Use of Volatile Organic Compounds Produced by Bacillus Bacteria for the Biological Control of Fusarium oxysporum

Abstract

Restricting the use of chemical pesticides in forestry requires the search for alternative solutions. These could be volatile organic compounds produced by three investigated species of bacteria (Bacillus amyloliquefaciens (ex Fukumoto) Priest, B. subtilis (Ehrenberg) Cohn and B. thuringiensis Berliner), which inhibit the growth of the pathogen F. oxysporum Schltdl. emend. Snyder & Hansen in forest nurseries. The highest inhibition of fungal growth (70%) was observed with B. amyloliquefaciens after 24 h of antagonism test, which had a higher content of carbonyl compounds (46.83 ± 8.41%) than B. subtilis (41.50 ± 6.45%) or B. thuringiensis (34.62 ± 4.77%). Only in the volatile emissions of B. amyloliquefaciens were 3-hydroxybutan-2-one, undecan-2-one, dodecan-5-one and tetradecan-5-one found. In contrast, the main components of the volatile emissions of F. oxysporum were chlorinated derivatives of benzaldehyde (e.g., 3,5-dichloro-4-methoxybenzaldehyde) and chlorinated derivatives of benzene (e.g., 1,4-dichloro-2,5-dimethoxybenzene), as well as carbonyl compounds (e.g., benzaldehyde) and alcohols (e.g., benzyl alcohol). Further compounds were found in the interactions between B. amyloliquefaciens and F. oxysporum (e.g., $\alpha$-cubebene, linalool, undecan-2-ol, decan-2-one and 2,6-dichloroanisole). Specific substances were found for B. amyloliquefaciens (limonene, nonan-2-ol, phenethyl alcohol, heptan-2-one and tridecan-2-one) and for F. oxysporum (propan-1-ol, propan-2-ol, heptan-2-one and tridecan-2-one). The amounts of volatile chemical compounds found in B. amyloliquefaciens or in the bacterium–fungus interaction can be used for further research to limit the pathogenic fungus. In the future, one should focus on the compounds that were found exclusively in interactions and whose content was higher than in isolated bacteria. In order to conquer an ecological niche, bacteria increase the production of secondary metabolites, including specific chemical compounds. The results presented are a prerequisite for creating an alternative solution or supplementing the currently used methods of plant protection against F. oxysporum. Understanding and applying the volatile organic compounds produced by bacteria can complement chemical plant protection against the pathogen, especially in greenhouses or tunnels where plants grow in conditions that favour fungal growth.

1. Introduction

Fusarium oxysporum Schltdl. emend. Snyder & Hansen is a dangerous forest plant pathogen and is listed among the ten most important fungal pathogens in plant pathology [1,2,3]. Fusarium oxysporum colonise plants at different growth stages and cause various forms of disease in economically important plants around the world [1,4,5]. Fusarium oxysporum can cause the damping-off, spotting, wilting and death of buds and whole plants with a fairly rapid progression, which can seriously reduce growth and yield. The species has been identified as the main cause of root rot in nurseries [4]. A mycelium can be observed on the surface of the infected plant, which can turn pink in an advanced stage due to the mass production of Fusarium spores. F. oxysporum can spread to neighbouring plants, so an infected plant can pose a significant threat to the entire crop [6,7,8]. F. oxysporum can also colonise plants asymptomatically, making it difficult to identify the cause of the disease [9]. For this reason, it is particularly important to prevent the occurrence of pathogens. Although diseases caused by Fusarium spread in the soil, they can also affect plants grown in a soilless system [10].

The most commonly used agents to control Fusarium are those containing chemicals from the group of triazoles [11], benzimidazoles [12], anilinopyrimidines [13] or quinazolines [14]. However, the chemical control of Fusarium on infected plants is difficult [10,15]. In addition, today’s society pays particular attention to the excessive chemicalisation of the environment.

The modern world faces the challenge of environmental pollution, including agriculture and forestry [16]. Legal regulations aim to limit excessive chemicalisation. Directive 2009/128/EC of the European Parliament and of the Council is based on the reduction in the use of chemical substances, the protection of biodiversity and the promotion of sustainable development. The most important measures include reducing the use of pesticides and restoring natural resources [17]. The reduction in chemicalisation can be achieved through the application of integrated pest management. This approach is based on minimising the use of pesticides and focuses instead on prevention. New, safer methods for the biological protection of plants against pathogens such as F. oxysporum are therefore being sought [18]. The use of biopesticides, which are produced from metabolic products of microorganisms, plays an important role in integrated pest management [19].

Some of the Bacillus bacteria can be used as biological control agents (BCAs) against plant pathogens. They produce volatile organic compounds. Volatile organic compounds (VOCs) are low-molecular-weight organic compounds (<300 Da) that generally have lipophilic properties and a low boiling point [20]. VOCs can be produced by bacterial and fungal microorganisms [18,20]. In this case, the term microbial VOCs (abbreviated mVOCs) is used to refer to this group of compounds. The importance of researching mVOCs is demonstrated by the fact that a database called mVOC Database 4.0 [21] has been created, which contains information on 3500 microbial VOCs produced by various species of bacteria and fungi. About 10% of all mVOCs can be assigned a biological function [22]; therefore, some mVOCs have the potential to be used in agriculture or forestry for the biological control of pathogens in the future. Microorganisms produce various inorganic and organic volatile compounds that can be widely utilised as infochemicals in interactions with plants, fungi and bacteria [16,23,24]

The aim of the present study was to determine the effect of volatile organic compounds produced by three species of Bacillus bacteria on the inhibition of F. oxysporum growth.

2. Materials and Methods

2.1. Bacillus spp. and F. oxysporum Strains

Strains of three species of Bacillus bacteria (B. amyloliquefaciens (ex Fukumoto) Priest, B. subtilis (Ehrenberg) Cohn and B. thuringiensis Berliner) and the fungus F. oxysporum came from the collection of the Forest Research Institute in Sękocin Stary (Poland).

Casein-Soy Broth TSB (Tryptic Soy Broth) (Cat. No. P-0120, BTL, Łódź, Poland) was used for the culture of Bacillus. Potato Dextrose Agar (PDA) (Cat. No. P-0134, BTL, Łódź, Poland) was used for F. oxysporum. The media were prepared according to the manufacturer’s instructions and then sterilised in an autoclave at 121 °C for 20 min.

The Bacillus bacteria were stored in liquid culture with TSB medium at 4 °C until analysed. The F. oxysporum fungus strain was stored in Petri dishes with PDA medium at 4 °C until analysis.

2.2. Test for Inhibition of F. oxysporum Growth by Volatile Organic Compounds Produced by Bacillus Bacteria

The inhibitory effects of volatile organic compounds (VOCs) produced by Bacillus strains in the antagonism assay were measured using the sealed-plate method described by Wu et al. [25], adjusting the composition of the culture medium and the incubation temperature. A 200 $\mathsf{\mu }$ Lcell suspension ($1\times {10}^8$ cfu/mL) of bacteria was spread on a TSB solid medium in a Petri dish (Figure 1) and incubated at 28 °C for 24 h. A 6 mm diameter mycelial disc of F. oxysporum was cut from an actively growing culture using a cork board and placed in the centre of a second Petri dish containing PDA medium (Figure 1). The lids were removed from both plates and the plate inoculated with Bacillus was inverted and placed on a plate with Fusarium mycelium (Figure 1).

The two plates were connected with parafilm to create a sealed two-plate chamber. A two-plate chamber without bacterial strains was used as a control. The average distance between the surface of the TSB agar and the PDA was 2 cm. The two-plate chamber was incubated at 25 °C for 96 h and the mycelial diameter was measured every 24 h. The degree of inhibition of mycelial growth (I%), expressed as a percentage, was then determined according to the formula [6]:

$$I\%=(A-B)/A\times 100,$$

with I%—inhibition of mycelium growth (%), A—mycelium diameter in the control sample (mm) and B—mycelium diameter in the test sample (mm).

The experiment was repeated three times for each variant; therefore, the mycelium diameters A and B are the average values of the three measurements.

2.3. Analysis of Volatile Organic Compounds Produced by Bacillus Bacteria and F. oxysporum Using Headspace Solid-Phase Microextraction and Gas Chromatography with Mass Spectrometry (HS-SPME/GC–MS)

Petri dishes with Bacillus bacteria and F. oxysporum fungi were closed and sealed with parafilm. In one side wall of each Petri dish there was a previously prepared hole with a diameter of 2 mm, which was also sealed with the parafilm. The volatile organic compounds emitted by Bacillus bacteria and F. oxysporum fungus were analysed 96 h after sealing the Petri dishes using headspace solid-phase microextraction and gas chromatography with mass spectrometry (HS–SPME/GC–MS).

A needle of the SPME equipment was inserted into the Petri dish containing the microorganism (Bacillus bacterium or F. oxysporum, or bacterium and fungus in interaction) and the adsorption fibre with divinylbenzene/carboxes/polydimethylsiloxane (DVB/CAR/PDMS) stationary phase was introduced. The sorption of volatiles was performed in the headspace for 30 min at 25 °C in a laboratory thermostat (Figure 2). Analyses were performed for 3 types of Bacillus bacteria, F. oxysporum fungi and 3 bacteria–fungus interactions (7 variants in total). All measurements were carried out in triplicate. A total of 21 analyses of the chemical composition of the volatiles produced by the microorganisms were performed.

The thermal desorption of the volatiles from the SPME fibre was carried out in an injection port of a gas chromatograph for 10 min at 250 °C. The injector operated in a split mode. Chemical composition analyses were performed using an Agilent 7890A gas chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA) with an Agilent 5975C mass spectrometer (Agilent Technologies Inc., Santa Clara, CA, USA). The chromatographic separation was performed on the HP-5MS capillary column (Agilent Technologies Inc., Santa Clara, CA, USA) (30 m × 0.25 mm × 0.25 $\mathsf{\mu }$m) with a stationary phase of 5%-phenyl-95%-methylpolysiloxane. The helium flow rate through the chromatography column was 1 mL/min. The temperature of the column was 35 °C at the beginning and increased to 250 °C at 5 °C/min, so that the total time for a single GC–MS analysis was 43 min. The temperatures of the ion source and the quadrupole were 230 °C and 150 °C, respectively. An ionisation energy of 70 eV was used. Detection was performed in a full scan mode in a range of 29–600 units.

The sum of the areas of all peaks in the chromatogram was assumed to be 100%. The percentage contribution of each chemical compound to the total ion current (%TIC) was calculated, i.e., to the signal recorded by the mass spectrometer. The identification of the chemical compounds was based on the mass spectra and the retention indices (RIs). The experimental mass spectra and retention indices were compared with literature data from the available databases [27,28] as well as NIST 2020 Mass Spectral Library, 2020 and Wiley Registry 12th Edition, 2020. The experimental retention indices ($R{I}_{exp.}$) of the compounds were calculated taking into account the retention times of the n-alkanes. The mixture of C5-C40 n-alkanes (1 $\mathsf{\mu }$L) was injected onto the chromatographic column and separated from volatiles under the previously described conditions for the GC–MS analyses. The linear temperature-programmed experimental retention indices ($R{I}_{exp.}$) were determined using the equation:

$$R{I}_{exp.}=100[n+(t_x-t_n)/(t_{n+1}-t_n)],$$

where n is number of carbon atoms in the n-alkane eluted immediately before the analyte, $t_x$ is the retention time of the analyte, $t_n$ is the retention time of the n-alkane eluted immediately before the analyte, $t_{n+1}$ is the retention time of the n-alkane that elutes directly after the analyte.

The inhibition of the fungal growth, caused by a given treatment, was calculated as the mean values for the data of the control samples (A) and the test samples (B) in the numerator of the printout: $I_{\%}=[(A-B)/A]\times 100$. That allowed the effect of bacteria on the growth of F. oxysporum but did not allow a comparison between the different treatments of the experiment, as was similarly presented by Munoz [29] and Santos et al. [30].

3. Results

3.1. Inhibition of F. oxysporum Growth by Volatile Organic Compounds Produced by Bacillus

The experiment analysed the effect of volatile compounds produced by bacteria of the genus Bacillus on the growth of the fungus F. oxysporum in a test with two connected plates over a period of 96 h. The interactions between F. oxysporum and bacteria of the genus Bacillus (B. amyloliquefaciens, B. subtilis and B. thuringiensis) were analysed.

The inhibition of the fungus F. oxysporum depended on the bacteria of the genus Bacillus used (Figure 3). The rate of mycelial inhibition of F. oxysporum varied with the duration of the antagonism test (Figure 3). Depending on the bacterial species and the duration of the test, the percentage of inhibition of mycelial growth ranged from 35% to 70% (Figure 3).

All Bacillus species showed fungistatic properties against F. oxysporum (Figure 3). The highest percentage of inhibition over the entire period of mycelial growth was recorded for B. amyloliquefaciens, which reduced the growth of F. oxysporum by approximately 70 % after 24 h of the test (Figure 3), while the inhibition was 45 % after 96 h of the experiment (Figure 3). The species B. subtilis and B. thuringiensis also showed high antagonistic properties towards F. oxysporum, and the calculated percentage of inhibition of the mycelial growth by these Bacillus species after 96 h was 35% and 40%, respectively (Figure 3).

The bacterium B. amyloliquefaciens, which showed the highest ability to inhibit mycelial growth after 96 h of the experiment, was selected for further testing.

3.2. Volatile Organic Compounds Produced by Bacillus Bacteria and F. oxysporum

Extensive experimental data were obtained as a result of the HS-SPME/GC–MS analyses of volatiles emitted by Bacillus bacteria and F. oxysporum. The detailed data included the names of the identified compounds, their chemical formulae and Chemical Abstracts Service numbers (CAS numbers). These data also contained the information used for identification of compounds by the GC–MS method, i.e., fragmentation ions (m/z), molecular ion (M+), retention time ($t_{ret.}$), experimental and literature retention indices ($R{I}_{exp.}$ and $R{I}_{lit.}$), peak area and relative content of the compound in the test sample (% TIC). To make this article clearer, the detailed data (Tables S1–S5) have been included in Supplementary Materials. However, the following subsections only contain the names of the compounds, their experimental retention indices and the relative contents (% TIC) in the test samples (

Table 1. Comparison of the chemical composition of volatile organic compounds emitted by B. amyloliquefaciens (I), B. subtilis (II) and B. thuringiensis (III).

Group of Compounds	${\mathit{RI}}_{\mathit{exp}.}$	Relative Content [% TIC]
		I	II	III
Carbonyl compounds, including the following:		46.83 ± 8.41	41.50 ± 6.45	34.62 ± 4.77
Acetone	480	6.92 ± 1.54	10.14 ± 0.52	8.70 ± 1.31
Butan-2-one	560	0.64 ± 0.19	1.01 ± 0.06	2.88 ± 0.49
1-Hydroxypropan-2-one	661	-	0.46 ± 0.10	-
Pentan-2-one	671	1.08 ± 0.27	0.56 ± 0.05	0.60 ± 0.08
3-Hydroxybutan-2-one	706	3.12 ± 0.72	-	-
3-Methylpentan-2-one	743	-	-	1.26 ± 0.22
5-Methylhexan-2-one	851	1.49 ± 0.16	1.89 ± 0.33	1.87 ± 0.31
Heptan-2-one	886	14.13 ± 2.01	8.43 ± 1.43	9.54 ± 0.91
6-Methylheptan-2-one	951	2.88 ± 0.71	3.22 ± 0.98	3.25 ± 0.51
Benzaldehyde	957	-	0.97 ± 0.14	0.78 ± 0.09
5-Methylheptan-2-one	961	3.33 ± 0.61	6.99 ± 1.62	1.40 ± 0.38
Nonan-5-one	1054	1.10 ± 0.14	1.35 ± 0.13	0.71 ± 0.12
Acetophenone	1065	1.22 ± 0.11	1.61 ± 0.08	1.87 ± 0.15
Nonan-2-one	1092	1.84 ± 0.31	1.17 ± 0.14	0.59 ± 0.07
Decan-5-one	1163	0.93 ± 0.16	0.79 ± 0.19	-
Undecan-2-one	1295	1.05 ± 0.14	-	-
Dodecan-5-one	1359	1.00 ± 0.17	-	-
2-Methyldecanal	1366	1.15 ± 0.15	0.69 ± 0.11	-
4-Acetyl-1-methylcyclohexene	1145	2.87 ± 0.55	1.17 ± 0.27	0.53 ± 0.07
Tridecan-2-one	1498	1.13 ± 0.30	1.04 ± 0.30	-
Tetradecan-5-one	1569	0.95 ± 0.16	-	-
Pentadecan-2-one	1700	-	-	0.64 ± 0.05
Alcohols, including the following:		4.58 ± 0.68	6.69 ± 1.27	3.44 ± 0.56
3-Methylbutan-1-ol	725	-	0.63 ± 0.06	0.45 ± 0.04
Heptan-2-ol	896	2.51 ± 0.39	2.63 ± 0.53	2.99 ± 0.52
5-Methyl-2-heptanol	968	-	1.28 ± 0.33	-
Nonan-2-ol	1100	1.41 ± 0.20	1.03 ± 0.21	-
Phenethyl alcohol	1114	0.66 ± 0.09	1.12 ± 0.15	-
Alkanes, including the following:		0.78 ± 0.20	1.49 ± 0.26	-
n-Hexadecane	1600	-	0.71 ± 0.09	-
n-Heptadecan	1700	0.78 ± 0.20	0.78 ± 0.17	-
Pyrazine derivatives, including the following:		32.76 ± 3.82	40.46 ± 3.38	48.98 ± 4.37
2-Methylpyrazine	818	1.42 ± 0.22	2.42 ± 0.27	1.71 ± 0.06
2,5-Dimethylpyrazine	906	23.94 ± 2.03	30.92 ± 1.74	34.17 ± 3.37
2,3,5-Trimethylpyrazine	998	6.96 ± 1.51	6.58 ± 1.25	11.09 ± 0.81
2-Methyl-3-izopropylpyrazine	1051	0.44 ± 0.06	0.54 ± 0.12	-
2,5-Dimethyl-3-ethylpyrazine	1078	-	-	2.01 ± 0.13
Amides, including the following:		-	-	3.54 ± 0.13
Acetamide	771	-	-	3.18 ± 0.11
Propanamide	855	-	-	0.37. ± 0.02
Sulfides, including the following:		-	0.73 ± 0.14	3.73 ± 0.26
1,2-Dimethylsulfide	735	-	-	2.69 ± 0.11
Dimethyl trisulfide	965	-	0.73 ± 0.14	1.04 ± 0.16
Monoterpenes, including:		3.12 ± 0.47	3.81 ± 0.66	2.99 ± 0.21
$\alpha$-Pinene	930	0.80 ± 0.04	1.15 ± 0.20	1.09 ± 0.06
$\delta$-3-Carene	1007	1.68 ± 0.28	1.89 ± 0.34	1.90 ± 0.15
Limonene	1027	0.65 ± 0.16	0.77 ± 0.11	-
Other compounds, including the following:		9.51 ± 1.39	4.35 ± 0.69	0.78 ± 0.13
Methyl N-hydroxybenzenecarboximidoate	903	3.66 ± 0.53	3.15 ± 0.35	-
Phenolic compound (C9H12O)	1260	3.92 ± 0.72	1.20 ± 0.34	0.78 ± 0.13
Phenolic compound (C9H12O)	1264	1.92 ± 0.14	-	-
Unidentified compounds, including the following:		2.42 ± 0.37	0.97 ± 0.16	1.91 ± 0.29
NN	1118	0.93 ± 0.14	-	-
NN	1150	-	0.97 ± 0.16	0.39. ± 0.04
NN	1724	1.49 ± 0.24	-	1.52 ± 0.24

,

Table 2. Chemical composition of volatile organic compounds emitted by F. oxysporum.

Group of Compounds	${\mathit{RI}}_{\mathit{exp}.}$	Relative Content [% TIC]
Sesquiterpenes, including the following:		8.49 ± 0.62
Cyclosativene	1370	0.10 ± 0.01
Spiroaxa-5,7-diene	1391	0.33 ± 0.03
$\beta$-Elemene	1391	0.10 ± 0.01
Sativene	1391	0.20 ± 0.01
$\beta$-Copaene	1435	0.11 ± 0.01
Sesquiterpene (C15H22)	1487	3.09 ± 0.07
$\alpha$-Murolene	1505	0.10 ± 0.01
$\delta$-Cadinene	1529	0.68 ± 0.03
Zonarene	1530	0.20 ± 0.02
$\beta$-Vatirenene	1556	2.44 ± o.31
Sesquiterpene (C15H24)	1574	0.53 ± 0.07
Glenol	1590	0.12 ± 0.01
1-epi-Cubenol	1635	0.27 ± 0.03
Cubenol	1646	0.21 ± 0.01
Alcohols, including the following:		14.53 ± 0.42
Ethanol	450	4.62 ± 0.01
Propan-1-ol	530	0.57 ± 0.04
Isobutanol	594	0.32 ± 0.03
2-Methylbutan-1-ol	728	0.84 ± 0.07
Benzyl alcohol	1030	8.18 ± 0.27
Carbonyl compounds, including the following:		26.69 ± 1.67
Isopentanal	626	0.54 ± 0.04
Benzaldehyde	959	25.83 ± 1.61
Acetofenone	1065	0.11 ± 0.01
Anisaldehyde	1247	0.09 ± 0.01
Undecan-2-one	1294	0.11 ± 0.01
Carboxylic acids, including the following:		0.58 ± 0.06
Acetic acid	612	0.10 ± 0.01
Isovaleric acid	830	0.35 ± 0.03
2-Methylbutanoic acid	835	0.13 ± 0.03
Esters, including the following:		0.72 ± 0.05
Methyl isopentanoate	770	0.38 ± 0.02
Methyl benzoate	1095	0.26 ± 0.01
Ethyl benzoate	1172	0.08 ± 0.01
Chlorinated benzene derivatives and chlorinated benzaldehyde derivatives, including the following:		48.45 ± 1.46
2-Chloro-1,4-dimethoxybenzene	1353	2.55 ± 0.17
1,4-Dichloro-2,5-dimethoxybenzene	1444	13.07 ± 0.43
3-Chloro-4-(methyloxy)benzaldehyde	1453	2.04 ± 0.02
3,5-Dichloro-4-methoxybenzaldehyde	1465	30.79 ± 0.83
Other compounds, including the following:		0.54 ± 0.04
Methyl N-hydroxybenzenecarboximidoate	903	0.19 ± 0.02
$\gamma$-Decalactone	1471	0.36 ± 0.02

,

Table 3. Volatile organic compounds produced by the interaction of B. amyloliquefaciens and F. oxysporum (I), as well as by pure cultures of B. amyloliquefaciens (II) and F. oxysporum (III).

Group of Compounds	${\mathit{RI}}_{\mathit{exp}.}$	Relative Content [% TIC]
		I	II	III
Sesquiterpenes, including the following:		50.99 ± 2.26	-	8.49 ± 0.62
Bicycloelemene	1341	0.28 ± 0.03	-	-
$\alpha$-Cubebene	1354	1.80 ± 0.15	-	-
Cyclosativene	1370	0.71 ± 0.06	-	0.10 ± 0.01
$\alpha$-Kopaene	1380	0.53 ± 0.03	-	-
Spiroaxa-5,7-diene	1391	16.06 ± 0.54	-	0.33 ± 0.03
$\beta$-Elemene	1393	0.96 ± 0.11	-	0.10 ± 0.01
Sativene	1394	2.54 ± 0.25	-	0.20 ± 0.01
Isosativene	1417	0.21 ± 0.01	-	-
Sesquiterpene (C15H24)	1420	0.14 ± 0.01	-	-
Sesquiterpene (C15H24)	1425	0.23 ± 0.03	-	-
Sesquiterpene (C15H24)	1429	0.16 ± 0.01	-	-
$\beta$-Copaene	1435	1.21 ± 0.07	-	0.11 ± 0.01
Aromadendrene	1443	0.25 ± 0.01	-	-
(E)-$\beta$-Famesen	1460	0.51 ± 0.02	-	-
Alloaromadendrene	1468	0.37 ± 0.03	-	-
trans-Cadina-1(6),4-diene	1479	0.73 ± 0.03	-	-
$\gamma$-Murolene	1482	0.38 ± 0.01	-	-
Sesquiterpene (C15H24)	1488	4.20 ± 0.09	-	3.09 ± 0.07
Alloaromadendr-9-ene	1493	1.79 ± 0.04	-	-
trans-Murola-4(14),5-diene	1497	1.10 ± 0.05	-	-
epi-Cubebol	1497	0.44 ± 0.01	-	-
Bicyclogermacren	1504	1.01 ± 0.03	-	-
$\alpha$-Murolene	1506	0.45 ± 0.04	-	0.10 ± 0.01
Germacrene A	1510	0.14 ± 0.02	-	-
$\gamma$-Cadinene	1519	0.33 ± 0.02	-	-
$\delta$-Cadinene	1529	4.83 ± 0.02	-	0.68 ± 0.03
Zonarene	1530	0.43 ± 0.04	-	0.20 ± 0.02
Cadina-1,4-diene	1539	0.38 ± 0.04	-	-
$\beta$-Vatirenen	1558	3.52 ± 0.26	-	2.44 ± 0.31
Sesquiterpene (C15H24)	1565	0.45 ± 0.02	-	-
Sesquiterpene (C15H24)	1574	0.38 ± 0.04	-	0.53 ± 0.07
Glenol	1591	0.59 ± 0.04	-	0.12 ± 0.01
1-epi-Cubenol	1635	3.43 ± 0.08	-	0.27 ± 0.03
Cubenol	1647	0.43 ± 0.02	-	0.21 ± 0.01
Monoterpenes, including the following:		1.68 ± 0.10	3.12 ± 0.47	-
$\alpha$-Pinene	930	0.27 ± 0.01	0.80 ± 0.04	-
$\delta$-3-Carene	1007	0.56 ± 0.02	1.68 ± 0.28	-
Limonene	1027	-	0.65 ± 0.16	-
Linalool	1097	0.85 ± 0.07	-	-
Alkanes, including the following:		0.58 ± 0.03	0.78 ± 0.20	-
n-Heptadecan	1700	0.58 ± 0.03	0.78 ± 0.20	-
Alcohols, including the following:		4.04 ± 0.28	4.58 ± 0.68	14.53 ± 0.42
Ethanol	450	0.47 ± 0.04	-	4.62 ± 0.01
Propan-1-ol	530	-	-	0.57 ± 0.04
Isobutanol	594	-	-	0.32 ± 0.03
Isopentanol	724	0.22 ± 0.01	-	-
2-Methylbutan-1-ol	728	-	-	0.84 ± 0.07
Butane-2,3-diol	785	0.26 ± 0.03	-	-
Heptan-2-ol	896	0.54 ± 0.01	2.51 ± 0.39	-
1-Octen-3-ol	976	0.29 ± 0.01	-	-
Benzyl alcohol	1030	1.75 ± 0.14	-	8.18 ± 0.27
Nonan-2-ol	1100	-	1.41 ± 0.20	-
Phenethyl alcohol	1114	-	0.66 ± 0.09	-
Undecan-2-ol	1301	0.51 ± 0.04	-	-
Carbonyl compounds, including the following:		27.70 ± 1.61	43.96 ± 7.85	27.04 ± 1.68
Acetone	480	3.55 ± 0.03	6.92 ± 1.54	-
Butan-2-one	560	1.29 ± 0.11	0.64 ± 0.19	-
Isopentanal	626	-	-	0.54 ± 0.04
Pentan-2-one	671	0.66 ± 0.01	1.08 ± 0.27	-
3-Hydroxybutan-2-one	708	0.18 ± 0.01	3.12 ± 0.72	-
3-Methylpentan-2-one	743	0.73 ± 0.01	-	-
Hexan-2-one	783	0.45 ± 0.01	-	-
5-Methylhexan-2-one	851	1.63 ± 0.11	1.49 ± 0.16	-
Heptan-2-one	886	-	14.13 ± 2.01	-
6-Methylheptan-2-one	951	2.54 ± 0.22	2.88 ± 0.71	-
Benzaldehyde	958	3.59 ± 0.14	-	25.83 ± 1.61
5-Methylheptan-2-one	961	3.40 ± 0.19	3.33 ± 0.61	-
Nonan-5-one	1054	0.61 ± 0.05	1.10 ± 0.14	-
Acetofenone	1065	1.25 ± 0.10	1.22 ± 0.11	0.11 ± 0.01
Nonan-2-one	1092	1.46 ± 0.11	1.84 ± 0.31	-
Decan-5-one	1163	0.87 ± 0.05	0.93 ± 0.16	-
Decan-2-one	1193	0.75 ± 0.07	-	-
Anisaldehyde	1251	0.54 ± 0.06	-	0.09 ± 0.01
Undecan-2-one	1294	1.32 ± 0.18	1.05 ± 0.14	0.11 ± 0.01
Dodecan-5-one	1359	0.98 ± 0.05	1.00 ± 0.17	-
2-Methyldecanal	1367	1.30 ± 0.09	1.15 ± 0.15	-
$\gamma$-Decalactone	1471	0.11 ± 0.01	-	0.36 ± 0.02
Tridecan-2-one	1498	-	1.13 ± 0.30	-
Tetradecan-5-one	1569	0.49 ± 0.02	0.95 ± 0.16	-
Carboxylic acids, including the following:		0.37 ± 0.01	-	0.58 ± 0.06
Acetic acid	612	0.37 ± 0.01	-	0.10 ± 0.01
Isovaleric acid	830	-	-	0.35 ± 0.03
2-Methylbutanoic acid	835	-	-	0.13 ± 0.03
Esters, including the following:		0.14 ± 0.02	-	0.72 ± 0.05
Methyl isopentanoate	770	-	-	0.38 ± 0.02
Methyl benzoate	1095	0.14 ± 0.02	-	0.26 ± 0.01
Ethyl benzoate	1172	-	-	0.08 ± 0.01
Benzene and benzaldehyde chlorine derivatives, including the following:		1.56 ± 0.12	-	48.45 ± 1.46
2-Chlorophenol	986	0.37 ± 0.02	-	-
2-Chloro-1,4-dimethoxybenzene	1353	0.85 ± 0.07	-	2.55 ± 0.17
1,4-Dichloro-2,5-dimethoxybenzene	1442	0.34 ± 0.03	-	13.07 ± 0.43
3-Chloro-4-(methyloxy)benzaldehyde	1453	-	-	2.04 ± 0.02
3,5-Dichloro-4-methoxybenzaldehyde	1465	-	-	30.79 ± 0.83
Pyrazine derivatives, including the following:		7.85 ± 0.49	32.76 ± 3.82	-
2-Methylpyrazine	820	0.58 ± 0.07	1.42 ± 0.22	-
2,5-Dimethylpyrazine	905	5.66 ± 0.35	23.94 ± 2.03	-
2,3,5-Trimethylpyrazine	1000	1.61 ± 0.06	6.96 ± 1.51	-
2-Methyl-3-izopropylpyrazine	1051	-	0.44 ± 0.06	-
Benzene derivatives, including the following:		2.84 ± 0.13	-	-
Styrene	886	1.57 ± 0.07	-	-
Phenol	980	1.13 ± 0.05	-	-
Methoxy-2-vinylbenzene	1153	0.13 ± 0.01	-	-
Anisole chlorine derivatives, including the following:		1.17 ± 0.08	-	-
4-Chloroanisole	1123	0.57 ± 0.02	-	-
2,6-Dichloroanisole	1199	0.60 ± 0.06	-	-
Other compounds, including the following:		1.08 ± 0.10	12.37 ± 1.94	0.19 ± 0.02
Methyl N-hydroxybenzenecarboximidoate	907	0.07 ± 0.01	3.66 ± 0.53	0.19 ± 0.02
Benzylnitrile	1139	0.33 ± 0.04	-	-
4-Acetyl-1-methylcyclohexene	1145	0.68 ± 0.05	2.87 ± 0.55	-
Phenolic compound (C9H12O)	1260	-	3.92 ± 0.72	-
Phenolic compound (C9H12O)	1264	-	1.92 ± 0.14	-
Unidentified compounds, including the following:		-	2.42 ± 0.37	-
NN	1118	-	0.93 ± 0.14	-
NN	1724	-	1.49 ± 0.24	-

).

3.2.1. Volatile Organic Compounds Produced by Bacillus Bacteria

The analysis of volatiles emitted by three species of Bacillus bacteria (i.e., B. amyloliquefaciens, B. subtilis and B. thuringiensis) revealed a total of 46 compounds, of which 41 were strictly identified, 2 were classified as phenolic compounds with the formula C₉H₁₂O, and the remaining 3 were not identified (Table 1).

In the volatile phase emitted by B. amyloliquefaciens, B. subtilis and B. thuringiensis, 30, 32 and 26 compounds were identified, respectively (Table 1). The analysed compositions of volatiles were rich in carbonyl compounds, of which heptan-2-one, acetone and two constitutional isomers of methylhepten-2-one, i.e., 5-methylhepten-2-one and 6-methylhepten-2-one, were found in the highest amount. B. amyloliquefaciens had the highest total content of carbonyl compounds (46.83 ± 8.41%). The presence of 3-hydroxybutan-2-one, undecan-2-one, dodecan-5-one and tetradecan-5-one was only found in B. amyloliquefaciens, 1-hydroxypropan-2-one only in B. subtilis and 3-methylpentan-2-one and pentadecan-2-one only in B. thuringiensis (Table 1).

The composition of volatile compounds emitted by Bacillus bacteria contained pyrazine derivatives, including in the highest amount 2,5-dimethyl-, 2,3,5-trimethyl- and 2-methylpyrazine. 2-Methyl-3-isopropylpyrazine was detected in B. subtilis and B. amyloliquefaciens, while 2,5-dimethyl-3-ethylpyrazine was only detected in cultures of B. thuringiensis. B. thuringiensis had the highest total content of pyrazine derivatives (48.98 ± 4.37%) (Table 1).

Alcohols (e.g., heptan-2-ol) and monoterpenes (e.g., $\delta$-3-carene) were also found in the samples. Amides such as acetamide and propanamide were exclusively produced by B. thuringiensis. Dimethyltrisulphide was detected in B. subtilis and B. thuringiensis, while dimethyldisulphide was only detected in B. thuringiensis. The volatile compositions of B. amyloliquefaciens and B. subtilis were also characterised by a content of about one percent of alkanes (Table 1).

In addition, selected information on the content of chemical compounds provided in the Table 1 is presented graphically in Figure 4. The graphical form of data presentation allows a more illustrative comparison of the content of individual chemical compounds in the composition of volatile compounds emitted by the three studied Bacillus species.

In Figure 4, chemical compounds were included whose content was more than 2% in at least one bacterial species. The names of the individual compounds are on the vertical axis and the relative content of the chemical compounds (% TIC) in the tested VOC mixture is indicated on the horizontal axis.

3.2.2. Volatile Organic Compounds Produced by F. oxysporum

In the volatile composition emitted by F. oxysporum, 36 compounds were found, among which 34 volatiles were identified, and 2 compounds were classified as sesquiterpenes, but their chemical structures were not determined (Table 2). The largest group were the sesquiterpenes, of which 14 were detected in the analysed samples. The total content of sesquiterpenes was 8.49 ± 0.62% (Table 2). The emissions from the fungus included five alcohols, five carbonyl compounds, three carboxylic acids and three esters (Table 2). It was particularly rich in benzaldehyde (25.83 ± 1.61%), benzyl alcohol (8.18 ± 0.27%) and ethanol (4.62 ± 0.01%). The total content of carboxylic acids and esters was relatively low (Table 2). The volatile emission of F. oxysporum was characterised by a high content of chlorinated benzene derivatives (1,4-dichloro-2,5-dimethoxybenzene, 2-chloro-1,4-dimethoxybenzene) and chlorinated benzaldehyde derivatives (3,5-dichloro-4-methoxybenzaldehyde, 3-chloro-4-(methyloxy)benzaldehyde), and the total content of these compounds was 48.45 ± 1.46% (Table 2). In addition, methyl N-hydroxybenzene carboximidoate and $\gamma$-decalactone were identified in the test samples (Table 2).

3.2.3. Volatile Organic Compounds Produced by the Interaction of B. amyloliquefaciens and F. oxysporum

Table 3 contains information on the chemical compounds emitted by the microorganisms during the interaction of B. amyloliquefaciens and F. oxysporum (I), as well as individual strain of B. amyloliquefaciens (II) and F. oxysporum (III). In the B. amyloliquefaciens + F. oxysporum interaction, 82 compounds were found, of which 76 were identified with specific chemical structures, and the remaining 6 compounds were classified as sesquiterpene hydrocarbons (Table 3, column I).

In the interaction between B. amyloliquefaciens and F. oxysporum, sesquiterpenes were the most abundant group of compounds, of which 34 were detected (Table 3). The total content of sesquiterpenes was 50.99 ± 2.26%, and the largest numbers were found for spiroax-5,7-diene (16.06 ± 0.54%), $\delta$-cadinene (0.33 ± 0.02%) and sesquiterpene C₁₅H₂₂ with $R{I}_{exp.}$ = 1488 (4.20 ± 0.09%). Sixteen sesquiterpenes were identified that occurred exclusively in the microbial interaction of B. amyloliquefaciens and F. oxysporum, including bicycloelemene, $\alpha$-cubebene or $\alpha$-copaene (Table 3).

The tested samples of B. amyloliquefaciens + F. oxysporum contained monoterpenes such as $\alpha$-pinene, $\delta$-3-carene and linalool, which were only present in the fungal–bacterial interaction (Table 3). The content of alcohols in B. amyloliquefaciens + F. oxysporum (I) and B. amyloliquefaciens (II) was about 4% and was more than three times lower than in the pure F. oxysporum culture (14.53 ± 0.42%).

In contrast, the content of carbonyl compounds in B. amyloliquefaciens + F. oxysporum (I) and F. oxysporum (III) varied around 27% and was significantly lower than in the pure B. amyloliquefaciens culture (43.96 ± 7.85%). A characteristic feature of the chemical composition of the VOCs present in the B. amyloliquefaciens + F. oxysporum interaction was the presence of alcohols such as isopentanol, butane-2,3-diol, 1-octen-3-ol and undecan-2-ol, as well as carbonyl compounds such as 3-methylpentan-2-one, hexan-2-one and decan-2-one (Table 3).

The chlorophenol and benzaldehyde content of the B. amyloliquefaciens + F. oxysporum interaction (1.56 ± 0.12%) was more than 30 times lower compared to pure F. oxysporum (48.45 ± 1.46%); however, 2-chlorophenol was only present in the bacteria–fungus interaction. In contrast, the content of pyrazine derivatives in B. amyloliquefaciens + F. oxysporum (7.85 ± 0.49%) was four times lower than in B. amyloliquefaciens (32.76 ± 3.82%). The specific group of compounds present in the B. amyloliquefaciens + F. oxysporum sample were benzene derivatives (styrene, phenol and methoxy-2-vinylbenzene), chlorinated anisole derivatives (4-chloroanisole and 2,6-dichloroanisole) and benzylnitrile (Table 3).

Additionally, selected information on the content of chemical compounds provided in Table 2 is presented graphically in Figure 5. The graph includes chemical compounds whose content in at least one of the samples tested was more than 2%.

4. Discussion

4.1. Test of Antagonism

The antagonism test was conducted to assess the ability of three bacterial species of the genus Bacillus (B. amyloliquefaciens, B. subtilis and B. thuringiensis) to inhibit the growth of the fungus F. oxysporum. Among them B. amyloliquefaciens exhibited the strongest inhibitory effect. Initially, the antagonism assay was performed using a two-compartment Petri dish, as described by Yuan et al. [31]. However, in the control setup, Fusarium colonies rapidly overgrew the partition of the Petri dish, making it impossible to accurately measure mycelial growth. Consequently, we adopted the sealed-plate method [25], which involves combining two Petri dishes of the same diameter. In this setup, the test fungus and bacterium are not in direct contact but are separated by approximately 2 cm, with the fungus placed on the lower plate and the bacterium on the upper one. It is worth noting that this method—also referred to in the literature as the double-plate method [25,30] or sandwich assay [29]—enables the evaluation of volatile organic compounds (VOCs) produced by antagonistic Bacillus strains and their inhibitory effects on F. oxysporum.

The maximum inhibition of Fusarium oxysporum growth (70%) by Bacillus amyloliquefaciens VOCs was observed after 24 h but dropped to 45% after 96 h. We hypothesize that this reduction may be attributed to one or more of the following factors:

Depletion or transformation of VOCs over time: VOCs are typically low-molecular-weight compounds with high volatility. As the antagonism test progresses, their concentration in the sealed environment may decline due to evaporation, adsorption to surfaces or microbial metabolism.

Adaptive fungal response: Prolonged exposure to sublethal concentrations of VOCs may induce stress response mechanisms in F. oxysporum, such as increased expression of detoxification enzymes or efflux transporters, leading to partial resistance [32,33].

Changes in Bacillus metabolic activity: As F. oxysporum continues to grow, it may compete for nutrients or create unfavourable conditions that suppress the bacterial production of VOCs.

4.2. Volatile Secondary Metabolites

The antagonism test analysed the interactions between the fungus F. oxysporum and the Bacillus species B. amyloliquefaciens, B. subtilis and B. thuringiensis. They were inspired by literature reports on the fungistatic activity of volatile organic compounds (VOCs) produced by bacteria of the genus Bacillus [23,34,35,36,37]. The ability of some strains to produce a unique and reproducible VOC profile under certain conditions is an important antagonistic mechanism [38]. The antagonistic effect of VOCs includes the inhibition of mycelial growth and spore germination [34] or the inhibition of the gene expression of some fungal species [39]. When mycelial growth is inhibited, certain volatiles are produced in response to the presence of a particular fungal species. The specific profile of VOCs produced may be due to factors such as the response of the fungi to the presence of different components of the volatile mixture, the interaction site or the different abilities of the fungi to detoxify volatile metabolites. The inhibitory effect of Bacillus species may be due to existing interactions between bacteria and fungi and belongs to species or even strain characteristics. The different degree of inhibition of mycelial growth depends, among other things, on the Bacillus species and Fusarium [34].

In the investigated case of the interaction between the bacterium B. amyloliquefaciens and the fungus F. oxysporum, an inhibition of mycelial growth was observed 24 h after the start of the measurement at a high level of 60%–70%. After 96 h, however, it was 40%–50%, indicating that the duration of the experiment was sufficiently long and no further extension was required. Similar results were obtained by Yuan et al. [31], who observed that the B. amyloliquefaciens NJN-6 strain they tested reduced the mycelial growth of F. oxysporum by 30%–40% after 3 days of the experiment compared to the control. In contrast, in the experiment by Wang et al. [40], B. amyloliquefaciens strain W19 inhibited the mycelial growth of F. oxysporum f. sp. cubense (FOC) by 21% during a 5-day antagonism test. Also, Santos et al. [30] confirmed the fungistatic properties of a Bacillus sp. strain (LPPC170) against F. kalimantanense, which is genetically closely related to F. oxysporum f. sp. cubense (FOC) and belongs to the F. oxysporum species complex (FOSC). In in vitro tests, Bacillus sp. (LPPC170) showed significant inhibition against Fusarium strain LPPC130 at 41.23%. The slightly lower inhibition values reported by Yuan et al., Wang et al. and Santos et al. [30,31,40] compared to the results obtained in this study may be due to differences in the composition of the culture media used and the particular characteristics of the strain used in this study.

4.3. Interactions Between Fungus and Bacteria

The in-house study showed that bacteria of the species B. thuringiensis and B. subtilis had a slightly lower inhibitory effect on the growth of the fungus F. oxysporum than that of B. amyloliquefaciens (about 45%). B. thuringiensis bacteria were able to inhibit mycelial growth by about 40%. He et al. [41] presented slightly different results on the effect of B. thuringiensis against F. oxysporum. The study of the cited authors showed that B. thuringiensis strain BCN10 had no antifungal properties against F. oxysporum. The differences could be due to the metabolic capacity of the strain or to the too short duration of the analysis, as they found that the inhibitory effect against the mycelium of F. oxysporum increased with the duration of the experiment [41].

On the other hand, Islam et al. [42] and Farag et al. [43] confirmed that the volatile organic compounds produced by B. subtilis could be used as biological control agents to protect against plant pathogenic microorganisms, including F. oxysporum. On the other hand, B. subtilis C9 showed potent antifungal activity in inhibiting the mycelium of F. oxysporum, which was 93.61% (for one of the compounds produced, the DG4 isomer of acetylbutanediol) [42]. In contrast, Tuyen et al. [44] tested the fungistatic activity of a B. subtilis suspension against F. oxysporum in in vitro and in vivo tests on maize. The VOCs were then purified and analysed for their antifungal properties in in vitro tests. The tests showed that B. subtilis bacteria exhibited high antifungal activity against F. oxysporum, and such a result gives hope for the use of the secondary metabolites of B. subtilis as biocontrol agents with high versatility without harming the environment [44].

The results of Li et al. [45] suggest that the VOCs produced by strain B. velezensis CT32 have broad-spectrum antifungal activity against F. oxysporum. The authors suggest that the volatile compounds produced by the tested Bacillus strain could potentially be used as an agent against the fungus F. oxysporum.

Many scientific reports indicate a strong inhibitory effect of the volatile organic compounds (VOCs) produced by Bacillus bacteria on the fungus F. oxysporum [23,25,46]. In contrast, the literature on the effects of VOCs is much sparser. Therefore, the analyses presented here are novel in terms of understanding the antagonistic properties of bacteria of the genus Bacillus against the fungus F. oxysporum and especially regarding the antagonistic properties of the VOCs they produce.

4.4. Chemical Compounds Detected by GC–MS

Based on HS-SPME/GC–MS analyses, it was found that the volatile emissions of the three investigated Bacillus species, i.e., B. amyloliquefaciens, B. subtilis and B. thuringiensis, contained different groups of chemical compounds, including carbonyl compounds, alcohols and alkanes, as well as pyrazine derivatives, amides, sulphides and monoterpenes. The presence of the above-mentioned groups of volatile compounds in the emissions of Bacillus cultures is also evident from the results of studies published by Wang et al. [40], Wu et al. [25], Farag et al. [43] and Chaves-López et al. [23]. Bacteria of the genus Bacillus produce a range of different carbonyl compounds, among which ketones predominate and aldehydes are less abundant. Wang et al. [40] identified heptan-2-one, nonan-2-one, tridecan-2-one and acetophenone in the VOC composition emitted by B. amyloliquefaciens. The presence of these ketones in B. amyloliquefaciens was also confirmed by the results of our work (Table 1). Wu et al. [25] also found the presence of 3-hydroxybutan-2-one (acetoin) and undecan-2-one in B. amyloliquefaciens, which was also confirmed by the results of our analyses (Table 1). In addition, Farag et al. [43] and Chaves-López et al. [23] detected acetone and butan-2-one in B. amyloliquefaciens and B. subtilis cultures, which is also consistent with the chemical results we obtained (Table 1).

Chaves-López et al. [23] found that benzaldehyde was present in the volatile emissions of B. subtilis and not present in B. amyloliquefaciens. This correlation was also confirmed by our results (Table 1). Farag et al. [43] also indicated the presence of benzaldehyde in B. subtilis. In contrast, Du et al. [47] identified heptano-2-one, 5-methylheptano-2-one and 6-methylheptano-2-one in B. subtilis, and the presence of the three mentioned ketones in B. subtilis was also confirmed by the presented results (Table 1). In addition, He et al. found [41] butan-2-one, 6-methylheptan-2-one, acetophenone and benzaldehyde in the chemical composition of the VOCs produced by B. thuringiensis, and ketones were also detected in B. thuringiensis cultures in our study (Table 1).

Ting et al. [48] detected acetone in the composition produced by Bacillus spp. and Guevara-Avendano et al. [49] identified butan-2-one, 5-methylheptan-2-one, 6-methylheptan-2-one and nonan-2-one, while the results of our analyses confirmed the presence of these ketones in all analysed Bacillus species, i.e., B. amyloliquefaciens, B. subtilis and B. thuringiensis (Table 1). Santos et al. [30] reported an acetoin content of 1.56 ± 0.09% in cultures of Bacillus sp. LPPC170, while the results of our study showed that the content of this ketone in B. amyloliquefaciens was 3.12 ± 0.72% and it was not present in the other two Bacillus species analysed, namely, B. subtilis and B. thuringiensis (Table 1). In addition, Velivelli et al. [50] and Guevara-Avendano et al. [49] identified tridecan-2-one in cultures of Bacillus sp., which was also confirmed by our studies on the products emitted by B. amyloliquefaciens and B. subtilis (Table 1). Guevara-Avendano et al. [49] also detected pentadecan-2-one in Bacillus sp., while their results confirmed the presence of this compound only in the volatile emission of B. thuringiensis (Table 1).

The second group of compounds found in the volatile emissions of Bacillus sp. were alcohols. Chaves-López et al. [23] found phenethyl alcohol in the composition emitted by B. amyloliquefaciens and B. subtilis. The same authors also identified 3-methylbutan-1-ol in B. subtilis. In contrast, Du et al. [47] detected heptano-2-ol in B. subtilis, and He et al. [41] detected the presence of 3-methylbutan-1-ol in B. thuringiensis. All the above information on the content of different alcohols in the different Bacillus species was also confirmed by the results of our study (Table 1). In addition, Santos et al. [30] found that the content of 3-methylbutan-1-ol in cultures of Bacillus sp. strain LPPC170, was 2.30 ± 0.59%, while our results showed that the content of this alcohol in the volatile composition emitted by the tested Bacillus species was lower at 0.63 ± 0.06% for B. subtilis and 0.45 ± 0.04% for B. thuringiensis, while no 3-methylbutan-1-ol was found in B. amyloliquefaciens (Table 1).

Wu et al. [51] identified n-heptadecane in a mixture of volatile organic compounds produced by B. amyloliquefaciens. Farag et al. [43] also confirmed the presence of saturated hydrocarbons in cultures of B. amyloliquefaciens and B. subtilis. The presence of alkanes in bacteria of the genus Bacillus was also confirmed by the results of our chemical analyses (Table 1). The composition of volatile compounds produced by Bacillus bacteria was found to be very rich in alkylpyrazine derivatives (e.g., 2-methylpyrazine) by Chaves-López et al. [23] in B. amyloliquefaciens and B. subtilis, while He et al. [41] found the presence of a large number of these pyrazine derivatives in B. thuringiensis. The presence of numerous representatives of this group of compounds in B. amyloliquefaciens, B. subtilis and B. thuringiensis is also evidenced by the results presented in Table 1 of this study. Velivelli et al. [50] and Guevara-Avendano et al. [49] identified 2,5-dimethylpyrazine in cultures of Bacillus sp., and in our analyses, we also found high levels of this compound (23.94%–34.17%) in the three Bacillus species studied (Table 1).

In addition, Farag et al. [43] identified di-methyltrisulphide in B. subtilis, which was also confirmed by the results of our analyses (Table 1). Guevara-Avendano et al. [49] also found this sulphide in Bacillus sp. In addition, Ting et al. [48], Velivelli et al. [50] and Guevara-Avendano et al. [49] identified 1,2-di-methylsulfide in Bacillus spp., while our studies showed that the sulphide in question was only present in B. thuringiensis (Table 1). Furthermore, Massawe et al. [52] identified $\alpha$-pinene in Bacillus spp. while the results of our work confirmed the presence of this monoterpene in cultures of the three Bacillus species analysed, i.e., B. amyloliquefaciens, B. subtilis and B. thuringiensis (Table 1). It should also be noted that Fiddaman and Rossall [53] showed that the type of culture medium also had a significant influence on the composition of volatile organic compounds produced by B. subtilis.

Based on a series of chemical analyses, it has been demonstrated that Fusarium fungi produce sesquiterpenes and monoterpenes as well as alcohols, carbonyl compounds, carboxylic acids and esters [20,30]. The volatile emissions of some Fusarium species have also been found to contain numerous benzene and benzaldehyde chlorine-derivatives. The volatile composition emitted by fungi of the genus Fusarium also includes monoterpenes, which are terpene formed by the combination of two isoprene units. Another group of volatile compounds found in the volatile emission of Fusarium fungi are low-molecular-weight alcohols.

Santos et al. [30] reported an isopentanol content of 21.17 ± 2.43% in F. oxysporum kalimantanense LPPC130, while our results did not confirm the presence of this alcohol in F. oxysporum cultures. Santos et al. [30] stated that the volatile emission of F. oxysporum kalimantanense LPPC130 had 3.67 ± 0.58% isobutanol and 13.97 ± 1.82% 2-methylbutanol. Our results confirmed the presence of these two alcohols in F. oxysporum, as their content in the cultures was the lowest at 0.32 ± 0.03% and 0.84 ± 0.07%, respectively (Table 2). Loulier et al. [20] identified benzaldehyde in the volatile emission of F. oxysporum, which was also found in our analyses, where a high content (25.83 ± 1.61%) of this aromatic ketone was found. Chlorobenzene and benzaldehyde derivatives are a characteristic group of volatile compounds found in the volatile composition of F. oxysporum. Loulier et al. [20] identified 3-chloro-4-methyloxybenzaldehyde in F. oxysporum, which was also confirmed by our results (Table 2). In addition, both the results of Loulier et al. [20] and our results indicate the presence of numerous chlorobenzene derivatives in F. oxysporum cultures (Table 2).

It should be noted that a large amount of information on volatile organic compounds produced by pure Bacillus cultures is available in the literature [23,25,30,40,41,43,47,48,49,50,52]. In contrast, literature data on volatiles emitted by fungi of the genus Fusarium are far less extensive [20,30], and information on volatile compounds produced during the interaction between bacteria of the genus Bacillus and fungi of the genus Fusarium is relatively scarce [30].

Santos et al. [30] found the presence of 14.56 ± 4.17% isopentanol in the composition of volatiles obtained from the interaction of Bacillus sp. LPPC170 and F. oxysporum kalimantanense LPPC130, while our results confirmed a 0.22 ± 0.01% content of this alcohol released during the interaction of B. amyloliquefaciens and F. oxysporum (Table 3). Santos et al. [30] also identified butane-2,3-diol (0.05 ± 0.10%) during the interaction of Bacillus sp. LPPC170 and F. oxysporum kalimantanense LPPC130, which was confirmed by our results indicating 0.26 ± 0.03% concentration of this diol produced during the interaction of B. amyloliquefaciens and F. oxysporum (Table 3). It is also worth noting that, based on our analyses, butane-2,3-diol was present during the interaction of B. amyloliquefaciens and F. oxysporum, whereas it was not found in pure B. amyloliquefaciens and F. oxysporum cultures (Table 3).

Other groups of compounds produced during the interaction of Bacillus bacteria and Fusarium fungi are ketones and carboxylic acids. Santos et al. [30] found nonan-2-one (0.08 ± 0.01%) in the interaction of Bacillus sp. LPPC170 and F. oxysporum kalimantanense LPPC130, and the presence of this ketone in the interaction of B. amyloliquefaciens and F. oxysporum (1.46 ± 0.11%) was also confirmed by our results (Table 3). Santos et al. [30] found that the interaction of Bacillus sp. LPPC170 and F. oxysporum kalimantanense LPPC130 showed an acetic acid content of 0.38 ± 0.12%, while our analyses showed a very similar acetic acid content (0.37 ± 0.01%) in the interaction of B. amyloliquefaciens and F. oxysporum (Table 3). In addition, Santos et al. [30] identified an anisole during the interaction studies of Bacillus sp. LPPC170 and F. oxysporum kalimantanense LPPC130, and our work confirmed the occurrence of anisole derivatives during the interactions of B. amyloliquefaciens and F. oxysporum (Table 3).

Santos et al. [30] concluded that Bacillus sp. LPPC170 and F. oxysporum kalimantanense LPPC130 interacted with each other via volatiles, leading to changes in the metabolism of both microorganisms. When the bacteria and the fungus interact, the production of some microbial VOCs can be increased, while other compounds can be produced in reduced quantities [30,54]. The same observations also emerged after a detailed analysis of the chemical composition of both pure Bacillus cultures and F. oxysporum fungi and the VOC compositions emitted during bacterial–fungal interactions (Table 3).

The use of individual pure volatile compounds to control Fusarium fungi is also interesting. The compounds used in such studies are identified by HS-SPME/GC–MS in the volatile emission of pure bacterial cultures of the genus Bacillus. Commercial standards of the identified compounds are then used in the studies, and the effect of a particular compound on the growth of a particular Fusarium fungus species is tested. Thus, by determining the degree of inhibition (%) in the antagonism test, the effect of a specific, single chemical compound is determined and not that of a mixture of volatile organic compounds produced by Bacillus bacteria. Yuan et al. [31] detected 36 chemical compounds in the volatile emissions of B. amyloliquefaciens NJN-6. They then used antagonism tests to show that 11 of the 36 compounds tested completely inhibited the growth of F. oxysporum f. sp. cubense [31]. Among others, Yuan et al. [31] identified seven ketones forming a homologous series from nonan-2-one to pentadecan-2-one in B. amyloliquefaciens NJN-6 cultures. They found that nonan-2-one and decan-2-one completely inhibited the growth of the fungal species F. oxysporum f. sp. cubense. Undecan-2-one, dodecan-2-one and tridecan-2-one only partially inhibited the growth of the test fungus, with the degree of inhibition (%) decreasing as the length of the ketone carbon chain increased. In contrast, tetradecan-2-one and pentadecan-2-one did not inhibit the mycelial growth of F. oxysporum f. sp. cubense [31]. In our study, the compounds nonan-2-one and undecan-2-one were identified both in the pure B. amyloliquefaciens culture and in the B. amyloliquefaciens + F. oxysporum interaction. In contrast, decan-2-one was only found in the B. amyloliquefaciens + F. oxysporum interaction, while tridecan-2-one was only detected in the pure B. amyloliquefaciens culture (Table 3). Based on the results of Yuan et al. [31] and the results of our study, it can be concluded that the bacterium produces a higher amount of decan-2-one in the interaction, which causes a significant inhibition of F. oxysporum growth. In contrast, the production of tridecan-2-one, whose inhibitory effect on the fungus is low, is reduced. The very different effects of ketones on the mycelial growth of F. oxysporum were also confirmed by the results of a study published by Li et al. [45], which showed that undecan-2-one inhibited the growth of F. oxysporum by 56.96 ± 1.95%, while tridecan-2-one inhibited growth by only 4.37 ± 1.26%.

The literature also indicates that a number of other volatile ketones inhibit the growth of F. oxysporum. Acetone, butan-2-one [23], heptan-2-one, acetophenone [40] and 3-hydroxybutan-2-one [47] have been shown to possess fungistatic properties. Our results confirm the presence of 3-hydroxybutan-2-one, commonly referred to as acetoin, when present in a pure culture of B. amyloliquefaciens, as well as in the interaction of B. amyloliquefaciens and F. oxysporum (Table 3). It is also worth mentioning that the hydroxycetone discussed not only causes a reduction in Fusarium infections but also those caused by other pathogenic fungi, e.g., Colletotrichum coccodes [55]. In addition, the volatiles produced by Bacillus are also effective against other pathogenic organisms, e.g., nematodes [56]. The authors showed that the volatile compounds secreted by the bacterium B. subtilis, such as nonan-2one and benzaldehyde, completely eliminated nematodes of the species Panagrellus redivivus and Bursaphelenchus xylophilus. Our analyses also found the two carbonyl compounds mentioned above in the volatile emission of B. subtilis (Table 1).

Small-molecule alcohols produced by bacteria of the genus Bacillus represent another group of chemical compounds that inhibit the growth of Fusarium fungi. Yuan et al. [31] showed that undecan-2-ol contained in the volatile emission of B. amyloliquefaciens NJN-6 completely inhibited the growth of F. oxysporum f. sp. cubense. Our results showed that this alcohol was present in the B. amyloliquefaciens + F. oxysporum interaction (0.51 ± 0.04%) and was not present in the pure B. amyloliquefaciens and F. oxysporum bacterial cultures (Table 3). Based on the information presented above, it can be surmised that B. amyloliquefaciens increases the production of undecan-2-ol to combat F. oxysporum.

The results available in the literature show that the growth inhibition of F. oxysporum is also influenced by other alcohols produced by Bacillus bacteria, such as isopentanol [23], heptan-2-ol [47] or phenethyl alcohol [34]. Our results confirm the presence of the three aforementioned alcohols in the volatile compositions of Bacillus (Table 1). In addition, Wei et al. [55] found that 2-ethylhexan-1-ol and butane-2,3-diol produced by B. mojavensis, a bacterial species closely related to B. subtilis [36], were effective in reducing the growth of the fungus Colletotrichum coccodes.

Li et al. [45] showed that n-hexadecane, which was also found in our analyses in the pure B. subtilis culture (Table 1), did not inhibit the growth of F. oxysporum. In contrast, n-heptadecane, whose presence in B. amyloliquefaciens and B. subtilis (Table 1) was confirmed by our analyses, inhibited the growth of the fungus F. oxysporum by only 1.29 ± 0.85% [45]. Guevara-Avendano et al. [49] found, that 2,3,5-tri-methylpyrazine completely inhibited the growth of F. soloni. Our studies confirmed the presence of this compound in the cultures of all bacterial species tested, namely, B. amyloliquefaciens, B. subtilis and B. thuringiensis (Table 1). In addition, Guevara-Avendano et al. [49] found that di-methyltrisulphide caused the complete growth inhibition of F. solani, while our analyses showed the presence of this sulphide in the volatile emissions of B. subtilis and B. thuringiensis (Table 1). In contrast, Yuan et al. [31] showed that styrene and phenol, which were present in the volatile emission of B. amyloliquefaciens NJN-6, reduced the growth of F. oxysporum f. sp. cubense. In our study, styrene and phenol were identified only for the interactions of B. amyloliquefaciens and F. oxysporum, while these compounds were not present in both pure B. amyloliquefaciens and F. oxysporum strains (Table 2).

Research by other authors also confirmed that short-chain fatty acids, which have between two and five carbon atoms in their backbone, effectively inhibited the growth of F. oxysporum [23,30,45]. Pentanoic acid and isopentanoic acid inhibited the growth of the fungus by 50%–70% [23,30]. For carboxylic acids with a shorter carbon chain, such as acetic acid, propanoic acid and butanoic acid, the inhibitory effect was lower at 20%–30% [30,45]. Zhou et al. [7] also demonstrated the antifungal properties of volatile carboxylic acids. They also demonstrated that acetic acid, propanoic acid, butanoic acid, pentanoic acid and isopentanoic acid had a synergistic effect [7].

To summarise, both our results and the data from the literature clearly indicate that volatile organic compounds released by bacteria of the genus Bacillus can be successfully used to control pathogenic fungi of F. oxysporum. Relevant information can be obtained by analysing the chemical composition of the volatile mixtures produced by both pure bacterial and fungal cultures, as well as those emitted during the interaction of the two microorganisms. Comparing the results of such analyses can provide valuable information on which chemical compounds are most effective against F. oxysporum. Both mixtures of VOCs produced by bacteria and individual compounds contained in these mixtures can have antifungal properties.

4.5. Future Work

Our study demonstrated the inhibitory potential of Bacillus-emitted VOCs against Fusarium oxysporum in vitro. We fully agree that to support their application in biological control, it is essential to validate this effect in a plant infection model.

In the next phase of our research, we plan to conduct in planta experiments using selected compounds identified via GC–MS. Specifically, we aim to test VOCs that were either exclusive to Bacillus–Fusarium interactions (e.g., $\alpha$-cubebene, linalool, undecan-2-ol, decan-2-one and 2,6-dichloroanisole) or significantly enriched in the interaction profiles (e.g., butan-2-one, 5-methylhexan-2-one, 5-methylheptan-2-one and 2-methyldecanal). These experiments will allow us to assess both the protective effects on plant hosts and the practicality of using VOCs as biological control agents in situ. This work will be accompanied by further exploration of the molecular mechanisms involved, such as transcriptomic responses of F. oxysporum to VOC exposure. We understand that chemical characterization alone, while informative, does not fully explain the biological implications of VOC–fungus interactions.

For instance, VOCs such as linalool and 2-methyldecanal are known to disrupt fungal membrane integrity and induce oxidative stress, leading to cell leakage and apoptosis-like responses [32,57]. Other compounds such as decan-2-one and butan-2-one have been shown to interfere with transcriptional regulation and signal transduction pathways, including the MAPK cascade and genes responsible for conidial germination [33,58]. Additionally, $\alpha$-cubebene and undecan-2-ol may suppress mycotoxin biosynthesis and modulate the expression of stress-related genes [59].

While these specific interactions have not yet been confirmed in our fungal model, they represent plausible avenues for future investigation. Accordingly, we are preparing transcriptomic studies, including RNA sequencing and qPCR validation, to evaluate the expression profiles of F. oxysporum exposed to VOCs, focusing on genes involved in membrane integrity (e.g., ergosterol biosynthesis), oxidative stress (e.g., catalases, superoxide dismutases) and signal transduction. Moreover, we plan to perform gene expression profiling (e.g., RNA-Seq, qPCR) of F. oxysporum exposed to VOCs to identify affected genes and pathways, focusing on membrane transporters, stress-related genes and transcriptional regulators.

5. Summary and Conclusions

Previous studies have extensively explored the antagonistic effects of Bacillus species and their VOCs against Fusarium oxysporum, including GC–MS metabolite profiling and various biocontrol assays. However, to the best of our knowledge, no study has focused specifically on the VOCs produced during the active interaction between Bacillus spp. and F. oxysporum.

Our research addressed this gap by comparing volatile emissions from monocultures of B. amyloliquefaciens and F. oxysporum to those released during their co-cultivation. We discovered that the VOC profile from the interaction was not a mere sum of the individual microbial emissions. Instead, it included novel compounds absent in either monoculture, such as $\alpha$-cubebene, linalool, undecan-2-ol, decan-2-one and 2,6-dichloroanisole. Additionally, several VOCs were enriched during co-cultivation, indicating the activation of specific metabolic pathways through microbial crosstalk.

This finding offers a new perspective: Bacillus bacteria may dynamically alter their VOC emission profile in response to fungal presence, potentially as a targeted defence strategy. Without this approach, such interaction-induced compounds would remain unidentified and untested for antifungal activity.

To build on these results, we are planning in vitro bioassays using standards of key VOCs identified in the interaction, in planta infection studies to validate biocontrol efficacy and transcriptomic analyses to investigate fungal responses to selected VOCs at the molecular level.

The following summarises the main results:

• Volatile organic compounds produced by three tested species of Bacillus bacteria (B. amyloliquefaciens, B. subtilis and B. thuringiensis) inhibited the growth of the fungus F. oxysporum.
• The highest inhibition of F. oxysporum’s growth (70%) was observed with B. amyloliquefaciens after 24 h of the antagonism test, while the inhibition decreased to 45% after 96 h. With the growth of F. oxysporum, the quantity of VOCs produced by B. amyloliquefaciens decreased in relation to the surface area of the fungus. This could be the reason for the decrease in inhibition over time.
• The quantitative and qualitative compositions of the VOCs emitted by the studied Bacillus bacteria were different. B. amyloliquefaciens was characterised by a higher content of carbonyl compounds (46.83 ± 8.41%) than B. subtilis (41.50 ± 6.45%) and B. thuringiensis (34.62 ± 4.77%). 3-Hydroxybutan-2-one, undecan-2-one, dodecan-5-one and tetradecan-5-one were only identified in the volatile emissions of B. amyloliquefaciens.
• The main components of the volatile emission of F. oxysporum were chlorinated benzaldehyde derivatives (e.g., 3,5-dichloro-4-methoxybenzaldehyde) and chlorinated benzene derivatives (e.g., 1,4-dichloro-2,5-dimethoxybenzene) as well as carbonyl compounds (e.g., benzaldehyde) and alcohols (e.g., benzyl alcohol).
• During the interaction of B. amyloliquefaciens and F. oxysporum, new compounds (e.g., $\alpha$-cubebene, linalool, undecan-2-ol, decan-2-one and 2,6-dichloroanisole) were found that were not present in the volatile emissions of the separated bacteria and fungal strains. On the other hand, some compounds found in the emissions of B. amyloliquefaciens (including limonene, nonan-2-ol, phenethyl alcohol, heptan-2-one and tridecan-2-one) or F. oxysporum (including propan-1-ol, 2-methylbutan-1-ol, isopentanal, 3-chloro-4-(methyloxy)benzaldehyde and 3,5-dichloro-4-methoxybenzaldehyde) were not found in the interaction of the microorganisms. The explanation for this could be that the two microorganisms interact via volatile compounds, thereby stimulating some metabolic pathways and inhibiting others.
• Standards of chemical compounds identified in the volatile emission of B. amyloliquefaciens or the interaction of the microorganisms can be used for further testing of the inhibition of F. oxysporum. In particular, it is worth focussing on compounds that occur exclusively in the interaction or on compounds whose content in the interaction was higher in relation to the content in B. amyloliquefaciens strains (i.e., butan-2-one, 5-methylhexan-2-one, 5-methylheptan-2-one and 2-methyldecanal). The bacteria can start producing a new compound or increase the production of a compound to combat the fungus.
• The results presented are a prerequisite for the creation of an alternative solution or supplement to the currently used methods of plant protection against F. oxysporum based on the application of volatile organic compounds produced by Bacillus bacteria.
• The application of selected species of Bacillus bacteria can be a method of biological protection of plants against the fungus F. oxysporum, especially in greenhouses or tunnels where plants are grown intensively.