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Article

Biocontrol and Growth-Promoting Potential of Antagonistic Strain YL84 Against Verticillium dahliae

by
Yuxin Tang
1,2,3,
Qinyuan Xue
1,2,3,
Jiahui Yu
1,2,3,
Zhen Zhang
1,2,3,
Zhe Wang
1,2,3,
Lan Wang
1,2,3,* and
Hongzu Feng
1,2,3,*
1
Laboratory of Green Pest Management, College of Agriculture, Tarim University, 705 Hongqiao South Road, Alar 843300, China
2
Key Laboratory of Integrated Pest Management in Southern Xinjiang, 1490 East Tarim Avenue, Alar 843300, China
3
National-Local Joint Engineering Laboratory for High-Efficiency Cultivation and Deep Processing of Characteristic Fruit Trees in Southern Xinjiang, 1490 East Tarim Avenue, Alar 843300, China
*
Authors to whom correspondence should be addressed.
Agronomy 2025, 15(8), 1997; https://doi.org/10.3390/agronomy15081997
Submission received: 11 July 2025 / Revised: 15 August 2025 / Accepted: 18 August 2025 / Published: 20 August 2025
(This article belongs to the Section Pest and Disease Management)
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Abstract

Cotton Verticillium wilt is a disease that significantly impacts the cotton industry, severely affecting cotton quality and the economic well-being of farmers. Bacillus atrophaeus YL84 is a biocontrol bacterium with broad-spectrum antagonistic and growth-promoting characteristics, previously isolated by our laboratory. This study aimed to elucidate the antagonistic effects of sterilized fermentation filtrate from Bacillus atrophaeus YL84 on cotton Verticillium wilt pathogen Verticillium dahliae and its growth-promoting effects on cotton. The experiments were conducted in vitro and in vivo to assess these effects comprehensively. Using the dual culture method, it was found that Bacillus atrophaeus YL84 exhibited a high inhibition rate on mycelial growth of V. dahliae, with an inhibition rate of 84.11%. The undiluted YL84 sterilized fermentation filtrate and its 10% volume fraction dilution (fermentation filtrate diluted to 10%) exhibited inhibition rates of 80.25% and 72.16% for conidial germination and mycelial growth of V. dahliae, respectively. Scanning electron microscopy showed increased branching, swelling, and shortened internodes in the antagonized mycelia. Conductivity measurements revealed a significant enhancement caused by the YL84 filtrate, with conductivity increasing by 8.94 times compared to the control at a 250 μg/mL concentration. Similarly, protein leakage peaked at 9.47 times the control level at 250 μg/mL, demonstrating the filtrate’s potent impact on mycelial cell membrane permeability. The enzymatic activities of polygalacturonase (PG), cellulase (CL), and β-glucosidase (β-GC) were significantly reduced following treatment with YL84 sterilized fermentation filtrate, with reductions from control levels of 15.78, 10.11, and 5.01 U/mL to treatment levels of 11.81, 6.96, and 1.44 U/mL, respectively. Indoor pot experiments demonstrated that different concentrations of YL84 sterilized fermentation filtrate significantly suppressed the occurrence of cotton Verticillium wilt while promoting plant growth. Compared to the control group, application of 250 μg/mL YL84 sterilized fermentation filtrate resulted in a control efficacy of 66.69% for cotton Verticillium wilt, with increases in plant height, root length, fresh weight, and dry weight of 9.36–33.85%, 17.33–29.49%, 16.79–28.24%, and 25–58.33%, respectively. These findings underscore the potential of the YL84 filtrate as both a biocontrol agent and a promoter of cotton plant growth in agricultural settings. These results indicate that Bacillus atrophaeus YL84 sterilized fermentation filtrate possesses both disease-suppressing and growth-promoting activities, making it a promising candidate for development and use as a biocontrol agent and plant growth promoter.

1. Introduction

Cotton (Gossypium spp.) is an important economic and oilseed crop, with China being one of the largest importers of cotton globally and a major producer [1]. The Xinjiang region accounts for 91% of the total national cotton yield, thus holding an extremely vital position in the country’s cotton production [2]. In recent years, the increasing planted area and the ecological adaptation pressures from climate and environmental changes have exacerbated the severity of Verticillium wilt caused by Verticillium dahliae in cotton crops [3,4]. This disease can harm cotton throughout its growth period, infecting the host’s vascular tissues via the roots and inducing plant disease [5]. During infection, V. dahliae produces enzymes and other substances that disrupt the normal physiological functions of the cotton plant, ultimately leading to wilting and reduced yields. The infection process of V. dahliae is complex, with a broad host range, diverse transmission pathways, and intricate pathogenic mechanisms, thereby significantly impacting cotton production and posing a serious threat to the sustainable development of cotton. Consequently, controlling cotton Verticillium wilt is paramount for ensuring the sustainable development of the entire cotton industry chain [6].
Currently, the control of this disease primarily involves crop rotation, breeding of resistant varieties, chemical control, and biological control to reduce the occurrence of cotton Verticillium wilt. However, the practical application of crop rotation is limited by planting structure constraints. The breeding of resistant varieties has faced challenges such as long breeding cycles and the potential for cultivar degradation, which can result in the loss of disease resistance. Although chemical agents can control the disease to some extent, their prolonged use may lead to the development of pathogen resistance and adverse effects on the soil’s ecological environment [7,8,9].
Compared to chemical control, biological control reduced environmental pollution and harm to humans, offering an environmentally friendly and sustainable method of disease management [10]. In the biological control of cotton Verticillium wilt, the selection and application of biocontrol strains were crucial [11]. An ideal biocontrol strain was needed to exhibit potent antagonistic effects against V. dahliae and promote plant growth, thereby simultaneously suppressing the disease and enhancing the stress resistance and yield of cotton [12]. As the focus on sustainable agriculture continues to deepen, recent studies have highlighted the significant potential of biocontrol agents in reducing the use of chemical pesticides. They also demonstrate their notable advantages in maintaining ecological balance, making them an ideal choice for traditional agricultural systems [13]. For instance, research has shown that biocontrol agents can enhance plant defenses against pathogen invasion through systemic resistance (ISR) induction, while simultaneously promoting plant growth through nitrogen fixation, phosphate solubilization, and the synthesis of plant hormones [14]. Additionally, field trials conducted by Ta et al. [15] revealed that the application of the biocontrol agent Bacillus velezensis F9 significantly alleviated the symptoms of stunting and wilting in cucumber caused by Fusarium oxysporum f. sp. cucumerinum, effectively increasing the plant height, fresh weight, and dry weight. This further underscores the potential of biocontrol agents in disease management and crop yield improvement. Bacillus spp. have attracted significant attention from researchers due to their advantages such as ease of cultivation and storage, suitability for biotechnological manipulation, and broad-spectrum antimicrobial activity, making them widely used in agricultural production [16]. For example, Zhang et al. [17] isolated an endophytic Bacillus strain T6 from the Verticillium wilt-resistant cotton variety ‘Xin Hai 15’, which exhibited a mycelial growth inhibition rate of 63.79% against cotton Verticillium wilt. Song et al. [18] isolated the strain Bacillus subtilis KRS015 from cotton seeds; treatment with its fermentation filtrate significantly reduced the incidence of Verticillium wilt in cotton seedlings, with a disease reduction rate of 62%, and it also promoted cotton growth. Gao et al. [19] identified Bacillus axarquiensis TUBP1 from the soil, with its fermentation filtrate showing a control effectiveness of 43% against cotton Verticillium wilt, and application resulted in a 40.6% increase in cotton yield. Although several antagonistic strains have been identified, they cannot fully meet the needs for controlling cotton Verticillium wilt in practical production. Through a review of the literature, it has been found that studies on the use of Bacillus atrophaeus for controlling cotton Verticillium wilt are relatively scarce. Therefore, selecting this strain for research could aid in exploring its unique broad-spectrum antagonistic and growth-promoting properties, while offering new perspectives and possibilities for the biological control of cotton diseases.
Bacillus atrophaeus YL84 was previously isolated from the Korla fragrant pear tree leaves by our laboratory. Preliminary experiments have demonstrated that this strain exhibits broad-spectrum antibacterial activity and possesses specific growth-promoting effects. Based on these findings, the present study focused on YL84 to investigate its inhibitory effects on the growth of V. dahliae and its growth-promotion effects on cotton. This study also aimed to preliminarily elucidate the antibacterial mechanism of the sterilized fermentation filtrate of YL84, thereby providing a reference for the biological control of cotton Verticillium wilt.

2. Materials and Methods

2.1. Media and Test Strains

Media: The composition of the potato dextrose agar (PDA) medium includes 200 g of potato, 20 g of dextrose, 15 g of agar, and 1 L of distilled water. The potato dextrose broth (PDB) medium is the same as PDA but without agar. The solid Luria–Bertani (LB) medium is composed of 10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, 15 g of agar, and 1 L of distilled water; the LB liquid medium is the same as the solid LB medium but without agar. Bacillus atrophaeus YL84 was isolated in April 2024 from healthy leaves of the Korla fragrant pear tree collected in Aral City, First Division of the Xinjiang Production and Construction Corps. The strain is identified as Bacillus atrophaeus and is deposited in the China Center for Type Culture Collection (CCTCC) under the accession number CCTCC M 2025267, with its sequence submitted to GenBank under the accession number PV748128. YL84 is stored in glycerol tubes containing 30% glycerol at −80 °C. Before use, YL84 is streaked onto Luria–Bertani (LB) medium and incubated in the dark at 28 °C for 2 d. The Verticillium dahliae strain used in this study is registered under GenBank accession number EU835817.1. V. dahliae is stored at −80 °C in the Green Pest Control Laboratory. Before use, V. dahliae is purified on PDA medium and cultured in the dark at 28 °C for 7 d.

2.2. In Vitro Efficacy Assessment of Strain YL84 Against V. dahliae

2.2.1. In Vitro Inhibitory Effect of Strain YL84 on V. dahliae

The antagonistic strains were screened using the dual culture assay. A 5 mm diameter plug of V. dahliae mycelium was extracted using a punch and inoculated at the center of a PDA plate. Four purified YL84 strain plugs were inoculated at equidistant positions, 2.5 cm from the edge of the V. dahliae plug. Plates inoculated only with V. dahliae served as controls. The plates were incubated in the dark at 28 °C for 7 d. The colony diameters were measured using the cross method, and the inhibition rate was calculated accordingly.
Inhibition rate (%) = [(Colony diameter of the control − Colony diameter of the treatment)/Colony diameter of the control] × 100%.

2.2.2. Inhibitory Effect of Sterilized Fermentation Filtrate of Strain YL84 on Conidial Germination of V. dahliae

Following the method of Zhang et al. [17], V. dahliae was inoculated onto PDA plates and incubated at a constant temperature of 28 °C for 15 d. The conidia were collected by carefully washing the plates with sterilized water and suspending them in the eluent. The suspension was then filtered using a sterilized sieve or gauze to remove residual medium and mycelium. The conidial concentration of the resulting spore suspension was determined using a hemocytometer under an optical microscope. Based on the counts, the spore suspension was appropriately diluted or concentrated to prepare a conidial suspension with a concentration of 1.0 × 106 CFU/mL for use in subsequent experiments.
The antagonistic strain YL84 was purified by streaking on LB solid medium, and a small amount of the purified colony was inoculated into 100 mL of LB liquid medium. The culture was incubated at 37 °C and 120 rpm for 2 d to obtain the fermentation broth of the strain. This fermentation broth was transferred to a sterilized 50 mL centrifuge tube and centrifuged at 13,000 rpm for 10 min at 4 °C. The supernatant was filtered using a 0.22 μm pore size membrane to obtain the original sterilized fermentation filtrate of the antagonistic strain.
Preliminary experimental results demonstrated that 100, 150, and 250 µg/mL concentrations exhibited significant inhibitory effects on the target strains in vitro. Additionally, these concentration ranges are widely used in the literature to evaluate the impact of biocontrol agents on different strains [20,21]. Therefore, we selected these three concentrations for subsequent experiments to ensure scientific rigor and reliability of the results.
The antagonistic bacterial strain’s sterilized fermentation filtrate stock solution was diluted using LB liquid medium to 100 µg/mL, 150 µg/mL, and 250 µg/mL, respectively. A total of 100 µL of each dilution was transferred into sterilized 1.5 mL centrifuge tubes. Subsequently, 100 µL of a V. dahliae conidial suspension at a concentration of 1.0 × 106 CFU/mL was added to each of the aforementioned centrifuge tubes. A conidial suspension without antagonistic sterile fermentation filtrate served as the control. After incubation at a constant temperature of 28 °C for 5 d, conidia germination was observed under an optical microscope. The number of germinated conidia was recorded, and the spore germination inhibition rate was calculated. Five fields of view were observed for each treatment, with 20 conidia reviewed per field, and this was repeated three times.
Inhibition rate of spore germination (%) = (Number of germinated conidia in the control − Number of germinated conidia in the treatment)/Number of germinated conidia in the control × 100%.

2.2.3. Inhibitory Effect of Sterilized Fermentation Filtrate of Strain YL84 on Mycelial Growth of V. dahliae

Following the method of Wei et al. [22], the sterilized fermentation filtrate of the antagonistic strain obtained in Section 2.2.2 was mixed at volume fractions of 2%, 4%, 6%, 8%, and 10% into 250 mL Erlenmeyer flasks containing 100 mL of PDA medium, maintained at a temperature of 55 °C. After thorough mixing, the medium was poured into plates. A 5 mm diameter plug of V. dahliae mycelium was placed at the center of each plate. PDA plates inoculated only with V. dahliae and without the sterilized fermentation filtrate of the antagonistic strain served as controls. The plates were incubated at a temperature of 28 °C for 7 d. The colony diameters were measured to calculate the inhibition rate. Each treatment was repeated five times.
Inhibition rate (%) = [(Colony diameter of the control − Colony diameter of the treatment)/Colony diameter of the control] × 100%.

2.2.4. Inhibitory Effect of Sterilized Fermentation Filtrate of Strain YL84 on Verticillium Wilt in Cotton

Following the method of Chen et al. [1], sterilized nutrient soil was mixed with vermiculite at a ratio of 3:1 and packed into nutrient pots (15 cm × 5 cm). Cotton seeds were surface-sterilized by soaking them in 1% sodium hypochlorite for 15 min, followed by several rinses with sterilized water. The disinfected seeds were then placed on moist sterilized gauze for germination at room temperature. Once the seeds germinated, three seeds were sown per nutrient pot. Seedlings were thinned 10 d after sowing. When two true leaves emerged, 15 mL of YL84 sterilized fermentation filtrate at concentrations of 100 μg/mL, 150 μg/mL, and 250 μg/mL was applied near the roots of each cotton seedling using a pipette. Seven days later, the lateral roots were wounded using sterilized bamboo skewers, and 15 mL of V. dahliae conidial suspension (1.0 × 106 CFU/mL) was inoculated near the root area of each seedling using a pipette, with sterilized water serving as a blank control. Each treatment was applied to three nutrient pots, with three replicates for each treatment. After 21 d of seedling growth, the disease severity index of Verticillium wilt was assessed based on a five-grade classification scale described by Chen et al. [1], and the control efficacy was calculated.
Disease severity index = ∑(Number of diseased plants at each level × Level)/(Total number of plants surveyed × Maximum level) × 100%.
Control efficacy (%) = (Disease severity index of control − Disease severity index of treatment)/Disease severity index of control × 100%.

2.3. Inhibitory Mechanism of Sterilized Fermentation Filtrate from Strain YL84 Against V. dahliae

2.3.1. Effects of Sterilized Fermentation Filtrate from Strain YL84 on Mycelial Morphology of V. dahliae

Following the method of Lan et al. [23], the mycelia of V. dahliae treated with a 10% volume fraction of the YL84 sterilized fermentation filtrate from Section 2.2.3 were collected and centrifuged at 10,000 rpm for 5 min. The supernatant was discarded, and the pellet was resuspended in 1 mL of sterilized phosphate-buffered saline (PBS) at a concentration of 10 mmol/L, followed by pipetting and centrifugation. This process was repeated three times. The mycelia were then evenly spread on aluminum foil, air-dried, and immersed in 2.5% glutaraldehyde solution for fixation at 4 °C for 3 h. The samples on the foil were rinsed with 10 mmol/L PBS solution twice, each for 15 min. Subsequently, the samples were dehydrated in a graded ethanol series of 50%, 60%, 70%, 80%, 90%, 95%, and 100%, each for 10 min. Finally, the samples were subjected to two exchanges with 100% isoamyl acetate, each for 15 min. After gold sputter-coating, the samples were observed under a scanning electron microscope (SEM) at an accelerating voltage of 12 kV.

2.3.2. Effects of Sterilized Fermentation Filtrate from Strain YL84 on the Cell Membrane Permeability of Mycelia in V. dahliae

Conductivity Measurement [24]: A 1 mL suspension of V. dahliae conidia was added to 150 mL PDB medium and incubated at 28 °C with shaking at 160 rpm for 7 d. The mycelia were filtrated and washed three times with 0.2 mol/L PBS at pH 7.0 before resuspending in sterilized water. The YL84 sterilized fermentation filtrate was added to achieve final concentrations of 100 μg/mL, 150 μg/mL, and 250 μg/mL, with an equal volume of sterilized water used as the control. Conductivity measurements were taken at 30, 60, 90, 120, 180, and 240 min for the control and treatment groups using a conductivity meter. Each treatment was replicated three times.
Protein Leakage Measurement [24]: Samples were centrifuged at 10,000 rpm for 5 min at 4 °C at 2, 4, 6, and 8 h of treatment. The mycelia were removed by filtration, and the supernatant was collected. The absorbance of the supernatant was measured at 280 nm. Each treatment was replicated three times.

2.3.3. Effects of Sterilized Fermentation Filtrate from Strain YL84 on Cell Wall-Degrading Enzymes of V. dahliae

A 0.1 g sample of V. dahliae mycelia from Section 2.3.2 was placed in a sterilized 1.5 mL centrifuge tube, adding 1 mL of YL84 sterilized fermentation filtrate at a concentration of 250 μg/mL. An equal amount of sterilized water was added to the control treatment. The samples were incubated in the dark at 28 °C for 7 d. On the 7 d, the activities of polygalacturonase (PG), cellulase (CL), and β-glucosidase (β-GC) in both the control and treatment groups were measured using enzyme activity assay kits, all of which were purchased from Solarbio Bio-technology Co., Ltd. (Beijing, China). Each treatment was replicated three times.

2.4. Growth-Promoting Effects of Sterilized Fermentation Filtrate from Strain YL84 on Cotton Plants

Following the method of Li et al. [25], an appropriate amount of cotton seeds was soaked for 12 h in YL84 sterilized fermentation filtrate at concentrations of 100 μg/mL, 150 μg/mL, and 250 μg/mL, followed by rinsing three times with sterilized water to remove the filtrate. The seeds were then air-dried and reserved for use. Control group seeds underwent the same treatment with LB liquid medium. Each treatment was replicated three times. The treated seeds were sown in paper pots filled with a substrate composed of nutrient soil and vermiculite at a volume ratio of 2:1, with three seeds per pot. The pots were placed in a greenhouse maintained at 28 °C with a photoperiod of 8 h of light and 16 h of darkness. Emergence rates were recorded 7 d after sowing. At 21 d, seedlings were carefully extracted, roots washed and air-dried, and plant height, root length, and fresh weight measurements were taken. The seedlings were then dried to constant weight in an oven set at 80 °C, and the dry weight was recorded.

2.5. Statistical Analysis

All experiments were conducted with a minimum of three replicates. Graphs were generated using GraphPad Prism 8 software. Statistical analyses were performed using SPSS 27.0 software. Prior to conducting Duncan’s new multiple range test to assess the significance of differences, we verified key assumptions, including homogeneity of variances and normality of data distribution. The homogeneity of variances was tested using Levene’s test, and normality was assessed via the Shapiro–Wilk test. A significance level of p < 0.05 was considered statistically significant.

3. Results

3.1. In Vitro Inhibitory Effects of Strain YL84 on V. dahliae

The dual culture plate method assessed the in vitro inhibitory activity of strain YL84 against V. dahliae. After 7 d of incubation, the average colony diameter of V. dahliae in the control group was 51.5 mm (Figure 1A). In contrast, the average colony diameter in the YL84 treatment group was reduced to 8.18 mm. The in vitro inhibition rate of strain YL84 against V. dahliae was calculated to be 84.11% (Figure 1B), indicating that YL84 effectively inhibited the growth of V. dahliae.

3.2. Inhibitory Effect of Sterilized Fermentation Filtrate from Strain YL84 on the Germination of Conidia in V. dahliae

The results indicated that sterilized fermentation filtrate from strain YL84, at various dilution levels, inhibited conidia germination in V. dahliae. Furthermore, the inhibition rate decreased as the dilution factor increased. At a concentration of 250 μg/mL, the highest inhibition rate was observed at 80.25%, whereas at 100 μg/mL, the inhibition rate was the lowest, at only 46.67% (Figure 2).

3.3. Inhibitory Effect of Sterilized Fermentation Filtrate from Strain YL84 on Mycelial Growth of V. dahliae

The results indicated that the sterilized fermentation filtrate from strain YL84 inhibited the mycelial growth of V. dahliae. This inhibitory effect increased with higher volume fractions of the filtrate. At a 10% volume fraction, the inhibition was most pronounced at 72.16%. Conversely, the inhibitory effect diminished as the volume fraction decreased, with the lowest inhibition observed at a 2% volume fraction, measuring 44.55% (Figure 3).

3.4. Efficacy of Sterilized Fermentation Filtrate from Strain YL84 in Controlling Verticillium Wilt in Cotton

The pot trial results indicated that varying concentrations of strain YL84 sterilized fermentation filtrate significantly impacted the disease index (a comprehensive index incorporating both disease incidence and severity) and control efficacy of cotton Verticillium wilt. The disease index decreased as the concentration of the filtrate increased. At a 100 μg/mL concentration, the disease index was 52.84 ± 1.95, with a control efficacy of 39.54 ± 2.25%. When the concentration of the YL84 filtrate was increased to 150 μg/mL, the disease index dropped to 41.23 ± 2.13, and the control efficacy improved to 53.01 ± 2.82%. At the highest concentration of 250 μg/mL, the disease index declined to 29.14 ± 2.12, achieving a maximum control efficacy of 66.69 ± 2.17%. These findings suggest that the YL84 fermentation filtrate can potentially enhance cotton’s resistance to Verticillium wilt (Table 1).

3.5. Effects of Sterilized Fermentation Filtrate from Strain YL84 on the Mycelial Morphology of V. dahliae

Under scanning electron microscopy, the fungal mycelia of the untreated control group exhibited an intact, slender morphology with complete structural integrity, resembling fine strands without noticeable depressions or protrusions (Figure 4A). In contrast, the mycelia treated with a 10% volume fraction of YL84 sterilized fermentation filtrate showed significant morphological abnormalities. The treated mycelia were characterized by breakage and twisting (Figure 4B) and swelling structures (Figure 4C), indicating the severe inhibition of V. dahliae mycelial growth.

3.6. Effects of Sterilized Fermentation Filtrate from Strain YL84 on Cell Permeability of V. dahliae

Measurement of conductivity revealed that three different concentrations of YL84 sterilized fermentation filtrate significantly increased the conductivity of V. dahliae mycelia. Post-treatment, conductivity showed a linear rising trend within the first 30 min and stabilized thereafter. At a concentration of 100 μg/mL, the conductivity of the treatment group at 240 min was 4.62 times that of the control group. At 150 μg/mL, the conductivity at 240 min was 5.79 times that of the control. When the concentration reached 250 μg/mL, the conductivity peaked at 180 min, with the treatment group measuring 8.94 times higher than the control (Figure 5A). Additionally, the absorbance measured at 280 nm indicated a significant increase in protein leakage from V. dahliae mycelia following YL84 filtrate treatment. This leakage rose rapidly within the first 2 h and then stabilized. At a 100 μg/mL concentration, protein leakage in the treatment group at 8 h was 4.47 times that of the control. At 150 μg/mL, the leakage at 8 h was 6.38 times greater, whereas at 250 μg/mL, protein leakage peaked at 6 h, reaching 9.47 times that of the control (Figure 5B). These findings suggest that YL84 sterilized fermentation filtrate significantly enhances the permeability of mycelial cell membranes in V. dahliae, promoting the leakage of intracellular biomacromolecules and exerting an inhibitory effect on the fungus. Furthermore, the conductivity and protein leakage increase correlates positively with the YL84 sterilized fermentation filtrate concentration.

3.7. Effects of Sterilized Fermentation Filtrate from Strain YL84 on Cell Wall-Degrading Enzymes in V. dahliae

Using spectrophotometry, the effects of strain YL84 sterilized fermentation filtrate on the enzymatic activities of polygalacturonase (PG), cellulase (CL), and β-glucosidase (β-GC) in V. dahliae were investigated. On the seventh day, the enzymatic activities of PG, CL, and β-GC in the treatment group were significantly lower than those in the control group (Figure 6). Specifically, PG activity was reduced to 11.81 U/mL in the treatment group compared to 15.78 U/mL in the control group. The activity of CL decreased from 10.11 U/mL in the control group to 6.96 U/mL in the treatment group. β-GC activity showed a significant reduction from 5.01 U/mL in the control group to 1.44 U/mL in the treatment group. These results indicate that the YL84 sterilized fermentation filtrate inhibits the production of cell wall-degrading enzymes in V.dahliae, potentially suppressing its infective capability.

3.8. Growth-Promoting Effects of YL84 Sterilized Fermentation Filtrate on Cotton Seedlings

As shown in Table 2, all three concentrations of YL84 sterilized fermentation filtrate significantly increased the plant height, root length, fresh weight, and dry weight of cotton plants to varying extents. Compared to the control group, the increase in plant height ranged from 9.36% to 33.85%, root length from 17.33% to 29.49%, fresh weight from 16.79% to 28.24%, and dry weight from 25% to 58.33%. Among the tested concentrations, the impact of the 250 μg/mL filtrate was the most pronounced, leading to the most significant increases in plant height, root length, fresh weight, and dry weight.

4. Discussion

V. dahliae can survive in the soil for many years, making implementing crop rotation difficult [26]. Additionally, the lack of effective resistant varieties and control agents has led to an increasing severity of cotton Verticillium wilt, posing a significant restriction on the healthy development of the cotton industry in Xinjiang [27]. Biological control, which utilizes beneficial organisms to suppress harmful ones, offers an environmentally friendly method with advantages such as effective control, safety to human health, and no pollution to the ecological environment. In plant disease biological control, selecting highly effective biocontrol strains is fundamental [28]. Various studies have leveraged biocontrol strains to manage plant diseases. For instance, Wang et al. [29] isolated 35 endophytic bacteria with antagonistic activity from healthy garlic bulbs, 16 of which exhibited more than 50% inhibition against the garlic white rot pathogen. Zhang et al. [10] isolated Chaetomium globosum CEF-082 from cotton, which not only significantly inhibited the mycelial growth and conidial production of the cotton Verticillium wilt pathogen but also induced the upregulation of resistance-related genes such as POD, PPO, and PAL in cotton. This study focuses on a strain of Bacillus atrophaeus YL84, which has been shown via the dual culture method to inhibit the mycelial growth and conidial germination of V. dahliae significantly. Furthermore, its sterilized fermentation filtrate caused abnormal morphological changes in V. dahliae mycelia, including breakage, twisting, and swelling. These observations highlight the potent inhibitory potential of Bacillus atrophaeus YL84.
Numerous studies have demonstrated that, besides directly inhibiting pathogen growth, biocontrol microorganisms can also exert their effects by impairing the functional integrity of the pathogen’s cell membrane system. In this study, treatment with YL84 sterilized fermentation filtrate led to a significant increase in conductivity and protein leakage in the mycelia of V. dahliae, indicating a change in cell membrane permeability. This alteration results in the loss of intracellular ions and vital biomolecules, disrupting the normal physiological functions of the pathogen and ultimately inhibiting its growth and reproduction. This may be attributed to the presence of bioactive metabolites in the YL84 sterilized fermentation filtrate, particularly lipopeptides (such as iturins or fengycins), polyketides, or volatile organic compounds (VOCs), which are commonly produced by Bacillus spp. and have been reported to disrupt fungal membranes or interfere with their development [30,31]. These findings are similar to those reported by Su et al. [32] with Brevibacillus laterosporus and Sun et al. [33] with Alternaria alternata, where microbial inhibition of pathogen growth was associated with compromised cell membrane permeability.
During infection of host plants, V. dahliae produces various cell wall-degrading enzymes, such as cellulase and polygalacturonase, which are crucial for breaking down the cell walls of host plants, thereby disrupting the physical barriers and facilitating pathogen colonization, dissemination, and expansion [34,35,36]. Carder et al. [37] demonstrated a positive correlation between pectinase activity and the virulence of cotton Verticillium wilt fungus, while Novo et al. [38] found a positive correlation between cellulase production and the infection capability of V. dahliae strains. These findings indicate that cell wall-degrading enzymes play a critical role in the infection process of V. dahliae. In this study, spectrophotometric analysis was employed to assess the impact of YL84 sterilized fermentation filtrate on the enzymatic activities of polygalacturonase (PG), cellulase (CL), and β-glucosidase (β-GC) in V. dahliae. Results revealed that by the seventh day, the enzymatic activities in the treatment group were significantly lower than those in the control group. This suggests that YL84 sterilized fermentation filtrate can effectively inhibit the production of cell wall-degrading enzymes in V. dahliae, potentially reducing its infection capability.
Microorganisms with both antagonistic and growth-promoting activities often demonstrate greater advantages in the biological control of plant diseases such as Bacillus spp. [39], Paenibacillus spp. [40], and Streptomyces spp. [41]. Bacillus atrophaeus exhibits distinct advantages in inhibiting the proliferation of pathogenic fungi due to its specific metabolites, and it has demonstrated significant antimicrobial activity in various studies. For instance, the study by Sheng et al. [30] demonstrated that Bacillus atrophaeus SHZ-24 can produce multiple antifungal compounds and significantly inhibit the mycelial growth of V. dahliae. In contrast, although Bacillus velezensis and Bacillus subtilis are widely used, their biocontrol efficacy may vary due to differences in environmental conditions and pathogen strains. Therefore, Bacillus atrophaeus might offer a more advantageous solution for controlling specific pathogens. In this study, pot trials were conducted to evaluate the efficacy of YL84 sterilized fermentation filtrate in controlling cotton Verticillium wilt and its growth-promoting effects on cotton plants. The results showed that the YL84 sterilized fermentation filtrate at a 250 μg/mL concentration significantly reduced cotton Verticillium wilt’s incidence and disease severity index, achieving a control efficacy of 66.69%. Furthermore, all tested concentrations of YL84 sterilized fermentation filtrate significantly enhanced the growth parameters of cotton plants, including plant height, root length, fresh weight, and dry weight. Compared to the control group, plant height, root length, fresh weight, and dry weight were increased by 9.36–33.85%, 17.33–29.49%, 16.79–28.24%, and 25–58.33%, respectively, with the 250 μg/mL concentration showing the most pronounced effects. These growth-promoting effects may be attributed to the bioactive compounds secreted by strain YL84, such as indole-3-acetic acid (IAA) or other plant growth-regulating substances. These metabolites are commonly found in Bacillus spp. [42]. Moreover, further studies are required to elucidate their specific mechanisms of action. These findings demonstrate that strain YL84 possesses significant disease-suppression capabilities and growth-promotion potential for cotton, highlighting its broad application prospects.

5. Conclusions

In summary, the sterilized fermentation filtrate of Bacillus atrophaeus YL84 effectively inhibits the mycelial growth and conidial germination of V. dahliae and significantly reduces the activity of cell wall-degrading enzymes. The inhibitory action is mediated through changes in cell membrane permeability. In pot trials, a 250 μg/mL concentration of YL84 fermentation filtrate increased disease control efficacy to 66.69%. It significantly enhanced the growth of cotton plants, including causing improvements in plant height, root length, fresh weight, and dry weight. Therefore, Bacillus atrophaeus YL84 shows promising potential for application in the management of cotton Verticillium wilt. Future research will focus on validating the efficacy of YL84 under greenhouse conditions, optimizing fermentation parameters to facilitate formulation development, and evaluating its biocontrol potential against a broader spectrum of phytopathogenic organisms. In addition, considering the potential risk of resistance development in V. dahliae with repeated application of the YL84 fermentation filtrate, future studies should also assess the long-term stability of its efficacy and explore strategies such as rotating treatments or combining multiple microbial agents to mitigate resistance development. Moreover, this study did not investigate changes in the rhizosphere microbiome following application of the YL84 fermentation filtrate. However, given the known ability of microbial metabolites to influence soil microbial communities, YL84 may affect the composition and diversity of the rhizosphere microbiome. Future studies using high-throughput sequencing techniques are needed to assess whether YL84 selectively inhibits pathogens while promoting beneficial microbes, and to evaluate its overall impact on the microbial ecosystem.

Author Contributions

Conceptualization, Y.T., Q.X., J.Y. and Z.Z.; methodology, Y.T., J.Y. and Z.Z.; software, Y.T., Q.X. and J.Y.; validation, L.W., H.F. and Y.T.; formal analysis, Y.T.; investigation, Y.T.; data curation, Y.T., Q.X. and J.Y.; writing—original draft preparation, Y.T., L.W., H.F. and Z.W.; writing—review and editing, Y.T., L.W. and H.F.; funding acquisition, L.W. and Z.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Joint Funds of the National Natural Science Foundation of China (U1903206); the Guided Science and Technology Program of Xinjiang Production and Construction Corps (2024ZD078); and the Scientific Research and Innovation Project for Postgraduates of Tarim University (TDGRI2024022).

Data Availability Statement

The data are available upon reasonable request to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Agronomy 15 01997 g001
Figure 1. In vitro antagonistic effect of strain YL84 against V. dahliae. (A) Colony of V. dahliae after 7 d of normal growth; (B) colony of V. dahliae after 7 d of antagonism by YL84.
Figure 1. In vitro antagonistic effect of strain YL84 against V. dahliae. (A) Colony of V. dahliae after 7 d of normal growth; (B) colony of V. dahliae after 7 d of antagonism by YL84.
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Agronomy 15 01997 g002
Figure 2. Inhibition rates of V. dahliae conidial germination by different concentrations of YL84 sterilized fermentation filtrate. “****” indicates a statistically significant difference (p < 0.0001). “***” indicates a statistically significant difference (p < 0.001). Circles represent data points in the graph.
Figure 2. Inhibition rates of V. dahliae conidial germination by different concentrations of YL84 sterilized fermentation filtrate. “****” indicates a statistically significant difference (p < 0.0001). “***” indicates a statistically significant difference (p < 0.001). Circles represent data points in the graph.
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Agronomy 15 01997 g003
Figure 3. Inhibition rate of V. dahliae mycelial growth by different concentrations of sterilized fermentation filtrate from strain YL84. Different lowercase letters in the same column indicate significant differences by Duncan’s new multiple range test (p < 0.05). Circles represent data points in the graph.
Figure 3. Inhibition rate of V. dahliae mycelial growth by different concentrations of sterilized fermentation filtrate from strain YL84. Different lowercase letters in the same column indicate significant differences by Duncan’s new multiple range test (p < 0.05). Circles represent data points in the graph.
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Agronomy 15 01997 g004
Figure 4. Effects of sterilized fermentation filtrate from strain YL84 on the mycelial morphology of V. dahliae. (A) Mycelia of V. dahliae under normal growth conditions; (B,C) mycelia of V. dahliae after treatment with YL84 sterilized fermentation filtrate, showing distortion, fragmentation, and swelling.
Figure 4. Effects of sterilized fermentation filtrate from strain YL84 on the mycelial morphology of V. dahliae. (A) Mycelia of V. dahliae under normal growth conditions; (B,C) mycelia of V. dahliae after treatment with YL84 sterilized fermentation filtrate, showing distortion, fragmentation, and swelling.
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Agronomy 15 01997 g005
Figure 5. Effects of different concentrations of YL84 sterilized fermentation filtrate on the conductivity (A) and protein leakage (B) of V. dahliae.
Figure 5. Effects of different concentrations of YL84 sterilized fermentation filtrate on the conductivity (A) and protein leakage (B) of V. dahliae.
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Agronomy 15 01997 g006
Figure 6. Effects of YL84 sterilized fermentation filtrate on the levels of cell wall-degrading enzymes in V. dahliae. “*” indicates a statistically significant difference (p < 0.05); “**” indicates a statistically significant difference (p < 0.01).
Figure 6. Effects of YL84 sterilized fermentation filtrate on the levels of cell wall-degrading enzymes in V. dahliae. “*” indicates a statistically significant difference (p < 0.05); “**” indicates a statistically significant difference (p < 0.01).
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Table 1. Effects of different concentrations of YL84 sterilized fermentation filtrate on the severity and disease index of cotton Verticillium wilt.
Table 1. Effects of different concentrations of YL84 sterilized fermentation filtrate on the severity and disease index of cotton Verticillium wilt.
TreatmentDisease IndexControl Efficacy (%)
100 μg/mL sterilized fermentation filtrate52.84 ± 1.95 d39.54 ± 2.25 c
150 μg/mL sterilized fermentation filtrate41.23 ± 2.13 c53.01 ± 2.82 b
250 μg/mL sterilized fermentation filtrate29.14 ± 2.12 b66.69 ± 2.17 a
CK87.41 ± 1.21 a—
Different lowercase letters (e.g., a, b, c, d) in the same column indicate significant differences by Duncan’s new multiple range test (p < 0.05).
Table 2. Promoting effect of different concentrations of the sterilized fermentation filtrate from strain YL84 on cotton growth.
Table 2. Promoting effect of different concentrations of the sterilized fermentation filtrate from strain YL84 on cotton growth.
ConcentrationPlant Height/cmRoot Length/cmFresh Weight/gDry Weight/g
CK10.91 ± 1.99 c12.75 ± 2.54 b1.31 ± 0.12 b0.12 ± 0.02 b
100 μg/mL11.93 ± 0.32 bc14.96 ± 1.18 ab1.53 ± 0.07 a0.15 ± 0.03 ab
150 μg/mL13.61 ± 0.49 ab16.91 ± 1.92 a1.64 ± 0.06 a0.18 ± 0.03 ab
250 μg/mL14.6 ± 0.48 a16.52 ± 0.58 a1.68 ± 0.07 a0.19 ± 0.02 a
Different lowercase letters (e.g., a, b, c) in the same column indicate significant differences by Duncan’s new multiple range test (p < 0.05).
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Tang, Y.; Xue, Q.; Yu, J.; Zhang, Z.; Wang, Z.; Wang, L.; Feng, H. Biocontrol and Growth-Promoting Potential of Antagonistic Strain YL84 Against Verticillium dahliae. Agronomy 2025, 15, 1997. https://doi.org/10.3390/agronomy15081997

AMA Style

Tang Y, Xue Q, Yu J, Zhang Z, Wang Z, Wang L, Feng H. Biocontrol and Growth-Promoting Potential of Antagonistic Strain YL84 Against Verticillium dahliae. Agronomy. 2025; 15(8):1997. https://doi.org/10.3390/agronomy15081997

Chicago/Turabian Style

Tang, Yuxin, Qinyuan Xue, Jiahui Yu, Zhen Zhang, Zhe Wang, Lan Wang, and Hongzu Feng. 2025. "Biocontrol and Growth-Promoting Potential of Antagonistic Strain YL84 Against Verticillium dahliae" Agronomy 15, no. 8: 1997. https://doi.org/10.3390/agronomy15081997

APA Style

Tang, Y., Xue, Q., Yu, J., Zhang, Z., Wang, Z., Wang, L., & Feng, H. (2025). Biocontrol and Growth-Promoting Potential of Antagonistic Strain YL84 Against Verticillium dahliae. Agronomy, 15(8), 1997. https://doi.org/10.3390/agronomy15081997

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