# Exploring Fusarium Biocontrol, drought Tolerance, and plant growth promotion by Bacillus strains from Cuban wheat varieties

Alexander Govin-Sanjudo, Marcia M Rojas Badia, Cédric Jacquard, Qassim Esmaeel

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## To cite this version:

Alexander Govin-Sanjudo, Marcia M Rojas Badia, Cédric Jacquard, Qassim Esmaeel. Exploring Fusarium Biocontrol, drought Tolerance, and plant growth promotion by Bacillus strains from Cuban wheat varieties. Biological Control, 2025, 205, pp.105776. 10.1016/j.biocontrol.2025.105776. hal-05210159

HAL Id: hal-05210159

https://hal.inrae.fr/hal-05210159v1

Submitted on 14 Aug 2025

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## Perspective

# Exploring Fusarium Biocontrol, drought Tolerance, and plant growth promotion by *Bacillus* strains from Cuban wheat varieties

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Alexander Govin-Sanjudo<sup>a,b</sup>, Marcia M. Rojas Badia<sup>b</sup>, Cédric Jacquard<sup>a</sup>, Qassim Esmaeel<sup>a,*</sup> ID

## HIGHLIGHTS

* 15 *Bacillus* strains isolated from Cuban wheat significantly inhibit the growth of *Fusarium* spp.

* The bacterial strains reduce Fusarium symptoms and incidence in wheat seedlings and leaves.

* Bacillus strains exhibit high tolerance to drought conditions.

* The strains produce divers metabolites with plant growth promoting potential.

* Promising *Bacillus* strains enhance wheat germination under both drought stress and Fusarium infection.

## GRAPHICAL ABSTRACT

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## ARTICLE INFO

**Keywords:**

*Bacillus*

Wheat

Plant growth promotion bacteria

Antagonistic activity

*Fusarium*

Drought stress

## ABSTRACT

Members of the Bacillaceae family are recognized for their ability to promote plant growth under drought stress and for their capability to produce bioactive metabolites displaying antagonistic activity against phytopathogenic fungi. In this study, we evaluated the potential of bacterial strains isolated from Cuban wheat accessions to inhibit *Fusarium* species, tolerate drought stress, and promote the germination of wheat under stress conditions. A total of 150 bacteria were isolated from three Cuban wheat accessions, and 15 isolates were selected based on their elevated antagonistic activity. These strains, belonging to the Bacillaceae family, effectively reduced *Fusarium* symptoms on wheat leaves. To determine tolerance to drought stress, the isolates were exposed to different concentrations of polyethylene glycol (PEG) –6000, showing tolerance to 20 % PEG-6000. Furthermore, the strains underwent *in vitro* evaluation for plant growth-promoting (PGP) and lytic enzymes production. Ammonia production, indoleacetic acid, amino cyclopropane carboxylate deaminase, and nitrogen fixation were detected in all bacteria; moreover, each strain produced at least two of the five lytic enzymes tested. The bacterial isolates promoted wheat germination under both drought stress and *Fusarium* infection; with the highest effects registered for strains TII-10, TII-19, TCG-6 and TMG-6. These findings highlight the potential of these strains as effective agents for the promotion of wheat growth under the dual stresses of *Fusarium* infection and drought, contributing to more sustainable wheat production.

<footnote><sup>a</sup> Université de Reims Champagne Ardenne, INRAE, RIBP, USC 1488 Reims, France</footnote>

<footnote><sup>b</sup> School of Biology. Universidad de La Habana, Cuba</footnote>

<footnote>* Corresponding author.
E-mail address: qassim.esmaeel@univ-reims.fr (Q. Esmaeel).</footnote>
# 1. Introduction

Wheat is one of the most widely consumed cereals in the world, providing vital nutrients such as protein to billions of people worldwide (Maulenbay and Rsaliyev, 2024). Its economic significance was substantial, with global wheat production reaching 798.98 million tonnes in 2023 (Ritchie et al., 2023). Europe contributed 269 million tonnes, accounting for 33.70 % of global output. Among European producers, France led with 13.36 % of the total output, followed by Ukraine (8.00 %) and Germany (7.99 %) (FAO, 2023). Wheat crops face continuous threats from several biotic and abiotic factors, leading to significant reduction in grain yield and quality. Among these, drought is a notable destructive abiotic stress. This challenge has intensified in recent years due to climate change, impacting millions of hectares worldwide (Fadiji et al., 2022). This stress triggers dramatic morphological, physiological, biochemical, and molecular changes in the plant. Thus, drought stress in plants is characterized by a decrease in water content, reduced leaf water potential, stomatal closure, loss of turgor, and a reduction in cell growth (Jaleel et al., 2008). Although wheat needs water at all growth stages, it is more critical during the tillering, jointing, and anthesis stages (Thapa et al., 2020), as water scarcity during these phases can result in delayed germination, tillering, booting, heading, anthesis, grain filling, and maturity (Camaille et al., 2021). The critical growth stages of wheat are the most vulnerable to water scarcity; any decrease in water supply at these stages leads to significant yield loss (Dhakal, 2021). Drought stress decreases the photosynthetic rate, disrupts the balance of carbon assimilation, and ultimately results in a higher nitrogen concentration in the grain (Yang et al., 2023). Additionally, it reduces the number of tillers, spikes, and seeds per plant and shortens the time of heading (Olumekun et al., 2023).

Fungal diseases are recognized as another significant yield-limiting factor in wheat, and the losses can research up to 50 % (Różewicz et al., 2021). One of the fungal genera that most significantly affects wheat is *Fusarium*. The *Fusarium* genus compromises a complex of species that impact cereal crops, including *F. graminearum*, *F. culmorum*, *F. pseudograminearum*, *F. avenaceum*, and *F. poae* (Karlsson et al., 2021). These fungi are also capable of producing mycotoxins, which contaminate grains and pose serious risks to human and animal health. Fusarium head blight (FHB) is known as one of the most destructive fungal diseases worldwide in wheat and other cereals, reducing yields by up 70 % (Semagn et al., 2023).

Conventional agriculture is characterized by using chemical fungicides to manage fungal diseases. This has led to higher production costs, environmental contamination, residues in harvested products, and the development of resistant pathogen populations (Penuelas et al., 2023). Furthermore, these compounds can affect human health. Hence, in recent years, the use of plant growth-promoting bacteria with antagonistic activity against phytopathogenic fungi has been increasingly explored (Jan et al., 2023).

Members of the Bacillaceae family are characterized by a close relationship with plants (Fazle Rabbee and Baek, 2020). Furthermore, these members are known to synthesize a diversity of secondary metabolites with the potential to inhibit fungal growth, such as lipopeptides, polyketides, siderophores, lytic enzymes, and inorganic and organic volatile compounds (Asaturova et al., 2022). Similarly, they can stimulate plant growth by solubilizing phosphorous and potassium, synthesizing phytohormones such as indol acetic acid (IAA), cytokines, and gibberellin (Shen et al., 2023). Moreover, they can enhance plant tolerance to drought by synthesis of exopolysaccharides, production of 1-aminocyclopropane-1-carboxylate (ACC) deaminase and proline, improving relative water content, total soluble sugar, total chlorophyll and carotenoid contents, and reducing lipid oxidation in stressed plants (Ahmad et al., 2022). *Bacillus*- based agricultural products have been developed and distributed worldwide (Miljaković et al., 2020). Thus, the isolation and selection of strains with antagonistic activity against phytopathogenic fungi and plant growth promoting traits constitute a

powerful tool for managing fungal diseases and improving plant tolerance to biotic and abiotic stress.

Wheat was introduced in Cuba in the Colonial Period and cultivated successfully until the first half of the 19th century (Moreno et al., 2001). However, production ceased due to the importation of flour from Castilla, the presence of pests, and the increase cultivation of sugar cane and tobacco. In the 20th century, wheat was reintroduced, but the cultivars lost their agronomic properties after several generations, necessitating the development of Cuban cultivars adapted to local climatic conditions (Moreno et al., 2001). Despite the existence of these cultivars, wheat production in Cuba nearly nonexistent, which explains the heavy reliance on imports for national production of different derivatives, around 700,000 tons of wheat were imported in 2020 (ONEI, 2022). Despite the topic is highly relevant, research in Cuba focusing on the isolation and selection of strains with potential for stimulating plant growth isolated from Cuban wheat crops are limited. In address this gap, this study aimed to evaluate the potential of bacterial strains isolated from Cuban wheat for drought stress tolerance, biocontrol and plant growth promotion under drought stress.

# 2. Materials and methods

## 2.1. Isolation of *Bacillus* from Cuban wheat varieties

Rhizospheric soil and plant material samples were taken from Cuban wheat accessions (CC-204, I-399 and M-04) grown in plots at the Grain Research Institute (IIG), Artemisa (GPS coordinates at 22°57'22.0" N and 82°28'14.4" W), and the Fundamental Research in Tropical Agriculture "Alejandro de Humboldt" (INIFAT), Havana (GPS coordinates at 22°58'35.8" N and 82°23'20.0" W), Havana, Cuba. The samples were stored in sterile bags, transported to the laboratory in ice containers, and kept at 4 °C until processing.

For the isolation of rhizosphere bacteria, 1 g of rhizosphere soil was added to 9 mL of sterile distilled water, and serial decimal dilutions were prepared. The 10⁻⁵ dilution was incubated at 80 °C for 10 min (Hellany et al., 2024) to eliminate the vegetative cells, and 100 µL was inoculated onto Tryptone Soy Agar (TSA, Difco™, Maryland, USA) plates. For the isolation of endophytic bacteria, the plant material was washed with tap water, immersed in 3 % (v/v) sodium hypochlorite for 3 min, and then washed five times with sterile distilled water. A 100 µL aliquot of the last rinse water was inoculated onto TSA plates as a disinfection control, and the plates were incubated at 28 °C for 48 h. Next, 1 g of disinfected plant material was macerated, and serial decimal dilutions were prepared. The 10⁻⁵ dilution was incubated at 80 °C for 10 min, and 100 µL was spread onto TSA plates. In both cases, the plates were incubated for 24 to 48 h at 28 °C. Once the growth was observed, colonies with distinct differences in morphology, size, and color were selected for purification. The isolates were purified and stored in 25 % glycerol at -80 °C. The phenotypic characteristics of the isolates were then determined.

## 2.2. Evaluation of antagonistic activity of bacterial isolates against *Fusarium* spp

Direct antagonism activity against *F. culmorum* SE-1 and *F. graminearum* SE-2, both of which are phylogenetically represented in Supplementary Fig. S1, was assessed through direct confrontation on solid media. A 7 mm diameter mycelial plug from a 7-day-old *Fusarium* colony was placed at the center of PDA (Potato Dextrose Agar, Difco™, Maryland, USA) plate. Then, 5 µL of bacterial culture was inoculated at four points, 2.5 cm away from the mycelial plug, with each point representing a different bacterial isolate. Control plates were inoculated with only the fungal strain. The plates were incubated at 25 °C for 7 days, and bacteria showing antagonistic activity were considered promising and selected for further study. In the subsequent evaluation of the direct antagonistic activity of promising bacteria, each strain was
inoculated individually on separated plates. The experience was performed in triplicate. After 7 days of incubation at 25 °C, the inhibition rate (IR) was calculated using the following formula (Tang et al., 2024a):

$$IR = \frac{\text{Control colony diameter} - \text{bacterial treatment colony diameter}}{\text{control colony diameter}} \times 100$$

Additionally, to assess the potential mycelial damage caused by bacterial strains, a fragment of the mycelium from the zone of inhibition was examined by an epifluorescence microscope.

## 2.3. Antagonistic effect of volatile organic compounds (VOCs) and hydrogen cyanide (HCN) production by bacterial strains

The double Petri-dish assay was used to determine whether bacterial isolated produce VOCs that could inhibit fungal growth (Tang et al., 2024b). Bacteria were inoculated on LB medium, while fungi were inoculated on PDA. The bottom of each plate was placed on top of its corresponding lid, and the bottoms of both plates were sealed with parafilm. Plates of PDA inoculated with fungal strains and non-inoculated LB agar plates were used as controls. The plates were incubated at 25 °C for 7 days. Fungal growth in the presence of bacteria was compared with that of the control.

Hydrogen cyanide production was assessed on Tryptone Soy agar supplemented with 4.4 g/l of glycine. Whatman filter paper No 1 was soaked in a solution containing 0.5 % picric acid and 0.2 % Na₂CO₃. The soaked filter paper was placed in the lid of the Petri plate, and the plates were sealed with Parafilm. The cultures were incubated at 28 °C for seven days. A color change of the filter paper from yellow to orange-brown indicated the production of HCN (Patel et al., 2023).

## 2.4. Antifungal activity of bacterial cell-free supernatant (CFS)

The antifungal activity of bacterial supernatant on the germination and growth of phytopathogenic fungi was assessed in both liquid and solid media. Cell-free supernatant was obtained from bacterial culture in Landy Medium broth (glutamic acid 5.0 g/L, tryptophan 0.016 g/L, KH₂PO₄ 0.5 g/L, K₂HPO₄ 0.5 g/L, MgSO₄·7H₂O 0.2 g/L, MnSO₄·H₂O 0.01 g/L, NaCl 0.01 g/L, FeSO₄·7H₂O 0.01 g/L, CuSO₄·7H₂O 0.01 g/L, CaCl₂·2H₂O 0.015 g/L) for 48 h at 28 °C and 150 rpm. Cultures were centrifuged at 10, 000 rpm for 10 min at 4 °C and the supernatant was filtered through a 0.20 µm filter.

For the liquid medium assay, the CSF was mixed at a 1:1 ratio with two times concentrated Potato Dextrose Broth (PDB, Difco™, Maryland, USA) (Besset-Manzoni et al., 2019) in 24 wells micro-plates. The plates were inoculated with fungal spore suspension (5000 spores/mL) and incubated at 25 °C and 250 rpm for 96 h. The experiment was carried out in triplicates. The absorbance was measured daily at an optical density (OD) of λ = 620 nm.

For the solid assay, the supernatant was mixed at a 1:1 with ration with two times concentrated PDA, and 24-well-plates were filled with 1.5 mL of the mixture. The plates were inoculated with 5 µL spore suspension (5000 spore/mL) and incubated at 25 °C for 7 days. The experiment was conducted in triplicates. Fungal morphological modifications in both assays were verified by observing the mycelium under an epifluorescence microscope.

To evaluate the production of biosurfactant, the drop collapse test was used. A 20 µl of supernatant was dropped onto parafilm and incubated for 1 h. The collapse of the drop was then compared with that of the control (non-inoculated medium) (Caulier et al., 2018).

## 2.5. Detached leaf assay

The biocontrol activity of bacterial strains isolated from Cuban wheat varieties against *F. culmorum* SE-1 and *F. graminearum* SE-2 was

evaluated using the detached leaf assay described by Dutilloy et al. (2024). The Calixo and Lennox wheat varieties were used in this test. Wheat seeds were planted in tray subtract and incubated in a green house until the 3-leaf stage (3-L stage) was reached. The second leaf was carefully removed, desinfected, and cut into 7 cm fragments. The leaf fragments were placed on water Agar (10 g/L) supplemented with kinetin (1 g/L). The leaves were injured with sterilized needles and inoculated with 5 µL of fungal spore suspension (5000 spores/mL) and 5 µL of bacterial inoculum (10⁸ cells/mL). The plates were incubated in a growth chamber at 18/20 °C with photoperiod of 16 h/8h light/darkness for 10 days. After incubation, Fusarium symptoms were examined, and lesion sizes were recorded. The experiments were performed in triplicate.

## 2.6. Tolerance to drought stress

Bacterial tolerance to drought stress was evaluated in Tryptone Soy broth medium (TSB, Difco™, Maryland, USA) supplemented with different concentrations of polyethylene glycol (PEG-6000): 0, 5, 10, 15, 20, 25 and 30 % (w/v). The osmotic potentials were calculated using the equation described by Michel and Kaufmann (1973):

$$\Psi = -1.18 \times 10^{-2} \cdot c - 1.18 \times 10^{-4} \cdot c^2 + 2.67 \times 10^{-4} \cdot c \cdot T + 8.39 \times 10^{-7} c^2 T$$

Where Ψ is the osmotic potential expressed in megapascals (MPa), c is the concentration of PEG-6000 in grams per kilogram of water and T is the temperature. The cultures were incubated at 25 °C, 150 rpm for 24 h, and the OD was determined at λ = 600 nm.

## 2.7. Exopolysaccharides production, biofilm formation and swarming motility

Exopolysaccharide (EPS) production was evaluated using two approaches. The first involved Congo Red medium (25.0 g/L LB broth, 50.0 g/L sucrose, 18.0 g/L agar, 0.8 g/L Congo Red, pH 7.0 ± 0.2). Bacterial strains were inoculated onto the medium, and the plates were incubated at 28 °C for 120 h (Shaffique et al., 2023). The presence of black colonies indicated EPS production. The second method was based on the medium described by Kavamura et al. (2013). Bacterial strains were inoculated onto 5 mm diameter paper discs dispersed in the medium, which consisted of: 20.0 g/L yeast extract; 15.0 g/L K₂HPO₄; 0.2 g/L MgSO₄; 0.015 g/L MnSO₄; 0.015 g/L FeSO₄; 0.03 g/L CaCl₂; 0.015 g/L NaCl; 15.0 g/L agar), supplemented with 10 % sucrose, pH 7.5. The plates were incubated at 25 °C for 48 h. EPS production was detected by the size of the halo produced and the slimy appearance around the discs. To confirm the presence of EPS, a portion of the mucoid substance was mixed with 2 mL of absolute ethanol, precipitation indicated the presence of EPS (Kavamura et al., 2013).

Biofilm production was assessed following the method of Dutilloy et al. (2024). Briefly, a bacterial pre-culture in LB broth was cultured for 18 h, then a suspension was prepared to an OD of 0.2 (λ = 600 nm). Aliquots of 200 µL were transferred to each well of a 96 wells plate. The plate was incubated for 48 h in static conditions at 28 °C. After incubation, the medium was removed, and the plate was carefully rinsed with distilled water and dried. 200 µL of crystal violet was added to each well and left for 10 min. After decanting the dye, the plate was rinsed with distilled water and left to dry for 1 h. Thereafter, 200 µL of 30 % acetic acid solution was added and incubated for 15 min at room temperature. Biofilm quantity was determined by measuring the OD at λ = 570 nm. The experiment was carried out five times.

Swarming motility was evaluated in LB medium supplemented with 0.7 % (w/v) agar. The plates were inoculated on the center with 5 µL bacterial suspension (OD 600 ~ 0.4). Cultures were incubated at 28 °C for 22 h and the diameter of bacterial growth was recorded (Cao et al., 2018; Shen et al., 2023).
## 2.8. Phosphate and potassium solubilization, ammonia and siderophore production

Phosphates solubilization was evaluated using NBRIP agar medium (5.0 g Ca₃(PO₄)₂, 10.0 g glucose, 5.0 g MgCl₂·6H₂O, 0.25 g MgSO₄·7H₂O, 0.2 g KCl, 0.1 g (NH₄)₂SO₄, 5.0 g Ca₃(PO₄)₂, 1 L distilled water) (Nautiyal, 1999). The plates were inoculated with 5 µL of bacterial suspension and incubated at 28 °C for 10 days. The development of a transparent halo around bacterial growth was considered as positive respond.

Potassium solubilization was assessed on Aleksandrov medium (5.0 g/L glucose, 0.5 g/L magnesium sulphate, 0.005 g/L ferric chloride, 0.1 g/L calcium carbonate, 2.0 g/L calcium phosphate, and 2.0 g/L KNO₃). The plates were inoculated with 5 µL of bacterial suspension and incubated at 28 °C for 72 h. Bacteria that produced a yellow halo around their growth were considered positive for potassium solubilizing (Rajawat et al., 2016).

Ammonia production was determined in Peptone-Water medium (10.0 g bacteriological peptone, 5.0 g NaCl, 5.0 g yeast extract, 1 L distilled water, pH 7.6). The medium was inoculated with 1 % (v/v) of bacterial suspension and incubated at 28 °C for 72 h. After incubation, the cultures were centrifuged at 10,000 rpm for 10 min, and 50 µL of supernatant was mixed with 100 µL of Nessler's reagent in 96 wellmicroplates. A color change from orange to brown was considered positive results. The ammonia quantity was determined by reading OD at λ = 450 nm, using a standard curve prepared with (NH₄)₂SO₄ (Abdelwahed et al., 2022).

Siderophores production was determined using the Chrome Azurol S (CAS) agar medium (Schwyn and Neilands, 1987) with modifications from Louden et al. (2011). The CAS agar medium was inoculated with 5 µL bacterial suspension and incubated for 96 h at 28 °C. A yellow orange halo around the colony indicated siderophores production. All experiments were performed in triplicate.

## 2.9. Nitrogen fixation

Nitrogen fixation was determined using nitrogen free medium (Nfb): 20.0 g mannitol, 0.2 g K₂HPO₄, 0.2 g NaCl, 0.2 g MgSO₄·7H₂O, 0.1 g K₂SO₄, 5.0 g CaCO₃, 20.0 g Agar, 1 L distilled water, pH 7.0 ± 0.2. Bacteria were inoculated three consecutive times on Nfb medium. The presence of bacterial growth was considered as positive indication of nitrogen fixation (Salem et al., 2024).

## 2.10. Production of indoleacetic acid (IAA)

The qualitative production of IAA was evaluated in Landy medium. The bacterial cultures were incubated at 28 °C for 72 h with continuous shaking at 150 rpm. Bacterial cultures were centrifuged, and 100 µL of supernatant were mixed with 100 µL of Salkowski reagent (Glickmann and Dessaux, 1995). The mixture was incubated in the dark at room temperature for 30 min. The development of a pink color indicated IAA production. IAA content was quantified by measuring the OD at λ = 520 nm, with a standard curve prepared using commercial IAA (Anguiano-Cabello et al., 2017). The experiment was conducted in triplicate.

## 2.11. Production of 1-aminocyclopropane-1-carboxylate (ACC) deaminase

The production of ACC deaminase was determined by assessing bacterial growth on Dworkin and Foster (DF) medium amended with 3 mM ACC (Gupta and Pandey, 2019). The cultures were incubated at 28 °C for 5 days, and bacterial growth was recorded. The experiment was carried out in triplicate.

## 2.12. Production of lytic enzymes

The ability of bacterial strains to produce lytic enzymes was evaluated on different culture media. A bacterial suspension was prepared, and 5 µL was inoculated into each medium. Protease production was evaluated on milk agar: 5.0 g/L tryptone, 2.5 g/L yeast extract, 1.0 g/L dextrose and 28.0 g/L skim milk powder. Milk was prepared independently and mixed with the base medium at 50 °C. Plates were incubated at 28 °C for 48 h. The presence of clear zones around the colony indicated protease activity (Syed-Ab-Rahman et al., 2018). Amylase activity was determined on Casein-Yeast extract agar (CYEA) (casein peptone, 5 g; yeast extract, 2.5 g; glucose, 1.0 g; agar, 18.0 g in 1 L of distilled water, pH 7.2 ± 0.2) supplemented by 0.5 % (w/v) starch. Plates were flooded with Lugol's iodine solution, and after a simple rinse, clear halos around the colony indicated amylase production.

Cellulase production was detected on carboxymethyl cellulose (CMC) agar: 10.0 g CMC disodium salt, 1.0 g K₂HPO₄, 1.0 g KH₂PO₄, 0.2 g MgSO₄·x7H₂O, 1.0 g NH₄NO₃, 0.05 g FeCl₃·6H₂O, 0.02 g CaCl₂, 20.0 g agar and 1 L distilled water. Agar plates were flooded with a 0.5 % (w/v) Congo Red for 15 min, then washed with 1 M NaCl solution for 15 min. Clear zones indicated cellulase production.

The detection of pectinases was evaluated using pectine-agar medium: Yeast extract 1.0 g, (NH₄)₂SO₄ 2.0 g, Na₂HPO₄ 6.0 g, KH₂PO₄ 3.0 g, Pectine 5.0 g, Agar 15.0 g. Cultures were incubated at 28 °C for 5 days. Hexadecyl trimethyl ammonium bromide (CTAB) solution was added for 15 min, followed by washing with NaCl solution (1 M) (Kenfaoui et al., 2023).

Lipases production was detected on Tween Agar, containing 10 g peptone, 5 g NaCl, 0.1 g CaCl₂·2H₂O, 15 g agar, 10 ml Tween 80, and 1 L distilled water. Plates were incubated at 28 °C for 96 h. The appearance of a milky white precipitate surrounding the colonies indicated lipases production.

The production of lytic enzymes was tested in triplicate, and both colony size and hydrolysis halo were measured. The hydrolytic index (HI) was calculated using the formula:

$$ HI = \frac{\text{diameter of halo (mm) + the diameter of a colony (mm)}}{\text{diameter of a colony (mm)}} $$

## 2.13. Biochemical and molecular identification of bacterial isolates

Bacterial isolates were identified by biochemical and molecular approaches. Biochemical characterization was determined by the API-50 CHB gallery. Molecular identification was carried out by sequencing the 16S rRNA gene with the universal primer: forward FD2 (AGAGTTT-GATCATGGCTCAG) and reverse RP1 (ACGGTTACCTTGTTACGACTT). Colony polymerase chain reaction (cPCR) was carried out to amplify the 16S rRNA gene. The PCR reaction mixture included 12.5 µL of PCR Master mix (2X) (Thermo Scientific Fermentas, Villebon sur Yvette, France), 1.5 µL of each primer, 3 µL of bacterial DNA template, Milli-Q water to make a final volume of 25 µL. The thermal cycling conditions were as follows: initial denaturation at 95 °C for 5 min, 35 cycles of 95 °C for 30 s, 56 °C for 30 s, 72 °C for 1.5 min, followed by a final extension at 72 °C for 10 min. PCR product was visualized using electrophoresis on a 1 % (w/v) agarose gel and purified by ExoSAP-ITTM (Applied Biosystem, Illkirch, France), following the manufacturer indications. Purified PCR products were sequenced by Sanger sequencing at Azenta Life Sciences (Leipzig, Germany). The sequences were analyzed using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) against the NCBI database. The 16S rRNA gene sequences of all strains have been deposited in GenBank, with the corresponding accession numbers provided in Supplementary Table S1. The phylogenetic tree was constructed using Geneious Prime® 2025.0.3 (https://www.geneious.com).
## 2.14. Influence of bacterial isolates on wheat germination under drought and Fusarium infection

The effect of bacterial strains on the germination of the Calixo wheat variety was evaluated. Seeds were disinfected in a 1 % (w/v) sodium hypochlorite solution for 5 min, then rinsed three times with sterile distilled water. Seeds were soaked in sterile distilled water for 24 h to accelerate the germination process. Different treatments were tested: non-inoculated seeds (control), seeds inoculated with bacteria, seeds infected with the fungus, and seeds co-inoculated with both bacteria and the fungus. Bacterial cultures were prepared in LB broth medium (incubated at 28 °C, 150 rpm, for 18 h), and the bacterial concentration was adjusted to $10^6$ cells/mL. Seeds were submerged in the bacterial suspension for 1 h for inoculation (Karimzad et al., 2023). A fungal spore suspension was prepared from a 5-day-old fungal culture of *F. culmorum* SE-1 and was adjusted to $10^6$ spores/mL. To infect seeds with the fungal strain, they were placed on a plate, sprayed with the fungal spore suspension using a handheld sprayer, and dried for 1 h (Kaur et al., 2020). Seeds were placed on water agar medium (12 seeds per plate) with different osmotic potentials (0, -0.5 and -1.5 MPa). Plates were incubated in a culture room at 20-22 °C with 16 h light period (12 000-16 000 lx) (Lastochkina et al., 2020). The experiment was designed as a completely randomized trial in triplicate. At the end of the experiment, the germination percentage was calculated, and three seedlings were randomly selected to measure their shoot and root size lengths.

## 2.15. Statistical analysis

All tests were carried out in triplicate and using a completely randomized design. Data were transformed to assess inhibition and germination percentage. Normality of the data was tested by

Shapiro-Wilk Test. Statistical significance was evaluated using a one-way analysis of variance (ANOVA), followed by Ducan test, with $p < 0.05$ considered statistically significant. When the data did not meet the assumptions required for ANOVA, the Kruskal Wallis test followed by Dunn test were applied. All analyses were performed using Infostat 2020 and GraphPad Prism 8.4.3 version.

# 3. Results

## 3.1. Isolation and screening of bacterial strains associated with Cuban wheat accessions

A total of 150 bacterial colonies were successfully isolated from the rhizosphere and endosphere of Cuban wheat accessions. The majority of isolates were obtained from the rhizosphere (104), 28 and 18 bacteria were also isolated from root-endosphere and endosphere respectively. All isolates were rod-shaped, Gram-positive, and endospore-forming.

To evaluate their antagonistic potential, the isolates were subjected to in vitro screening against *F. graminearum* and *F. culmorum* using dual culture assays. Fifteen isolates exhibited significant differences compared with the controls, with fungal growth reduced by over 50 % of both pathogens (Figs. 1A1-3 and 1B1-3). The strain TMG-6 displayed the highest inhibition rate, with 74.6 % against *F. graminearum* and 75.8 % against *F. culmorum*. Additionally, strains TII-10 and TCG-6 demonstrated notable inhibition percentage, higher than 69 % against both phytopathogenic fungi. While most strains had similar inhibition percentages for both fungi, strains TIG-13, TMG-19, and TMG-43 exhibited differences exceeding 8 % between the two pathogens.

To reveal the potential damage caused by the bacterial strains, a fragment of mycelium from inhibition zone was examined under the microscope and compared with the control. The control displayed active

<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Antagonistic potential of bacterial strains isolated from Cuban wheat varieties against *F. graminearum* (A) and *F. culmorum* (B). Inhibition percentage of fungal growth by bacteria, the bar represents the means of three determinations ± standard deviation, different letters indicate significant differences according to ANOVA, Ducan's test ($p < 0.05$, $n = 3$). Dual cultures bioassay of bacterial isolates and phytopathogenic fungi (1-3); control (1), representative bacterial isolates TCG-6 (2), TMG-6 (3); the bar represents 1 cm. Microscopic observations of morphological changes in the mycelium (4-6), control (4), representative bacteria: TCG-6 (5), TMG-6 (6); the arrows indicate cytological modifications and the bar represents 20 µm.</figcaption></figure>
branching with normal hyphal structure (Fig. 1A4 1B4). In contrast, bacterial strains induced significant hyphal modifications, including swelling, deformation with distorted and condensed structures, vacuolation, mycelium degradation, and the formation of structures resembling chlamydospores (Fig. 1A5-6 and 1B5-6).

Additionally, to evaluate whether bacterial strains produce volatile metabolites with antifungal potential, a double Petri-dish bioassay was carried out. No significant differences were observed between the bacterial treatments and the control for both fungi (Supplementary Fig. S2A, S2B, S2C and S2D), as fungal inhibition was not detected. Microscopic observation showed active branching and normal hyphal structure in the bacterial treatments (Supplementary Fig. S2E, S2F, S2G and S2H). Moreover, all bacterial strains showed a negative response to HCN production. These findings indicate that the antagonistic activity is not attributed to volatile compounds.

## 3.2. Direct antagonism of bacterial cell-free supernatant (CFS)

The antifungal activity of the CSF was evaluated in both liquid and solid media. In liquid medium, the CSF significantly delayed fungal germination and reduced fungal growth after 96 h compared to the control (Fig. 2A and 2B). The supernatants of strains TII-10 and TMG-6 exhibited the strongest anti-germination effects, with absorbance values 40 to 20 times lower for *F. graminearum* and *F. culmorum*, respectively. Moreover, the CSF from TII-19 had a potent inhibitory effect against *F. graminearum*, while metabolites from TCG-6 significantly inhibited *F. culmorum*.

On solid medium, the CSF demonstrated antifungal potential, but inhibition percentage was approximately 15 % lower than those observed in the dual culture assay (Fig. 2C and 2D). Statistically significant differences were observed among treatments, with strains TMG-6, TCG-6, TII-10, TII-3, TII-19, and TMG-37 achieving inhibition rates exceeding 40 % against both pathogens. The highest inhibition was recorded by TMG-6, with 48.9 % against both fungi.

Microscopic observations in both assays allowed to identify

significant hyphal alterations in *F. graminearum* and *F. culmorum*. These included hyphal swelling, granule formation, massive conglобation along the hyphae, macroconidia degradation, formation of chlamydospore-like structures, and the presence of macroconidia (Fig. 2A1, 2A2, 2B1 and B2). These cytological modifications highlight the potent inhibitory effects of bacterial metabolites on fungal development and reinforce the potential of Cuban wheat-associated *Bacillus* strains as biocontrol agents. Further studies are necessary to confirm their efficacy and to reveal their mechanism of action against *Fusarium* sp.

## 3.3. *Bacillus* strains significantly reduce Fusarium-related necrosis and growth in detached leaves

The ability of bacterial strains to control Fusarium diseases was evaluated using the detached leaf bioassay. Two wheat varieties, Calixo and Lennox, were tested. Bacterial and fungal cultures were inoculated simultaneously. In the positive control, Fusarium-related necrosis and fungal growth became visible on leaves of both wheat varieties three days post-infection. However, in leaves treated with some bacterial strains, symptoms appeared only 5dpi, indicating a delay in necrosis and fungal growth (Fig. 3). By the end of the test, wheat leaves treated with *Bacillus* strains exhibited reduced lesion sizes and fungal growth, with significant differences compared to the positive control. Greater inhibition was detected against *F. culmorum* compared to *F. graminearum*. Specifically, 10 and 11 bacteria inhibited *F. culmorum* symptoms in Calixo and Lennox varieties, respectively, whereas 6 and 5 for *F. graminearum*. Notably, strains TII-3, TII-10, TII-19, TCG-6, TMG-6 and TMG-37 demonstrated significant disease control, reducing lesion sizes by more than 40-fold compared to control. These results highlight the antifungal potential of bacterial strains under study in mitigating Fusarium- disease severity.

<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Effect of bacterial cell-free supernatants on fungal germination and growth, against *F. graminearum* (A, C) and *F. culmorum* (B, D). Microscopic observations of morphological alterations induced by bacterial metabolites from the representative strain TMG-6 (A2, B2, C2 and D2) were compared to Control (A1, B1, C1 and D1). The test was evaluated in liquid (A and B) and solid medium (C and D). M: macroconidia. The bars represent the mean value of three determinations ± standard deviation. Different letters indicate significant differences, Dunnett test (n = 3, p < 0.05). The arrow indicated morphological alterations. The white bars represent 275 µm and black bars represent 20 µm.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Detached leaves assay (A-B: Calix variety and C-D: Lennox variety). Fungal lesion size (A-C: leaves infected with *F. graminearum*), (B-D: leaves infected with *F. culmorum*). The bars represent the mean value of three determinations ± standard deviation. Asterisk (*) indicates significant differences, Kruskal Wallis, Dunn's test (n = 9, p < 0.05). Visual observations, C-, Neg. Control: negative control (non-infected leaves) C+, Pos. Control: positive control (leaves inoculated with *Fusarium* spp.); TMG-6 and TMG-19: leaves inoculated with bacterial strains. The plates were incubated under a photoperiod of 16 h light/ 8 h dark for a duration of 10 days. Bar represents 2 cm. The arrows indicate fungal growth on wheat leaves.</figcaption></figure>

**3.4. High drought tolerance of *Bacillus* strains under PEG-induced osmotic stress**

To evaluate drought tolerance, bacterial isolates were subjected to different levels of osmotic stress using LB broth supplemented with increasing concentration of PEG-6000 (0—25 %, w/v). Higher PEG-6000 concentrations negatively impacted bacterial growth, revealing a pronounced osmotic effect (Fig. 4). Based on OD values, isolates were categorized into 4 groups: highly sensitive (OD < 0.3); sensitive (OD 0.3 to 0.39); tolerant (OD 0.4 to 0.5), and completely tolerant (OD > 0.5) (Ashry et al., 2022). All tested strains were completely tolerant to 20 % of PEG-6000, while five strains (TII-19, TCG-6 TMG-3, TMG-6, and TMG-19) exhibited tolerance up to 25 % of PEG-6000. These findings provide strong evidence of the potential drought tolerance of *Bacillus* strains isolated from Cuban varieties, suggesting their potential role in enhancing drought stress tolerance in wheat.

**3.5. Comprehensive identification and functional characterization of bacterial strains**

The 15 bacterial strains that exhibited promising antagonistic activities against *Fusarium* were identified through 16S rRNA sequence analysis. These strains belonged to the *Bacillus* genus and shared high similarities with different *Bacillus* species. A subset showed close identity with *Bacillus velezensis*, while others aligned more closely with *B. amyloliquefaciens*, *B. spizizenii*, *B. subtilis*, *B. halotolerans*, and *B. cereus*. (Fig. 5). Moreover, the bacterial isolates were biochemically characterized using the API 50 CHB system, with results are presented in the Supplementary Table S2. All strains were able to metabolize D- glucose, D- fructose, D- cellobiose, and esculin ferric citrate. However, they did not assimilate 21 other compounds as carbon sources. Distinct metabolic

patterns were detected among the strains, even within those belonging to the same species, confirming their classification as different bacterial strains.

**3.6. Plant growth promoting (PGP) characteristics**

Bacterial strains were investigated *in vitro* for different PGP traits, including phosphate and potassium solubilization, nitrogen fixation, siderophores production, amino cyclopropane carboxylate (ACC) deaminase, indoleacetic acid (IAA), ammonia, biosurfactant, exopolysaccharides, and biofilm production. All strains exhibited at least four PGP characteristics (Table 1). Phosphate solubilization was detected in the strains TII-3, TII-10, TMG-2, and TMG-19 strains, while potassium solubilization was observed in 14 strains, with TMG-43 strain showing no response (Supplementary Fig. S3A and S3B). Siderophores production was identified in 13 strains (Supplementary Fig. S3C). The production of ACC deaminase was confirmed by bacterial growth on DF medium, with all strains showing positive results (Supplementary Fig. S3D). Nitrogen fixation was observed in all strains, while biosurfactant production was detected in 13 bacterial strains, with TMG-3 and TMG-43 showing no activity (Supplementary Fig. S3E and S3F). Additionally, 13 strains produced EPS, as indicated by black, and mucoid colonies on Congo red medium and halo formation on EPS inductor medium (Supplementary Fig. S3G and S3H). Strains TCG-17 and TMG-43 did not show EPS production.

IAA production ranged from 7.8 µg/L –39.9 µg/L, with TII-15 strain having the highest and TMG-43 the lowest production (Table 1). Ammonia production was detected in all strains, with concentrations ranging from 4789.6 to 9218.4 µmol/L, being TMG-22 strain which registered the higher production and TII-3 which showed the lowest production. These findings highlight the potential of bacterial strains
<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Bacterial growth in TSB medium amended with different concentrations of PEG-6000. The cultures were incubated at 200 rpm, 28 °C for 24 h. The bars represent the mean value of three determinations ± standard deviation. Different letters indicate significant differences, Duncan and TMG-43 was analyzed by Dunnett's test (n = 3, p < 0.05).</figcaption></figure>

isolated from Cuban varieties for plant growth promotion.

Regarding biofilm formation, 12 strains exhibited higher OD values than negative control. Strain TMG-19 displayed the strongest biofilm formation, with an OD value 89 times greater than the control (Table1). Based on OD values, strains were classified as non-adherent (TII-15, TCG-17, TMG-43, OD ≤ 0.202), moderately adherent (TCG-6, OD: 0.845 ± 0.098), and strongly adherent (the 11 other strains, OD > 0.808). Bacterial EPS and biofilm are recognized for their high-water content and their ability to enhance tolerance to drought. These findings highlight the potential of bacterial isolates to induce drought stress tolerance.

The swarming motility ability was observed in 14 isolates, in which bacterial growth was expanded, covering the entire culture medium (Supplementary Fig. S3I1). A negative response was detected by strains TMG-43, in which the growth was spread only a few millimeters from the point of inoculation (Supplementary Fig. S3I2).

### 3.7. Production of lytic enzymes

To gain insight into the possible antagonistic activity mechanisms of the bacterial strains, the production of lytic enzymes was studied. Amylases, glucanases, proteases, pectinases and lipases were detected on specific media (Supplementary Fig. S4A-S4E), with all strains producing proteases (Table 2). While, all strains produced proteases, amylases production was detected in 13 isolates, glucanases in 11, pectinases in 10, and lipases in 14 strains. The production of all enzymes was

observed in six bacteria (TII-3, TCG-6, TCG-17, TMG-3, TMG-6 and TMG-37).

Hydrolytic indices varied among strains: the amylolytic index ranged from 2.0 to 2.5, with TMG-19 exhibiting the highest index (2.5 ± 0.0). The cellulolytic index ranged from 2.1 to 3.5, with the TMG-6 having the highest value (3.5 ± 0.2). The proteolytic index spanned from 2.0 to 2.5, with TCG-17 recording the highest value (2.5 ± 0.1), while the pectinolytic index extended from 2.1 to 3.6, with TCG-6 and TMG-15 strains both having the highest value (3.6 ± 0.3). Additionally, no HCN production was detected in any of the strains.

### 3.8. Bacterial strains enhance wheat germination and growth while mitigating drought and Fusarium stress

The effect of bacterial strains on wheat germination and growth were evaluated under different osmotic potential and *Fusarium* infection (Table 3). At higher osmotic potentials, wheat germination rates decreased. Under 0 and -0.5 MPa, no significant differences were detected between bacterial treatments and controls. However, at -1.5 MPa, seven strains (TII-19, TCG-6, TCG-17, TMG-6, TMG-15, TMG-19, and TMG-37) enhanced germination. The highest rates were recorded for TMG-15 (0.78 ± 0.13), TII-19 (0.75 ± 0.00), and TCG-17 (0.75 ± 0.00), indicating these bacteria promote germination under drought stress conditions.

When subjected to both *Fusarium* infection and drought stress, no significant differences were observed at 0 and -0.5 MPa. At -1.5 MPa,
<figure><img src="image_9.png" /><figcaption>**Fig. 5.** Phylogenetic trees of *Bacillus* strains. The tree was built using Geneious Prime® 2025.0.3 (https://www.geneious.com). *Clavibacter michiganensis* LMG 7333 was used as an outgroup. Type strains are indicated a superscript “T”.</figcaption></figure>

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

*In vitro* plant growth promotion traits of bacterial isolates from Cuban wheat varieties.</caption><thead><tr><th>Strains</th><th>PS</th><th>KS</th><th>SP</th><th>AP</th><th>NF</th><th>BP</th><th>EP</th><th>SW</th><th>IAA (µg/L)</th><th>NH<sub>4</sub> (µmol/L)</th><th>BFP (OD)</th></tr></thead><tbody><tr><td>TII-3</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>18.0 ± 1.9 (bc)</td><td>4789.6 ± 205.4 (g)</td><td>3.8 ± 2.0 (cd)</td></tr><tr><td>TII-10</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>15.1 ± 1.6 (cd)</td><td>7818.1 ± 640.2 (bc)</td><td>9.2 ± 4.8 (b)</td></tr><tr><td>TII-15</td><td>—</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>39.9 ± 1.7 (a)</td><td>7385.3 ± 534.5 (cd)</td><td>0.2 ± 0.1 (e)</td></tr><tr><td>TII-19</td><td>—</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>12.5 ± 1.1 (cde)</td><td>7697.7 ± 634.5 (bc)</td><td>5.1 ± 2.5 (c)</td></tr><tr><td>TCG-6</td><td>—</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>12.8 ± 1.9 (cde)</td><td>5038.0 ± 269.4 (fg)</td><td>0.8 ± 0.1 (de)</td></tr><tr><td>TCG-17</td><td>—</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>—</td><td>+</td><td>8.3 ± 2.5 (f)</td><td>6397.3 ± 240.0 (e)</td><td>0.3 ± 0.1 (e)</td></tr><tr><td>TIG-13</td><td>—</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>18.4 ± 0.4 (bc)</td><td>6744.8 ± 672.9 (de)</td><td>3.7 ± 1.8 (cd)</td></tr><tr><td>TMG-2</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>18.4 ± 2.0 (bc)</td><td>8392.9 ± 489.9 (ab)</td><td>5.5 ± 1.5 (c)</td></tr><tr><td>TMG-3</td><td>—</td><td>+</td><td>+</td><td>+</td><td>+</td><td>—</td><td>+</td><td>+</td><td>15.3 ± 1.2 (cd)</td><td>6236.3 ± 809.9 (e)</td><td>4.4 ± 1.2 (cd)</td></tr><tr><td>TMG-6</td><td>—</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>12.1 ± 4.3 (def)</td><td>5775.6 ± 88.7 (ef)</td><td>5.7 ± 1.6 (c)</td></tr><tr><td>TMG-15</td><td>—</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>13.3 ± 3.7 (cde)</td><td>6644.9 ± 379.4 (de)</td><td>1.3 ± 0.4 (de)</td></tr><tr><td>TMG-19</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>23.7 ± 0.6 (b)</td><td>6100.0 ± 1068.3 (e)</td><td>19.3 ± 3.2 (a)</td></tr><tr><td>TMG-22</td><td>—</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>10.2 ± 2.9 (ef)</td><td>9218.4 ± 46.1 (a)</td><td>1.3 ± 0.9 (de)</td></tr><tr><td>TMG-37</td><td>—</td><td>+</td><td>—</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>16.3 ± 0.1(bcd)</td><td>6522.0 ± 381.1 (de)</td><td>10.7 ± 2.2 (b)</td></tr><tr><td>TMG-43</td><td>—</td><td>—</td><td>—</td><td>+</td><td>+</td><td>—</td><td>—</td><td>—</td><td>7.8 ± 0.3 (f)</td><td>7992.0 ± 474.0 (bc)</td><td>0.1 ± 0.0 (e)</td></tr></tbody></table>

Legend: PS: phosphate solubilization, KS: potassium solubilization, SP: siderophores production, AP: amino cyclopropane carboxylate deaminase production, NF: nitrogen fixing, BP: biosurfactant production, EP: exopolysaccharides production, SW: swarming motility, IAA: indoleacetic acid production, NH₃: ammonia production, and BFP: biofilm production. IAA, NH₄, and BFP are presented as the means of three determinations ± standard deviation. The different letters represent significant differences; Duncan (n = 3, p < 0.05).

the same seven bacterial isolates decreased the negative impact of combined stress, with TII-15 (0.75 ± 0.00) and TII-19 (0.71 ± 0.11) achieving the highest germination rates.

Furthermore, fungal growth appeared on control plates 4 dpi, while bacterial treatments delayed it until 6 dpi, highlighting their

antagonistic potential against *Fusarium*.

Additionally, seedling growth was also evaluated after 10 days, and their size was determined (Fig. 6). Under 0 MPa (Fig. 6A), bacterial treatments with strains TCG-6, TII-19, TII-10, TMG-6, TII-15, and TMG-15 significantly enhanced seedling growth, with TMG-6 producing the
<caption>**Table 2**</caption>

Production of lytic enzymes and HCN by bacterial strains isolated from Cuban wheat.

<table><thead><tr><th>Strains</th><th>AI</th><th>CI</th><th>ProI</th><th>PectI</th><th>LP</th></tr></thead><tbody><tr><td>TII-3</td><td>2.0 ± 0.0<br>(a)</td><td>2.5 ± 0.1 (abc)</td><td>2.1 ± 0.1<br>(ab)</td><td>2.1 ± 0.0<br>(abc)</td><td>+</td></tr><tr><td>TII-10</td><td>2.1 ± 0.0<br>(a)</td><td>0.0 ± 0.0 (d)</td><td>2.2 ± 0.1<br>(ab)</td><td>2.1 ± 0.0<br>(ab)</td><td>+</td></tr><tr><td>TII-15</td><td>2.1 ± 0.0<br>(a)</td><td>2.1 ± 0.0 (cd)</td><td>2.1 ± 0.0<br>(ab)</td><td>0.0 ± 0.0 (c)</td><td>+</td></tr><tr><td>TII-19</td><td>2.1 ± 0.1<br>(a)</td><td>2.4 ± 0.1<br>(abcd)</td><td>2.3 ± 0.0<br>(abc)</td><td>0.0 ± 0.0 (c)</td><td>+</td></tr><tr><td>TCG-6</td><td>2.2 ± 0.0<br>(a)</td><td>2.7 ± 0.1 (ab)</td><td>2.4 ± 0.2<br>(ab)</td><td>3.6 ± 0.1 (a)</td><td>+</td></tr><tr><td>TCG-17</td><td>2.3 ± 0.1<br>(a)</td><td>2.6 ± 0.2 (abc)</td><td>2.5 ± 0.1 (a)</td><td>2.1 ± 0.0<br>(abc)</td><td>+</td></tr><tr><td>TIG-13</td><td>2.0 ± 0.0<br>(a)</td><td>0.0 ± 0.0 (d)</td><td>2.1 ± 0.1<br>(ab)</td><td>0.0 ± 0.0 (c)</td><td>+</td></tr><tr><td>TMG-2</td><td>2.0 ± 0.0<br>(a)</td><td>0.0 ± 0.0 (d)</td><td>2.1 ± 0.0<br>(ab)</td><td>2.1 ± 0.0 (bc)</td><td>+</td></tr><tr><td>TMG-3</td><td>2.0 ± 0.0<br>(a)</td><td>2.5 ± 0.1 (abc)</td><td>2.0 ± 0.0 (b)</td><td>2.1 ± 0.0<br>(abc)</td><td>+</td></tr><tr><td>TMG-6</td><td>2.3 ± 0.1<br>(a)</td><td>3.5 ± 0.2 (a)</td><td>2.3 ± 0.1<br>(ab)</td><td>3.3 ± 0.1<br>(ab)</td><td>+</td></tr><tr><td>TMG-15</td><td>0.0 ± 0.0<br>(b)</td><td>2.5 ± 0.2 (abc)</td><td>2.2 ± 0.1<br>(ab)</td><td>3.6 ± 0.3 (a)</td><td>+</td></tr><tr><td>TMG-19</td><td>2.5 ± 0.0<br>(a)</td><td>0.0 ± 0.0 (d)</td><td>2.3 ± 0.3<br>(ab)</td><td>3.1 ± 0.1<br>(ab)</td><td>—</td></tr><tr><td>TMG-22</td><td>0.0 ± 0.0<br>(b)</td><td>2.2 ± 0.0<br>(bcd)</td><td>2.1 ± 0.0<br>(ab)</td><td>0.0 ± 0.0 (c)</td><td>+</td></tr><tr><td>TMG-37</td><td>2.0 ± 0.0<br>(a)</td><td>2.4 ± 0.1<br>(abcd)</td><td>2.1 ± 0.0<br>(ab)</td><td>2.5 ± 0.3<br>(abc)</td><td>+</td></tr><tr><td>TMG-43</td><td>2.0 ± 0.0<br>(a)</td><td>2.1 ± 0.1 (cd)</td><td>2.1 ± 0.1<br>(ab)</td><td>0.0 ± 0.0 (c)</td><td>+</td></tr></tbody></table>

The values registered are the mean values of hydrolytic index ± standard deviation of three replicates. The different letters indicate significant differences Kruskal Wallis, Dunn't test (n = 3, p < 0.05). Legend: AI: amylolitic index, CI: cellulolytic index, ProI: proteolytic index, PectI: pectinolytic index and LP: lipases production. +, and - means positive and negative response at the test respectively.

largest increase (33.1 ± 1.4 cm compared to 25.5 ± 3.2 cm in controls). At -0.5 MPa, strains TMG-6, TCG-6, TII-10, and TII-19 improved seedling size by more than 5 cm, with TMG-6 showing the highest growth rates (34.3 ± 1.9 cm). Even under severe drought stress (-1.5 MPa), strains TII-19, TMG-19, TCG-6, TMG-3, and TMG-6 enhanced the seedling size by more than 4 cm compared to the control (19.4 ± 2.2 cm).

The measurements of seedlings under *Fusarium* infection and different osmotic potential (Fig. 6B) revealed that under 0 MPa and -1.5 MPa, the bacterial treatment with strains TCG-6, TMG-6, TMG-15, TMG-3, TII-19, TMG-37 and TII-3 increased the seedling size by more than 5 cm and 4 cm, respectively compared to the control (18.9 ± 1.2 cm and 18.0 ± 2.7 cm). However, under combined stress (drought stress -1.5 MPa and *Fusarium* infection) only the treated seedlings with TII-9, TMG-6, TII-10, TMG-43 and TCG-6 showed an increase of more than 4 cm compared to the control (14.2 ± 0.8).

On the other hand, all control plants infected with *F. culmorum* exhibited pronounced Fusarium root rot symptoms, including light brown and brown lesions on the roots and root zone of the shoot. Seedlings also showed yellowing and wilting leaves, with symptoms becoming noticeable 9 to 10 dpi and more severe under -0.5 and -1.5 MPa. In contrast, bacterized seedlings significantly exhibited reduced disease severity. Eight strains (TMG-6, TII-10, TCG-6, TII-19, TMG-22, TII-15, and TII-3) reduced Fusarium root rot incidence by more than 50 % across different osmotic potentials, showing significant differences compared to the non-bacterized control (Fig. 7). Notably, TII-10, TII-19, TCG-6 and TMG-6 exhibited the strongest effects. These results highlight the potential of *Bacillus* strains as plant growth promoting bacteria under drought stress conditions and as effective agents for *Fusarium* biocontrol.

<caption>**Table 3**</caption>

Germination rate of wheat under different osmotic potential and *Fusarium* infection.

<table><thead><tr><th rowspan="2">Mean</th><th colspan="3">Non- Fusarium infected</th><th colspan="3">Fusarium infected</th></tr><tr><th>0 MPa</th><th>-0.5 MPa</th><th>-1.5 MPa</th><th>0 MPa</th><th>-0.5 MPa</th><th>-1.5 MPa</th></tr></thead><tbody><tr><td>Control</td><td>0.92 ± 0.08</td><td>0.86 ± 0.09</td><td>0.41 ± 0.01 (ab)</td><td>0.84 ± 0.08</td><td>0.83 ± 0.08</td><td>0.36 ± 0.04 (ab)</td></tr><tr><td>TII-3</td><td>0.91 ± 0.01</td><td>0.96 ± 0.04</td><td>0.426 ± 0.15 (abc)</td><td>0.83 ± 0.08</td><td>0.74 ± 0.16</td><td>0.47 ± 0.05<br>(abcde)</td></tr><tr><td>TII-10</td><td>0.83 ± 0.08</td><td>0.68 ± 0.09</td><td>0.36 ± 0.04 (a)</td><td>0.74 ± 0.07</td><td>0.74 ± 0.07</td><td>0.34 ± 0.01 (ab)</td></tr><tr><td>TII-15</td><td>0.92 ± 0.08</td><td>0.88 ± 0.04</td><td>0.58 ± 0.14<br>(abcde)</td><td>0.81 ± 0.05</td><td>0.86 ± 0.09</td><td>0.75 ± 0.00 (f)</td></tr><tr><td>TII-19</td><td>0.98 ± 0.03</td><td>0.93 ± 0.09</td><td>0.75 ± 0.00 (e)</td><td>0.94 ± 0.10</td><td>0.84 ± 0.08</td><td>0.71 ± 0.11 (ef)</td></tr><tr><td>TCG-6</td><td>0.94 ± 0.05</td><td>0.86 ± 0.09</td><td>0.74 ± 0.07 (cde)</td><td>0.91 ± 0.08</td><td>0.78 ± 0.18</td><td>0.63 ± 0.11 (cdef)</td></tr><tr><td>TCG-17</td><td>0.81 ± 0.05</td><td>0.89 ± 0.05</td><td>0.75 ± 0.00 (e)</td><td>0.75 ± 0.00</td><td>0.67 ± 0.00</td><td>0.53 ± 0.05<br>(bcdef)</td></tr><tr><td>TIG-13</td><td>0.91 ± 0.01</td><td>0.87 ± 0.04</td><td>0.34 ± 0.08 (a)</td><td>0.77 ± 0.12</td><td>0.74 ± 0.16</td><td>0.52 ± 0.12<br>(bcdef)</td></tr><tr><td>TMG-2</td><td>0.97 ± 0.06</td><td>0.92 ± 0.08</td><td>0.3 ± 0.14<br>(a)</td><td>0.73 ± 0.11</td><td>0.63 ± 0.15</td><td>0.44 ± 0.05 (abcd)</td></tr><tr><td>TMG-3</td><td>0.93 ± 0.02</td><td>0.81 ± 0.05</td><td>0.41 ± 0.01 (ab)</td><td>0.9 ± 0.06</td><td>0.84 ± 0.10</td><td>0.49 ± 0.07<br>(abcdef)</td></tr><tr><td>TMG-6</td><td>0.96 ± 0.04</td><td>0.87 ± 0.13</td><td>0.72 ± 0.05 (cde)</td><td>0.88 ± 0.13</td><td>0.88 ± 0.13</td><td>0.63 ± 0.13 (def)</td></tr><tr><td>TMG-15</td><td>0.89 ± 0.05</td><td>0.89 ± 0.05</td><td>0.78 ± 0.13 (de)</td><td>0.72 ± 0.05</td><td>0.69 ± 0.05</td><td>0.39 ± 0.05 (abc)</td></tr><tr><td>TMG-19</td><td>0.89 ± 0.05</td><td>0.92 ± 0.00</td><td>0.67 ± 0.08<br>(bcde)</td><td>0.67 ± 0.08</td><td>0.67 ± 0.08</td><td>0.56 ± 0.05 (cdef)</td></tr><tr><td>TMG-22</td><td>0.86 ± 0.05</td><td>0.92 ± 0.00</td><td>0.44 ± 0.05<br>(abcd)</td><td>0.83 ± 0.00</td><td>0.56 ± 0.17</td><td>0.22 ± 0.05 (a)</td></tr><tr><td>TMG-37</td><td>0.89 ± 0.10</td><td>0.92 ± 0.08</td><td>0.61 ± 0.05<br>(bcde)</td><td>0.67 ± 0.14</td><td>0.67 ± 0.08</td><td>0.53 ± 0.10 (cdef)</td></tr><tr><td>TMG-43</td><td>0.92 ± 0.00</td><td>0.83 ± 0.08</td><td>0.47 ± 0.10<br>(abcde)</td><td>0.72 ± 0.10</td><td>0.72 ± 0.10</td><td>0.56 ± 0.05 (cdef)</td></tr></tbody></table>

The values were registered as means of three repetitions ± standard deviation, different letters indicate significant differences, Kruskal Wallis and Dunn's test (n = 3, p < 0.05).

## 4. Discussion

Wheat is a globally significant cereal crop, but its production is severely impacted by abiotic stresses such as drought and biotic stresses caused by *Fusarium* diseases. *Fusarium* pathogens, including *F. graminearum* and *F. culmorum* cause devastating diseases like *Fusarium* head blight, *Fusarium* root rot, *Fusarium* crown rot and *Fusarium* stem rot (Chin et al., 2023). These pathogens also produce harmful mycotoxins such as deoxynivalenol (DON), posing significant food safety risk (Alisaac and Mahlein, 2023). The use of plant growth-promoting bacteria (PGPB), particularly members of the Bacillaceae family, has emerged as a promising sustainable strategy for alleviating these stresses and improving crop yields.

In this study, 150 bacterial isolates from Cuban wheat varieties were screened, and 15 isolates demonstrated strong antifungal activity against *F. graminearum* and *F. culmorum*.

The dominant genus identified was *Bacillus*, aligning with its common presence in wheat microbiome. This prevalence is attributed to its stress tolerance, rapid growth, and ability to produce a wide range of metabolites. *Bacillus* has been found in both the rhizosphere and the endosphere of plants (Chen et al., 2022). In this sense, Pang et al. (2022)
<figure><img src="image_10.png" /><figcaption>**Fig. 6.** Effect of bacterial strains over wheat seedling size under different osmotic potentials (A) and under different osmotic potentials and *Fusarium* infection (B). The plates were incubated for 12 days. The bars represent the mean value of three determinations ± standard deviation. Different letters for each strain indicate significant differences, Duncan (n = 6, p < 0.05). White bars represent 2 cm.</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>**Fig. 7.** *F. culmorum* incidence in wheat seedlings under different osmotic potentials. The bars represent the mean value of three determinations ± standard deviation. Different letters for each strain indicate significant differences, Dunn test (n = 3, p < 0.05).</figcaption></figure>

isolated a total of 610 endophytic bacteria from the roots, stems, leaves, and seeds of three wheat cultivars in China and identified *Bacillus* as the most predominant bacterial taxon in winter wheat, representing more than five species of this genus.

Members of Bacillaceae family are among the bacteria with the most

potential due to its high thermal tolerance, enabled by spore-forming ability, and rapid growth (Mawar et al., 2021). Its intrinsic properties provide several mechanisms that contribute to biostimulation, biocontrol of phytopathogenic fungi, and help the plant tolerance to abiotic stress (Cherif-Silini et al., 2016). These characteristics make Bacillaceae
as valuable candidates for studying its effects on promoting wheat growth under both biotic and abiotic stress conditions.

## 4.1. Antifungal mechanisms and biocontrol potential of bacterial strains

This study highlights the antifungal potential of bacterial strains through the production of bioactive metabolites. While volatile organic compound test from bacterial strains showed no significant effect on fungal growth, cell free supernatant significantly inhibited fungal germination and induced cytological changes at the hyphal level. These findings align with the documented ability of *Bacillus* spp. to produce a diversity of fungitoxic metabolites, including lipopeptides, lytic enzymes, and polyketides (Fazle Rabbee and Baek, 2020; Yi et al., 2024).

Structure changes in fungal hyphae, such as vacuolation and cellular damage, are consistent with the findings by Asaturova et al. (2022), who reported similar effects caused by lipopeptides, particularly iturin A and surfactin, produced by *B. velezensis* BZR336 and BZR 517. Moreover, Zhao et al. (2014) demonstrated that metabolites like chitinase, fengycins and surfactins produced by *Bacillus subtilis* SG6 disrupted the hyphal structure of *F. graminearum*. Similarly, Zalila-Kolsi et al. (2016) reported antifungal metabolites, including iturin, surfactin, fengycin, fusaricidin, and polymyxin, produced by *B. amyloliquefaciens* BLB369, *B. subtilis* BLB277, and *Paenibacillus polymyxa* BLB267, inhibited *F. graminearum* and induced morphological alterations such hyphal swelling and chlamydospores production.

Additionally, the antifungal properties of bacterial strains are also translated into effective biocontrol against Fusarium-induced symptoms in wheat. The detached leaf assay is a simple commonly used to assess host responses to *Fusarium* both barley and wheat (Kumar et al., 2011; Perochon and Doohan, 2016). In our study, *Bacillus* strains delayed the appearance of symptoms and reduced *Fusarium* lesions, indicating their potential for *Fusarium* biocontrol. Our observations align with data reported by Dutilloy et al. (2024), who found that *B. velezensis* BE2 and its supernatant significatively reduced *Pyrenophora teres* symptoms on barley leaves. Moreover, Surovy et al. (2023) observed that *B. subtilis* BTS-3, *B. velezensis* BTS-4, and *B. velezensis* BTLK6A significantly reduced *Magnaporthe oryzae* *Triticum* lesions on the wheat cultivar BR 18 leaves in the detached leaf assay.

## 4.2. Enhanced drought tolerance in bacterial strains

Bacterial strains under study were completely tolerant to 20 % PEG-6000, while strains TII-19, TCG-6 TMG-3 and TMG-6 were tolerant up to 25 % PEG-6000. These results align with previous research. For example, Yadav et al. (2022) reported that 113 bacteria isolated from wheat and the wild plant *Argemone mexicana* were able to survive 5–8 % PEG-6000, highlighting the tolerance of *B. paramycoides* DT-85 and *B. paranthracis* DT-97, which grew on LB amended with 30 % PEG-6000. Moreover, Rashid et al. (2022) characterized strains *B. megaterium* and *B. licheniformis*, which tolerated 25 % PEG-6000. Additionally, Bouremani et al. (2024) described that *Bacillus* sp. D13 tolerated up to 30 % PEG-6000. Latif et al. (2022) isolated 30 bacteria from the rhizospheric soil of wheat in different fields, and all isolates tolerated 4 % PEG-6000, while *Chryseobacterium* sp. LEW3, *Acinetobacter* sp. LEW9, and *Klebsiella* sp. LEW16 tolerated 9 % PEG-6000.

Bacterial tolerance to drought conditions may be attributed to the production of exopolysaccharides and biofilm. In the current study, exopolysaccharides production was observed in 13 bacteria, while 12 bacterial strains produced biofilm. Exopolysaccharides have a significant water retention capacity (up to 70 g water per g polysaccharide), and as a result, they have garnered interest for their protective properties against desiccation in bacteria (Camaille et al., 2021). Literature reports that, under drought stress, the inoculation of exopolysaccharide-producing bacteria can improve plant tolerance to drought conditions. Additionally, they can induce changes in soil structure and enhance root growth (Ilyas et al., 2020).

Biofilm, a complex matrix composed of polysaccharides, DNA, and proteins, protects bacteria against drought stress and enhances root colonization ability (Li et al., 2024). The beneficial effect of biofilm-producing bacteria in agriculture has been studied in recent years. For example, Karimi et al. (2022) reported that biofilm-forming bacterial strains *Peribacillus simplex* B1, *B. pumilus* B2, and *Peribacillus simplex* B3 increased relative water content and harvest index in two wheat varieties under both moderate and severe water deficit conditions. Also, Ahmad Ansari et al. (2023) reported that biofilm-forming strains *B. subtilis* FAB1 and *Pseudomonas azotoformans* FAP3 improved drought tolerance in wheat, while a bacterial consortium containing both strains significantly improved wheat survival during drought conditions. Recently, Rafique et al. (2024) found that biofilm-forming rhizobacteria *Brucella* sp. BF10 and *Lysinibacillus macroides* BF15, isolated from the wheat rhizosphere, improved various wheat characteristics, such as: roots and shoots length, plant height, number of tillers, leaf pigmentation, and relative water content. Consequently, the findings in the current study highlight the potential for *Bacillus* strains to induce drought tolerance in wheat.

## 4.3. Biostimulant and biocontrol potential of bacterial strains for wheat under stress conditions

The effect of bacterial strains on germination and wheat growth was evaluated, revealing that the most significant impact on germination was observed under drought stress conditions. These results align with those of Karimzad et al. (2023), who reported that three *Bacillus wiedmannii* strains, isolated from the wheat rhizosphere, increased germination percentage, germination rate, and seed vigor index under drought stress. Moreover, we observed a notable reduction in *F. culmorum* incidence under drought conditions, indicating the potential of bacterial strains for biocontrol in stress environments. The overcome observed in this study are consistent with those of Lastochkina et al. (2020), who evaluated *Bacillus subtilis* 10-4 mixed with salicylic acid to enhance growth and tolerance of *Triticum aestivum* L. plants subjected to combined drought and *Fusarium culmorum*-induced root rot (FRR) stresses. They reported that pre-sowing treatment with *B. subtilis* 10-4, SA, and *B. subtilis* 10-4, SA, and the combination of both significantly reduced fungal disease incidence in wheat plants under normal and combined drought conditions. Moreover, bacterial treatments and combined with salicylic acid notably enhanced plant growth under both normal and stress conditions, reducing stress in wheat plants. Hence, the results obtained in the study highlight the potential application of bacterial strains as promising biostimulants for wheat under stress conditions.

## 4.4. Plant growth-promoting traits of bacterial strains

The bacterial strains exhibited several PGP traits. In the present study the IAA-production levels by bacterial strains (7.8 µg/L –39.9 µg/L) are in correspondence with those reported by Gul et al. (2023), who reported 31.68 µg/mL of IAA produced by *B. subtilis* in the medium supplemented with tryptophan. Similarly, Cherif-Silini et al. (2016) detected IAA production in 35 *Bacillus* strains isolated from wheat, in Algeria and the IAA production varied between 10–19 µg/mL. Ji et al. (2020) found that *B. methylotrophicus* M4-1 strain, isolated from the rhizosphere of wheat plants in China, produced 25.07 µg/mL under control conditions. IAA is a key phytohormone involved in regulating plant growth (Tang et al., 2023).

Ammonia production, a critical source of nitrogen for plant, and production levels registered by bacterial strains (4789.6–9218.4 µmol/L) correspond to those of Sherpa et al. (2021), who detected ammonia production between 2000–61000 µmol/L in eight rhizospheric bacteria isolated from the organic paddy fields of Sikkim in India. Additionally, Rajkumar et al. (2024) observed ammonia production ranging from 1700 to 5200 µg/mL in bacteria isolated from little millet
rhizosperes (Panicum sumatrense L.).

Bacterial strains also exhibited ACC deaminase activity, which plays a key role in reducing ethylene-induced stress by breaking down the ethylene precursor ACC into ammonia and α-ketobutyrate. This action can mitigate plant senescence under stress conditions such as drought (Gupta and Pandey, 2019). In this study, the production of ACC deaminase further underlies the potential of bacteria to enhance plant stress tolerance.

Nitrogen fixation, another important PGP trait, was also present in these strains, contributing to soil fertility and reducing the need for synthetic fertilizers (Salem et al., 2024). Phosphorous and potassium are important nutrients for plants, but their soil- availability is low. However, some microorganisms can solubilize P and K by production of organic acids enhancing their uptake by plants (Nawaz et al., 2023; da Silva et al., 2023). PGP traits registered by bacterial strains in the current study are comparable to those obtained by Salem et al. (2024), who isolated three *Bacillus* strains and one *Myroides* sp. from wheat rhizosphere soil in Egypt.

Swarming motility in *Bacillus* spp. has a crucial role in biocontrol allowing the bacterial growth faster and space competition against pathogenic microorganisms. Morales Sandoval et al. (2024) obtained the *Bacillus cabrialesii* TE3T-UV25 mutant by UV, which exhibited a stronger antifungal activity against *B. sorokiniana* TPQ3 by nutrient and space competition and diffusible metabolites compared with the wild type strains *B. cabrialesii* TE3T. Moreover, the swarming motility promotes bacteria- plant interactions, and enhance the bacterial colonization (Shen et al., 2023).

Current study, the siderophores production was detected in 13 strains, while the lytic enzyme production varied among strains. Our observations align with the data reported by Ayaz et al. (2024) who informed that *B. subtilis* BS-2301 produced siderophores, amylases, cellulases and proteases and inhibited the *S. sclerotiorum* growth. Moreover, (Yi et al., 2024) informed that *B. amyloliquefaciens* ZK-9 strains produced pectinases, proteases, cellulases, amylases and it showed a strong antifungal activity against *F. graminearum*. Siderophores are organic molecules with low molecular weight, produced by some microorganisms. They have a high affinity for iron, limiting its availability for pathogens, thus they can reduce different plant fungal diseases (Khan et al., 2022). The mode of action of lytic enzymes is disintegrating cell wall and cell membrane through degradation of molecules including glycoprotein, polysaccharides, lipids, chitin, and other compounds (Prasad et al., 2023). On the other hand, biosurfactants are amphiphilic molecules with surface active properties, they can insert into lipid bilayers and destabilize the cell membrane inducing cell lysis. Thus, the use of bacterial biosurfactant in biocontrol have gain attention in recent years (Sani et al., 2024). For instance, Ben Khedher et al. (2020) reported that biosurfactants produced by *Bacillus subtilis* V26 inhibited *Botrytis cinerea* growth leading to the swelling and the deformation of fungal hyphae. Moreover, biosurfactant exhibited significant protection against gray mold severity. The bacterial strains TII-10, TII-19, TCG-6 and TMG-6 were considered promising according to their antifungal potential, drought tolerance, and ability to promote plant growth, confirmed their value for inclusion in future research aimed at developing bacterial products to improve wheat production under biotic and abiotic conditions.

## 5. Conclusion

Bacteria, isolated from Cuban wheat, belonging to the Bacillaceae family, demonstrated strong inhibitory potential against *F. graminearum* and *F. culmorum*. Moreover, they induced cytological modifications in both phytopathogenic fungi, highlighting their potential as biocontrol agents. The strains exhibited tolerance to 20 % PEG-6000, indicating their resilience under drought stress conditions. The strains TII-19, TII-10, TCG-6, and TMG-6 enhanced wheat germination and growth under both drought and *Fusarium* infection conditions, confirming their

biostimulant potential. Further studies are necessary to validate these results and elucidate the mechanisms by which these strains enhance wheat growth under stress conditions.

## CRediT authorship contribution statement

**Alexander Govin- Sanjudo:** Writing – original draft, Software, Methodology, Formal analysis, Data curation, Conceptualization. **Marcia M. Rojas Badia:** Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization. **Cédric Jacquard:** Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization. **Qassim Esmaeel:** Writing – review & editing, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Data curation, Conceptualization.

## Funding

This research was funded by the University of Reims Champagne-Ardenne (France), the University of Havana (Cuba), and the Grand Reims through the Fusablé project.

## 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.

## Acknowledgments

We gratefully acknowledge the financial support provided by the University of Reims Champagne-Ardenne (France), the University of Havana (Cuba) through Cuban PSSAV project PS223MY003-124, and the Grand Reims through the Fusablé project.

## Author statement.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.biocontrol.2025.105776.

## Data availability

All data supporting the findings of this study are available within the paper and within its supplementary materials published online. The sequence data have been submitted to NCBI, and the accession numbers are provided.

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