# A workflow for sourcing and characterizing wheat field microorganisms for the biocontrol of Fusarium head blight

Toan Bao Hung Nguyen, Flora Pensec, Jérôme Mounier, Monika Coton, Amandine Henri-Sanvoisin, Massilia Mouzaoui, Adeline Picot

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Toan Bao Hung Nguyen, Flora Pensec, Jérôme Mounier, Monika Coton, Amandine Henri-Sanvoisin, et al.. A workflow for sourcing and characterizing wheat field microorganisms for the biocontrol of Fusarium head blight. Biological Control, 2025, 209, pp.105856. 10.1016/j.biocontrol.2025.105856. hal-05386700

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# A workflow for sourcing and characterizing wheat field microorganisms for the biocontrol of Fusarium head blight

Toan Bao Hung Nguyen, Flora Pensec, Jérôme Mounier, Monika Coton, Amandine Henri-Sanvoisin, Massilia Mouzaoui, Adeline Picot*

Univ Brest, INRAE, Laboratoire Universitaire de Biodiversité et Écologie Microbienne, F-29280 Plouzané, France

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

## HIGHLIGHTS

* A workflow to screen field microorganisms for FHB biocontrol was developed.

* A large collection of 1231 bacteria, 365 molds, and 39 yeasts was built.

* Intra and interspecific *Fusarium* diversity was considered during screening.

* One *Trichoderma gamsii* isolate allowed complete inhibition of *Fusarium*.

* Other isolates exhibited promising activity albeit a narrower spectrum.

## ARTICLE INFO

**Keywords:**

Screening

Antagonistic agents

Microbiota

*Trichoderma*

Phytomicrobiome

## ABSTRACT

Fusarium head blight (FHB), caused by numerous *Fusarium* species, is a major fungal disease affecting wheat and other food staples. Biological control agents (BCAs) are promising alternatives to synthetic fungicides for integrated disease management. In the present study, a workflow was developed to isolate and screen wheat field-associated microorganisms for their antagonistic activities against FHB causal agents, by taking into account both intra- and interspecific diversity of pathogen complexes. Non-selective and selective isolation methods were used to collect 1231 bacteria, 365 molds, and 39 yeasts from wheat grains, maize residues, and soil samples. From this collection, microbial antagonistic activities were first determined against a *Fusarium graminearum* strain, followed by taxonomic identification of positive isolates. Their activity spectrum was also determined against additional *Fusarium graminearum* strains, as well as *Fusarium avenaceum* and *Fusarium poae*. Then, screening of the ten most effective isolates (4 bacteria, 1 yeast-like, and 5 molds: *Rahnella aceris*, *Streptomyces* sp., 2 *Pantoea agglomerans* isolates, *Cistella* sp., *Trichoderma paraviridescens*, *Trichoderma gamsii*, *Epicoccum layuense*, *Podila minutissima*, and *Penicillium virgatum*) on surface-disinfected grains confirmed their ability to control or delay pathogen growth. Significant variations in BCA antagonistic activities were observed among isolates across different *Fusarium* species and/or strains, which reinforced the importance of including intra- and interspecific pathogen diversity during the screening steps. One *T. gamsii* isolate emerged as the most promising candidate, achieving complete inhibition of *Fusarium* growth (100%) and preventing or dramatically reducing mycotoxin production (up to 100% reduction of deoxynivalenol production). This study highlights that wheat fields serve as a valuable reservoir for potential BCAs against FHB control.

## 1. Introduction

Fusarium head blight (FHB) is one of the most devastating diseases affecting wheat and small grain cereals worldwide, leading to significant yield and quality losses (Summerell, 2019). FHB also poses serious food safety concerns due to the accumulation of mycotoxins, particularly

trichothecenes such as deoxynivalenol (DON) and modified forms of DON, nivalenol (NIV), as well as zearalenone (ZEA) and fumonisins. For instance, the presence of DON in wheat grains is a primary concern for both human and animal health as it can induce dermal toxicity, toxic aleukia, food refusal, vomiting, and suppress immunity (Knutsen et al., 2017). Consequently, DON levels are regulated by the European Union

<footnote>* Corresponding author.
E-mail address: adeline.picot@univ-brest.fr (A. Picot).</footnote>
in various commodities, with a limit of 1000 µg kg⁻¹ for unprocessed wheat grains (European Commission, 2024).

At least seventeen *Fusarium* species have been identified as causal agents of FHB and other root, stalk, and ear rot diseases (van der Lee et al., 2015). In Europe, *F. graminearum*, *F. poae*, and *F. avenaceum* are commonly identified as the major pathogens associated with this disease (Hellin et al., 2016; Nguyen et al., 2024). FHB infections primarily occur during wheat flowering, typically through the dissemination of airborne or splash-dispersed spores, often originating from infected crop residues (Dill-Macky and Jones, 2000; Fernando et al., 2000).

Early detection and control of *Fusarium* infection and mycotoxin production are crucial to prevent toxins from entering the food chain (Moretti et al., 2014). Various strategies can be employed to mitigate the impact of FHB, including crop rotation with non-cereal crops, tillage practices, fungicide application, and the use of resistant cultivars. However, none of these methods taken alone or in combination can completely control this disease (Hollins et al., 2003; Shah et al., 2018). Furthermore, the high diversity of *Fusarium* populations together with their rapid adaptability to environmental changes and agronomic practices, such as the use of resistant cultivars, fungicide applications, and climate change, pose significant challenges for disease management strategies. Although fungicide application has proven to be an effective method (Mesterházy et al., 2011), complete disease control remains challenging, especially under conducive weather conditions, such as high precipitation levels during flowering (Birr et al., 2020; Nguyen et al., 2024; Rossi et al., 2002). Fungicide efficacy is also influenced by other factors, including application timing, fungicide class, cultivar resistance level, and pathogen sensitivity (Bolanos-Carriel et al., 2020; Mesterházy et al., 2011). With the declared ambition of the European Union to achieve a sustainable reduction in pesticide use due to their negative impact on the environment and human health, biological control agents (BCAs) are considered promising alternatives to chemical treatments and as part of an integrated FHB management program.

Stenberg et al. (2021) defined biological control as an environmentally friendly strategy that uses living organisms to reduce or prevent a targeted pathogen or pest, while other definitions also include the use of non-living natural substances (Collinge et al., 2022). Biological control of phytopathogens, based on living organisms, is achieved through various types of interactions between the BCA and their target species, including direct and/or indirect effects. BCAs can directly attack the pathogen through parasitism, antibiosis, or predation mechanisms, notably by producing antagonistic compounds or lytic enzymes (Pal and Gardener, 2006). Moreover, BCAs can indirectly control pathogen development through competition for nutrients and space, thus reducing the pathogenic population, or by inducing host plant resistance. BCA efficacy depends on its mode of action and the environmental conditions in which it is applied. BCAs were hypothesized to be more efficient when multiple mechanisms were combined simultaneously (Pal and Gardener, 2006). Despite the identification of numerous promising antagonistic agents in the scientific literature, only a few are now commercially available (Legrand et al., 2017), primarily due to unstable efficacies under field conditions (Murphy et al., 2018; Parnell et al., 2016). The use of native organisms may provide more reliable field performance as they are supposedly well-adapted to local conditions, including climate, substrates, and microbiota. For these reasons, they may colonize the treated substrates more efficiently. Moreover, due to concerns about import and release of exotic natural antagonists, as well as increased evaluation and registration demands, indigenous antagonistic agents are usually preferred (van Lenteren, 2012).

Furthermore, a significant limitation of current screening assays is the insufficient accounting for the extensive diversity present within target pathogen populations. Most studies focus on a small number of reference strains, typically *F. graminearum*, without accounting for the broader *Fusarium* pathobiome involved in FHB disease. This oversight is critical, as intra- and interspecific diversity within *Fusarium* populations can strongly affect the efficacy of BCAs and the potential development of

pathogen resistance (Carter et al., 2002; Mahmoud, 2016; McDonald and Mundt, 2016).

In a previous study (Nguyen et al., 2024), we determined that *F. graminearum* and *F. avenaceum* (to a lesser extent) were predominant in wheat grains and maize residues, while *F. poae* was primarily found in grains and thrived under warmer and drier conditions during flowering. We also provided evidence that residues were likely sources contributing to grain infection by *F. graminearum*, especially when climatic conditions are favorable during flowering (i.e., rainfall during flowering), in agreement with many previous studies (Dong et al., 2016; Scala et al., 2016; Schöneberg et al., 2018). Based on these findings, the predominant FHB causal species that should be targeted for BCA selection were identified, namely *F. graminearum*, *F. avenaceum*, and *F. poae*. Additionally, we hypothesize that BCA application is most likely to impact pathogen population, whether applied directly on crop residues to reduce primary inoculum load and/or onto wheat heads before or during wheat flowering.

With this knowledge in mind, the purpose of this study was to develop a workflow for sourcing and characterizing wheat field microorganisms with antagonistic activities towards FHB causal agents. To achieve this goal, bacterial and fungal isolates were collected from soil, maize residues, and wheat grains in wheat fields in Brittany, France, using both non-selective and selective isolation methods based on their antagonistic activities. Then, in vitro and subsequent microcosm assays were conducted to further characterize the antagonistic capacity of these isolates against three predominant *Fusarium* species in residues and/or grains (Nguyen et al., 2024) and four *F. graminearum* strains, accounting for intra- and interspecific diversity.

## 2. Materials and methods

### 2.1. *Inoculum preparation of Fusarium species*

Four *F. graminearum* strains (Fg1, Fg2, Fg3, Fg4), one *F. avenaceum* isolate (Fa), and one *F. poae* isolate (Fp) were selected to screen and evaluate the antagonistic efficacy of candidate isolates. These *Fusarium* strains were previously isolated from wheat grains and maize residues collected from various wheat fields at different stages of the wheat growth cycle (Table 1). *Fusarium graminearum* strains were purified through single-spore isolation, and their genotypes were described using a sequence-based microsatellite genotyping method based on 34 SSR markers (data not shown). The Fg1 strain, identified as a unique genotype (data not shown), was selected for its high sporulation rate. The other *F. graminearum* strains (i.e., Fg2, Fg3, and Fg4) were selected to capture intraspecific diversity, with the Fg2 strain being among the most frequently detected genotypes (data not shown) (Table 1). All *F. graminearum* strains exhibited the 15ADON chemotype and were also able of producing DON in wheat grains under controlled microcosm conditions. *Fusarium avenaceum* and *F. poae* isolates were selected as

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

List of target *Fusarium* isolates used to evaluate the antagonistic activity of bacterial and fungal isolates.</caption><thead><tr><td>Strain number</td><td>UBOCC number</td><td>Species</td><td>Isolation date</td><td>Substrate of origin</td></tr></thead><tbody><tr><td>Fg1</td><td>UBOCC-A-124194</td><td>F. graminearum</td><td>November 2020</td><td>Maize residues</td></tr><tr><td>Fg2</td><td>UBOCC-A-124195</td><td>F. graminearum</td><td>January 2021</td><td>Maize residues</td></tr><tr><td>Fg3</td><td>UBOCC-A-124196</td><td>F. graminearum</td><td>June 2021</td><td>Wheat grains</td></tr><tr><td>Fg4</td><td>UBOCC-A-124199</td><td>F. graminearum</td><td>August 2021</td><td>Wheat grains</td></tr><tr><td>Fa</td><td>UBOCC-A-124197</td><td>F. avenaceum</td><td>February 2022</td><td>Maize residues</td></tr><tr><td>Fp</td><td>UBOCC-A-124198</td><td>F. poae</td><td>June 2021</td><td>Wheat grains</td></tr></tbody></table>
they produced high levels of NIV and enniatins (ENNs) in wheat grains under controlled microcosm conditions. Pure cultures were maintained on potato dextrose agar (PDA; BD Difco, Sparks, MA, USA) at 4 °C. To prepare spore suspension, *Fusarium* strains were cultured on PDA at room temperature for 7 days under daylight conditions. Then, conidia were recuperated by washing the culture surface with 5 mL of sterile demineralized water supplemented with 0.01 % Tween 80 (Sigma-Aldrich, MO, USA). After spore enumeration using a Malassez counting chamber, conidial suspensions were adjusted to a final concentration of 10⁷ spores mL⁻¹ in glycerol 10 % (v/v) and stored at -80 °C until use. For supplementation to a solidified medium, spore suspensions were added to the liquid medium before it solidified. All *Fusarium* isolates are available at the UBO culture collection (UBOCC, Plouzané, France).

## 2.2. Bacterial and fungal isolation from field samples

Samples of soil, maize residues, and wheat grains collected from wheat fields in Brittany, France, at different stages of the wheat development cycle were used for microbial isolation. Field sampling was detailed in a previous study (Nguyen et al., 2024). Upon collection, samples (i.e., soil, maize stalks cut into pieces, and wheat spikes) were either placed in tubes containing 20 % (v/v) glycerol and stored at -80 °C until further analysis or were directly processed for microbial isolation. Two different isolation procedures were used: the first approach, hereafter referred to as “blind isolation”, consisted in the isolation of a subsample of microbial isolates grown on the tested media, while the second one, hereafter referred to as “selective isolation”, consisted in a subsequent selection of isolates showing antagonistic activity against *F. graminearum* after pouring a second layer containing spores of Fg1 on top of the first one, as further described below. In these procedures, grain and soil-based media were used as substrates to simulate natural environments from which the microorganisms were collected, thereby promoting the growth of microbial communities adapted to these substrates, where *Fusarium* species and potential antagonists naturally coexist and interact.

For grain-based medium preparation, 20 g of ground wheat grains were mixed with 1 L of demineralized water. After decanting, the liquid phase was supplemented with 1.5 % agar (w/v), and the medium was autoclaved at 121 °C for 20 min. For the soil-based medium, 500 g of air-dried, silty clay soil was mixed with 1 L of demineralized water, and the suspension was autoclaved at 121 °C for 1 h. Following centrifugation at 5000 rpm for 10 min, the collected supernatant was mixed with 1.5 % agar (w/v) and then autoclaved at 121 °C for 15 min. Media were supplemented with 50 mg L⁻¹ of amphotericin (Sigma-Aldrich, St. Louis, MO, USA) for bacterial isolation or a mixture of 50 mg L⁻¹ of streptomycin, penicillin, and chlortetracycline (Sigma-Aldrich, St. Louis, MO, USA) for fungal isolation.

The blind isolation of microbial representatives of field samples consisted of vortexing 6 g of wheat heads, maize residues, or soil with 20 mL of a sterile peptone-salt solution (bioMérieux, Marcy l'Etoile, France) before inoculating 1 mL of 10-fold dilutions with sterile peptone-salt solution (i.e., 10⁻³ and 10⁻⁴ for bacteria, and 10⁻² and 10⁻³ for fungi) in grain and soil-based media, using the pour plate method. Then, plates were incubated at 20 °C in darkness for 4 days and the resulting individual bacterial isolates were placed on 96-well microplates containing 200 µL of Brain Heart Infusion broth (BHI, Biokar Diagnostic, Beauvais, France), incubated at 20 °C in darkness for 7 days followed by storage in 10 % (v/v) glycerol at -80 °C until use. Fungal isolates were subcultured on Petri dishes with PDA, checked for purity, and mycelial plugs were placed in tubes containing 10 % (v/v) glycerol and stored at -80 °C until use.

For the selective isolation approach, the 4-day-old agar plates produced for the blind method (containing either grain or soil medium) were further overlaid with 8 mL of soft agar PDA medium (0.8 % agar, w/v) containing 10⁴ spores mL⁻¹ of Fg1. Agar plates were then incubated at 20 °C in darkness, resulting in the development of a pink mat on

the Petri dish within 3-7 days due to Fg1 growth. The isolates exhibiting positive activity, as indicated by a non-pink halo zone around bacterial or fungal colonies (Fig. S1), were isolated, purified, and cryopreserved as described above.

## 2.3. Workflow for sourcing and characterizing wheat field microorganisms

The workflow for sourcing and characterizing wheat field microorganisms with antagonistic activities against FHB causal agents is illustrated in Fig. 1.

### 2.3.1. First stage: Active isolates screened against Fusarium graminearum Fg1

All fungal isolates collected using blind and selective isolation methods were screened for antagonistic activity on square Petri plates (12 × 12 cm, Greiner Bio-One, Frickenhausen, Germany), divided into 4 × 4 squares. Antagonistic activity of microbial isolates was evaluated after adding 55 mL of grain-based medium supplemented with 10⁵ spores mL⁻¹ of Fg1 (Fig. 2A). For these antagonistic experiments, the grain-based medium containing 10⁵ spores mL⁻¹ was incubated at 20 °C in darkness for one day. Then, each square of the plates was subsequently inoculated centrally with 5-mm diameter plugs from the growing margin of a 5- to 7-day-old fungal thallus. A control, inoculated with a PDA plug, was included in each plate. Inoculated plates were then incubated at 20 °C in darkness for 6 days and the antagonistic activity of isolates against Fg1 was scored based on a 4-point scale score (Fig. 2B), with 0 corresponding to no inhibition and 4 corresponding to the total inhibition of Fg1 or the total growth of the candidate over Fg1.

The antagonistic activity of bacterial isolates, previously stored in 96-well microplates at -80 °C, was first assessed on the square Petri plates (Fig. 2C). A subculture step on Plate Count Agar (PCA, Biokar Diagnostic, Beauvais, France) was performed to purify the bacterial isolates prior to screening. After a revivification step in BHI broth at 20 °C in darkness for two days, bacterial cultures were placed on the surface of grain-based medium containing 10⁵ spores mL⁻¹ of Fg1 using a sterile 96-pin replicator (Boekel Scientific, PA, USA). Active bacterial isolates were selected for their inhibitory activity against Fg1, as indicated by the presence of a halo around the colonies after 5 days of incubation at 20 °C in darkness (Fig. 2C). If bacterial colonies were too close to be precisely identified as antagonistic isolates, a second screening step was performed to distinguish them. The selected bacterial isolates associated with the halo were first subcultured on PCA for 2-3 days, and a colony of each isolate was individually screened in 48-well microplates containing 500 µL of grain-based medium supplemented with 10⁵ spores mL⁻¹ of Fg1, followed by incubation at 20 °C for 7 days in darkness (Fig. 2D). A control without a bacterial isolate was included in each screening plate. Bacterial isolates were scored for their inhibitory activity against Fg1, compared to the control, on a scale of 0 to 2, where 0 corresponded to no inhibition, 1 to a weak inhibition, and 2 to total inhibition of Fg1 growth.

### 2.3.2. Second stage: Taxonomic identification of active microbial isolates

Active bacterial (n = 111) and fungal isolates (n = 74) were taxonomically identified. Bacterial isolates showing antagonistic activity against Fg1 were identified based on 16S rRNA gene and/or gyrB gene sequence analysis. Bacterial genomic DNA was extracted using the FastDNA® SPIN Kit (MP Biomedicals, Irvine, CA) following the manufacturer's instructions. 16S rRNA and gyrB gene PCR amplification was carried out as described by Weisburg et al. (1991) and Dauga (2002), respectively. Note that gyrB gene sequencing was only conducted on bacterial isolates that were further tested in microcosm assays (detailed below) to improve taxonomic resolution for these specific antagonistic candidates (B35, B52, and B81, Table 2). Fungal isolates showing a moderate or strong antagonistic effect against Fg1 (inhibition score ≥ 3 or presence of inhibition halo) were identified based on preliminary
<figure><img src="image_3.png" /><figcaption>Fig. 1. Workflow to select antagonistic agents from wheat field microbiota to control *Fusarium* infection and mycotoxin contamination of wheat grains.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>Fig. 2. Screening of fungal and bacterial isolates with antagonistic activity against *Fusarium graminearum* Fg1 strain. On square Petri plates containing wheat grain-based medium supplemented with 10⁵ spores mL⁻¹ of strain Fg1, the score of competitive inhibition (A) was assessed after 6 days of incubation at 20 °C in darkness, using a 4-point scale (B). (C) The bacterial isolates associated with an inhibition halo against Fg1 were subsequently tested in 48-well microplates (D) to confirm their antagonistic activity. Bacterial isolates were scored for their inhibitory activity against Fg1, compared to the positive control (antagonistic isolate, T + ) and the negative control (only containing Fg1 spores, T-), on a scale of 0 to 2, where 0 corresponded to no inhibition, 1 to a weak inhibition, and 2 to total inhibition of Fg1 growth.</figcaption></figure>
<caption>**Table 2**</caption>

Identification of the bacteria, yeast-like, and mold isolates of the best antagonists used in the microcosm assay. Two negative controls were also included.

<table><thead><tr><th>Isolates</th><th>Substrates</th><th>Species designation</th><th>UBOCC number</th></tr></thead><tbody><tr><td>B35</td><td>Residues</td><td><i>Rahnella aceris</i></td><td>UBOCC-A-323012</td></tr><tr><td>B48</td><td>Soil</td><td><i>Streptomyces</i> sp.</td><td>UBOCC-A-323018</td></tr><tr><td>B52</td><td>Grains</td><td><i>Pantoea agglomerans</i></td><td>UBOCC-A-323032</td></tr><tr><td>B81</td><td>Grains</td><td><i>Pantoea agglomerans</i></td><td>UBOCC-A-323038</td></tr><tr><td>Y49</td><td>Residues</td><td><i>Cistella</i> sp.</td><td></td></tr><tr><td>C101</td><td>Residues</td><td><i>Trichoderma paraviridescens</i></td><td>UBOCC-A-124189</td></tr><tr><td>C165</td><td>Grains</td><td><i>Epicoccom layuense</i></td><td>UBOCC-A-124190</td></tr><tr><td>C222</td><td>Residues</td><td><i>Penicillium virgatum</i></td><td>UBOCC-A-124191</td></tr><tr><td>C224</td><td>Residues</td><td><i>Trichoderma gamsii</i></td><td>UBOCC-A-124192</td></tr><tr><td>C371</td><td>Residues</td><td><i>Podila minutissima</i></td><td>UBOCC-A-124193</td></tr><tr><td>B10 (negative control)</td><td>Residues</td><td><i>Pseudomonas fragi</i></td><td></td></tr><tr><td>C080 (negative control)</td><td>Soil</td><td><i>Purpureocillium lilacinum</i></td><td></td></tr></tbody></table>

morphological and further gene sequence analyses. Genomic DNA of molds was automatically extracted from 7-day-old cultures on PDA using KingFisher™ Duo Prime (Thermo Fisher Scientific, MA, USA) and Mag-Bind® Universal Pathogen DNA 96 Kit (Omega Bio-Tek, GA, USA). For fungal isolates morphologically identified as *Penicillium* or *Epi-coccum*, partial β-tubulin (β-tub) gene sequencing was performed using primers Bt2a and Bt2b (Glass and Donaldson, 1995). For fungal isolates morphologically identified as *Fusarium* or *Trichoderma*, partial trans-lation elongation factor 1α (tef1) gene sequencing was performed using primers EF1-728F and EF1-986R (Carbone and Kohn, 1999). Other fungal isolates were identified based on ITS gene sequence analysis using primers ITS4 and ITS5 (White et al., 1990). Yeast isolates were identified after sequencing of the D1/D2 domain of the 26S rRNA gene using primers NL1 and NL4 (Kurtzman and Robnett, 1998; O'Donnell, 1993). PCR was performed using the GoTaq® kit and dNTP mix (Promega, WI, USA). Sequencing was performed by Eurofins Genomics (Ebersberg, Germany). The obtained bacterial and fungal sequences were assembled into contigs using DNA Baser (Heracle BioSoft, Arges, Romania) and compared to the Genbank database using the “Basic Local Alignment Search Tool” (BLAST). For isolates belonging to the *Tricho-derma* genus and for bacterial isolates further tested in the microcosm assay (B35, B52, and B81) phylogenetic analyses (multiple alignment using ClustalW with default parameters and phylogenetic reconstruction using the neighbor-joining method) were performed with sequences retrieved from Genbank using the molecular evolutionary genetics analysis (MEGA) software version 11 (Tamura et al., 2021) (Figs. S2 to S5).

Based on the obtained knowledge of their taxonomic identity, all known human or plant pathogens (*Sarocladium*, *Chaetomium*, *Erwinia*, and *Fusarium*) were discarded. Additionally, only one representative per identified species was kept when isolates originated from the same substrate and sampling period. Lastly, taxonomic information also helped us further prioritize genera or species that were previously identified as predominant in soil, maize residues, and wheat grains, and/or negatively correlated with *Fusarium* species based on co-occurrence networks and/or associated with mycotoxin-free grain samples (Nguyen et al., 2025). Overall, a total of 29 fungal (28 molds and 1 yeast-like isolate) and 45 bacterial isolates were selected to determine the breadth of their activity spectrum.

### 2.3.3. Third stage: Activity spectrum of active microbial isolates

The antagonistic efficacy of this subset of isolates was further evaluated against the following *Fusarium* isolates: three strains of *F. graminearum* (Fg2, Fg3, Fg4), *F. poae* (Fp), and *F. avenaceum* (Fa). This step was added to account for the intra- and interspecific diversity of pathogenic populations. The same screening methods, using square Petri plates for molds and 48-well microplates for yeast and bacteria, as described above, were employed. Each screening plate also contained a negative (i.e., a PDA plug for molds and without an isolate for bacteria and yeast) and a positive control (i.e., highly antagonistic isolates, *Trichoderma velutinum* C205 and *Streptomyces cirratus* B47). The inhibitory activity was scored after 7 days of incubation at 20 °C in darkness. It is worth noting that the *F. avenaceum* strain grew slowly and produced white, sterile mycelia on a grain-based medium, making it difficult to differentiate between no or weak inhibition. Consequently, we evaluated the antagonistic activity against this strain, focusing solely on complete or no inhibition.

### 2.3.4. Fourth stage: Microcosm assay on surface-disinfected wheat grains

Based on the activity spectrum assay, isolates having broad-spectrum anti-*Fusarium* activity were further selected. We evaluated their efficacy against *F. graminearum* (Fg1, Fg2, Fg3, Fg4), *F. poae* (Fp), and *F. avenaceum* (Fa) directly on living wheat grains. One fungal and one bacterial isolate, which were antagonistic against Fg1 but inactive against the other tested *Fusarium* strains, were used in the experiments as controls (Table 2). Prior to treatment, wheat grains (organic wheat purchased locally from Magasin Vert, Plouzané, Brittany, France) were surface-disinfected by immersion in a 1 % (v/v) sodium hypochlorite solution for 10 min, followed by 3 rinses with sterile demineralized water. The disinfected grains were aseptically dried before use.

About 4 g of disinfected wheat grains were mixed with 4 mL of sterile demineralized water in a Petri dish (55 mm in diameter). Selected fungal and bacterial isolates were inoculated on the grains at a final concentration of 1250 spores g⁻¹ or 5.10⁶ UFC g⁻¹ of grains, respectively. For bacteria, we inoculated 4 g of grains with 200 µL of bacterial suspension at approximately 0.5 optical density (OD), corresponding to 10⁸ UFC mL⁻¹. For inoculum preparations, bacterial and yeast suspensions were revived on PCA at 20 °C for 2–3 days and in BHI broth for 2–3 days at 20 °C, before centrifugation (9000 rpm, 5 min), the pellet was washed with peptone-salt solution, and resuspended in 2 mL of peptone-salt solution. For the *Streptomyces* sp. B48 and *Cistella* sp. Y49 isolates, the inoculum suspension was additionally homogenized for 40 s using a GentleMacs™ Dissociator (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) due to the formation of filamentous particles (pellets). The resulting suspension was then used to inoculate grains, with 200 µL of suspension at approximately 0.4 OD for *Streptomyces* sp. B48 and 0.5 OD for *Cistella* sp. Y49, each corresponding to 10⁸ UFC mL⁻¹. Plates containing wheat grains inoculated with BCA were sealed with parafilm to maintain a relative humidity of 65–75 % and incubated for 3 days at 20 °C in darkness. After 3 days of incubation, the *Fusarium* inoculum was added at a final concentration of 1250 spores g⁻¹ (50 µL of a fungal spore suspension at 10⁵ spores mL⁻¹), prepared as described above, before incubation at 20 °C in darkness. Control treatments consisted of plates inoculated with either *Fusarium* or the BCA alone, where the BCA or *Fusarium* inoculum was replaced with a peptone-salt solution. Additionally, control plates without BCA or *Fusarium* inoculum were included to verify the absence of *Fusarium* and mycotoxins in wheat grains. Five replicated plates were used for each inoculated and control treatment. Anti-*Fusarium* activities were assessed based on the growth of *Fusarium* species on wheat grains at 4, 7, 10, and 14 days after inoculation (dai) of the *Fusarium* inoculum, using a 3-point scale, where 0 corresponded to no growth of *Fusarium* on grains, 1 corresponded to > 0–50 % of infected grains, 2 corresponded to 50–70 % of infected grains, and 3, to *Fusarium* growth exceeding 70 % of grains (Fig. 6A). Complementarily, to observe further development of *Fusarium*, the percentage of grains contaminated with *Fusarium* species was also
determined at 7 and 14 days after inoculation of the *Fusarium* inoculum. To do so, five grains per plate were placed on PDA supplemented with 50 mg L⁻¹ streptomycin, penicillin, and chlortetracycline. The identification of *Fusarium* species was carried out after 5–7 days of incubation at room temperature under natural light exposure, based on the morphological characteristics of the species (Fig. 6B). All selected antagonistic isolates were first screened against *F. graminearum* Fg1, and a subset of effective isolates was then tested against the other *Fusarium* strains, i.e., *F. graminearum* (Fg2, Fg3, and Fg4), *F. avenaceum* Fa, and *F. poae* Fp.

## 2.4. Quantitation of mycotoxins

At the end of the assay on disinfected grains (14 days post inoculation), plates were stored at -20 °C until use. These samples were then used to evaluate mycotoxin production in grains inoculated with *Fusarium* strains, including four *F. graminearum* strains (Fg1, Fg2, Fg3, and Fg4), one *F. avenaceum* strain (Fa), and one *F. poae* strain (Fp), with or without prior treatment with BCA. Mycotoxin analysis was only carried out for the most effective antagonistic isolate. Targeted fusariotoxins, including DON, 15ADON, ZEA, NIV, and ENNs, were quantified using an HR-Q-TOF LC/MS method, previously described in Nguyen et al. (2024).

## 2.5. Statistical analyses

A chi-square test of independence was used to compare the number of isolates obtained with the two isolation methods, as well as the number of isolates from different substrates, using the stats (version 4.1.3) and chisq.posthoc.test (version 0.1.2) R packages. Colonization scores and the number of contaminated grains during microcosm assays were compared to those of the *Fusarium* target at each time point using a non-parametric Kruskal-Wallis test, followed by a Dunn's test (rstatix package version 0.7.2).

# 3. Results

## 3.1. Culture collection of microbial isolates from field samples

Microbial isolations from wheat field substrates, including soil, maize residues, and wheat grains, were carried out using grain and soil-based media. Two isolation strategies were employed: a blind isolation method, in which microorganisms were non-selectively isolated, and a

selective isolation approach based on *F. graminearum* Fg1 growth inhibition (Fig. S1). A total of 1541 and 94 isolates were collected using the blind versus selective isolation methods, respectively. A total of 1213 and 18 bacterial, 291 and 74 mold, 37 and 2 yeast isolates were respectively obtained (Fig. 3). Based on the blind method, 500, 405, and 636 microbial isolates (both bacteria and molds) were obtained from soil, residue, and grain samples, respectively, while 54, 22, and 18 microbial isolates were collected from soil, residues, and grains, respectively, using the selective isolation approach (Fig. 3). A significantly higher proportion of mold isolates was collected using the latter method, while a significantly higher proportion of bacteria was isolated using the blind method ($p < 0.001$).

## 3.2. Stage 1: Selection of antagonistic isolates against Fusarium graminearum Fg1

Among the 1541 isolates belonging to the blind isolation culture collection, 121 molds and 109 bacteria, representing 42 % and 9 % of total mold and bacterial isolates, inhibited Fg1 mycelial growth in grain-based medium after 7 days of incubation at 25 °C (Fig. 3A). Similarly, 44 molds, 2 bacteria, and 2 yeasts, representing 60 %, 11 %, and 100 % of total molds, bacteria, and yeasts from the selective isolation method, showed inhibition activity against Fg1 (Fig. 3B). The selective method was significantly more effective for isolating antagonistic fungi, including both molds and yeasts (chi-squared test, $p < 0.001$).

Following this first screening stage, all bacterial and yeast isolates displaying antagonistic activity (111 bacteria and 2 yeasts), as well as molds with inhibition scores exceeding 3 (63 isolates) or exhibiting a clearing zone (halo) around their colonies (9 isolates), were selected for further characterization. In total, 111 bacteria, 2 yeasts, and 72 molds were taxonomically identified, based on the sequencing of 16S rRNA for bacteria, ITS for molds, *tef1* for *Fusarium* and *Trichoderma*, *β*-tub for *Penicillium*, and the D1-D2 domain of the 26S rDNA for yeasts (Fig. 4). The two isolation methods yielded different bacterial and fungal genera, except *Trichoderma*, which were commonly found in both methods (Fig. 4). The 111 antagonistic bacterial isolates were affiliated with sixteen different genera, including *Pantoea* (33 isolates), *Pseudomonas* (29), *Rahnella* (16), *Arthrobacter* (6), *Stenotrophomonas* (5), *Curtobacterium* (4), *Pseudarthrobacter* (3), *Serratia* (3), *Acinetobacter* (2), *Erwinia* (2), *Sphingobacterium* (2), *Duffyella* (1), *Frigoribacterium* (1), *Frondihabitans* (1), *Staphylococcus* (1), and *Streptomyces* (2). The 72 mold isolates

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Bacterial and fungal isolate distribution according to their substrate of origin (soil, maize residues, wheat grains). (A) Microbial isolates were randomly selected on grain and soil-based media using the blind isolation approach. (B) Microbial isolates showing an inhibition halo on the overlaying soft potato dextrose agar medium containing $10^5$ spores mL$^{-1}$ of *Fusarium graminearum* strain Fg1 for the selective isolation approach. The innermost to outermost circles illustrate the number of isolates categorized by microorganism type (bacteria, mold, yeast), substrate (wheat grains, maize residues, soil), and antagonistic activity against *F. graminearum* strains Fg1 in screening assays. The number of isolates is given for each group.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Genus level distribution according to isolation substrates (grains – red, residues – green, and soil – blue) for bacteria (n = 111, names in orange) and fungi (n = 74 including 72 molds and 2 yeasts, names in green) exhibiting strong inhibitory effect against *Fusarium graminearum* Fg1 (score ≥ 3 for molds and score ≥ 1 for bacteria and yeasts). Bacteria, yeast, and mold isolates were respectively identified using 16S rDNA, D1D2, ITS, *β*-tub, and *tef1* gene analysis.</figcaption></figure>

were affiliated with fifteen genera, including *Trichoderma* (47), *Penicillium* (7), *Akanthomyces* (3), *Talaromyces* (3), *Sarocladium* (2), *Albophoma* (1), *Chaetomium* (1), *Diaporthe* (1), *Epicoccum* (1), *Fusarium* (1), *Mortierella* (1), *Plectosphaerella* (1), *Podila* (1), *Purpureocillium* (1), and *Pyrenochaetopsis* (1). The two yeast-like isolates were identified as belonging to the genus *Cistella*. Based on the presumptive identification at the species level, 41 out of 109 bacterial and 28 out of 54 fungal species were identified from blind isolation collection, while 2 out of 2 bacterial and 10 out of 20 fungal distinct species were identified from the selective isolation collection. A high dominance of *Pantoea agglomerans* (21 isolates), *Rahnella aceris* (12), and *Pseudomonas allii* (7) was observed among the antagonistic bacterial isolates. Similarly, the majority of anti-*Fusarium* fungal isolates were *Trichoderma velutinum* (8 isolates), *T. viride* (6), and *T. gamsii* (6). It is also worth mentioning that more than 65 % of *Trichoderma* isolates were affiliated with the Viride (including *T. gamsii*, *T. hamatum*, *T. viride*, *T. ovalisporum*, *T. samuelsii*, and *T. neokoningii*) and Viridescens clade (*T. viridescens*, *T. viridialbum*, and *T. paraviridescens*). The remaining isolates were identified as members of the *T. harzianum* clade, including *T. velutinum*, *T. cerinum*, *T. neotropicale*, and *T. tomentosum* (Figs. S2 and S3). Although all three substrates (soil, residues, grains) provided multiple antagonistic isolates, active bacterial isolates primarily originated from wheat grains (68 out of 111 isolates), while the majority of active fungal cultures were isolated from soil samples (82 out of 167 isolates) (chisq-test, *p* < 0.001). Most antifungal *Pantoea* and *Pseudomonas* isolates were isolated from wheat grains (97 % and 72 %, respectively), while 68 % of *Trichoderma* isolates were obtained from soil. Crop residues contributed to the

isolation of 19 % bacterial and 29 % fungal antagonists.

### 3.3. Stage 2: Selection based on field microbiome composition

Results from a previous study, in which maize residue, wheat grain, and soil microbiomes were identified using metabarcoding sequencing, were further used as a selection criterion (Nguyen et al., 2025) (Table S1). Our rationale, here, was to further select, among antagonistic isolates to Fg1, microbial genera that were identified as predominant by metabarcoding (namely *Curtobacterium*, *Frdndihabitans*, *Pantoea*, *Pseudomonas*, and *Serratia* in grains; *Epicoccum*, *Pyrenochaetopsis*, *Curtobacterium*, *Pseudarthrobacter*, *Pseudomonas*, and *Rahnella* in residues; *Epicoccum*, *Mortierella*, and *Podila* in soil) and/or negatively correlated to *Fusarium* taxa based on Sparse Correlations for Compositional (SparCC) co-occurrence networks (*Epicoccum*, *Pyrenochaetopsis*, *Curtobacterium*, *Pantoea*, and *Pseudomonas*) and/or found as an indicator of mycotoxin-free grain samples (*Pantoea*). Additionally, when isolates originating from the same substrate and sampling stage shared the same phylogenetic affiliation, only one representative was retained. Notably, the isolation method (i.e., blind or selective) was not taken into consideration during this selection process. Moreover, besides exhibiting antagonistic activity, some isolates belonging to *Sarocladium*, *Chaetomium*, *Erwinia*, and *Fusarium* were discarded as they are considered plant pathogenic genera.
### 3.4. Stage 3: Broad-spectrum activity assay

A total of 28 molds, one yeast-like fungus, and 45 bacteria were screened against three other strains of *F. graminearum* (Fg2, Fg3, Fg4), one strain of *F. poae* (Fp), and one strain of *F. avenaceum* (Fa). The antagonistic activity varied greatly among *F. graminearum* strains and *Fusarium* species (Fig. 5 A and B), with a few exceptions, including Y49 (Cistella sp.), B47 (S. cirratus), B48 (Streptomyces sp.), and C152 (T. samuelsii), which showed broad-spectrum activity against all targets. Among 45 bacterial isolates, 33 exhibited inhibition activity against at least two *Fusarium* strains, among which 19 were antagonistic towards two different *Fusarium* species. The remaining 12 bacterial isolates, which were antagonistic to Fg1, did not impact the growth of the other tested *Fusarium* strains (Fig. 5B). In contrast, all 28 tested mold isolates were antagonistic against Fg1 and *F. poae*, with variations observed between the other *F. graminearum* strains. Strong antagonistic activities, with an inhibition score of 4 against 5 out of 6 targets, were observed for five molds, including *T. gamsii* (C224), *T. ovalisporum* (C330 and C093), *T. paraviridescens* (C101), *T. samuelsii* (C152), and *T. velutinum* (C205), which were isolated from maize residues and soil. Other fungal isolates that did not belong to *Trichoderma* spp. exhibited lower inhibition against *Fusarium* species as compared to the latter isolates. Indeed, *E. layuense* C165, *Penicillium virgatum* C222, *Podila minutissima* C371, and *Talaromyces amestolkieae* C017 moderately inhibited the growth of certain *F. graminearum* strains as well as another *Fusarium* species. All mold isolates that were obtained using the selective isolation method (*T. amestolkieae* C017, *T. cerinum* C065, *T. neotropicale* C176, and *T. velutinum* C205) exhibited a strong to moderate inhibition towards *Fusarium* growth. The yeast-like isolate *Cistella* sp. Y49, isolated from maize residues using the selective method, totally inhibited all *Fusarium* strains (Fig. 5A). Based on their anti-*Fusarium* activity spectrum, 4 bacteria, 1 yeast-like, and 5 mold isolates were further selected for screening tests on wheat grains and maize residues (Table 2). Preference

was given to isolates obtained from grain and residue substrates over those isolated from soil, since BCA treatments are meant to be applied to grains and/or residues but not soils. Isolates B10 (*Pseudomonas fragi*) and C080 (*Purpureocillium lilacinum*), which exhibited low or no inhibition against *F. graminearum* strains (Fg2, Fg3, Fg4), *F. poae*, and *F. avenaceum*, were included as negative controls in these microcosm assays.

### 3.5. Stage 4: Antagonistic activity of microbial isolates on natural substrates

#### 3.5.1. Microcosm assay on surface-disinfected wheat grains

The antagonistic efficacy of selected isolates (Table 2) on wheat grains was first assessed against *F. graminearum* Fg1 (Fig. 6). In the control plates, where surface-disinfected wheat grains were inoculated with a peptone-salt solution, no grains were contaminated with *F. graminearum*, although little fungal growth could be observed, while grains were able to germinate, suggesting that the grains and endophytic mycobiota were still alive. Additionally, *F. graminearum* Fg1 grew well under controlled conditions, with colonization scores reaching 3 (i.e., *Fusarium* growth exceeding 80 % of grains) at 4 dai and all grains becoming contaminated by 7 dai. The negative controls (*P. fragi* B10 and *P. lilacinum* C80) exhibited no antagonistic effect against *F. graminearum* Fg1.

The four selected bacterial isolates did not effectively control Fg1 growth in terms of the number of *Fusarium*-infected grains (Fig. 6B). However, Fg1 colonization was delayed when grains were pre-treated with *Streptomyces* sp. B48 or with *P. agglomerans* B52, to a lesser extent (Fig. 6A). Among the fungal isolates tested, *T. gamsii* C224 was the most promising candidate, as it completely prevented Fg1 growth on grains (colonization score of 0 maintained at all-time points). Although less effective, *Cistella* sp. Y49, *T. paraviridescens* C101, and to a lesser extent, *E. layuense* C165 were able to significantly delay Fg1 growth. Low colonization scores as low as 0.9 ± 0.5 at 14 dai were observed.

<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Activity spectrum of bacterial, yeast (A), and mold (B) isolates against *Fusarium graminearum* strains (Fg1, Fg2, Fg3, Fg4), *F. poae* (Fp), and *F. avenaceum* (Fa). The names of isolates obtained by the blind isolation approach are colored in black, and those obtained by the selective isolation approach are colored in orange. Isolates in bold text were selected for microcosm assays.</figcaption></figure>

<figure><img src="image_8.png" /></figure>
<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Antagonistic activity of four bacterial isolates and six fungal isolates against *Fusarium graminearum* Fg1 in microcosm assay on wheat grains based on a colonization score and number of infected grains at four time points (4, 7, 10, and 14 days after Fg1 inoculation, dai). (A) Antagonistic activity based on visual observation of *Fusarium* growth on wheat grains using the 3-point scale of colonization score (A), where 0 corresponds to no growth of *Fusarium* on grains, 1 corresponds to > 0–50 % of infected grains, 2 corresponds to 50–70 % of infected grains, and 3, to > 70 % infected grains. Each dot represents the score of each of the five replicates. (B) The antagonistic activity was also assessed based on the number of grains infected by *Fusarium* species after placing five grains per plate on potato dextrose agar (PDA) supplemented with 50 mg L⁻¹ streptomycin, penicillin, and chlortetracycline and incubating for 5–7 days at room temperature. Barplots represent the sum of infected grains of five replicates. NA: not assessed. TS (peptone salt), *Pseudomonas fragi* B10, and *Purpureocillium lilacinum* C80 were used as negative controls and did not exhibit any antagonistic effect. Fg1: *F. graminearum* strain Fg1; B35: *Rahnella aceris* B35; B48: *Streptomyces* sp. B48; B52: *Pantoea agglomerans* B52; B81: *P. agglomerans* B81; B10: *Pseudomonas fragi* B10; Y49: *Cistella* sp. Y49; C101: *Trichoderma paraviridescens* C101; C165: *Epicoccum layuense* C165; C222: *Penicillium virgatum* C222; C224: *Trichoderma gamsii* C224; C371: *Podila horticola* C371; C80: *Purpureocillium lilacinum* C80.</figcaption></figure>

These isolates, as well as *T. gamsii* C224, were subsequently screened against other *Fusarium* strains to evaluate the broadness of their activity spectrum.

Among the four selected isolates, *T. paraviridescens* C101, *E. layuense* C165, and *Cistella* sp. Y49 exhibited inconsistent antagonistic activity against different *F. graminearum* strains and different *Fusarium* species (*F. graminearum*, *F. avenaceum*, and *F. poae*) (Fig. 7 and S6). *Trichoderma*

gamsii C224 was the most effective, displaying broad-spectrum anti-*Fusarium* activity. Indeed, visual evaluation showed no signs of *Fusarium* development, which was confirmed by the absence of *Fusarium*-contaminated grains after plating on PDA agar plates, regardless of *Fusarium* strains (Fig. 7A). In contrast, other tested isolates were effective in controlling or delaying the infection of certain *Fusarium* strains, but not all. Specifically, we observed that *T. paraviridescens* C101
<figure><img src="image_10.png" /><figcaption>**Fig. 7.** Activity spectrum of fungal isolates, including (A) *Trichoderma gamsii* C224, (B) *Trichoderma paraviridescens* C101, (C) *Epicoccum layuense* C165, and (D) *Cistella* sp. Y49, against *Fusarium* spp. (*F. graminearum* Fg1, Fg2, Fg3, Fg4; *F. avenaceum* Fa; and *F. poae* Fp) on wheat grains. Boxplots illustrate the number of infected grains at two time-points: 7 and 14 days after *Fusarium* inoculation (dai). The efficacy of each antagonistic isolate was evaluated by comparing its performance to the target alone at each time point using a non-parametric Kruskal-Wallis test followed by a post hoc Dunn's test. Asterisks represent significance levels, \* = 0.05 and \*\* = 0.01.</figcaption></figure>
significantly reduced *F. graminearum* Fg1, Fg2, and Fg3 infection at both 7 and 14 dai (Fig. 7B), while *Cistella* sp. Y49 effectively controlled *F. graminearum* Fg1, *F. avenaceum* Fa, and *F. poae* Fp infection (Fig. 7D). *Epicoccum layense* C165 was effective in delaying *F. graminearum* Fg1, Fg2, and *F. poae* Fp growth (Fig. 7C).

### 3.5.2. Inhibition of mycotoxin production

Subsequently, the ability of the most antagonistic biocontrol strain, *T. gamsii* C224, to inhibit mycotoxin production by different *Fusarium* species was evaluated (Fig. 8). In the absence of the antagonistic strain C224, all *Fusarium* strains produced high levels of mycotoxins under the tested conditions. Specifically, the four *F. graminearum* strains were DON, 15ADON, and ZEA producers, while *F. avenaceum* and *F. poae* strains produced NIV and/or ENNs (including ENN B, B1, A, and A1). DON levels produced by different *F. graminearum* strains varied between replicate plates, with a maximum production of up to 1791 µg g⁻¹ of dried grains, while the four strains also produced ZEA and 15ADON, but only in some replicates. When *T. gamsii* C224 was inoculated on grains three days prior to *Fusarium*, reduced mycotoxin production or undetectable levels were observed (detection limits of 96.21 ng g⁻¹ for DON, 11.92 ng g⁻¹ for ZEA, and 41.69 ng g⁻¹ for NIV) for all *F. graminearum* strains, except Fg2 and Fg4. These strains produced detectable but not quantifiable levels of DON and 15ADON. ZEA was also detected for Fg4. *Trichoderma gamsii* C224 also significantly reduced *F. poae* NIV production. However, *T. gamsii* C224 was not entirely effective in inhibiting ENN production by *F. avenaceum*, as ENN B and B1 were detected in one out of five treated plates.

## 4. Discussion

In the present study, we developed a workflow for sourcing and selecting potential BCAs from different field substrates to prevent or at least largely reduce dominant *Fusarium* species causing FHB of cereals. These species included *F. graminearum*, *F. poae*, and *F. avenaceum*. To reach this goal, we used soil, maize residues, and wheat grains collected from wheat fields to directly isolate potential BCAs to control FHB infections. BCA treatments are expected to reduce FHB infection, either after application on crop residues to reduce primary inoculum loads or after direct application on wheat heads before or during wheat flowering. Two isolation strategies were applied to efficiently select anti-*Fusarium* candidates for FHB disease control. A non-selective method, referred to as “blind isolation”, was used to collect a large number of microorganisms, whereas the “selective” approach enabled the preselection of isolates directly showing antagonistic activity against one *F. graminearum* strain on a wheat grain-agar-based medium, before further assessing their broader antagonistic activity. Combining the isolation method with a preselection step significantly enhanced the efficacy of sourcing active microorganisms, in particular for fungal isolates, since 60% had antagonistic activity against one *F. graminearum* strain versus 42% of fungal isolates collected with the blind isolation method. However, nearly 40% of active isolates obtained from the selective isolation approach no longer showed antagonistic properties in the subsequent individual screening assay, although we used similar experimental conditions to determine their activity (grain-based medium inoculated with Fg1 spores). This loss in efficacy may be attributed to the complex synergistic effects of multiple colonies found in the Petri dish simultaneously. Alternatively, we cannot rule out the fact that several bacterial isolates co-occurred within a single colony and that an inactive one may have been subsequently purified.

In addition, distinct bacterial and mold genera were isolated with each approach, except for *Trichoderma*. These findings suggest that the combined use of both non-selective and selective methods is helpful to achieve a more complete collection of potential BCAs, covering higher taxonomic and strain-level diversities. However, the full breadth of cultivable microorganisms was likely underexplored here, as our culture conditions tended to select dominant and non-fastidious organotrophs

and facultative anaerobic microorganisms. As an example, more fastidious and/or subdominant microorganisms such as lactic acid bacteria or *Bacillus* spp. were not isolated despite the fact that these taxa are often present in the studied field substrates and are known to possess antifungal activities. However, our previous metabarcoding analysis identified these taxa as minor rather than dominant in soil and maize residues and were not detected in grains (Nguyen et al., 2025).

Overall, 111 bacteria, 72 molds, and 2 yeast-like fungi capable of inhibiting *F. graminearum* Fg1 growth on grain-based medium were obtained using our screening method. These active isolates were identified, primarily belonging to *Pantoea*, *Pseudomonas*, and *Rahnella* spp. for bacteria, and *Trichoderma* spp. for fungi. These results are in agreement with previous studies where species belonging to these genera were among the most commonly reported antagonistic agents against FHB and other plant diseases (Ganeshan and Manoj Kumar, 2005; Jiang et al., 2019; Karimi et al., 2012; Matarese et al., 2012; Palmieri et al., 2017; Xu et al., 2022). Additionally, *Pseudomonas* and *Pantoea*, as well as other genera such as *Epicoccum*, *Curtobacterium*, *Erwinia*, and *Pyr- enochaetopsis*, for which antagonistic activities were observed in the present study, were previously identified to be negatively correlated with *Fusarium* species in co-occurrence networks of grain and residue-associated microbiomes. *Pantoea* genus was also associated with fusariotoxin-free wheat grains (Nguyen et al., 2025). The predominance of specific native genera in maize residues (e.g., *Epicoccum*, *Pyr- enochaetopsis*, *Curtobacterium*, *Pseudomonas*, and *Rahnella*) and wheat grains (e.g., *Curtobacterium*, *Pantoea*, and *Pseudomonas*), as described in Nguyen et al. (2025), supports their promising potential as BCAs in wheat fields. Their predominance in these substrates indicates their strong adaptability to local conditions, thereby facilitating their application and integration into agricultural practices with the aim of enhancing plant health and resilience against pathogens.

The screening method using a grain-based medium containing *F. graminearum* spores was proven to be a fast and straightforward approach for selecting microorganisms with confirmed in vitro biocontrol activity. Additionally, this method can also be adapted to rank active isolates according to their efficacy, including their effect on pathogen growth (Islam et al., 2018; Ramesh and Phadke, 2012; Sales et al., 2016), sporulation (Zhao et al., 2014), and mycoparasitic interactions (Bhat, 2017; Ramírez-Olier et al., 2019). Despite being one of the most reported screening methods, the dual culture assay used for BCA screening is restricted to those with direct activity against the pathogen, such as parasitism, excretion of antimicrobial compounds, secretion of enzymes, or competition for nutrients and expressed under in vitro conditions (Raymaekers et al., 2020). This screening approach thus limits the possibility of discovering other potential antagonistic agents that indirectly exert their biocontrol activity by inducing plant-mediated responses, thereby enhancing host defense mechanisms (Raymaekers et al., 2020). Also, expression of these mechanisms under in vitro conditions does not necessarily mean that they will remain active in planta. To limit these flaws, various novel screening approaches have been implemented to identify endophytic fungi or bacteria from healthy wheat spikes exposed to high FHB pressure (Deroo et al., 2022; Rojas et al., 2020). Deroo et al. (2022) introduced an innovative workflow for discovering novel bacterial BCAs in wheat targeting FHB infection in wheat. This approach involves enriching wheat spikes through successive passages of microbial communities extracted from four distinct cultivars in the presence of *F. graminearum*. The bacterial community exhibiting a reduction in FHB symptoms was subsequently selected for isolation and further screening. This workflow has proven effective in enhancing the success rate of biocontrol isolates compared to the overall number screened.

In the third screening phase, we sought to identify antagonistic isolates with broad-spectrum activity against various *F. graminearum* strains isolated in a previous study, as well as against other *Fusarium* species. Indeed, because of the high genetic diversity within *F. graminearum* populations (Vogelgsang et al., 2019; Zhang et al.,
<figure><img src="image_11.png" /><figcaption>**Fig. 8.** Inhibitory effect of *Trichoderma gamsii* C224 on mycotoxin production of *Fusarium graminearum* (strains Fg1, Fg2, Fg3, and Fg4), *F. avenaceum* Fa, and *F. poae* Fp after inoculation on wheat grains under controlled conditions. Pairwise comparisons were carried out using a non-parametric Kruskal-Wallis test followed by a post hoc Dunn's test. Asterisks represent significance levels, \* = 0.05 and \*\* = 0.01. ZEA: zearalenone, 15ADON: 15-acetyldeoxynivalenol, DON: deoxynivalenol, NIV: nivalenol, ENNs: sum of enniatin A, A1, B, and B1 concentrations.</figcaption></figure>
2023), isolates exhibiting consistent effectiveness across multiple pathogens have a better potential for use as biocontrol solutions. In this regard, we found that substantial variations in the antagonistic efficacy of active isolates occurred within *Fusarium* species (*F. graminearum*, *F. poae*, and *F. avenaceum*) and *F. graminearum* strains. Several isolates that exhibited high antagonistic capacity against *F. graminearum* Fg1 showed no inhibitory effect against the other tested haplotypes (Fg2, Fg3, and Fg4). This observation suggests that the latter *F. graminearum* strains may possess greater competitive abilities or resistance towards BCA as compared to the other haplotype. Among bacterial isolates, two *Streptomyces* isolates were able to inhibit the growth of all tested *Fusarium* strains in Petri plate screening assays, suggesting their significant potential in controlling FHB infection. The high efficacy of *Streptomyces* species as BCAs against plant pathogenic fungi has often been reported because of their ability to produce a large number of antifungal secondary metabolites (Chen et al., 2018; Wei et al., 2020; Yun et al., 2018; Zou et al., 2021).

Additionally, isolates of *R. aceris* and *P. agglomerans* were also effective against *F. graminearum* and *F. poae*. Several studies have demonstrated the antagonistic effect of *Pantoea* species against a wide range of phytopathogenic organisms, attributing these properties to antifungal compound production (*P. agglomerans*, Kim et al., 2012; Pusey et al., 2011; P. brenneri, Itkina et al., 2021; Suleimanova et al., 2024). Although *R. aceris* was recently described as a novel plant growth-promoting rhizobacterium (Xu et al., 2022), its inhibitory activity observed in our screening suggests the potential production of anti-*Fusarium* compounds.

Unlike bacteria, all tested mold isolates, among which the majority were identified as *Trichoderma* spp., exhibited broad-spectrum antagonistic activity against *F. graminearum*, *F. poae*, and *F. avenaceum*. This result was not surprising as accumulating evidence indicates that *Trichoderma* spp. are effective BCAs against various pathogens, including *F. oxysporum* (Mohapatra and Mittra, 2017), *F. graminearum* (Ferrigo et al., 2020; Foroutan, 2013; Sarrocco et al., 2021; Tian et al., 2018; Xue et al., 2017), and *F. verticillioides* (Ferrigo et al., 2020). The antagonistic properties of *Trichoderma* result from a combination of several mechanisms such as mycoparasitism (Nguyen et al., 2020; Nofal et al., 2021; Rajani et al., 2021), antibiosis (Kumari and Srividhya, 2020; Saravanakumar et al., 2018), and competition for space and nutrients (Wang et al., 2023). Furthermore, the yeast-like isolate *Cistella* sp., isolated from maize residues, also showed a strong and consistent inhibitory effect against *Fusarium* species. To our knowledge, this is the first time that this genus is reported as a potential BCA against *Fusarium* species.

In vitro screening assays provide a high-throughput method to verify the antagonistic activity of isolates, but this approach is often artificial and does not reproduce natural field conditions. Therefore, subsequent in vivo tests are recommended to assess their biocontrol efficiency in more realistic scenarios, starting with microcosm experiments. We thus selected the best candidates isolated primarily from wheat heads and maize residues, which are the targeted substrates for FHB treatment, and performed a microcosm assay on wheat grains. Besides, we used surface-disinfected grains and unsterilized grains because the endophytic microbiota is known to potentially restrict BCA colonization (Comby et al., 2017; Legrand et al., 2017). Surface-sterilization was mandatory as, otherwise, fast-growing molds (e.g., *Rhizopus*) arose quickly, preventing us from any observation. Preliminary experiments showed an effect of treatment timing on disease reduction, which is why BCAs were applied three days before *Fusarium* inoculation (data not shown). Indeed, except *Trichoderma* isolates, application of antagonistic agents three days prior to *Fusarium* inoculation resulted in a significantly higher inhibitory or delay effect on *Fusarium* growth on wheat grains as compared to simultaneous inoculation (data not shown). The timing of application at least two days before pathogen attack was previously reported as exhibiting higher efficacy in a recent study investigating the biocontrol activity of wheat fungal endophytes against *F. graminearum*

(Rojas et al., 2020). This timing effect, also observed with fungicide treatment (González-Domínguez et al., 2021), improves antagonistic efficiency against FHB and therefore requires application at the early anthesis stage of wheat. In the present study, the bacterial isolates were much less effective than fungi in inhibiting or delaying *F. graminearum* Fg1 under the tested conditions, potentially due to a too short establishment period (3 days). The absence or reduced efficacy may also result from potential inhibitors produced by BCAs not being synthesized or at insufficient concentrations, to inhibit fungal growth under the tested conditions. *Trichoderma gamsii* C224 showed the highest potential for controlling FHB disease because of its capacity to completely inhibit the growth of all *Fusarium* strains in both inoculation conditions (delayed and simultaneous inoculation, data not shown). The application of this strain also significantly reduced mycotoxin production, including DON, ZEA, 15ADON, and NIV of all *Fusarium* strains. In addition, spores of this *T. gamsii* isolate were able to survive on maize residues under microcosm assay and subsequently showed a total inhibition of *F. graminearum* after maize residue incubation on PDA medium (data not shown). These results confirmed *T. gamsii* C224 as a promising candidate for BCA for further greenhouse and field trials. Numerous previous studies have demonstrated the effective antagonism of *T. gamsii* against phytopathogens such as *F. sambucinum* on potato (Aydin, 2019), *Phytophthora capsici* on zucchini (Gilardi et al., 2015), *F. oxysporum* on black gram (Valan Arasu et al., 2023), and *F. graminearum* on wheat (Risoli et al., 2023) and crop residues (Sarrocco et al., 2021). The biocontrol properties of *T. gamsii* are mainly linked to its production of cell wall-degrading enzymes (Bedine Boat et al., 2020; Valan Arasu et al., 2023), various metabolites with antifungal properties (Marik et al., 2018), and plant growth-promoting substances (Rinu et al., 2014). In addition to *T. gamsii*, five other tested isolates effectively controlled or delayed the growth of several strains of *Fusarium* (*Cistella* sp. Y49, *E. layuense* C165, *P. virgatum* C222, *P. minutissima* C371, and *T. paraviridescens* C101). One possible strategy to enhance that narrow spectrum activity is the application of a cocktail of BCAs (Kannan and Sureendar, 2009). For example, combining *T. paraviridescens* C101, which inhibited the growth of all *F. graminearum* strains, with *Cistella* sp. Y49, which reduced the infection of *F. avenaceum* and *F. poae*, could provide a broader antagonistic activity spectrum. The selected broad-spectrum anti-*Fusarium* isolates will be further investigated in order to evaluate their efficacy against other important FHB causal agents (e.g., *F. culmorum*) as well as under wheat field conditions. Furthermore, the abundance of antagonistic isolates found in maize residues suggests potential for treating *Fusarium* inoculum and/or reducing pathogen dispersal on infected crop residues as a promising conservation strategy (Gimeno et al., 2020). In vitro assays on crop residues are thus necessary to further evaluate this approach.

## 5. Conclusion

The purpose of this study was to isolate and characterize anti-*Fusarium* bacterial and fungal isolates from field substrates meant to be used as part of the biocontrol strategy against *Fusarium* Head Blight. We developed a workflow that took into consideration the intra- and interspecific diversity of pathogens as well as their life cycles, which are central for the selection of appropriate biocontrol strategies.

We isolated a high diversity of native microorganisms with potent antagonistic activity from soil, maize residues, and wheat grains. A selective isolation method, involving a preselection step, proved to be highly effective in identifying biocontrol agents. However, it is noteworthy that specific criteria drive each selection method and consequently introduce inherent biases, which may lead to the oversight of certain microorganisms with potential suitability as biocontrol agents. Substantial variations in inhibitory effects of antagonistic isolates across different strains of *F. graminearum* and among various *Fusarium* species highlight the need to incorporate both inter- and intraspecific diversity into screening methods. *Trichoderma gamsii* C224 emerged as the most
efficient agent, demonstrating complete control over the growth and highly reduced mycotoxin production of all tested *Fusarium* strains, both in vitro and in vivo. Other antagonistic isolates, in particular *T. paraviridescens* C101 and *Cistella* sp. Y49 also exhibited promising activity, albeit with a narrower spectrum. Applying these isolates in combination could enhance their effectiveness. The use of native BCAs such as *T. gamsii* offers an environmentally friendly alternative to synthetic fungicides, aligning with the principles of integrated pest management and contributing to reducing chemical inputs in cereal crop systems. Further investigations will focus on elucidating the mechanisms underlying antagonistic interactions among the selected bioactive isolates and the pathogen. Additionally, future research will need to evaluate their efficacy against an even broader range of FHB causal agents and under field conditions, as well as explore formulation strategies to ensure stable activity without negatively impacting plant growth. These efforts are critical for translating the promising in vitro and in vivo results into practical and sustainable biocontrol applications in real agricultural environments.

## Funding

This research was financially supported by the Agence Nationale de Recherche, grant number ANR-20-CE32-0008.

## CRediT authorship contribution statement

**Toan Bao Hung Nguyen:** Writing – original draft, Visualization, Investigation, Formal analysis, Data curation, Conceptualization. **Flora Pensec:** Writing – review & editing, Visualization, Supervision, Methodology, Investigation, Conceptualization. **Jérôme Mounier:** Writing – review & editing, Visualization, Supervision, Methodology, Investigation, Conceptualization. **Monika Coton:** Writing – review & editing, Methodology. **Amandine Henri-Sanvoisin:** Investigation. **Massilia Mouzaoui:** Investigation. **Adeline Picot:** Writing – review & editing, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization.

## 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

We extend our gratitude to the farmers for their participation and for granting us access to their fields for sample collection. We also would like to thank the Agence Nationale de Recherche for funding this work (grant number ANR-20-CE32-0008).

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.biocontrol.2025.105856.

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