Effect of Bacillus velezensis GHt-q6 on Cucumber Root Soil Microecology and Root-Knot Nematodes

Abstract

Root-knot nematode (RKN) causes severe yield loss in cucumber. Understanding the interactions of biocontrol agent–soil microbiomes and RKNs is essential for enhancing the efficacy of biocontrol agents and nematicides to curb RKN damage to cucumber. The field experiment in this work was conducted to determine the ability of Bacillus velezensis GHt-q6 to colonize cucumber plants, investigate its effect on the control of RKNs, and assess its influence on soil microbiology in the inter-root zone of cucumber plants. After 10 days post-treatment (DPT), GHt-q6-Rif could stably colonize the roots (4.55 × 10⁴ cfu·g⁻¹), stems (3.60 × 10³ cfu·g⁻¹), and leaves (3.60 × 10² cfu·g⁻¹) of cucumber. The high-throughput sequencing results suggested that the bacterial community diversity increased at the late development phase (p > 0.05). The strain GHt-q6 increased the relative abundance of beneficial bacteria (Gemmatimonadaceae, Sphingomonadaceae, Pseudomonadaceae). Throughout the complete cucumber growth period, strain GHt-q6 significantly increased soil urease, sucrase, accessible potassium, and phosphorus (p < 0.05). However, strain GHt-q6 had a minimal effect on catalase activity. At the pulling stage, strain GHt-q6 exhibited 43.35% control effect on cucumber RKNs, which was 7.54% higher than that of Bacillus subtilis. The results highlighted the significant potential of the strain GHt-q6 to manage cucumber RKNs and improve soil microecology. Hence, the applications of B. velezensis GHt-q6 can enhance the nematicidal action to curb RKN infecting cucumber.

1. Introduction

Cucumber, known for its crisp texture and refreshing taste, is highly favored in the global market. In China, the continuous development of facility agriculture has led to an annual expansion in the planting area and scale of cucumber cultivation, contributing significantly to agricultural revenue. The specificity of the greenhouse microclimate and the lack of natural enemies lead to the accumulation of pests and diseases in cucumbers. RKN disease affects almost all vegetable species worldwide, with a global host range exceeding 5500 plant species, and it has become a major limiting factor in the protected vegetable production systems [1,2]. RKNs induce overgrowth of root cells, which leads to the formation of galls on plant roots. The nematode damages the vascular tissue of the roots, thereby interfering with the normal movement of water and nutrients [3]. In such a condition, the yield of plants can drop by 50–80%, and in some cases, there is an entire yield loss [4]. Additionally, RKNs can aggravate plant diseases by synergizing with other pathogens such as Thielaviopsis, Fusarium, Phytophthora, and Ralstonia solanacearum, leading to severe soil-borne plant ailments [5,6,7,8]. Currently, RKNs are primarily controlled through chemical treatments. However, the pesticide residues pose serious risks to vegetable safety, human health, and the environment [9,10].

Soil microorganisms are a vital component of soil and plant health. Naturally, soil contains a vast and diverse community of microorganisms, which governs both direct and indirect roles in the ecosystem to promote plant growth and suppress the proliferation of soil-borne plant pathogens through antagonistic activity [11]. Consequently, using bioagents compatible with the plant-beneficial microbes in the rhizosphere can positively impact the environment and become an effective, sustainable strategy for managing plant parasitic nematodes [12]. Bacillus have been demonstrated to be effective against RKNs due to their capacity to colonize the rhizosphere and secrete nematicidal secondary metabolites via direct antagonism [13,14]. Additionally, Bacillus can indirectly mitigate RKNs by promoting plant growth and inducing plant systemic resistance [15,16,17]. Previous studies have determined that Bacillus velezensis can increase the relative abundance of Bacillus, Pseudomonas, and Sphingomonas in soils and has the potential to promote crop growth and effectively inhibit RKNs [18,19]. Therefore, elucidating the role of biocontrol bacteria in rhizosphere microecology is essential for the sustainable management of RKNs.

The strain GHt-q6 was isolated from the walnut epidermis; through pre-experiments, it was found that the corrected mortality rate of RKNs in the fermentation broth was about 90% at 3~4 h. Our study aimed to (i) define the colonization ability of strain GHt-q6 and (ii) determine the prevention and control effect of strain GHt-q6 and (iii) the role of strain GHt-q6 in soil microbial communities. The experiments were performed in continuous cropping sheds where cucumber RKN disease occurs year-round to study the effects of this strain on soil microbiology and cucumber RKN. This study could provide a theoretical basis for the effective control of cucumber RKN disease in agricultural production.

2. Materials and Methods

2.1. Materials and Greenhouse Conditions

The strain GHt-q6 was isolated and preserved by the Plant Pathology Laboratory of Shanxi Agricultural University. The control agent, Bacillus subtilis wettable powder (10 billion/g), was purchased from Shandong Jingbo Agrochemicals Technology Co., Ltd. (Binzhou, China).

The field experiment was performed in the greenhouse in Lixiu village, Taigu County. The greenhouse is a winter shed, daylight greenhouse, it had been continuously cultivated with cucumbers, tomatoes, and peppers for over 20 years, and RKN occurs every year. The greenhouse adopted water and fertilizer integration management, medium management level, and the soil texture was sandy loam. Experiment date was January 2024.

2.2. Greenhouse Experiment Design

Rifampicin solution was added to nutrient agar (NA) in an Erlenmeyer flask cooled to 50 °C with a filling volume of 50 mL using a pipette gun to prepare rifampicin containing agar plates with concentrations of 2, 10, 25, 75, 150, 250, and 300 µg·mL⁻¹. After 24 h of incubation in NB, 0.1 mL of GHt-q6 culture was spread onto NA plate containing 2 µg·mL⁻¹ rifampicin. After 36 h incubation, the mutant strains on the plate were transferred to the same concentration of the drug-containing plate and incubated for two generations. Then, the mutant strains were progressively transferred to plates supplemented with higher concentrations of the drug in the same way. The final screened mutant strain was able to grow consistently on NA plates containing 300 µg·mL⁻¹ rifampicin, and its physiological traits and antagonistic effects on pathogenic bacteria were consistent with those of the original strain, denoted as GHt-q6-Rif.

A single colony of GHt-q6 and GHt-q6-Rif activated in NA at 28 °C for 36 h was inoculated in nutrient broth (NB) medium and cultured at 28 °C and 180 rpm. Then, 2 mL of the inoculant was transferred to fresh NB medium and cultured at 28 °C and 180 rpm for 48 h and diluted with sterile water to 10⁸ colony-forming units per milliliter (CFU/mL) for planting irrigation.

The cucumber plants were moved to the greenhouse, and after the plants had grown three or four true leaves (approximately twenty days), they were irrigated with various treatments (100 mL per plant): GHt-q6 (Treat 1), B. subtilis (Treat 2), clean water (Treat 3), and GHt-q6-Rif (it was used for colonization of strains).

The experiment was conducted in a completely randomized design with four replicates (n = 4) for each treatment and 20 plants for each replicate.

2.3. Determination of Soil Microorganisms and Rhizosphere Colonization Capacity of GHt-q6-Rif

At 2, 6, 10, 14, 20, 33, and 48 days after treatment with GHt-q6-Rif, 2.0 g of roots, stems, and leaves were disinfected with 75% alcohol for 30 min. Add 10 mL of sterile water, grind into a homogenate, take 100 μL of slurry and its 10-fold and 100-fold dilutions, coat them on an NA plate containing 300 μg mL⁻¹ rifampicin, incubate at 28 °C for 48 h, and count the number of colonies in each petri dish.

Soil samples were collected using five-point sampling method. Surface debris was first removed, followed by collection of soil from 5–10 cm depth within a 2.5 cm radius of cucumber roots. Weigh 2 g of sieved soil sample into a triangular flask containing 20 mL of sterile water, oscillate for 30 min (190–220 r·min⁻¹), and allow to stand for 10 min to form a soil suspension recorded as 10⁻¹. The soil suspension was sequentially diluted into a solution on a 10⁻²–10⁻⁶ gradient. The number of colonies within each plate was recorded and the microorganisms contained per gram of dry soil were calculated.

2.4. Effects of Soil Enzyme Activity and the Physicochemical Properties of the Strain GHt-q6

Soil sucrase activity was measured according to the 3.5-dinitrosalicylic acid colorimetric method [20], urease activity was measured according to indophenol blue colorimetric method [21], and catalase activity was measured according to the potassium permanganate titration method [22]. Atomic absorption spectrophotometer was used for the determination of fast potassium [23], sodium bicarbonate leachate for fast phosphorus [24], and potassium dichromate oxidation–volumetric method for organic matter [25].

2.5. High-Throughput Sequencing and Data Analysis

Fresh soil samples collected at the end of cucumber fertility (60 d) were sent to Nanjing Personal Gene Technology Co., Ltd., (Nanjing China). for bacterial diversity analysis. PCR amplification of the bacterial 16 S rRNA genes’ V4b region was performed using the forward primer 515F (5′-GTGYCAGCMGCCGCGGTAA-3′) and the reverse primer 806R (5′-GGACTACNVGGGGTWTCTAAT-3′). Sample-specific 7 bp barcodes were incorporated into the primers for multiplex sequencing. After the individual quantification step, amplicons were pooled in equal amounts, and pair-end 2 × 250 bp sequencing was performed using the lllumina NovaSeqplatform with NovaSeq 6000 SP Reagent Kit (500 cycles) at Shanghai Personal Biotechnology Co., Ltd. (Shanghai, China).

Vsearch (v2.13.4_linux_x86_64) software was used to control the quality of the raw sequences, and Cutadapt (v2.3) software was used to cluster the sequences into OTUs based on 97% similarity. QIIME2 (2019.4) software was used to perform species annotation analysis of OTUs using the Greengenes database (Release 13.8, http://greengenes.secondgenome.com/, 15 April 2025). The software also counted the community composition at each taxonomic level and analyzed and evaluated dilution curves to determine how different the samples were in terms of sequencing intensity. QIIME2 (2019.4) and the R packages (v3.2.0) were utilized to calculate diversity indices such as the Chao1, ACE, Shannon, and Simpson indices and to evaluate the diversity of sequence libraries.

2.6. Effect of the Strain GHt-q6 on the Soil Root-Knot Nematodes Population Density and Control Efficacy

After 30 d, 45 d, and 60 d of cucumber root irrigation, RKN population density was quantified per 100 g of soil using the Bermann funnel method. The control efficacy was assessed at the harvest stage by calculating the disease index and percent reduction in RKN infestation. Disease assessment was performed according to the Chinese National Standard GB/T 17980. 38-2000, and the symptoms and characteristics of cucumber RKN disease were combined and investigated to the following grading standards [26]:

Grade 0, a root structure free of nodules and in good health;

Grade 1, minimal quantity of root knots inside the root system, with a percentage of less than 25%;

Grade 2, 25% to 50% of the total number of galls in the root system were medium-sized;

Grade 3, a high percentage of root nodules (between 50% and 75%) throughout the root system;

Grade 4, a proportion of root nodules of at least 75%, and the number of nodules on the root system was abnormally high.

$$\mathrm{P}\mathrm{r}\mathrm{e}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{t} \mathrm{a}\mathrm{n}\mathrm{d} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l} \mathrm{e}\mathrm{f}\mathrm{f}\mathrm{e}\mathrm{c}\mathrm{t}\left(\%\right)=\left[1-{\displaystyle \frac{\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t} \mathrm{d}\mathrm{i}\mathrm{s}\mathrm{e}\mathrm{a}\mathrm{s}\mathrm{e} \mathrm{i}\mathrm{n}\mathrm{d}\mathrm{e}\mathrm{x}}{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l} \mathrm{d}\mathrm{i}\mathrm{s}\mathrm{e}\mathrm{a}\mathrm{s}\mathrm{e} \mathrm{i}\mathrm{n}\mathrm{d}\mathrm{e}\mathrm{x}}}\right]\times 100$$

(1)

$$\mathrm{Disease} \mathrm{index}\left(\%\right)=\left[\sum {\displaystyle \frac{\mathrm{representative} \mathrm{value} \mathrm{at} \mathrm{all} \mathrm{levels}\times \mathrm{number} \mathrm{of} \mathrm{disease} \mathrm{strains} \mathrm{of} \mathrm{this} \mathrm{level}}{\mathrm{total} \mathrm{number} \mathrm{of} \mathrm{plants} \mathrm{investigated}\times \mathrm{highest} \mathrm{representative} \mathrm{value}}}\right]\times 100$$

(2)

2.7. Data Analysis

Statistical data processing was performed by standard methods using MS Excel (2016) and SPSS 22.0. All data were expressed as mean from triplicate samples ± standard deviation (n = 3). Duncan test was used, and differences were considered statistically significant at p < 0.05 level. Plotting was carried out using Origin software (version 2021).

3. Results

3.1. Colonization of Cucumber Plants by the Strain GHt-q6-Rif

At 2 DPT, root colonization peaked at (5.89 ± 0.32) × 10⁵ cfu·g⁻¹, and after 10 days, the colonization rate stabilized at (4.62 ± 0.21) × 10⁴ cfu·g⁻¹ (Figure 1). At 20 DPT, (3.69 ± 0.03) × 10³ cfu·g⁻¹ was the stable colonization amount for stems, while (3.12 ± 0.4) × 10³ cfu·g⁻¹ was the stable colonization level for leaves. Cucumber plants treated with GHt-q6-Rif by root irrigation showed stable colonization after 20 days. This suggests GHt-q6-Rif stably colonized the cucumber tissues. The degree of colonization decreased as follows: roots > stems > leaves.

3.2. Effects of the Strain GHt-q6 on the Soil Microbial Population

The abundance of actinomycetes and fungi significantly decreased after treatment with strain GHt-q6 and B. subtilis. At 7 DPT, the number of fungi was lowest in the strain GHt-q6 and B. subtilis treatment groups, at (1.49 ± 0.24) × 10⁶ cfu·g⁻¹ and (2.48 ± 0.25) × 10⁶ cfu·g⁻¹, respectively (p < 0.05). Throughout the reproductive period of cucumber, actinomycetes counts were significantly lower in the strain GHt-q6 treatment group than in the control group (p < 0.05) (

Table 1. Variation in the number of soil microbes after inoculation with strain GHt-q6.

Microbe	Disposal	2 d	7 d	20 d	34 d	48 d
Bacteria (108 cfu·g−1)	GHt-q6	3.47 ± 0.19 a	5.35 ± 0.15 a	2.39 ± 0.16 a	2.50 ± 0.15 a	3.94 ± 0.12 a
	B. subtilis	2.00 ± 0.09 b	3.30 ± 0.12 b	2.00 ± 0.19 b	1.77 ± 0.12 b	3.20 ± 0.19 b
	CK	1.24 ± 0.14 c	2.01 ± 0.04 c	0.84 ± 0.19 c	2.05 ± 0.14 c	3.50 ± 0.14 b
Fungi (106 cfu·g−1)	GHt-q6	1.74 ± 0.07 a	1.49 ± 0.24 a	7.11 ± 0.86 a	1.86 ± 0.33 a	4.05 ± 0.31 a
	B. subtilis	3.39 ± 0.22 b	2.48 ± 0.25 b	7.77 ± 0.51 a	3.07 ± 0.53 b	3.96 ± 0.40 a
	CK	3.98 ± 0.78 b	4.21 ± 0.61 c	8.38 ± 0.91 a	1.22 ± 0.23 a	3.32 ± 0.67 a
Actinomycetes (107 cfu·g−1)	GHt-q6	1.30 ± 0.06 a	1.05 ± 0.03 a	1.01 ± 0.09 a	0.63 ± 0.06 a	2.66 ± 0.11 a
	B. subtilis	1.03 ± 0.09 b	0.64 ± 0.08 b	1.86 ± 0.16 b	0.08 ± 0.01 b	2.54 ± 0.17 b
	CK	1.08 ± 0.09 b	1.44 ± 0.18 c	2.44 ± 0.13 c	2.12 ± 0.07 c	4.05 ± 0.06 c

Statistical analysis results are presented as the mean ± standard deviation (df = 2). Statistical analysis was conducted using ANOVA, with Duncan’s post hoc test (p < 0.05). Different letters in the same column represent significant differences among treatments at a significance level of p < 0.05.

). The results demonstrated the inhibitory effects of B. subtilis and the strain GHt-q6 treatment group on actinomycetes and fungus. Changes in bacterial volume in the strain GHt-q6 and B. subtilis treatment groups were similar to the control group, indicating an initial increase in bacterial volume followed by a decrease, and finally, an increase. After strain GHt-q6 treatment, the bacterial counts were all significantly higher than the control (p < 0.05), and the promotion effect was better than the B. subtilis treatment group. At 7 DPT, the bacterial count reached a maximum value of (5.35 ± 1.56) × 10⁸ cfu·g⁻¹. The results demonstrated that B. subtilis and strain GHt-q6 may stimulate bacterial growth in the soil surrounding cucumber roots, and strain GHt-q6 may have a stronger promoting effect.

3.3. Effects of the Strain GHt-q6 on Soil Bacterial Community Diversity

According to the α-diversity study, the Shannon and Simpson indices of species diversity for the treated soil flora were greater than those of the control. Furthermore, the bacterial community of the control group displayed a relatively high species abundance (Chao1 index), while no discernible variations were observed (Figure 2A). The microbial communities of the control and GHt-q6 treatment groups showed significant compositional differences based on beta diversity analysis (STRESS value < 2, indicating the reliability of the NMDS analysis results) (Figure 2B). This suggests that the introduction of biocontainment bacteria resulted in a substantial alteration in the root soil microbial community.

By categorizing and estimating the relative abundance of the OTUs derived from the sequences, the composition of the bacterial groups was determined. The beneficial bacteria with increased relative abundance were as follows: Gemmatimonadaceae (8.04% in the control group and 8.25% in the GHt-q6 group), Vicinamibacteraceae (4.77% in the control group and 4.90% in the GHt-q6 group), Sphingomonadaceae (2.55% in the control group and 2.57% in the GHt-q6 group), and Pseudomonadaceae (1.53% in the control group and 1.83% in the GHt-q6 group) dominated the core bacterial groups in both the treatment and control groups. Conversely, the relative abundance of the pathogenic bacterium Xanthomonadaceae decreased (2.60% in the control group and 2.37% in the GHt-q6 group) (Figure 3A). In comparison with the control, subgroup22, PAUC26f, and subgroup2 showed high enrichment of the Acidobacteria phyla, according to the MetagenomeSeq study (Figure 3B). Functional prediction of 16S rRNA gene soil bacterial composition data was performed using PICRUSt 2. Compared with the control group, carbohydrate degradation, secondary metabolite degradation, fatty acid and lipid degradation, metabolic regulator biosynthesis, secondary metabolite degradation, and fermentation were all higher in the strain GHt-q6 treatment group.

3.4. Effect of the Strain GHt-q6 on Soil Enzyme Activity

The enzyme activity of the strain GHt-q6 treatment group was consistently greater than that of the control group and B. subtilis treatment group. In 7 DPT, the sucrase activity in strain GHt-q6 showed a significant increase relative to the control (p < 0.05). However, sucrase activity was reduced after 48 DPT, although the reduction was not significant (

Table 2. Changes in soil enzymes activity after inoculation with strain GHt-q6.

Soil Enzymes	Disposal	2 d	7 d	20 d	34 d	48 d
Soil urease activity (mg·g−1)	GHt-q6	6.75 ± 0.19 a	5.38 ± 0.61 a	4.33 ± 0.47 a	3.75 ± 0.33 a	5.50 ± 0.09 a
	B. subtilis	5.41 ± 0.09 ab	5.08 ± 0.20 ab	5.80 ± 0.08 b	4.21 ± 0.03 b	5.63 ± 0.02 b
	CK	4.28 ± 0.01 b	4.51 ± 0.03 b	5.14 ± 0.14 c	3.05 ± 0.07 c	5.37 ± 0.01 c
Soil sucrase activity (mg·g−1)	GHt-q6	1.74 ± 0.04 a	1.94 ± 0.01 ab	1.50 ± 0.07 b	2.14 ± 0.04 a	1.20 ± 0.01 a
	B. subtilis	1.22 ± 0.06 a	1.88 ± 0.01 b	1.42 ± 0.01 a	1.97 ± 0.03 b	1.42 ± 0.08 b
	CK	1.12 ± 0.03 b	1.07 ± 0.00 a	1.05 ± 0.05 b	1.73 ± 0.05 c	1.24 ± 0.01 a
Soil catalase activity mg H2O2·(g·20 min) −1	GHt-q6	4.52 ± 0.08 a	3.40 ± 0.12 ab	2.65 ± 0.11 a	3.78 ± 0.15 a	3.93 ± 0.14 a
	B. subtilis	4.70 ± 0.11 a	3.32 ± 0.09 a	2.98 ± 0.16 b	4.61 ± 0.17 b	3.60 ± 0.15 b
	CK	5.10 ± 0.11 b	3.64 ± 0.17 b	2.57 ± 0.18 a	3.41 ± 0.09 c	4.00 ± 0.14 a

Statistical analysis results are presented as the mean ± standard deviation (df = 2). Statistical analysis was conducted using ANOVA, with Duncan’s post hoc test (p < 0.05). Different letters in the same column represent significant differences among treatments at a significance level of p < 0.05.

). After 20 days, urease activity in strain GHt-q6 was generally lower than that in the control group (p < 0.01). Catalase activity was negligible throughout the course of the cucumber growth period and was comparable to the trend of the control.

3.5. Effects of Strain GHt-q6 on Soil Physicochemical Properties

During the course of the growth cycle, the strain GHt-q6 and B. subtilis treatment groups had higher levels of soil organic matter, available phosphorus, and available potassium than did the control group (

Table 3. Effect of the strain GHt-q6 on the physicochemical properties of soil in the cucumber root system.

Growing Period	Disposal	AP (mg·kg−1)	AK (mg·kg−1)	SOM (g·kg−1)
Seedling stage	GHt-q6	150.25 ± 7.62 b	448.80 ± 12.75 a	35.14 ± 0.40 a
	B. subtilis	129.45 ± 7.41 ab	313.37 ± 10.81 b	29.53 ± 0.53 a
	CK	104.90 ± 8.63 a	207.40 ± 10.14 c	24.71 ± 0.47 b
Flowering stage	GHt-q6	113.75 ± 11.13 a	401.20 ± 5.91 a	33.45 ± 1.73 a
	B. subtilis	100.45 ± 6.89 a	309.97 ± 17.74 ab	27.71 ± 0.04 ab
	CK	106.27 ± 7.75 a	272.00 ± 16.43 b	23.45 ± 0.76 b
Fruiting stage	GHt-q6	127.88 ± 8.29 a	307.40 ± 3.05 a	34.03 ± 0.81 a
	B. subtilis	101.46 ± 2.88 a	296.93 ± 15.82 ab	30.33 ± 0.76 a
	CK	99.75 ± 7.62 a	253.30 ± 4.14 b	23.13 ± 0.87 b
Pull stage	GHt-q6	192.69 ± 8.29 a	233.60 ± 6.05 a	17.47 ± 0.93 a
	B. subtilis	187.42 ± 8.91 a	210.80 ± 10.89 a	14.41 ± 0.96 a
	CK	166.00 ± 11.12 a	158.10 ± 18.25 b	13.09 ± 0.72 a

Statistical analysis results are presented as the mean ± standard deviation (df = 2). Statistical analysis was conducted using ANOVA, with Duncan’s post hoc test (p < 0.05). Different letters in the same column represent significant differences among treatments at a significance level of p < 0.05.

). The application of biocontrol bacteria can swiftly alter the physical and chemical properties of soil, as evidenced by the promotion effect of these microorganisms, which is more noticeable at the seedling stage (p < 0.05). It is evident that available phosphorus, available potassium, and organic matter are all positively impacted by biocontrol bacteria, and that this effect is more pronounced than that of B. subtilis.

3.6. Efficacy of the Strain GHt-q6 Against Cucumber Root-Knot Nematodes

The RKN incidence index of the control group was significantly higher than that of the two treatment groups, and the control effect of GHt-q6 reached (43.35 ± 5.46)%, which was 7.54% higher than that of the B. subtilis treatment group (

Table 4. The control efficiency of GHt-q6 cucumber root-knot nematodes.

Disposal	Disease Index	Control Effect (%)
Treat 1	13.13 ± 1.61 b	43.35 ± 5.46 a
Treat 2	15.00 ± 1.02 b	35.81 ± 3.31 a
Treat 3	23.13 ± 0.72 a

Statistical analysis results are presented as the mean ± standard deviation (df = 2). Statistical analysis was conducted using ANOVA, with Duncan’s post hoc test (p < 0.05). Different letters in the same column represent significant differences among treatments at a significance level of p < 0.05.

and Figure 4). The number of RKNs in the soil of the GHt-q6 treatment group was lower than that of the control group at different periods. At the fruiting stage, the RKN population in the control group was 2.09 times (p < 0.05) higher than that in the strain GHt-q6 treatment group and 1.62 times higher than that in the B. subtilis treatment group (Figure 5. These results suggest that the strain GHt-q6 was able to reduce the reproduction rate of RKNs and reduce the damage to plant roots by reducing the invasion of RKNs into the root system.

4. Discussion

As an important biological control agent, Bacillus has been widely used in the prevention and control of plant diseases. Bacillus can increase plant resistance to soil pathogens by competing for niches and nutrients, producing antagonists, and inducing plant system resistance. It is generally believed that biocontrol bacteria colonize and establish in plant root systems preferentially, forming a complex ecosystem with the native inter-root microorganisms and the roots of host plants. They compete with pathogenic bacteria in plant roots and induce plant disease resistance, thus exhibiting stable and efficient biocontrol capabilities [27,28]. In this study, strain GHt-q6 also showed good inter-root colonization compared with previous findings [29,30].

Soil microbial population size and community composition are closely related to the occurrence of plant diseases, and in soil ecosystems, the abundance of soil microbes and the diversity of community structure are the basis for maintaining the stability of soil ecosystems. The greater the diversity of soil microorganisms, the greater the species richness and homogeneity are, the more stable the microbial ecosystem is, and the stronger the resistance to pathogenic microorganisms is [31]. It has been reported that Bacillus can recruit beneficial bacteria and lead the development of soil bacterial communities toward stabilization and diversification [32,33]. This is consistent with our results. In this paper, we found that the strain GHt-q6 favorably affected the dominant Pseudoalteromonas and Gemmatimonadaceae. Pseudoalteromonas and Gemmatimonadaceae have demonstrated the ability to control various pathogens via mechanisms such as antimicrobial production, induction of systemic resistance, promotion of plant growth, siderophore production, and competition for nutrients [34]. There was a significant increase in Sphingomonas in the phylum Ascomycota compared with that in the control group, and it was found that this bacterium functions as an efficient metabolic regulatory mechanism to promote the growth of plants that can improve soil pollution and degrade harmful substances [35]. Our findings were consistent with evidence from numerous reports [18,19,36,37]. To gain insight into the functioning of the rhizosphere microbial community, we used PICRUSt 2. Metabolism (carbohydrate metabolism, energy metabolism, metabolism of other amino acids, and nucleotide metabolism) in the bacterial community as a core function of the bacterial community plays an important role in promoting the cycling of plant soil matter and promoting plant growth [38].

Soil nutrients are an important factor in sustainable agricultural production and ecosystem functioning, affecting plant growth and rhizosphere microorganism proliferation. [39,40]. Bacillus can improve soil nutrient availability by improving nitrogen supply and phosphorus and potassium solubilization, and it can also secrete metabolites related to root development, improving plants’ absorption of nutrients. Soil sucrase is an important driving force of ecosystem metabolism and promotes soil nutrient cycling. It has also been suggested that nutrient levels play an important role in the development of diseases. Tian et al. reported that biocontrol bacteria increased soil urease, catalase, and phosphatase activity and decreased soil cellulase activity [41]. In this study, we found that the application of strain GHt-q6 altered soil enzyme activities and physicochemical properties. Therefore, improving the soil nutrient level can improve the survival and reproduction ability of the strain GHt-q6 in the soil and can also stimulate the reproduction of antagonistic microorganisms living in the soil, thereby further inhibiting the reproduction of root knot nematodes [42,43].

Bacillus can act as a nematode antagonist by inhibiting J2 hatching from eggs, motility, and viability [44,45]. Therefore, many researchers have noted the ability of Bacillus strains to reduce the number and damage of plant-parasitic nematodes attacking crops [46,47]. Hu et al. reported that Bv-DS1 has dual effects on plant growth promotion and protection against RKNs, possibly related to the regulation of water and solute transport via TIPs [48]. The research group has analyzed the whole genome of biocontrol bacteria and found that B. velezensis can produce metabolites such as cyclolipid peptides (LPs) (surfactin, fengycin, planttazolicin) and polyketones (bacillibactin, bacillibactin, difficidin, macrolactin), which play a key role in pathogen inhibition, stimulating plant defense mechanisms and biofilm formation, and promoting plant growth. In addition, this strain has been shown to have a good preventive and therapeutic effect on cucumber wilt. This suggests that strain GHt-q6 can reduce RKN damage to cucumber plants by competing with fungi for nutrients and niches in the soil, improving soil nutrient cycling and microbial community structure and function in the soil, secreting nematicides, and inhibiting fungal growth. The results of this study provide a scientific basis for developing an efficient bio-based nematicide, and whether the nematicidal effect arises from GHt-q6 metabolites or the bacterium itself requires further investigation.

5. Conclusions

This study highlighted the significant potential of the strain GHt-q6 to manage cucumber RKNs and improve soil health. The successful colonization of GHt-q6 in rhizosphere soils and cucumber roots creates a complex ecosystem with native microorganisms, and competition for space and resources in this ecosystem is critical for biocontrol effectiveness. The present study showed that GHt-q6 has stable rhizosphere colonization, which is consistent with the previous results and shows promising application prospects. The application of GHt-q6 had a positive effect on soil microbiology, which is critical for plant health. Strain GHt-q6 significantly reduced the population density of RKNs in cucumber rhizosphere soil. This reduction was attributed to the antagonistic properties of Bacillus that inhibit nematode hatching, motility, and survival. Strain GHt-q6 mediated the interaction between soil microorganisms in the root zone, root-dominant bacteria, and RKNs, which ultimately reduced cucumber RKN disease. The mechanism may be as follows: GHt-q6 triggered complex interactions between cucumber plants, soil microorganisms, soil physicochemical properties, and nematodes in the root zone soil microcosm system, leading to a reduction in RKN infestation and/or an increase in cucumber disease resistance. In conclusion, this study lays the foundation for the development of effective biological control strategies that provide sustainable alternatives to chemical pesticides and contribute to soil health and agricultural productivity. However, further studies are needed to determine whether the nematicide effect of GHt-q6 is due to its secreted metabolites or the bacterium itself alongside the application of its biological agent potential in agricultural production.