and spatially well-defined, with a velvety to cottony texture. Initially, they were white to pale cream, and as they matured, they developed a darker hue, ranging from light brown to deep brown or even black. Over time, a bluish area appeared towards the periphery, which expanded to some extent and eventually became whitish. The growth rate on PDA medium was moderate, with radial growth reaching approximately 2–5 cm in diameter within 7–10 days at 25 °C.

Under the microscope, the hyphae of *P. macrospinosa* PmsSV01 were septate and slightly pigmented, appearing branched and forming dense mycelial mats (Fig. 1B). The septate mycelium was 2.9 µm–3.3 µm wide. Conidia (asexual spores) were spherical to ovoid, approximately 14.5 µm in diameter. These conidia were produced on short conidiophores that arose directly from the hyphae. Although *Periconia* species (including *P. macrospinosa*) are known to form microsclerotia (which are aggregations of hyphae that can act as survival structures) these were not observed under our culture conditions. All cell structures (hyphae, conidiophores, and conidia) exhibited dark pigmentation. In this respect, Periconiaceae are part of an artificial group of dark septate endophytes (DSEs). The pigmentation is attributed to the accumulation of melanin in the hyphae and conidia. It exhibits beneficial physical and chemical properties to fungi, and its high resistance enables organisms to survive in harsh environments [32]. Additionally, *P. macrospinosa*

PmSV01 lacked sexual structures, a characteristic shared by all *Periconia* species, which are classified as anamorphic fungi, i.e. those that reproduce asexually [33]. All these morphological characteristics align with the *Periconia* subphylum [13], and complement the phylogenetic analyses (Fig. 1C).

BLAST results of the ITS and SSU sequences unequivocally supported the placement of the strain *P. macrospinosa* PmsSV01 within the genus *Periconia*. The phylogenetic tree based on these sequences showed that *P. macrospinosa* pmSV01 forms a clade with *Periconia macrospinosa* ZMXR33, exhibiting 100 % identity in the ITS and SSU regions, and receiving high bootstrap support (Fig. 1C). Additionally, *P. macrospinosa* PmsSV01 is closely related to *P. igniaria* CBS 298.66, with 98 % and 99 % identities in the ITS and SSU regions, respectively, suggesting they share a recent common ancestor [34,35]. They also exhibit similar morphological and microscopic characteristics [35]. This analysis highlights the genetic similarity and confirms the taxonomic classification of *P. macrospinosa* pmSV01, contributing to our understanding of the diversity and phylogenetic relationships within the *Periconia* genus.

### 3.1.2. *P. macrospinosa* PmSV01 is facultative endophyte

Dark septate endophytes are a common and widely distributed group of fungi found in both natural and anthropogenic landscapes, including several species capable of colonizing plant roots with significant physiological impacts on the host [14]. The colonization capacity of *P. macrospinosa* PmSV01 and its potential to invade wheat roots as a fungal endophyte were tested using a scanning electronic microscope (SEM). Fifteen days after inoculation under axenic conditions, roots of control plants that grew from seeds not primed with *P. macrospinosa* PmSV01 showed no evidence of natural endophytic microorganisms either on the surface or inside (Fig. 2AB). In contrast, roots grown from seeds inoculated with *P. macrospinosa* PmSV01 displayed abundant mycelial development around the roots, particularly at the base of the root hairs (Fig. 2CD). More importantly, branching hyphae were also

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Morphological and molecular characterizations of *Periconia macrospinosa* PmSV01. (A) Macroscopic morphology of *P. macrospinosa* PmSV01 after 3 weeks of growth on PDA medium at 25 °C in the dark. The scale bar represents 1 cm. (1) View from above the Petri dish, (2) view from below (B). Optical microscopy images of hyphae (magnification × 400) after staining with toluidine blue (0.5 % w/v). The scale bar represents 50 µm. Co, Conidia; CpC, Conidiogenous cells; Cpho, Conidiophores; Hy, Septate hyphes. (C) ITS-SSU-concatenated molecular phylogenetic analysis of *P. macrospinosa* PmSV01 (red triangle) with closely related sequences retrieved from NCBI. The DSE Dothideomycetes *Phoma eupyrena* and *Arthopyrenia salicis* were used as outgroup sequences. The analysis was performed using the maximum likelihood method implemented in the PhyML program. Bootstrap values (1000 repetitions) are shown at the nodes. The scale bar represents 0.05 nucleotide substitutions per sequence position.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Invasion of *Periconia macrospinosa* PmSV01 on the surface and inside wheat roots. Observations were made using scanning electron microscopy (SEM). Root sections were taken from roots grown on PDA in axenic medium at 25 °C, from control seeds treated with sterile distilled water (A, B), or inoculated with *P. macrospinosa* PmSV01 (C, D, E). Red arrows indicate the presence of PmSV01 hyphae. St: stele; Pc: cortical parenchyma; Rd: rhizodermis; RH: root hair. (B) is an enlarged view of (A), (D) of (C), and (E) of (D). The regions of interest in the zoomed images are outlined in red.</figcaption></figure>

observed inside peripheral root cells, specifically in the rhizodermis and parenchyma regions (Fig. 2DEF). This finding is consistent with the fact that *P. macrospinosa* is a well-known pleosporalean root-colonizing fungi belonging to the Periconiaceae family within the Pleosporales order [13].

Fungi exhibit remarkably diverse and versatile metabolic profiles, ranging from neutral to beneficial or pathogenic behaviors, and some experimental studies have suggested that fungal root endophytes may act as latent pathogens [14,36]. The impact of these root-colonizing endophytes on their hosts varies depending on the host's
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Asymptomatic (non-pathogenic) interaction of *Periconia macrospinosa* PmSV01 on wheat seed during the seed germination process. (A) Symptoms were investigated after 2 days of seed culture on 1 % water agar medium. Seeds were categorized as follows: (a) seeds that had not yet germinated, (b) seeds showing seed coat re-imbibition and germ revival, and (c) seeds with early coleoptile and root development. *P. macrospinosa* PmSV01 was inoculated using a solution of crushed mycelium from a 2-week-old culture. The numbers on each bar indicate the number of germinated seeds. The *p* value was calculated using a Kruskal-Wallis test (*p* < 0.05), from a total of 55 plants per biological condition. (B) The black bodies appearing on the agar medium correspond to mycelial fragments of *P. macrospinosa* PmSV01. The bar scale represents 1 cm. The complete images, including the morphological aspect of 2-week-old seedlings, are shown in Supplemental Fig. S1.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** *Periconia macrospinosa*'s PmSV01 PGP activity on the growth dynamics of wheat seedlings over 21 days of interaction (A), and at 21 days after culture (B). *P. macrospinosa* PmSV01 was inoculated with a solution of crushed mycelium from a 2-week-old culture. The scale bar on the micrographs represents 5 cm. Data are presented as means and standard errors from 55 biological replicates. Asterisks indicate significant differences from the controls, according to One-way ANOVA with Tukey's HSD test (*p* < 0.05), further refined by Bonferroni-adjusted *p*-values, where "*" ≤ 0.05, "**" ≤ 0.01, "***" ≤ 0.001, and "****" ≤ 0.0001.</figcaption></figure>

developmental stage, physiological and genetic status, as well as the availability of organic and inorganic nutrients, and specific environmental pressures or experimental conditions [14,37–39]. In this context, although DSEs are predominantly saprophytic and/or endophytes by nature, some *Periconia* species have been described as plant pathogens, primarily in graminaceous plants. For example, *P. macrospinosa* causes leaf necrosis in pointed gourd (*Trichosanthes dioica*) [40], and leads to reduced root and shoot growth and Crater disease in wheat [41,42]. *P. igniaria* is responsible for leaf spot on Yellow Starthistle [43], and *P. circinata* causes blackening and rotting of wheat roots [44]. Accordingly, it was crucial to ensure that the close and beneficial interaction established by *P. macrospinosa* PmSV01 on cv. Recital remained stable and did not progress towards a deleterious, pathogenic state (i.e., disease), especially when the fungus was restricted to seeds as its sole organic resource in a nutrient-limited environment such as 1 % water-agar. Pathogenicity was assessed through a germination test, in which seeds were inoculated three times at 24-h intervals, followed by screening for potential symptoms (necrosis, chlorosis, or spotting) on

seeds, young germinants, and developing seedlings. The results showed that *P. macrospinosa* PmSV01 did not affect the germination process (i.e., swelling of the seed coat and physiological revival of the embryo) or the general appearance of the young growing tissues compared to control plants, where no morbid symptoms were observed (*p* value = 0.571) (Fig. 3; Supplemental Fig. S1). Consequently, its non-pathogenic nature on wheat allows us to further explore its potentially endophytic behavior and its PGP and BCA attributes.

## 3.2. *P. macrospinosa* PmSV01 is a beneficial PGP endophyte for wheat

### 3.2.1. *P. macrospinosa* PmSV01 promotes seedling growth and related physiological processes

The plant growth-promoting (PGP) activity of *P. macrospinosa* PmSV01 was observed through an *in planta* experiment conducted in growth chamber. The length of the aerial parts was measured at different developmental time intervals to assess seedling growth dynamics (Fig. 4). Following germination, the length of the aerial organs in primed
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Inhibition actions of *Periconia macrospinosa* PmSV01 against *Fusarium graminearum* Fg1 in an axenic environment. Inhibitions were assessed through dual confrontation using three culture modalities: direct physical contact on the same PDA media (*physical contact*), no physical contact on separated PDA media (*VOCs*), and no physical contact with successive mycelial growths on the same PDA medium (*Non-volatile secondary metabolites-SMs*). Photographs show cultures after 4 days (*B*) and 7 days (*C*) of *F. graminearum* Fg1 growth in the presence or absence of *P. macrospinosa* PmSV01. Cultures were maintained at 25 °C in the dark. Data (*A*) are presented as means and standard errors from 7 biological replicates. Asterisks indicate significant differences from the controls, based on One-way ANOVA with Tukey's HSD test (*p* < 0.05), further refined by Bonferroni-adjusted *p*-values, where "*" ≤ 0.05, "**" ≤ 0.01, "***" ≤ 0.001, and "****" ≤ 0.0001. Scale bars in (*B*) and (*C*) represent 1 cm. BS, back side of the Petri dish; US, upper side. The evolution of fungal cultures after 1 month of confrontation is detailed in Supplemental Fig. S2.</figcaption></figure>

seedlings was initially reduced by approximately 10 % (p = 0.015) compared to control seedlings (Fig. 4A). This reduction was evident immediately after the seedling emerged from the soil, but it was transitory. After 6 days of growth, the opposite effect was observed: the length of the primed seedlings became significantly greater than that of the control plants, showing an increase of approximately 10 % after one (p = 0.017) and two weeks (p = 0.00057) of growth. Interestingly, similar trends were observed 21 days post-inoculation, with fresh and dry weights of the aerial parts increasing by 15 % (p = 0.021) and 33 % (p = 4.01 × 10⁻⁰⁴), respectively (Table 2). Likewise, root length showed a significant increase of 15 % (p = 0.0017), resulting in a 13 % increase in fresh weight (though not statistically significant, p > 0.05) and a 24 % increase in dry weight (p = 0.0035), compared to the control seedlings' roots. Additionally, the average number of tillers increased by 19 % (p = 5.12 × 10⁻⁰⁵). These increases reflect findings from several studies involving various PGPMs, whether described as endophytes or not [23, 45,46]. All these morphometric variables, enhanced by *P. macrospinosa*

PmSV01, are visually represented in Fig. 4B.

*P. macrospinosa* PmSV01 enhances systemic plant morphological performance, which is driven and regulated by numerous intricate physiological processes and underlying molecular mechanisms. The results showed that these positive effects co-varied with a significant increase in chlorophyll by 18 % (*p* < 10⁻¹²), epidermal flavonol by 22 % (*p* < 10⁻¹²), and the nitrogen balance index by 18 % (*p* = 1.25 × 10⁻⁰⁸) after 21 days of seedling growth (Table 2). These increases were associated with enhanced photosynthetic and nitrogen assimilation efficiencies, which likely explain the observed improvements in seedling growth, both in the aerial and root systems, as shown in Fig. 4 and Table 2. These biostimulatory effects on wheat growth are promising. From a broader, holistic perspective, and particularly in light of increasing concerns over the substantial impact of global climate change on agricultural productivity, recent studies emphasize the potential of incorporating dark septate endophytes (DSEs) into sustainable, microbe-based agricultural strategies to promote plant growth, including certain
<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Protective ability of *Periconia macrospinosa* PmSV01 against *Fusarium graminearum* Fg1 on the growth dynamics of wheat seedlings over 21 days of interaction (A), and at 21 days after culture (B, C). *P. macrospinosa* PmSV01 was inoculated with a solution of crushed mycelium from a 2-week-old culture, and *F. graminearum* Fg1 was inoculated at $10^5$ spores ml$^{-1}$. The scale bar in the micrographs represents 5 cm. C, control seedlings. Data are presented as means and standard errors from 55 biological replicates. Asterisks denote significant differences from the controls, according to One-way ANOVA with Tukey's HSD test ($p < 0.05$), further refined by Bonferroni-adjusted *p*-values, where "*" ≤ 0.05, "**" ≤ 0.01, "***" ≤ 0.001, and "****" ≤ 0.0001.</figcaption></figure>

*P. macrospinosa* strains [16–18]. As observed with many beneficial fungi, the underlying mechanisms responsible for this positive effect are multifaceted and are often associated with enhanced nutrient acquisition by the host plant [47,48]. This is largely attributed to the secretion of a diverse enzymatic arsenal capable of degrading plant cell walls, along with the production of various phytohormone analogs. These enzymatic activities facilitate the breakdown of complex organic substrates, which are ultimately assimilated through a network of transporters, including pumps and channels, and processed via downstream metabolic pathways, thereby supporting nutrient uptake under conditions of severe nutrient limitation.

### 3.2.2. The initiation of the wheat/PmSV01 interaction is a high-energy physiological event for the host

Our results showed that *P. macrospinosa* PmSV01 reduced seedling growth velocity immediately after seed germination, a priori during the initial phase of the interaction. Nevertheless, this effect proved to be transient, with a rapid reversal observed after six days of post-germination development, as previously reported in the analysis of Fig. 4A. Very few studies report such a marked reduction in plant growth within the context of a beneficial interaction [49,50], and none involving a DSE. Several hypotheses can be proposed, but it is highly probable that during the establishment of this interaction between plants and beneficial endophytes, the plants exude a wide array of chemicals, some of which play key roles in the recruitment of microorganisms [51]. Additionally, during the interaction, the endophyte, which is a carbon-heterotrophic organism, exchanges minerals for sugars derived from a rerouting of the carbon reserves in the seed endosperm and/or the photosynthetic activities initiated by the very young seedlings, which are also potentiated by the endophyte [52]. The redistribution of organic matter between partners, along with the host's production of exudates, imposes a significant carbon and nitrogen energy cost on the plant, leading to a slight but temporary reduction in post-germination growth [49,50]. Similarly, this reduction may also be explained by the induction of plant defense mechanisms, which imposes a substantial energetic demand on the host [53,54].

It is noteworthy that the trade-off between effective endophyte control, cost-effectiveness, and minimal growth inhibition remains a major challenge in fully exploiting the potential of a PGP candidate for agricultural applications. However, this negative effect is transient, and *P. macrospinosa* PmSV01 can still be considered as a promising plant growth-promoting candidate.

### 3.3. Biocontrol activity of *P. macrospinosa* PmSV01 on *F. graminearum* Fg1 in axenic condition and in planta

Beneficial microorganisms display several antagonistic strategies against plant pathogens. These strategies include two main actions: (i) a direct antagonistic action, by limiting the pathogen's development, and (ii) an indirect effect on the phytopathogen, through the elicitation of an effective "host immunization", resulting in the potentiation of the plant's overall physiological status and the transcriptional expression of a wide range of resistance-related genes. These two non-exclusive actions are studied in an axenic environment and in planta, respectively.

#### 3.3.1. *P. macrospinosa* PmSV01 exhibits bio-fungicidal abilities against *F. graminearum* Fg1 in axenic conditions

A dual confrontation assay was conducted to investigate the antagonistic activity of *P. macrospinosa* PmSV01 against *F. graminearum* Fg1. This assay facilitated direct interaction between the two organisms, sharing the same culture medium and atmosphere, allowing for potential physical contact. The results showed a significant inhibition of *F. graminearum* Fg1 mycelial growth, with an inhibition rate of 39 % ($p = 5.02 \times 10^{-14}$) compared to the control plates (Fig. 5ABC). The growth rate of the *F. graminearum* Fg1 mycelium progressively decreased over time and ceased upon physical contact with the mycelium of *P. macrospinosa* PmSV01. Interestingly, neither fungus outcompeted nor overgrew the other during the course of the assay (Supplemental Fig. S2).

Additional tests were conducted to further elucidate this antagonistic activity, focusing on the emissions of volatile organic compounds (VOCs) and non-volatile or diffusible secondary metabolites (SMs) (Fig. 5AB). Each assay demonstrated a significant inhibitory effect on the mycelial growth of the pathogen after 9 days of incubation. The results confirmed the diffusion of non-volatile secondary metabolites in the medium, with an inhibition rate of 85 % ($p < 10^{-15}$) (Fig. 5AB), and the production of volatile organic compounds, resulting in an inhibition rate of 28 % ($p = 1.69^{-11}$) (Fig. 5AB). These findings highlight the multifaceted antagonistic mechanisms employed by *P. macrospinosa* PmSV01.

In addition to melanin production, which diffuses slightly into the medium, more than a hundred bioactive secondary metabolites from the genus *Periconia* have been discovered. Some of these organic compounds exhibit substantial antiseptic and antioxidant activities [55–58]. *P. macrospinosa* PmSV01 regulates the mycelial growth of
<figure><img src="image_10.png" /><figcaption>**Fig. 7.** Transcript abundance of defense-encoding genes in wheat. Transcript abundances were measured in crown and root tissues of wheat at 21 days after inoculation with *P. macrospinosa* PmSV01 and/or *F. graminearum* Fg1. Gene transcript levels were quantified by real-time qRT-PCR and are presented relative to control levels using the 2⁻⁵ΔΔCT method. Color scales represent level of transcriptional expression, with green indicating up-regulation and red indicating down-regulation. Different letters denote significant differences between untreated and treated samples for each organ, based on four independent biological replicates, according to One-way ANOVA with Tukey's HSD test (*p* < 0.05), further refined by Bonferroni-adjusted *p*-values. Values relative to means and standard errors are detailed in Fig. S3.</figcaption></figure>

*F. graminearum* Fg1 through the production of both non-volatiles and volatiles secondary metabolites. It may also inhibit growth by occupying space. Although these confrontations tests were conducted *ex planta*, the results suggest that *P. macrospinosa* PmSV01 can act as both a physical and chemical barrier to the growth of *F. graminearum* Fg1. Given that this beneficial fungus is capable of being a facultative endophyte, it may thus inhibit (or delay) the pathogen's penetration into the root vasculature.

### 3.3.2. *F. graminearum* Fg1 aggressiveness on wheat

Inoculation of *F. graminearum* Fg1 significantly affected seed germination and plant growth over the 21-days of the experiment.

Germination performance of the Fg1-infested seeds decreased by 22%, as evidenced by the failure of integument swelling or rapid germinant collapse (Fig. 6AB). Among the surviving seedlings, most exhibited a pronounced slowdown in aerial growth: a reduction of 49% (*p* < 10⁻¹⁵) at 4 days, and around 25% (*p* < 10⁻¹⁵) at 8 and 21 days post-infestation. Root length also decreased significantly, by around 24% (*p* = 3.73 × 10⁻⁰⁷) at 21 days (Table 3). This negative effect was particularly pronounced in the fresh and dry weights of these organs, with average reduction of around 56% (*p* = 1.33 × 10⁻¹⁵) and 46% (*p* = 6.99 × 10⁻⁰⁷), respectively, for the aboveground parts, and around 66% (*p* = 1.33 × 10⁻¹⁵) and 62% (*p* = 4.76 × 10⁻¹³), respectively, for the roots. Similarly, the number of tillers dropped significantly by 29% (*p* = 1.21
<table><caption>**Table 2**
Effect of *Periconia macrospinosa* (PmSV01) on ecophysiological variables in wheat seedlings after 21 days of culture. Controls seedlings were treated with sterile PDB medium. *P. macrospinosa* PmSV01 was inoculated at a concentration of < 10³ spores ml⁻¹. A total of 55 plants per biological condition were assessed for measurements. Asterisks indicate significant differences from the controls, based on one-way ANOVA with Tukey's HSD test (p < 0.05), further refined by Bonferroni-adjusted *p*-values, where "³" ≤ 0.05, "**" ≤ 0.01, "***" ≤ 0.001, and "****" ≤ 0.0001.</caption><thead><tr><th>Ecophysiological variables</th><th>Control</th><th>PmSV01</th></tr></thead><tbody><tr><td>Leaf fresh weight (mg)</td><td>2.106 ± 0.034</td><td>2.433 ± 0.048<sup>a</sup></td></tr><tr><td>Leaf dry weight (mg)</td><td>0.219 ± 0.034</td><td>0.291 ± 0.047***</td></tr><tr><td>Root fresh weight (mg)</td><td>1.347 ± 0.038</td><td>1.529 ± 0.052<sup>ns</sup></td></tr><tr><td>Root dry weight (mg)</td><td>0.161 ± 0.038</td><td>0.201 ± 0.052**</td></tr><tr><td>Root length (cm)</td><td>16.28 ± 0.19</td><td>18.75 ± 0.27**</td></tr><tr><td>Tiller number per seedling</td><td>3.34 ± 0.09</td><td>3.98 ± 0.09***<sup>a</sup></td></tr><tr><td>Chlorophyll (µg cm<sup>-2</sup>, Dualex®)</td><td>32.33 ± 0.58</td><td>38.29 ± 0.57***<sup>a</sup></td></tr><tr><td>Flavonols (Index, Dualex®)</td><td>0.549 ± 0.009</td><td>0.667 ± 0.007***<sup>a</sup></td></tr><tr><td>NBI<sup>a</sup> (Index, Dualex®)</td><td>51.89 ± 0.84</td><td>61.44 ± 0.89***<sup>a</sup></td></tr></tbody></table>

<table><caption>**Table 3**
*Periconia macrospinosa* PmSV01's protective ability against *Fusarium graminearum* Fg1 through modulation of ecophysiological variables in wheat seedlings after 21 days of culture. Controls seedlings were treated with sterile PDB medium. *P. macrospinosa* PmSV01 was inoculated with a solution of crushed mycelium from a 2-week-old culture, and *F. graminearum* Fg1 at 10⁵ spores ml⁻¹. A total of 55 plants was evaluated under each biological condition. Asterisks indicate significant differences from the controls, according to one-way ANOVA with Tukey's HSD test (p < 0.05), further refined by Bonferroni-adjusted *p*-values, where "³" ≤ 0.05, "**" ≤ 0.01, "***" ≤ 0.001, and "****" ≤ 0.0001.</caption><thead><tr><th>Ecophysiological variables</th><th>Control</th><th>Fg1</th><th>PmSV01 + Fg1</th></tr></thead><tbody><tr><td>Leaf fresh weight (mg)</td><td>2.106 ± 0.034</td><td>0.946 ± 0.054***<sup>a</sup></td><td>1.88 ± 0.051<sup>ns</sup></td></tr><tr><td>Leaf dry weight (mg)</td><td>0.219 ± 0.034</td><td>0.118 ± 0.054***<sup>a</sup></td><td>0.209 ± 0.050<sup>ns</sup></td></tr><tr><td>Root fresh weight (mg)</td><td>1.347 ± 0.038</td><td>0.457 ± 0.023***<sup>a</sup></td><td>0.948 ± 0.024***<sup>a</sup></td></tr><tr><td>Root dry weight (mg)</td><td>0.161 ± 0.038</td><td>0.062 ± 0.022***<sup>a</sup></td><td>0.119 ± 0.023**</td></tr><tr><td>Root length (cm)</td><td>16.28 ± 0.19</td><td>12.35 ± 0.35**</td><td>14.35 ± 0.28<sup>a</sup></td></tr><tr><td>Tiller number per seedling</td><td>3.34 ± 0.09</td><td>2.38 ± 0.11***<sup>a</sup></td><td>3.54 ± 0.12<sup>ns</sup></td></tr><tr><td>Chlorophyll (µg cm<sup>-2</sup>, Dualex®)</td><td>32.33 ± 0.58</td><td>16.36 ± 0.99***<sup>a</sup></td><td>35.36 ± 0.65<sup>a</sup></td></tr><tr><td>Flavonols (Index, Dualex®)</td><td>0.549 ± 0.009</td><td>0.441 ± 0.013***</td><td>0.61 ± 0.008***</td></tr><tr><td>NBI<sup>a</sup> (Index, Dualex®)</td><td>51.89 ± 0.84</td><td>28.11 ± 1.66***<sup>a</sup></td><td>56.28 ± 1.02<sup>a</sup></td></tr></tbody></table>

× 10⁻⁰⁸). These symptoms of reduced seedling growth are plausibly due to colonization of the stem by *F. graminearum* mycelium, which has been reported to inhibit the flow of water and nutrients [59]. Collaterally, these inhibitions impacted several physiological processes, as shown by the decreased chlorophyll and flavonol contents: 49 % (p < 10⁻⁰⁵) and 19 % (p = 1.51 × 10⁻¹²), respectively, and NBI: 46 % (p < 10⁻¹⁵) (Table 3).

These initial morphological symptoms are followed as necrosis at the basal part of the leaf sheaths, which extends to the base of the crown and the young internode, eventually affecting all internodes and roots. In this experiment, we focused specifically on the early events associated with the onset of Fusarium crown rot (FCR), characterized initially by a slowdown in plant growth (S1 score), followed by the appearance of the first necrotic lesions at the base of the crown and on the young internode (S2 score) (Fig. 6C). S1 symptoms emerged immediately after the germination of certain seeds and rapidly became predominant, with 58 % of seedlings exhibiting infection symptoms by 8 days post-

inoculation. Progression to S2 symptoms was observed in 9 % and 57 % of seedlings at 14 and 21 days post-inoculation, respectively. Subsequently, symptom development intensified markedly, with all seedlings inoculated with *F. graminearum* Fg1 exhibiting characteristic FCR symptoms by the end of the experiment.

### 3.3.3. *P. macrospinosa* PmSV01 significantly mitigates *F. graminearum* Fg1 aggressiveness on wheat

*F. graminearum* Fg1 manifested only very slight aggressiveness on PmSV01-primed seedlings, highlighting the ability of *P. macrospinosa* PmSV01 to significantly protect seedlings against FCR disease. When PmSV01 and Fg1 were co-inoculated and compared to control seedlings treated with sterile water, a slight alteration in the growth dynamics of the aerial part was observed throughout the experiment: approximately 20 % reduction (p = 1.49 × 10⁻⁰⁷) at 4 and 8 days, 5 % (not statistically significant; p > 0.05) at 2 weeks, and 7 % (p = 6.91 × 10⁻⁰⁵) at 3 weeks (Fig. 6AB, Table 3). This negative effect was observed only with the fresh and dry weights of the roots, with reductions of 29 % (p = 6.88 × 10⁻⁰⁵) and 26 % (p = 0.0015), respectively, at 21 days. Despite these negative trends, the fresh and dry weights of the aerial parts did not statistically differ from those of the control seedlings. Similarly, root length decreased slightly by 12 % (p = 0.024). The number of tillers remained similar to that of control seedlings. Interestingly, these protective effects were even more evident at the ecophysiological level, as exemplified by increased chlorophyll and flavonol contents: 9 % (p = 0.038) and 11 % (p = 3.53 × 10⁻⁰⁵), respectively, and NBI: 8 % (p = 0.031), compared to the control seedlings.

This slight reduction in growth, corresponding to the S1 score, was attributable to the aggressiveness of *F. graminearum* Fg1. However, it remained very limited compared to plants infected with the pathogen alone. Indeed, symptom severity did not increase in most plants, and fewer than 3 % of PmSV01-primed seedlings infested by *F. graminearum* Fg1 developed extensive necrosis on their crowns (S2 score) at 21 dpi (Fig. 6C).

Collectively, the morphometric and ecophysiological data clearly provide clear evidence that *P. macrospinosa* PmSV01 is capable of delaying and mitigating the onset of FCR symptoms in wheat, including the absence of necrotic tissue collapse in the host. While interactions between the fungal partners may occur both externally and within host tissues -and while it is plausible that *P. macrospinosa* PmSV01 negatively modulates the molecular mechanisms underlying the aggressiveness of *F. graminearum* Fg1-one potential explanation for this protective effect lies in the enhancement of overall seedlings physiological performance, as initially reflected by improved growth. Nonetheless, a key physiological process to further explore in the context of plant-beneficial microbe interactions is the regulation of plant defense pathways, which will be addressed in the following paragraph.

### 3.3.4. *P. macrospinosa* PmSV01 reorganizes the plant's defensive machinery initially deregulated by *F. graminearum* Fg1

Transcriptional regulation of candidate genes involved in stress defense and growth processes was quantified in the crown and roots at 21 days under five biological conditions: SDW control seedlings, PmSV01-primed seedlings, Fg1-infected seedlings displaying S1 or S2 symptom scores, and Fg1-PmSV01-co-inoculated seedlings (S1 score only). Several common gene markers were studied, covering different defense pathways such as oxidative stress (involving ROS generation and antioxidative machinery), accumulation of secondary metabolites (phenylpropenes, terpenes, cyanogens, and PR-proteins), cell wall strengthening, water stress, and phytohormone biosynthesis and signaling associated with abscisic acid (ABA), cytokinins (CK), salicylic acid (SA), ethylene (Et), and jasmonic acid (JA).

Plant defenses are highly diverse, and the regulatory pathways involved are also divers and inherently complex. Their regulation depends on the recognition of bioagents, which in our study includes both beneficial (*P. macrospinosa* PmSV01) and pathogenic (*F. graminearum*

<footnote>a NBI, Nitrogen Balance Index.</footnote>

<footnote>a NBI, Nitrogen Balance Index.</footnote>
Fg1) agents that interact either alone or synergistically with the host. In this context, the effective promotion of immunity and the appropriate cellular response to disease, depending on whether the interaction is with beneficial endophytes or pathogens, involves the contrasting up-regulation or down-regulation of numerous genes operating within a highly complex network. It would be inappropriate to interpret the relationship based on any single marker, especially since most belong to multigene families with members that may exhibit contrasting expression levels. Nevertheless, each trend remains highly informative. Consequently, the modulation of these markers was analyzed within a broader functional context, considering the various metabolic pathways to which they are linked and, when applicable, the probable interactions between them (Fig. 7, Fig. S3).

Four general observations can be highlighted: (i) all gene markers were modulated by both fungi, (ii) *F. graminearum* Fg1 induced gene expression differently depending on the severity of seedling infection (i. e., scores 1 and 2) over time, (iii) *P. macrospinosa* PmSV01, whether inoculated alone or co-inoculated with *F. graminearum* Fg1, attenuated or reversed several transcriptional expression trends induced by *F. graminearum* Fg1 when inoculated alone, and (iv), very few transcriptional modulations were tissue-dependent.

In light of this preamble, certain transcriptional modulations are worth noting, as they offer insight into the molecular mechanisms underlying the potentiation of immune responses, potentially driven by *P. macrospinosa* PmSV01. For example, oxidative stress and water stress are considered driving forces in Fusarium disease [60–63]. In our pathosystem, a significant increase in reactive oxygen species (ROS), particularly oxygen radicals, can be hypothesized. This is illustrated by the transcriptional up-regulation of genes involved in superoxide anion (O₂⁻) accumulation (NADPH oxidase, *nox*) in the apoplast, and the transcriptional down-regulation of genes involved in ROS scavenging (Superoxide dismutase, *sod*; Catalase, *cat*; Glutathione S-transferase, *GST*; and Ascorbate peroxidase, *apx*) and diffusion (Aquaporin, *aqp*). These down-modulations coincide with a reduction in flavonol content (Table 3), as these plant pigments have pleiotropic actions, such as protection against oxidative damage. Oxidative stress is a complex chemical and physiological phenomenon resulting from the overproduction and accumulation of ROS, including superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂), and hydroxyl radicals (OH⁺). While ROS are constitutively produced under normal circumstances, excessive ROS production that overloads the cell’s antioxidant machinery can cause cellular damage through lipid peroxidation, protein oxidation, and DNA damage. Depending on the extent of oxidation, this can lead to a substantial reduction in the plant’s metabolic performance, including growth and immunity, and ultimately result in cell death. The elicitation of oxidative cell collapse is a characteristic of several necrotrophic pathogens, which hijack and exploit the primary function of oxidative stress in the establishment of resistance -namely, the hypersensitive reaction-to colonize the host tissues and extract cytosolic content for trophic purposes [62–65]. *P. macrospinosa* PmSV01 alone is more likely to induce these oxidative markers, although the increase in their expression was not significantly different from that in control seedlings. In fact, *P. macrospinosa* PmSV01 behaves like an avirulent symbiont, theoretically part of an incompatible -and therefore resistant-interaction. It is noteworthy that under co-inoculation conditions with *F. graminearum* Fg1, oxidative stress appears to be considerably attenuated, with inhibition of *nox* and activation of markers involved in the Ascorbate-Glutathione cycle (*apx* and *gr*). This creates a suitable oxidative environment that hinders the detrimental pro-oxidative molecular mechanisms in host cells, thereby affecting the establishment of FCR disease. Furthermore, the expression of ROS producers such as Oxalate oxidase (*oxo*) and Peroxidase (*pod*) was either slightly affected or not impacted, while *sod* was up-regulated when *cat* was down-regulated. This molecular configuration would theoretically lead to H₂O₂ accumulation. Among ROS, H₂O₂ is particularly significant: while it acts as a biocide at high concentrations, at low

concentrations it integrates into the complex phytohormonal balance that regulates several physiological processes, including plant immunity [66]. The moderate levels of transcriptional regulation of the genes encoding these enzymes would favor a putative scenario where a cell-lethal oxidative burst is attenuated, allowing for the elicitation of H₂O₂-dependent signaling that contributes to effective resistance.

The second driving force behind the Fusarium diseases is related to water stress [60,61]. Water, along with a plethora of low-molecular-weight solutes, crosses membranes through specialized channels called aquaporins [67]. The *aqp*-encoding gene was significantly inhibited by *F. graminearum* Fg1, but remained non-affected by *P. macrospinosa* PmSV01, whether inoculated alone or co-inoculated with the pathogen. This result suggests that a pathogen-induced water stress occurs very early in the compatible interaction. As observed for oxidative stress, the beneficial partner mitigates this disease mechanism. This hypothesis is supported by the expression of three gene markers that are involved in maintaining water homeostasis in stressed cells: Late embryogenesis abundant (*lea*), Dehydrin 9 (*dhn9*), and Salt stress protein (*sst*). These genes encode stress proteins that help preserve cell homeostasis and membrane fluidity, which are significantly compromised during salinity- or drought-induced water stress [8]. This also explains why PmSV01-primed seedlings exhibited similar development to that of control seedlings. A final point to emphasize concerns AQPs, particularly those with peroxyporin activities, which regulate the diffusion of hydrogen peroxide (H₂O₂) [67]. H₂O₂ is a small, uncharged, and relatively long-lived molecule produced in the apoplast under physio-phytopathological conditions. Although it is relatively poor at spontaneously permeating membranes, peroxyporins facilitate its transport [68,69]. Thus, it is highly relevant to correlate the disease mechanisms associated with pro-oxidant environment and water deprivation –with water stress itself triggering ROS overproduction-with the gene reprogramming mechanisms employed by the beneficial biocontrol agent, enabling the plant to resist *F. graminearum* Fg1.

Reactive oxygen species are part of the array of potentially toxic secondary metabolites and proteins produced by infested plants to inhibit pathogen establishment and/or development. Three metabolic pathways were studied: phenylpropanoid (Phenylalanine ammonia lyase, *pal*; Chalcone synthase, *chs*; Benzoate 4-monoxygenase, *b4mo*; Polyphenol oxidase, *ppo*; Caffeic acid O-methyltransferase, *comt*; Cinnamyl alcohol dehydrogenase 1, *cad1*; Cinnamyl Coenzyme Reductase3, *ccr3*; and Glucan synthase-like 22, *gsl22*), terpenoid (Farnesyl pyrophosphate synthase, *fps*; and Sesquiterpene Synthase, *sqts*), and cyano-genesis (β-Glucosidase, *bdg2*). In addition, the accumulation of Pathogenesis-related (PR) proteins (Oxalate oxidase, *oxo*; *pr-1*; Glucanases, *glu*; Chitinases, *cht3*; Thaumatin like-protein, *pr-5*; Peroxidase, *pod*; Defensins, *def*; and Lipid transfert proteins, *ltp*) was quantified. All these defensive mechanisms were significantly modulated by *F. graminearum* Fg1, with markedly contrasting patterns depending on the degree of the disease. They were mainly up-regulated in seedlings showing growth slowdown (S1 score) and down-regulated in seedlings showing the first necrosis at the base of the crown and in the young internode (S2 score). However, some gene markers were up-regulated (*pr-1*, *cht3*, *ltp*, *pod*, *pal*, *ppo*, *comt*, *bdg2*, *cad1*, and *ccr3*) or strongly down-regulated (*ltp*, *comt*, and *cad1*) under both biological conditions. These genes are presumably involved in defense activities against *F. graminearum*, as demonstrated by previous study [70]. Interestingly, some of these markers were significantly modulated by *P. macrospinosa* PmSV01, with some being up-regulated (*cht3*, *pr-5*, *def*, *b4mo*, *fps*, and *sqts*) or down-regulated (*oxo*, *pr-1*, *glu*, and *ccr3*). These modulations differed substantially from those caused by *F. graminearum* Fg1 alone (with the exception of *def*) and more closely resembled those observed when the beneficial fungus was co-inoculated with the pathogen. As observed with oxidative stress responses, it can be hypothesized that gene modulation triggered by the beneficial agent impacts *F. graminearum* Fg1 pathogenicity, thereby contributing to seedling resistance.
Pathogens and beneficial microorganisms profoundly influence the overall phytohormonal balance in plants, affecting both local and systemic signaling responses to stress and development. These responses are orchestrated by a complex phytohormonal network that supplements or amplifies the primary signals arising from direct microbial recognition. This network includes SA- and JA/Et-mediated signaling, which are responsible for Systemic Acquired Resistance (SAR) and Induced Systemic Resistance (ISR), respectively, making plants resistant to a broad spectrum of pathogens. Additionally, ABA and CKs are involved in growth and tolerance to abiotic stress, such as salinization and drought. *F. graminearum* Fg1 and *P. macrospinosa* PmSV01 modulated genes encoding enzymes involved in their metabolism and signaling. Reprogramming of gene expression by *P. macrospinosa* PmSV01 in the presence of *F. graminearum* Fg1 primarily involved the JA pathway. Additionally, we can highlight plausible connections between several molecular events that may initially appear independent. The inhibition of the antioxidant system induces severe oxidative damage, leading to lipid peroxidation of membranes [71]. The breakdown of lipid membranes releases fatty acid fragments that serve as substrates for Lipoxygenases (*lox*) [72]. In our assay, the *lox* marker was strongly induced by *F. graminearum* Fg1 and to a lesser extent by *P. macrospinosa* PmSV01. Similar patterns were observed for Allene oxide cyclase (*aoc*) and a Mitogen-activated protein kinase (*wck1*), both of which are part of the metabolic pathway linked to the production of JA and their conjugated forms (methyl jasmonate and jasmonic acid isoleucine), and the JA-signaling pathway, respectively. This JA-signaling trio plays an essential role in plant defense mechanisms by regulating the expression of several JA-associated genes that confer resistance to biotic stress [73]. However, the fact that *F. graminearum* Fg1 up-regulated JA markers more strongly than the beneficial agent indicates that certain *Fusarium* spp. may hijack the JA-signaling pathway for disease development [74–76]. This could occur by blocking the SA-Et signaling pathways, as observed in our assay with the strong inhibition of related markers (*npr3*, *eds1*, *mapk3*, *aco*, and *erf3* genes), particularly at the S2 disease score, or by providing necessary intermediates required by the pathogen for disease progression [77,78].

We observed that some markers were specifically inhibited by *P. macrospinosa* PmSV01 in the crown and/or roots. This is exemplified by three PR-proteins: *oxo*, *pr-1*, and *glu*, as well as three enzymes involved in cell wall reinforcement (*cad*, Cinnamyl alcohol dehydrogenase, and *ccr3*, Cinnamyl coenzyme reductase 3) and callose apposition (gsl22, Glucan synthase-like 22). These strong inhibitions, along with defensive genes that were weakly or not modulated in PmSV01-primed seedlings but were strongly activated by the pathogen, could be associated with the successful, long-lasting establishment of *P. macrospinosa* PmSV01 on and/or in these organs; this also echoes its facultative epi-/endophytic behavior (Fig. 3). These transcriptional responses are highly instructive and clearly demonstrate *P. macrospinosa* PmSV01's abilities to (i) adapt and perpetuate appropriate cellular responses in its host over time, as an avirulent symbiont would, and (ii) reprogram pathogen-elicited cellular susceptibility responses to support resistance. These results further underscore the complexity of the molecular dialogues that shape the relationships between plants and their beneficial microorganisms.

Overall, we present some host molecular responses that help explain the establishment of FCR disease by *F. graminearum* Fg1. Interestingly, when these responses are reprogrammed by a “third-party” beneficial agent, they may contribute to the development of plant resistance against the same pathogen. Therefore, some of these markers, which can be considered potential plant susceptibility genes, may be key targets for manipulation to enhance crop resistance against *Fusarium* [79,80]. Likewise, these transcriptomic results further our understanding of *P. macrospinosa*'s ability to improve overall plant physiological performance, particularly in terms of immunity and resistance to *F. graminearum*. These findings should be viewed within the context of broader phytoprotective abilities. For example, *P. macrospinosa*

enhances nitrogen uptake from various organic source [16], and alleviate drought and salinity stresses by promoting the synthesis of free osmoprotectants and antioxidants in plants [17,18].

## 4. Conclusion

Nature offers a remarkable diversity of beneficial microorganisms (PGP and BCA) and eco-friendly phytosanitary solutions that could potentially replace chemical inputs (fertilizers and pesticides) for regulating plant diseases. In this study, we demonstrated that priming wheat seedlings through the seed inoculation with *P. macrospinosa* PmSV01 provided significant advantages under cryptogamic disease conditions, as evidenced by a marked reduction in the physiological symptoms of FCR caused by *F. graminearum* Fg1. Multiple physiological mechanisms likely contribute to this observed FCR resistance. They may include the ability of *P. macrospinosa* PmSV01 to colonize the host's roots in a manner analogous to that of an avirulent symbiont, to enhance the host's overall physiological status, to elicit or reprogram defense responses that would otherwise be hijacked by the pathogen. These *in planta* findings are supported by axenic culture experiments, which highlight the ability of *P. macrospinosa* PmSV01 to produce secondary metabolites capable of inhibiting the mycelial growth of *F. graminearum* Fg1. While these findings are promising, they remain preliminary. The complementary mechanisms identified in this study now require correlative validation to determine whether they act synergistically *in planta* to establish a broadly mycotoxic environment that impedes root colonization by *F. graminearum* Fg1. Future studies are required to gain more comprehensive understanding of the individual mechanisms through which *P. macrospinosa* PmSV01 exerts its PGP and BCA activities in wheat, particularly under field conditions. This will ultimately allow for the assessment of the broader applicability of these capabilities across a range of major agronomic crops exposed to both biotic and abiotic stresses.

### CRediT authorship contribution statement

**Mouadh Saadaoui:** Writing – original draft, Methodology, Investigation, Conceptualization.
**Théo Morlevat:** Writing – original draft, Formal analysis, Data curation.
**Olivier Loison:** Methodology, Formal analysis, Data curation.
**Christelle Blavignac:** Methodology, Formal analysis, Data curation.
**Mohamed Faize:** Writing – review & editing.
**Lalla Aicha Rifai:** Writing – original draft.
**Chaimae Nouri:** Methodology, Formal analysis, Data curation.
**Jamila Dich:** Methodology, Formal analysis, Data curation.
**Ninon Magliaraschi:** Methodology, Formal analysis, Data curation.
**Géraldine Philippe:** Methodology, Formal analysis, Data curation.
**Ludovic Bonhomme:** Writing – review & editing, Data curation.
**Jean-Stéphane Venisse:** Writing – review & editing, Supervision, Resources, Project administration, Methodology, Funding acquisition.

### Ethics approval and consent to participate

There are no ethical concerns regarding the organisms and the topic of this research: this article does not contain any studies with human participants or animals (vertebrates) performed by any of the authors.

### Availability of data and materials

The microbiological material and all the data generated or analyzed as part of this study are fully available to scientists from the corresponding author on request.

### Funding

*P. macrospinosa* PmSV01 strain was isolated and molecularly characterized as part of a PhD thesis (Mouadh SAADAoui, 2023/16/11),
under the project "Pack Ambition International 2019" [ANR grant No. P0100003] co-funded by the University of Clermont Auvergne and the French Auvergne-Rhônes-Alpes Region. The authors also acknowledge financial support from the Fédération des Recherches en Environnement - Clermont-Ferrand. Pr Mohamed FAIZE was supported by funding from the Ministère de l'Education Nationale, de la Formation Professionnelle, de l'Enseignement Supérieur et de la Recherche Scientifique (MENF- PESRS, Morocco; Agreement No. 3), within the framework of a PRIMA Project (UToPIQ-PCI2021-121941). Chaimae NOURI was supported by funding from the "Centre Nationale de la Recherche Scientifique et Technique" (CNRST, Morocco), within the framework of PhD Associate Scholarship (PASS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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

The authors are grateful to Céline SAC for their technical assistance with plant cultures and molecular biology, respectively, and Jérôme FRANCHEL for his technical assistance in the molecular characterization of *P. macrospinosa* PMSV01. The authors express their sincere gratitude to all financial partners: Fédération des Recherches en Environnement, University of Clermont Auvergne, Auvergne-Rhônes-Alpes Region, Ministère de l'Education Nationale, de la Formation Professionnelle, de l'Enseignement Supérieur et de la Recherche Scientifique, and PASS. We are indebted to the two anonymous reviewers for their helpful suggestions.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.pmpp.2025.102995.

Data availability

Data will be made available on request.

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