Bacillus velezensis LT-22 Volatiles for the Biocontrol of Phytophthora capsici: Antifungal Action and Underlying Mechanisms

Abstract

Phytophthora capsici, an invasive oomycete pathogen causing blight in crops like cucurbits, tomatoes, and peppers, has led to significant economic losses. Due to the limitations of conventional control methods, research has focused on effective and eco-friendly biocontrol alternatives. The bacterial strain LT-22, identified as Bacillus velezensis, exhibits broad-spectrum antifungal activity and plant growth-promoting potential. This study investigated the biocontrol efficacy of volatile organic compounds (VOCs) from strain LT-22. LT-22 VOCs strongly inhibited P. capsici PC153 growth in vitro and reduced disease symptoms in pepper fruits. Microscopic observations showed that LT-22 VOCs caused significant morphological changes in P. capsici PC153 mycelium by disrupting cell wall and membrane integrity. The treatment increased malondialdehyde (MDA) levels and reduced cellulose content, further indicating that LT-22 VOCs compromise cell integrity to exert antifungal effects. Furthermore, 2-methylbutanoic acid was identified as the primary antifungal VOC, with an EC₅₀ value of 0.095 µL/mL against P. capsici PC153, lower than the reported plant-derived compounds. Together, these results indicate that strain LT-22 and its VOCs, especially 2-methylbutanoic acid, have significant potential as an effective biocontrol agent for managing Phytophthora blight.

1. Introduction

Peppers (Capsicum annuum L.), which are nutrient-rich and economically valuable, are widely cultivated globally [1,2]. However, they are highly susceptible to Phytophthora blight caused by the invasive oomycete pathogen Phytophthora capsici [3], which causes crown and fruit rot, foliar blight, stem necrosis, wilting, and water-soaked spots on fruit [4]. P. capsici also threatens crops such as tomato, eggplant, cucumber, and melons, resulting in annual economic losses of USD 1–10 billion [5,6]. Current control measures rely on chemical fungicides, such as mefenoxam and metalaxyl, which pose risks of resistance, environmental toxicity, and chemical pesticide residues [7,8]. Therefore, identifying effective and safe novel fungicides for managing P. capsici is crucial.

Biopesticides are seen as a sustainable alternative to chemical pesticides because of their minimal environmental impact and lower resistance risk [9]. Bacillus species, as antagonistic microorganisms, are widely used biopesticides against pathogenic fungi due to niche competition, antifungal compound production, and plant systemic resistance induction [8,10,11]. Among them, volatile organic compounds (VOCs) produced by Bacillus species act as key antifungal agents by inhibiting fungal growth without direct contact. For example, B. subtilis CL2 releases 2,3-butanedione and 3-methylbutyric acid, which deform and shrink the mycelium of plant pathogenic fungi, reducing disease severity [12]. Additionally, 2,3-butanedione inhibits spore germination of Aspergillus flavus, decreasing peanut infestation [13]. Similarly, VOCs from B. subtilis 0618A inhibit mycelial growth and sclerotia germination of Sclerotium rolfsii, improving soil health during biocontrol of peanut southern blight [9]. These findings highlight the potential of VOCs in biological control, acting over distances without direct contact with plants or pathogens [14]. Moreover, VOCs promote plant growth, enhance stress resistance, and provide pest resistance, demonstrating diverse benefits in agriculture [15].

Bacillus velezensis, a representative species within the Bacillus genus, has been widely studied for its potential in developing biocontrol agents in the field of plant disease control [16,17,18,19]. It has been reported that B. velezensis soluble metabolites, such as lipopeptide antibiotics and peptides, can disrupt the cell membrane integrity of pathogenic fungi and thus be used for the management of plant diseases [20,21]. Recent research has focused on this bacterium and its soluble metabolites. However, there is limited reporting on the antifungal activity of B. velezensis VOCs at present.

In this study, a B. velezensis strain LT-22 was isolated from soil, and its VOCs exhibited strong inhibitory activity against P. capsici PC153 in vivo and in vitro. The study aims to systematically evaluate the effects of strain LT-22 on mycelial morphology, cellular ultrastructure, and cell wall and membrane integrity of P. capsici PC153. Meanwhile, the key effective components of the VOCs were analyzed and identified, and their antifungal efficacy against P. capsici PC153 was assessed in vitro. Although Bacillus-derived VOCs are known to combat plant pathogens, studies specifically on B. velezensis controlling P. capsici are first reported. This research provides a new case for the biological control of Phytophthora blight and offers potential substances for eco-friendly fungicide development.

2. Materials and Methods

2.1. The Reagents and Pathogenic Fungi

The Oxford cups were purchased from Shanghai Jingyang Electronic Technology Development Co., Ltd., Shanghai, China. 2-Nonanone was purchased from MACKIN, Beijing, China. 6-Methyl-2-heptanone was purchased from Adamas, Beijing, China. 2-Dodecanone was purchased from J&K Scientific LLC., San Jose, CA, USA. Dimethyl trisulfide was purchased from Beijing Jinming Biotechnology Co., Ltd., Beijing, China. 2-Methylbutanoic acid was purchased from MREDA, Beijing, China. Tetradecane, decanal, 2,5-dimethylpyrazine, 2-methylpyridine, nonanal, N-methylaniline and benzaldehyde were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China. Other drugs were purchased from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China.

P. capsici PC153, Fusarium graminearum PH-1, Colletotrichum gloeosporioides BW22, Botryosphaeria dothidea TD14, and Magnaporthe oryzae Guy11 were obtained from Institute of Plant Protection, Jiangsu Key Laboratory for Food Quality (Nanjing, China). The pathogens were cultured on potato dextrose agar (PDA) medium at 30 °C for 5–7 days.

2.2. Isolation, Screening, and Identification of Strain LT-22

Antagonistic microorganisms with the ability to inhibit P. capsici were isolated from soil samples collected at Changshu Linglong Farm (Changshu, China) according to a previously described method [9]. A purified single colony of antagonistic bacterial strain LT-22 was obtained, and its morphology was examined microscopically. Gram staining, the Voges–Proskauer test, citrate utilization, starch hydrolysis, the indole test, the methyl red test, and sugar metabolism tests were performed to evaluate physiological and biochemical characteristics. Strain LT-22 was identified through 16S rDNA gene sequencing. Bacterial universal primers 27F and 1492R were used to amplify partial 16S rDNA gene sequences [16]. PCR products were analyzed on a 1% agarose gel and sequenced by Genewiz Biotechnology Co., Ltd., Suzhou, China. Sequences were compared against the GenBank database using NCBI BLAST. against NCBI data, and phylogenetic trees were constructed using MEGA 6.0. Strain LT-22 was deposited in the China Center for Type Culture Collection (CCTCC No. M2023779).

2.3. Assessment of Antifungal Activity and Plant Growth-Promoting Properties of Strain LT-22

The antifungal activity of strain LT-22 was assessed using the Oxford cup method [17]. Mycelial plug (8 mm diameter) of P. capsici PC153, F. graminearum PH-1, C. gloeosporioides BW22, B. dothidea TD14, and M. oryzae Guy11 were placed at the center of PDA plates, respectively, and 50 µL of LT-22 fermentation broth was added 2.5 cm away from each plug. Plates were incubated at 30 °C and tested in triplicate.

Cellulase, amylase, and protease activities were assessed using established methods [17,22]. Siderophore production, phosphate solubilization, and nitrogen fixation were evaluated in specific detection media.

2.4. Effect of the Strain LT-22 VOCs on P. capsici PC153 Growth

The antifungal activity of the VOCs produced by strain LT-22 was evaluated using a dual-culture method in sealed Petri dishes [23]. A 9 cm blank Petri dish was filled with 15 mL of PDA or Luria-Bertani (LB) solid medium. A 100 µL aliquot of LT-22 bacterial suspension at a concentration of 10⁷ CFU/mL was evenly spread on an LB plate, with sterile water used as a control. Concurrently, an 8 mm-diameter agar plug containing P. capsici PC153 was placed at the center of a PDA plate. The PDA plate inoculated with the P. capsici plug was then inverted and positioned over the LB agar plate seeded with the bacterial suspension. Both plates were sealed together with Parafilm. Each experiment was performed in triplicate and incubated at 30 °C for 5–7 days to observe the antagonistic effect and calculate the mycelial growth inhibition rate. Inhibition rate (%) = [(diameter of control colony − diameter of treated colony)/(diameter of control colony − initial diameter of pathogen)] × 100.

2.5. Effects of VOCs from Strain LT-22 Against P. capsici PC153 in Pepper Fruits

The control efficacy of VOCs produced by strain LT-22 against P. capsici PC153 in pepper fruits was evaluated as previously described [19,24,25]. Six-millimeter plugs were taken from the edge of 5-day-old PDA cultures and stored for later use. Healthy cayenne pepper fruits were surface-sterilized via immersion in 75% ethanol for 30 s, rinsed three times with sterile water, treated with 2% sodium hypochlorite for 5 min, and rinsed again three times. A cross-shaped wound was made on the underside of each fruit using a sterilized needle, and a fungal plug was placed at the center of the wound. The LT-22 strain was evenly spread on LB agar and placed at the top of a transparent plastic container, while pepper fruits were positioned at the bottom to expose them to VOCs. All containers were sealed with parafilm to prevent VOCs leakage. Lesion diameters were measured after incubation at 30 °C for 3 and 7 days. Disease inhibition rate (%) = [(lesion diameter of control groups − lesion diameter of treated groups)/lesion diameter of control groups] × 100.

2.6. Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) Observations

The morphological and ultrastructural changes of P. capsici PC153 in control groups or those exposed to VOCs from strain LT-22, were examined using SEM and TEM. P. capsici PC153 was co-cultured with VOCs from strain LT-22 for 5 d at 30 °C using the face-to-face Petri dish method. Samples were fixed in 2.5% glutaraldehyde for 4 h, rinsed 5–6 times with PBS buffer (pH 6.8) for 20 min each, then post-fixed in 1% osmium tetroxide and rinsed again with PBS buffer (pH 6.8) for 90 min, 5–6 times. Dehydration was performed with alcohol concentrations of 30%, 50%, 70%, and 100% for 20–30 min per step. The samples were vacuum-dried for 2 h before observation by scanning electron microscopy (Regulus 8100, Hitachi, Tokyo, Japan).

TEM observations were conducted using the method described by Li et al. (2023) [26]. The dehydrated samples were subsequently transferred to absolute acetone for further processing. Following this, the samples underwent treatment with varying ratios of Spurr resin and acetone mixtures. Then, the samples were immersed in Spurr resin for an extended period (overnight) to ensure thorough infiltration. Ultrathin sections were subsequently prepared and examined using transmission electron microscopy (HT-7700, Hitachi, Tokyo, Japan).

2.7. Effect of LT-22 VOCs on Cell Wall and Membrane Integrity of P. capsici PC153

The effect of VOCs produced by strain LT-22 on the cell wall and membrane integrity of P. capsici PC153 was evaluated using a modified version of the method described by Li et al. (2023) [26]. The P. capsici PC153 mycelial disc (6 mm in diameter) was placed in the center of the PDA plate, which was incubated at 30 °C for 3 days. Subsequently, a PDA plate was inverted and placed over LB agar plates on which strain LT-22 has been evenly spread at 10⁷ CFU/mL, and then incubated in the dark for 24 h. After treatment, the mycelia were collected and stained with Calcofluor White (CFW) and propidium iodide (PI) at room temperature in the dark for 20 min. Observations and imaging were conducted using a laser scanning confocal microscope (Olympus FV3000, Olympus Corporation, Tokyo, Japan). All treatments were replicated three times.

2.8. Determination of Relative Conductivity, MDA, and Cellulose Content

The mycelial samples of P. capsici PC153 were prepared according to the above-described method. The same amount of mycelium was suspended in sterile distilled water, and electrical conductivity was measured at 0, 10, 20, 40, 80, 100, and 120 min using a conductivity meter (DDS-11A, Shanghai Leici Instrument Inc., Shanghai, China). After the final measurement, samples were boiled, and a final conductivity reading was recorded. Relative conductivity (%) = (conductivity/final conductivity) × 100 [27].

Mycelia were cultured as described above. The collected mycelium was ground with liquid nitrogen. The malondialdehyde (MDA) and cellulose contents of P. capsici PC153 mycelia were measured using MDA (BC0025, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) and cellulose (BC4280, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) detection kits.

2.9. Identification of the Volatile Compounds from Strain LT-22

To identify the VOCs released by strain LT-22, GC/MS analysis was conducted using SPME for extraction. Specifically, 5 mL of the sample in a headspace vial was equilibrated at 40 °C for 1 min and extracted at 40 °C for 20 min. The SPME fibers were then introduced into the injection port of the GC-MS-TQ8040 Ultra (Shimadzu, Kyoto, Japan), where VOCs were thermally desorbed at 250 °C for 5 min. Gas chromatographic separation was performed with helium as the carrier gas (1 mL/min) on a DB-WAX column (30 m × 0.25 mm × 0.25 µm). The oven temperature started at 40 °C for 2 min, increased to 240 °C at a rate of 5 °C/min, and was held at 240 °C for 5 min. The mass spectrometer operated in EI mode (70 eV) with a source temperature of 230 °C, scanning from 35 to 450 m/z.

2.10. Effect of Pure VOCs on P. capsici PC153 Growth

The antifungal activities of 11 VOCs against P. capsici PC153 were initially evaluated using a modified protocol adapted from Cheng et al. (2025) [9]. A 9 cm blank Petri dish was filled with 10 mL of water-agar (0.18% w/v) medium. For the liquid VOCs, 50 µL of the standards was evenly coated onto the water-agar plate. Subsequently, a 6 mm diameter agar plug containing P. capsici PC153 was placed at the center of a fresh PDA medium. Then, the two PDA plates were sealed together and incubated at 30 °C. Water–agar plates without added VOCs served as controls. After 5 days, the mycelial growth inhibition rate was determined as described in Section 2.4. The experiment was repeated three times.

2.11. Effect of 2-Methylbutanoic Acid on P. capsici PC153 Growth

Acknowledged as a food additive in numerous countries, 2-Methylbutanoic acid has been confirmed to effectively inhibit the growth of Ceratocystis fimbriata [28]. The effect of 2-methylbutanoic acid on P. capsici PC153 growth was further studied. The treatment and cultivation were the same as described in Section 2.10, with the 50 µL of the standards being replaced by different volumes (3 µL, 6 µL, 12 µL, 24 µL, and 48 µL) of 2-methylbutanoic acid. For the control group, no 2-methylbutanoic acid was applied. Each experiment was replicated three times. The inhibition rate was calculated as described in Section 2.4. The median effective concentration (EC₅₀) value was determined using GraphPad Prism 10.0 by plotting the inhibition rate against the logarithm (base 10) of the 2-methylbutanoic acid concentration [6]. The void volume in the central cavity of the sealed double-dish system was 216 mL.

To test the broad-spectrum antifungal activity of 2-methylbutanoic acid on plant-pathogenic fungi, F. graminearum PH-1, C. gloeosporioides BW22, B. dothidea TD14, and M. oryzae Guy11 were used as test strains. The treatment and cultivation were the same as described in Section 2.10, with P. capsici PC153 being replaced by the above-mentioned four plant-pathogenic fungi. Each experiment was replicated three times.

3. Results

3.1. Broad-Spectrum Antifungal Activity and Plant Growth-Promoting Properties of Strain LT-22

The strain designated LT-22 was isolated, purified, and selected through antagonistic screening. It exhibited broad-spectrum antifungal activity, suppressing the growth of various phytopathogens, such as P. capsici PC153, B. dothidea TD14, M. oryzae Guy11, C. gloeosporioides BW22, and F. graminearum PH-1 (Figure 1A). Furthermore, the plant-growth-promoting properties of strain LT-22 were evaluated in vitro. Strain LT-22 was found to produce lytic enzymes, such as cellulase, protease, and amylase. Additionally, it exhibited the capacity to solubilize phosphate, release potassium, mobilize chelated iron, and perform nitrogen fixation ability under in vitro conditions (Figure 1B).

3.2. Identification of Strain LT-22

On LB medium, strain LT-22 formed semi-transparent, smooth, creamy-white, circular colonies with well-defined edges. As the culture progressed to later stages, the colony surface gradually developed wrinkles and exhibited a viscous consistency (Figure 2A). Morphological observations revealed that the cells were short rods and Gram-positive, as indicated by purple staining (Figure 2B). The strain utilized glucose, maltose, lactose, fructose, and xylose as sole carbon sources. It was positive for the VP reaction, citrate utilization, indole production, gelatin liquefaction, and nitrate reduction, but negative for H₂S production and methyl red test. Moreover, 16S rDNA gene sequencing identified the strain as B. velezensis, with 99% sequence homology to B. velezensis YH21 (Figure 2C).

3.3. Antifungal Activity of LT-22 VOCs Against P. capsici PC153 In Vitro and In Vivo

The double-Petri dish method was employed to assess the antifungal activity of VOCs from strain LT-22 against P. capsici PC153 in vitro (Figure 3A). The LT-22 VOCs inhibited P. capsici PC153 mycelial growth by 60.78% (Figure 3B,C). Results from in vivo pepper fruit inoculation experiments showed that the LT-22 VOCs effectively suppressed disease development and enhanced biocontrol. Three days post-inoculation, symptom development and pathogen spread were clearly evident in control fruits. By day seven, untreated control fruits exhibited severe lesions, while treated fruits showed significantly less disease progression and smaller lesions (Figure 4A,B). The disease inhibition rates in the LT-22 VOCs-treated group were 60.29% and 75.36% at three days and seven days post-inoculation, respectively (Figure 4C). Collectively, these findings indicate that LT-22 VOCs represent a promising and effective alternative strategy for controlling P. capsici-induced diseases in pepper fruits.

3.4. Effects of LT-22 VOCs on the Morphology and Ultrastructure of P. capsici PC153

Morphology in P. capsici PC153 cells exposed to LT-22 VOCs was observed via SEM. In the control group, P. capsici PC153 hyphae exhibited a smooth and uniform surface. In contrast, after treatment with LT-22 VOCs, the mycelia exhibited abnormalities in surface morphology, including irregular surfaces, pronounced folds, swelling, and thickening (Figure 5A). Furthermore, mycelial ultrastructure variations between untreated samples and those treated with LT-22 VOCs were investigated using TEM. The results indicated that the mycelia of normally growing P. capsici PC153 were intact, whereas treatment with VOCs produced by strain LT-22 severely disrupted the ultrastructure of P. capsici PC153 hyphae. In the treated samples, the cytoplasmic membrane separated from the cell wall, organelles ruptured, large vacuoles formed within the cytoplasm, and most of the plasma membrane dissolved, with partial degradation of the cell walls (Figure 5B). Taken together, these results demonstrate that LT-22 VOCs not only inhibit hyphal expansion but also compromise the structural integrity of P. capsici PC153 hyphae, thereby impeding its growth.

3.5. Impact of LT-22 VOCs on the Cell Wall of P. capsici PC153

As a non-specific fluorescent brightener, CFW binds to cellulose and chitin in the cell wall, emitting intense blue fluorescence, which facilitates the visualization of alterations in cell wall architecture [29]. In Figure 6A, treatment with LT-22 VOCs markedly altered cellulose distribution in P. capsici PC153 mycelia, as indicated by a pronounced reduction in blue fluorescence intensity relative to the control. Given that cellulose is the primary structural component of the oomycete cell wall [27], cellulose content was conducted to further assess the effect of LT-22 VOCs. Exposure to LT-22 VOCs resulted in a 57.09% reduction in cellulose levels in P. capsici PC153 compared to the control (Figure 6B). These findings indicate that LT-22 VOCs reduce cellulose biosynthesis or deposition in P. capsici PC153, potentially compromising the mechanical stability and integrity of the cell wall.

3.6. Effect of LT-22 VOCs on the Cell Membrane of P. capsici PC153

PI is a fluorescent dye that easily permeates dead cell membranes, making it useful for distinguishing between dead and living cells. As illustrated in Figure 7A, the mycelia of P. capsici PC153 in the control group exhibited no noticeable fluorescence, likely because PI cannot penetrate cells with intact membranes. In contrast, after exposure to LT-22 VOCs, the mycelia displayed strong red fluorescence. The impact of LT-22 VOCs on the cell membrane permeability of P. capsici PC153 was evaluated by assessing the extracellular conductivity and MDA levels. Compared with the control, LT-22 VOCs treatments resulted in a leakage of mycelial electrolytes in P. capsici PC153, with higher electrical conductivity (Figure 7B). MDA is a typical product of lipid oxidation and serves as a key indicator of cell membrane damage. Exposure to LT-22 VOCs significantly increased MDA content by 1.96-fold relative to the control group (Figure 7C). These findings indicate that LT-22 VOCs disrupt cell membrane permeability and compromise the structural integrity of P. capsici PC153.

3.7. Identification of of VOCs Produced by Strain LT-22

The VOCs emitted by strain LT-22 were comprehensively analyzed via SPME-GC/MS. A total of 24 VOCs were identified and listed in

Table 1. Volatile organic compounds species.

Chemicals	Remote Time (min)	Peak Area	Relative Percentage (%)
5-methyl-5-propyl-nonane	5.696	116,962	0.26
5-methyl-2-hexanone	7.108	88,050	0.20
2-methylpyridine	9.179	908,617	2.03
6-methyl-2-heptanone	9.501	2,802,991	6.26
5-methyl-2-heptanone	9.915	1,519,833	3.40
methyl-pyrazine	10.24	800,840	1.79
octanal	10.648	225,688	0.50
tridecane	10.821	129,246	0.29
2,5-dimethylpyrazine	11.468	3,255,639	7.28
6-methyl-5-hepten-2-one	11.767	467,212	1.04
dimethyl trisulfide	12.556	675,814	1.51
2-nonanone	12.826	696,450	1.56
nonanal	12.899	1,951,281	4.36
tetradecane	13.086	1,097,432	2.45
2-methyl-3-isopropylpyrazine	13.293	1,279,440	2.86
2-ethyl-1-hexanol	14.814	530,838	1.19
decanal	14.948	1,082,734	2.42
benzaldehyde	15.385	12,123,912	27.10
2-methylpropanoic acid	16.335	2,497,806	5.58
2-dodecanone	18.889	1,394,826	3.12
2-methylbutanoic acid	19.042	7,384,587	16.50
N-methylaniline	20.556	2,343,586	5.24
aniline	21.472	304,758	0.68
4-(1,1,3,3-tetramethylbutyl)phenol	39.649	1,066,227	2.38

. Among these, nine compounds, each with a relative area greater than 3.0%, emerged as significant contributors to the VOC profile. These included benzaldehyde (27.10%), 2-methylbutanoic acid (16.50%), 2,5-dimethylpyrazine (7.28%), 6-methyl-2-heptanone (6.26%), 2-methylpropanoic acid (5.58%), N-methylaniline (5.24%), nonanal (4.36%), 5-methyl-2-heptanone (3.40%), and 2-dodecanone (3.12%). Furthermore, five compounds with area percentages between 2.0% and 3.0% were identified, including 2-methyl-3-isopropylpyrazine (2.86%), tetradecane (2.45%), decanal (2.42%), 4-(1,1,3,3-tetramethylbutyl)phenol (2.38%), and 2-methylpyridine (2.03%).

3.8. In Vitro Antagonistic Effect of Pure VOCs Against P. capsici PC153 Growth

To assess the effects of VOCs produced by strain LT-22 on P. capsici PC153, 11 individual VOCs were tested in vitro (Figure 8A). The results revealed that seven of these compounds—namely, 2-methylbutanoic acid, dimethyl trisulfide, nonanal, 2-nonanone, 6-methyl-2-heptanone, N-methylaniline, and benzaldehyde—at a concentration of 50 µL per plate, completely inhibited the growth of P. capsici PC153. Furthermore, 2-methylpyridine, 2,5-dimethylpyrazine, and 2-dodecanone exhibited significant inhibitory effects on mycelial growth, with inhibition rates of 98.25%, 61.40%, and 55.56%, respectively (Figure 8B). The remaining compounds showed weak or negligible antagonistic activity against P. capsici PC153.

3.9. Antifungal Spectrum of 2-Methylbutanoic Acid

Among the LT-22 VOCs, 2-methylbutanoic acid as the main composition, showed excellent antibacterial activity against P. capsici PC153. The inhibitory potential activities of 2-methylbutanoic acid against P. capsici PC153 at varying concentrations were further explored. At the concentration of 3, 6, 12, and 24 µL per plate, the growth inhibition rates of P. capsici PC153 were recorded as 12.42%, 26.14%, 52.94% and 83.01%, respectively (Figure 9A). Remarkably, when the concentration increased to 48 µL per plate, 2-methylbutanoic acid achieved complete inhibition of P. capsici PC153 growth. The inhibitory effects were dose dependent. The EC₅₀ value of 2-methylbutanoic acid against P. capsici PC153 was determined to be 0.095 µL/mL (20.54 µL per plate) (Figure 9B). Furthermore, the VOC 2-methylbutanoic acid exhibited broad-spectrum antifungal activity against plant pathogens. At a dosage of 50 µL per plate, it completely inhibited the mycelial growth of F. graminearum PH-1 and C. gloeosporioides BW22, and significantly suppressed that of M. oryzae Guy11 and B. doth idea TD14 (Figure 9C), with inhibition rates of 96.75% and 74.89%, respectively.

4. Discussion

P. capsici poses a substantial threat to agricultural productivity, resulting in significant economic losses [2]. The extensive reliance on chemical fungicides for controlling P. capsici has led to several critical issues, including the development of resistance, pesticide residues in vegetables, and environmental contamination [7,8]. Therefore, investigating biological control agents, such as Bacillus species that exhibit antagonistic activity against P. capsici, represents a promising strategy for managing diseases caused by this pathogen. In this study, a bacterial strain designated LT-22 was isolated from the soil of Changshu Linglong Farm and evaluated for its antagonistic activity against P. capsici PC153. Based on morphological characteristics, cultural properties, and 16S rDNA sequence analysis, strain LT-22 was identified as Bacillus velezensis. Recent studies have highlighted Bacillus spp. as promising biocontrol agents. Strains such as B. subtilis CL2, ZD01, and CF-3 have demonstrated significant antagonistic activities against various plant pathogens in vitro, including Fusarium arcuatisporum, Alternaria solani, and Monilinia fructicola, respectively [12,30,31]. Moreover, B. velezensis L1 and BV01 have been shown to inhibit the mycelial growth of multiple phytopathogens, such as Alternaria iridiaustralis, Bipolaris sorokiniana, Botrytis cinerea, and Colletotrichum capsici [32,33]. However, research on the antifungal potential of Bacillus species against P. capsici is currently limited.

Strain LT-22 has attracted considerable attention due to its strong antifungal activity against P. capsici PC153 as well as a broad spectrum of other pathogenic fungal species. Moreover, our research indicates that LT-22 is capable of producing extracellular hydrolytic enzymes and biocontrol-related regulatory substances. Numerous antagonistic bacterial strains can produce and secrete a variety of extracellular hydrolytic enzymes, which play a crucial role in degrading fungal cell walls [17,34]. Enzymes such as cellulase and proteases play a key role in controlling agricultural diseases by degrading essential components of fungal cell walls, including glucan and glycoproteins. Understanding the mechanisms of pathogen inhibition is crucial for selecting effective biocontrol agents. Strain LT-22 shows strong antagonistic activity against various pathogenic fungi, significantly suppressing mycelial growth. Its production of biocontrol-related enzymes was therefore investigated, confirming enzymatic activities that support its potential as a promising biological control agent.

Several studies have demonstrated that VOCs produced by Bacillus spp. possess significant antifungal activity [35,36]. The small molecular size of VOCs allows for long-distance dispersion, enabling indirect interactions between organisms and offering a novel avenue for biological control [28]. B. subtilis 0618A produces VOCs that effectively suppress S. rolfsii and enhance soil health, thereby contributing to the biological control of peanut southern blight [9]. However, it remains unclear whether it can be effectively utilized to control P. capsici, the causal agent of Phytophthora blight. This study demonstrates that LT-22 VOCs significantly inhibit the mycelial growth of P. capsici PC153, with fumigation exhibiting greater inhibitory efficacy than direct contact. The efficacy of a microorganism as a biocontrol agent depends on its performance both in vivo and in vitro. In vivo experiments confirmed that VOCs reduce disease incidence on detached pepper fruits by suppressing the growth of P. capsici PC153. This is the first report demonstrating that VOCs from B. velezensis exhibit biocontrol activity against this pathogen, highlighting their potential as a novel biological control agent. In this study, we focus on a specific strain of P. capsici. Future research could further evaluate the biocontrol efficacy of strain LT-22 by testing it against a larger number of wild-type P. capsici strains.

Some biocontrol bacterial strains inhibit fungi by targeting cell membranes and walls, increasing permeability and weakening structural integrity, leading to leakage of cellular contents, disrupted homeostasis, and cell death [18,19,22]. Given the antifungal activity of LT-22 VOCs against P. capsici PC153, this study investigated the underlying mechanisms. Exposure to LT-22 VOCs significantly compromised cell wall integrity, as shown by reduced cellulose content and weaker CFW staining. Cellulose is a key structural component of oomycete cell walls [27]. Its reduction likely explains the observed structural disruption. Similar effects were reported in S. rolfsii exposed to B. subtilis 0618A VOCs [9] and in P. capsici treated with rhein [37]. Additionally, LT-22 VOCs severely damaged cell membrane integrity in P. capsici PC153 mycelia, impairing function—evidenced by increased PI staining and elevated MDA levels, indicating lipid peroxidation and oxidative damage. These findings align with those of Cheng et al. (2025) [9]. SEM and TEM analyses further revealed significant alterations in mycelial morphology and ultrastructure after VOC exposure. These changes may result from increased membrane permeability, causing efflux of small molecules and heightened lipid peroxidation, ultimately promoting cell death. Thus, LT-22 VOCs inhibit P. capsici PC153 primarily by disrupting plasma membrane function and compromising cell wall structure, leading to cell death. This dual-targeting mechanism reduces the likelihood of resistance development and offers strategic advantages in combating resistance to chemical fungicides. However, the specific target of LT-22 VOCs against P. capsici PC153 requires further experimental validation.

To identify the main antifungal compound in LT-22 VOCs that is active against P. capsici PC153, SPME coupled with GC-MS was used for analysis. A total of 24 VOCs were identified, and this study focuses on those with a relative abundance above 2.0% to assess their impact on P. capsici PC153. Compounds including dimethyl trisulfide, nonanal, 2-nonanone, 6-methyl-2-heptanone, N-methylaniline, benzaldehyde, and 2-methylbutanoic acid showed strong in vitro antifungal activity against P. capsici PC153, consistent with previous findings on their inhibitory effects against other fungal pathogens [3,13,38]. The detection of these compounds suggests that strain LT-22 has strong potential as a biological control agent.

As a food additive recognized for its high biosafety, 2-Methylbutanoic acid exhibits potential antifungal activity [28,39,40]. At present, many plant-derived natural compounds have shown strong antifungal activity and are considered potential agents for managing P. capsici. For example, Euphorbia factor L3 inhibits P. capsici with an EC₅₀ of 2.96 µg/mL [27], while 10-Deacetyl-bacatin III, a taxane diterpenoid, shows even greater potency at 1.46 µg/mL [41]. Other plant metabolites—citral, carvacrol, and trans-2-decenal—inhibit mycelial growth with IC₅₀ values of 17.33, 5.67, and 8.66 µg/mL, respectively [42]. Cuminic acid from Cuminum cyminum L. seeds has an average EC₅₀ of 14.54 µg/mL [43], and 1,6-O,O-diacetylbritannilactone (ABLOO) from Inula britannica shows an EC₅₀ of 93.18 µg/mL [44]. In this study, 2-methylbutanoic acid from strain LT22 exhibited strong antifungal activity against P. capsici PC153 with an EC₅₀ of 0.095 µL/mL, lower than that of all the above-mentioned plant-derived compounds. Its low EC₅₀ supports practical application, and its volatility enables fumigation use, enhancing its utility. In addition, 2-methylbutanoic acid showed significant antifungal activity against F. graminearum PH-1, C. gloeosporioides BW22, B. dothidea TD14, and M. oryzae Guy11, which cause diseases in fruits, vegetables, and crops. These results further indicate that 2-methylbutanoic acid serves as an effective antifungal agent for biocontrol. However, the underlying mechanism of 2-methylbutanoic acid against P. capsici needs to be further investigated in future studies.

5. Conclusions

The study shows that Bacillus velezensis LT-22 exhibits broad-spectrum antifungal activity and plant growth-promoting potential. The VOCs emitted by strain LT-22 effectively inhibited P. capsici PC153 in vivo and in vitro. The antifungal activity of these VOCs against P. capsici PC153 likely stems from direct effects on the cell membrane and wall, leading to increased membrane permeability, higher MDA levels, loss of cell wall integrity, and reduced cellulose content. Future studies could integrate multi-omics approaches to more comprehensively explore the underlying molecular mechanisms. In addition, 2-methylbutanoic acid is the main antifungal VOC produced by strain LT-22. It shows strong efficacy and a lower EC₅₀ value against P. capsici PC153, suggesting its potential as a novel biofungicide for Phytophthora blight. These findings provide valuable resources for the biological control of P. capsici, support eco-friendly biocontrol strategies, and help reduce chemical fungicide residues in agriculture, enhancing food supply chain safety.