Fermentation Conditions and Wettable Powder Formulation of Biocontrol Agent Bacillus atrophaeus YL84 in Control of Pear Valsa Canker

TL;DR

This study optimizes the fermentation and formulation of Bacillus atrophaeus YL84 to enhance its effectiveness as a biocontrol agent for pear canker disease.

Contribution

The paper introduces an optimized fermentation process and wettable powder formulation for Bacillus atrophaeus YL84.

Findings

Optimal fermentation conditions achieved an 83.71% inhibition rate and OD600 of 1.758.

The developed wettable powder formulation showed strong antagonistic activity against Cytospora pyri.

The WP formulation demonstrated biocontrol efficacy in detached-branch assays.

Abstract

Bacillus atrophaeus has considerable potential for development as a microbial pesticide. Optimization of fermentation conditions and the wettable powder (WP) formulation is critical for its industrialization and application in sustainable agriculture. In this study, the fermentation of B. atrophaeus YL84 was optimized using single-factor experiments and response surface methodology. Based on these results, a WP formulation was developed and further optimized. The optimal carbon, nitrogen, and inorganic salt sources were sucrose (13.9 g·L−1), tryptone (11.8 g·L−1), and MgSO4 (5.9 g·L−1), respectively; optimal fermentation conditions were pH 7.0, 32 °C, and 210 r·min−1. After optimization, the inhibition rate and OD600 reached 83.71% and 1.758, respectively. The optimized formulation comprised attapulgite-based powder (79%, as carrier), sodium alkyl naphthalene sulfonate (5.4%) as a wetting agent, PEG-6000 (12.6%), CaCO3 (2%), and vitamin C (1%). The resulting WP exhibited a spore viability of 2.63 × 109 CFU·g−1, and its 50-fold dilution demonstrated antagonistic activity in vitro against Cytospora pyri (Korla pear valsa canker agent) and biocontrol efficacy in vivo on detached-branch assays. These findings demonstrate that the YL84 WP is a promising candidate for the biological control of Korla pear valsa canker.

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Full text

1. Introduction

Korla pear valsa canker, caused by the cytosporaceous fungus Cytospora pyri Fuckel 1860, infects host tissues through wounds. The disease leads to tissue decay, progressive tree decline, yield losses, and deterioration of fruit quality [1,2]. It is severe, is difficult to control, and has a broad host range [3]. Notably, this pathogen is capable of infecting more than 70 plant species, including economically important Rosaceae fruit trees such as apple, peach, plum, hawthorn, and crabapple, thereby posing a serious threat to orchard production systems beyond Central Asia [4,5]. Current management relies primarily on chemical measures, such as lime–sulfur preparations and fungicides like tebuconazole applied by trunk drenching [6]. However, chemical control poses risks of environmental pollution and pesticide residues, highlighting the need for safer, sustainable alternatives.

Biological control offers a promising approach. Bacillus species, in particular, are advantageous due to their stress tolerance, antimicrobial metabolites, and plant growth-promoting abilities [7,8,9]. Biocontrol typically causes minimal disruption to non-target organisms and presents lower risks of environmental pollution and phytotoxicity, which underpins its ecological value and biosafety. Nevertheless, novel biopesticides face constraints such as long development timelines, limited mechanistic evidence for some strains, formulation and stability challenges, and inconsistent field performance; therefore, improving formulation performance, standardizing evaluation, and conducting demonstration trials are necessary to promote adoption.

Although numerous Bacillus-based wettable powder (WP) products are registered in China (≈200 products as of September 2025), reports indicate that various B. atrophaeus can inhibit plant pathogens and produce bioactive metabolites. For example, Valdivia et al. [10] reported that B. atrophaeus strain B5, isolated from cherimoya (Annona cherimola) and avocado (Persea americana), produced cell-free metabolites that effectively inhibited mycelial growth and conidial germination of Colletotrichum spp. and significantly reduced the incidence of postharvest fruit diseases. Xue et al. [11] found that B. atrophaeus NX12 secretes fengycin, inhibiting Fusarium oxysporum f. sp. cucumerinum. Rajaofera et al. [12] demonstrated that B. atrophaeus HAB-5 exhibited strong inhibitory activity against Colletotrichum gloeosporioides and that crude antimicrobial extracts retained potent and stable bioactivity after exposure to different treatment conditions. However, a scoping search of publicly accessible pesticide registration records (ICAMA) and patent databases (up to September 2025) indicated that no commercially registered WP products list B. atrophaeus as the active microorganism, and 58 related patents were identified. In contrast, B. subtilis has 67 registered WP products, with 6108 associated patents. More importantly, no publicly available reports have explicitly documented product-oriented WP research targeting Korla pear valsa canker caused by Cytospora. pyri. Therefore, B. atrophaeus remains underdeveloped with respect to systematic WP formulation research and translation toward commercialization. This gap is particularly relevant for Korla pear valsa canker, for which no dedicated microbial WP product has been developed.

B. atrophaeus YL84, isolated from healthy Korla pear leaves, was selected from 30 antagonistic isolates because it exhibited the most potent inhibition of C. pyri, showed broad-spectrum antagonistic activity, and promoted host growth, thereby offering strong potential for biocontrol application [13]. In practical application, fermentation conditions and formulation quality are key factors influencing microbial efficacy and field performance [14,15,16,17,18,19,20,21].

To address the lack of B. atrophaeus WP products and to facilitate potential industrialization, this study optimized fermentation conditions and evaluated the biocontrol efficacy of strain YL84 against Korla pear valsa canker. Hypothesis: Bacillus atrophaeus YL84 can be optimized into a stable and effective wettable powder (WP) formulation for the biocontrol of C. pyri. Specifically, the objectives were to enhance antagonistic activity through fermentation optimization and to assess the efficacy of the resulting formulation in vitro and on detached branches, thereby providing a scientific basis for further development and field validation.

2. Materials and Methods

2.1. Strains and Culture Media

Bacillus atrophaeus YL84 was isolated in May 2024 from a Korla fragrant pear orchard located in Huaqiao Town, Alar City, Xinjiang, China (81°35′22″ E, 40°32′53″ N). The isolate was deposited in the China Center for Type Culture Collection (CCTCC) in March 2025 under the accession number CCTCC M 2025267 with its sequence submitted to the NCBI GenBank under the accession number PV748128 [22]. Prior to experiments, YL84 was streaked onto LB agar and incubated at 28 °C for 48 h in the dark. The pathogenic fungus Cytospora pyri (the causal agent of Korla pear valsa canker) was isolated from a diseased Korla fragrant pear orchard in Nankou Town, Alar City, Xinjiang, China (81°21′12″ E, 40°28′37″ N) in July 2023. It was identified and preserved by the Green Control Laboratory at Tarim University, and its sequence was deposited in NCBI GenBank under accession number PP494037 [23].

To select the optimal basal medium for subsequent fermentation and medium optimization, we evaluated the growth and antagonistic activity of B. atrophaeus YL84 in seven commonly used bacterial media:

Potato Dextrose Agar (PDA): 200 g potato, 20 g dextrose, and 15 g agar per liter of distilled water. It was sterilized for 25 min. The dextrose was purchased from Xinbote Chemical (Tianjin, China), and the agar was purchased from Lanjie Ke Technology (Beijing, China).

Luria–Bertani (LB) Agar: 10 g tryptone, 5 g yeast extract, 5 g NaCl, and 15 g agar per liter of distilled water. It was sterilized for 30 min. The tryptone was purchased from Aoboxing BIO-TECH (Beijing, China), the yeast extract was purchased from Yongda Chemical (Tianjin, China), and the NaCl was purchased from Yongda Chemical (Tianjin, China).

LB Broth: 10 g tryptone, 5 g yeast extract, and 5 g NaCl per liter of distilled water. It was sterilized for 30 min.

Nutrient Broth (NB): 10 g peptone, 3 g beef extract, 10 g glucose, and 1 g yeast extract per liter of distilled water; pH was adjusted to 7.0–7.2. It was sterilized for 25 min. The peptone was purchased from Yuanye Bio-Tech (Shanghai, China), and the beef extract was purchased from Yuanye Bio-Tech (Shanghai, China).

SOB Medium: 20 g peptone, 5 g yeast extract, 0.5 g NaCl, 0.18 g KCl, and 0.95 g MgCl_2_·H_2_O per liter of distilled water; pH was adjusted to 7.0–7.2. It was sterilized for 25 min. The KCl was purchased from Yongda Chemical (Tianjin, China), and the MgCl_2_·H_2_O was purchased from Yuanye Bio-Tech (Shanghai, China).

BPY Medium: 5 g beef extract, 10 g peptone, 5 g yeast extract, and 5 g glucose per liter of distilled water; pH was adjusted to 7.0–7.2. It was sterilized for 25 min.

NYBD Medium: 8 g beef extract, 5 g yeast extract, 10 g glucose, and 5 g NaCl per liter of distilled water; pH was adjusted to 7.3. It was sterilized for 25 min.

2.2. Carriers and Adjuvants

To develop a stable and effective wettable powder (WP) formulation of B. atrophaeus YL84, formulation components including carriers, wetting agents, dispersants, stabilizers, and UV protectants were systematically screened and optimized. Selection was based on their compatibility with bacterial spores and their effects on key formulation quality parameters such as wetting time, suspensibility, and spore viability. Carriers: kaolin, diatomite, talcum powder, precipitated silica, and attapulgite were purchased from Yuanye Bio-Tech (Shanghai, China). Wetting agents: sodium alkyl naphthalene sulfonate was purchased from Lanjie Ke Technology (Beijing, China), sodium dodecyl sulfate (SDS) was purchased from Sinopharm Chemical Reagent (Shanghai, China), and tween 60 was purchased from Yuanye Bio-Tech (Shanghai, China). Dispersants: sodium lignin sulfonate, polyethylene glycol 6000 (PEG 6000), and sodium tripolyphosphate were purchased from Yuanye Bio-Tech (Shanghai, China). Stabilizers: K_2_HPO_4_ was purchased from Aoboxing BIO-TECH (Beijing, China), and CMC-Na and CaCO_3_ were purchased from Yuanye Bio-Tech (Shanghai, China). Protectants: ascorbic acid (vitamin C, VC), dextrin, and humic acid were purchased from Xinbote Chemical (Tianjin, China).

2.3. Preparation of Seed Inoculum

A single colony of isolate YL84 from an LB agar plate was inoculated into 50 mL of LB broth and cultured at 30 °C with shaking (Neo 400, Herry Tech, Shanghai, China) at 200 r/min^−1^ for 24 h to prepare the seed inoculum.

2.4. Effect of Fermentation Media on Bacterial Growth and Antagonistic Activity

For each of the five liquid media tested, 50 mL aliquots were dispensed into 150 mL Erlenmeyer flasks. The seed inoculum was added at 3% (v/v), followed by incubation at 30 °C and 200 r·min^−1^ for 24 h. After incubation, OD_600_ (9230G, Aucy Scientific instrument, Shanghai, China) was recorded and antifungal activity was assessed via the plate confrontation assay. When the pathogen in the control plates (85 mm) fully covered the medium surface, the mycelial diameters in each treatment and the control were measured using a perpendicular cross method to calculate mycelial growth inhibition rate:

$$[eqn]$$

2.5. Single-Factor Optimization of Fermentation Medium Components

To obtain the carbon source that maximized OD_600_ and the mycelial growth inhibition rate of the antagonist, the following carbon sources were evaluated as candidates through carbon source screening: glucose, sucrose, soluble starch, fructose, mannitol, and yeast extract, with standard LB broth serving as the control. The fermentation and subsequent measurement of OD_600_ and mycelial growth inhibition rate were performed as described in Section 2.4. The optimal carbon source was subsequently tested at concentrations of 0, 5, 10, 15, 20, 25, and 30 g·L^−1^ to determine its optimal concentration.

To obtain the nitrogen source that maximized OD_600_ and the mycelial growth inhibition rate of the antagonist, the following nitrogen sources were evaluated as candidates using nitrogen source screening and the same procedure: beef extract, NH_4_NO_3_, tryptone, (NH_4_)2_SO_4, soybean meal, and KNO_3_. The optimal nitrogen source was then tested at concentrations of 0, 6, 8, 10, 12, 14, and 16 g·L^−1^.

To obtain the inorganic salt that maximized OD_600_ and the mycelial growth inhibition rate of the antagonist, the following inorganic salts were evaluated as candidates through inorganic salt screening: MgCl_2_, K_2_HPO_4_, CaCl_2_, MnCl_2_, FeSO_4_·7H_2_O, and NaCl. They were separately supplemented into the LB broth. Following fermentation, OD_600_ and the mycelial growth inhibition rate were measured. The optimal inorganic salt was further examined at concentrations of 0, 0.2, 0.6, 1.0, 1.4, 1.8, 2.2, and 2.6% (w/v, equivalent to 0, 2, 6, 10, 14, 18, 22, and 26 g·L^−1^).

2.6. Response Surface Optimization of Fermentation Medium Components

Based on the single-factor experiments [24], the three most influential factors were selected. A three-factor, three-level Box–Behnken design (BBD) was employed. The factors and their levels were as follows: sucrose concentration (A): 10, 15, and 20 g·L^−1^; tryptone concentration (B): 10, 12, and 14 g·L^−1^; MgSO_4_ concentration (C): 2, 6, and 10 g·L^−1^ (for coded levels, see Table 1). The design comprised 17 experimental runs, including five replicates at the center point (0, 0, 0) to estimate pure error. All fermentation experiments were conducted with three biological replicates. The mycelial growth inhibition rate (Y) was used as the response variable, and a Box–Behnken design was constructed using Design-Expert 13 to determine the optimal composition of the fermentation medium.

2.7. Optimization of Fermentation Conditions

Under the optimal medium combination, cultures were incubated at different pH values (4, 5, 6, 7, 8, 9, 10, and 11), temperatures (22, 25, 28, 32, and 35 °C) and shaking speeds (120, 150, 180, 210, and 240 r·min^−1^). After 24 h, OD_600_ and mycelial growth inhibition rate were measured to identify the optimal fermentation conditions.

2.8. Evaluation of Fermentation Outcomes

Plate confrontation assays were performed to measure OD_600_ and mycelial growth inhibition rate of YL84 fermentation broths before and after optimization against the pathogen of Korla pear valsa canker.

2.9. Screening of Wettable Powder Carriers and Adjuvants

2.9.1. Carrier Selection and Preparation of Original Powder

The carrier adsorption capacity was determined according to Shen et al. [25]. Briefly, 100 g of sterilized carrier was placed in an Erlenmeyer flask, and the YL84 fermentation broth was slowly added under continuous stirring until the carrier reached a state of being free-flowing, non-caking, and without visible free water. The volume of broth absorbed (V) was recorded, and the maximum adsorption capacity (H, in kg·L^−1^) was calculated as H = V/0.1. To assess wetting time and suspensibility, the fermentation broth was uniformly mixed with each carrier and spray-dried (H-Spray, Holves Bio-Tech Co., Ltd., Beijing, China) with an inlet temperature of 175 °C and an outlet temperature of 60 °C to obtain an original powder. The wetting time and suspensibility of this concentrate were then determined following the quality-testing methods described in Section 2.10. The final carrier was selected based on a comprehensive evaluation of the wetting time, suspensibility, adsorption capacity, and cost.

2.9.2. Screening of Wetting and Dispersing Agents

Following the method of Wen et al. [26], the YL84 original powder was blended with candidate wetting or dispersing agents at a concentration of 12% (w/w). The mixtures were pulverized using an ultrafine mill and homogenized to WP samples. Each treatment was prepared in triplicate. The wetting time, suspensibility, and spore viability of the samples were determined as outlined in Section 2.10 to identify the optimal wetting and dispersing agents.

2.9.3. Optimization of the Wetting-to-Dispersing Agent Ratio

Using wetting time and suspensibility as the primary indices, the selected wetting and dispersing agents were combined at nine different mass ratios (from 1:9 to 9:1). The YL84 original powder was thoroughly mixed with each agent blend after ultrafine pulverization to prepare WP samples. Each formulation was replicated three times. Wetting time, suspensibility, and spore viability were measured, and the optimal ratio was selected by integrating the results of all three performance indicators.

2.9.4. Effect of Total Adjuvant Dosage on Wettable Powder Performance

The selected wetting and dispersing agents were pulverized, blended, sieved through a 325-mesh screen, and then incorporated into the YL84 original powder at total dosages ranging from 2% to 18% (w/w). Each dosage level was prepared in triplicate. Wetting time, suspensibility, and spore viability were measured to determine the optimal total adjuvant dosage based on dispersibility, suspension stability, and spore viability.

2.9.5. Stabilizer Screening

The YL84 original powder was combined with the selected wetting agent, dispersing agent, and candidate stabilizers at concentrations of 1%, 2%, or 3% (w/w). The mixtures were then ultrafine-milled and homogenized into the final WP. Treatments without any stabilizer served as controls, with three replicates for each. All samples were subjected to an accelerated storage test at (54 ± 2 °C) for 14 d. This protocol followed the Chinese national standard GB/T 19136-2021 [27] for evaluating WP storage stability. Spore viability was measured after this storage period to evaluate the protective effect of each stabilizer.

2.9.6. UV Protectant Screening

The original powder was blended with the selected wetting, dispersing, and stabilizing agents, along with candidate UV protectants at concentrations of 0.5%, 1.0%, or 1.5% (w/w). After ultrafine milling and homogenization, the WP samples were exposed to a 254 nm UV lamp (20 W) at a distance of 60 cm for 12 h. Treatments without a UV protectant served as controls. Spore viability was subsequently calculated to identify the most effective UV protectant.

2.9.7. Formulation of the Final YL84 Wettable Powder

The selected carrier, wetting agent, dispersing agent, stabilizer, and UV protectant were blended with the YL84 fermentation broth at their respective optimal ratios. The mixture was continuously stirred and then spray-dried using a two-fluid nozzle (atomization pressure 0.1MPa) with a inlet temperature of 120 °C, an outlet temperature of 50–55 °C, a feed rate of 720 mL·h^−1^, and a drying-air flow of 30 m^3^·h^−1^ to obtain the final WP formulation.

2.9.8. Quality Evaluation of the Wettable Powder

A 10× standard hard-water stock solution was prepared by dissolving 6.08 g of anhydrous CaCl_2_ and 2.78 g of MgCl_2_·6H_2_O in sterile distilled water, bringing the final volume to 2 L. This stock was diluted 1:10 with sterile distilled water to obtain the standard hard water for testing.

Suspensibility was assessed to determine the proportion of wettable powder that remained in stable suspension after standard agitation, indicating its redispersibility during application: approximately 2.00 g (recorded as w) of the WP sample was added to a 250 mL graduated cylinder containing 250 mL of standard hard water. The cylinder was inverted 30 times within 1 min. After allowing the suspension to settle, 225 mL of the supernatant was carefully decanted, leaving approximately 25 mL of suspension. This remaining suspension was dried at 105 °C to a constant weight (recorded as w_1_). The suspensibility (X, %) was calculated using the following formula: X = (10/9) × (w − w_1_)/w × 100%.

Wetting time was measured to evaluate the rate at which the powder was fully wetted upon water contact, a key factor for field usability: five grams of the WP was gently sprinkled along the inner wall of a 250 mL beaker containing 100 mL of standard hard water, ensuring the water surface was not disturbed. The time required for the entire powder sample to penetrate the water surface was recorded as the wetting time. This was repeated four times, and the average value was calculated.

Spore viability was monitored to ensure the formulation process maintained the biological activity of B. atrophaeus YL84: one gram of the WP was suspended in 10 mL of sterile water in a 50 mL flask and shaken at 120 r·min^−1^ for 1 h. A series of ten-fold dilutions were prepared, and 100 µL aliquots from two appropriate dilutions were spread onto LB agar plates. The colonies were counted after incubation to determine the viable spore count.

2.9.9. Inhibition of Mycelial Growth of Cytospora pyri by the Wettable Powder

The inhibitory activity of 50-fold and 100-fold dilutions of the YL84 WP against the pathogen was evaluated using dual-culture assays. When the mycelium of the pathogen fully covered the medium surface in the control plates, the colony diameters in all treatments were measured using the perpendicular cross method. The mycelial growth inhibition rate was then calculated.

2.9.10. Detached-Branch Assay Against Korla Pear Valsa Canker

Annual Korla pear shoots of uniform vigor were collected from the Tarim University horticultural station. Branch segments (15 cm) were rinsed, sterilized (75% ethanol, 1 min; 1% NaOCl, 3 min), rinsed with sterile distilled water, dried, and sealed with paraffin at both ends. A 5 mm hole was bored to the xylem at the center. Four treatments were applied: (1) protective assay—60 µL of 50-fold YL84 WP was added to the wound and incubated at 25 °C for 1 d before placement of a 5 mm C. pyri plug; (2) therapeutic assay—the pathogen plug was applied for 1 d, removed, and replaced with 60 µL of 50-fold YL84 WP; (3) pathogen control—only C. pyri; (4) blank control—60 µL of 50-fold YL84 WP only. Branch segments were randomly assigned. To maintain humidity, sterile absorbent cotton moistened with distilled water was wrapped 2 cm above and below each hole. Branches were incubated at 25 °C in darkness for 7 d, lesion lengths were measured, and control efficacy was calculated as [(lesion length of control − lesion length of treatment)/lesion length of control] × 100%. Each treatment had three replicates.

2.10. Statistical Analysis

Data are expressed as mean ± standard deviation (SD) and each treatment was performed in triplicate (n = 3). Treatment effects were analyzed by one-way analysis of variance (ANOVA) using the model Yij = μ + τi + εij, where Yij is the response of the jth replicate of treatment i, μ is the overall mean, τi is the fixed effect of treatment i, and εij is the residual error. For comparisons among more than two groups, post hoc multiple comparisons were conducted using Duncan’s new multiple range test. Pairwise comparisons were performed by independent sample t-tests. Prior to parametric testing, assumptions of normality and homogeneity of variance were assessed by the Shapiro–Wilk test and Levene’s test, respectively. For response surface analysis, a Box–Behnken design was fitted with a second-order polynomial model in Design-Expert 13.0, and model adequacy was evaluated by ANOVA, the lack-of-fit test, and R^2^ and adjusted R^2^. Data processing was supported by Excel and SPSS Statistics 20.0, and figures were prepared with GraphPad Prism 8.1.2 and Origin 2022. Statistical significance was set at α = 0.05, p-values are presented as threshold categories consistent with the software output, and the type of test applied is stated in the corresponding figure legends or table notes [22].

3. Results

3.1. Effects of Basal Fermentation Media on YL84 Growth and Antagonistic Activity

After 24 h of cultivation at 30 °C and 200 rpm, the OD_600_ of B. atrophaeus YL84 across the five basal media ranged from 0.327 to 1.669, with inhibition rates between 55.11% and 80.26% (Figure 1). YL84 exhibited the most robust growth and antagonistic activity in LB medium, achieving the highest OD_600_ (1.669) and inhibition rate (80.26%). This was followed by NB, NYBD, and BPY media, while the slowest growth was observed in SOB medium. Consequently, LB was selected as the optimal basal medium for subsequent experiments.

3.2. Determination of Optimal Carbon, Nitrogen, and Inorganic Salt Sources and Loading Levels

Bacillus atrophaeus YL84 grew in the presence of all six carbon sources. Under identical conditions, sucrose produced the highest OD_600_ (1.464) and inhibition rate (82.37%), significantly exceeding the other carbon sources (Figure 2A). Fructose also supported strong growth, whereas glucose resulted in lower values (OD_600_ 0.925; inhibition rate 59.02%). Sucrose was consequently chosen. As shown in Figure 2B, both OD_600_ and inhibition rate increased with sucrose concentrations from 0 to 15 g·L^−1^, peaked at 15 g·L^−1^, and then decreased at higher levels, indicating that excessive carbon may impede growth or antagonism.

Evaluation of six nitrogen sources showed that all promoted growth, but tryptone yielded the highest OD600 (1.509) and inhibition rate (80.56%) (Figure 3A), whereas KNO_3_ produced the lowest. Accordingly, tryptone was selected. With increasing tryptone concentration from 0 to 12 g·L^−1^, OD600 and inhibition rate rose to a peak at 12 g·L^−1^ and decreased thereafter (Figure 3B), suggesting that excess tryptone is inhibitory.

Among the inorganic salts, MgSO_4_ resulted in the highest OD600 (1.810) and inhibition rate (80.95%) (Figure 4A) and was therefore selected. Both OD600 and inhibition rate increased with MgSO_4_ concentration, reaching a maximum at 6 g·L^−1^ (Figure 4B).

3.3. Response Surface Methodology for Medium Optimization

Based on the single-factor experiments, the inhibition rate (Y) was used as the response variable, and three significant factors were selected: A (sucrose concentration), B (tryptone concentration), and C (MgSO_4_ concentration) (Table 1). A Box–Behnken design was generated using Design-Expert 13 (Table 2). Multiple regression of the data produced the following quadratic polynomial equation:

$$[eqn]$$

Response surface and contour plots were generated using Origin 2022 to visualize the interactions between the factors (Figure 5, Figure 6 and Figure 7). The surfaces for AB, AC, and BC showed pronounced gradients, indicating their significant effects on the inhibition rate. All 3D surfaces were convex and downward, demonstrating the presence of maxima, and the elliptical contours confirmed significant interactions.

To validate the adequacy of the regression model, verification experiments were conducted under the model-predicted optimal conditions. The predicted optimal medium composition was sucrose 13.954 g·L^−1^, tryptone 11.823 g·L^−1^, and MgSO_4_ 5.93 g·L^−1^, corresponding to a predicted inhibition rate of 82.146%. For practical operation, these values were adjusted to sucrose 13.9 g·L^−1^, tryptone 11.8 g·L^−1^, and MgSO_4_ 5.9 g·L^−1^. Three parallel experiments were performed under the adjusted conditions, yielding an average inhibition rate of 83.01%. The close agreement between the experimental and predicted values confirmed the reliability of the model for predicting the optimal fermentation conditions.

3.4. Effects of Fermentation Conditions on YL84

The effects of pH, temperature, and shaking speed are shown in Figure 8, Figure 9 and Figure 10. With an increase in pH from 4 to 7, both the inhibition rate and OD_600_ increased, peaking at pH 7, after which they decreased (Figure 8). When the temperature was raised from 22 °C to 32 °C, the inhibition rate and OD_600_ increased and reached a maximum at 32 °C; further increases led to a decline (Figure 9). An increase in shaking speed from 120 rpm to 210 rpm also increased both parameters, which peaked at 210 rpm; speeds beyond this point caused a reduction (Figure 10).

3.5. Inhibition of Korla Pear Valsa Canker Mycelia Under Optimized Conditions

As demonstrated in Table 3, when cultured in the optimized medium and under the optimized conditions, the OD_600_ and inhibition rate of the YL84 fermentation broth increased to 1.758 and 83.71%(Figures S1 and S2), respectively. These validation results confirm that the optimization process enhanced the broth’s capacity to suppress the mycelial growth of the pathogen.

3.6. Carrier Selection for the YL84 Wettable Powder

Carriers were screened based on adsorption capacity, wetting time, and suspensibility (Table 4). Precipitated silica exhibited the highest adsorption capacity (2.26 mL/g), followed by attapulgite (1.86 mL/g), diatomite (1.42 mL/g), and kaolin (0.88 mL/g). Diatomite demonstrated the shortest wetting time (19 s), while attapulgite achieved the highest suspensibility (48.6%), significantly outperforming the other carriers. Considering the overall balance of these key properties, attapulgite was selected as the optimal carrier. The attapulgite carrier was mixed with the YL84 fermentation broth, followed by drying and milling to obtain the original powder (Figure 11).

3.7. Screening of Wetting and Dispersing Agents

Candidate wetting and dispersing agents were individually incorporated into the carrier–fermentation broth mixture at a 12% mass fraction. The resulting WP formulations were evaluated for the wetting time, suspensibility, and spore viability (Table 5). Among the formulations, those containing sodium alkyl naphthalene sulfonate as a wetting agent (Formulations 2, 4, and 9) showed the shortest wetting times (26.8–35.6 s). Based on its superior overall performance, Formulation 4 was selected for further optimization.

3.8. Optimization of the Wetting-to-Dispersing Agent Ratio

The selected wetting agent (sodium alkyl naphthalene sulfonate) and dispersant (PEG 6000) were blended at different mass ratios. As presented in Table 6, the ratio of 3:7 (wetting agent to dispersant) provided the best compromise, yielding a wetting time of 33.0 s, a suspensibility of 71.56%, and a spore viability of 2.87 × 10^9^ CFU/g. Consequently, this ratio was adopted.

3.9. Determination of the Total Adjuvant Dosage

The effect of the total adjuvant (wetting agent + dispersant) dosage, ranging from 2% to 18% (w/w), was investigated (Table 7). While a 4% dosage resulted in the shortest wetting time (26.6 s), the 18% dosage yielded the highest suspensibility (72.22%) and the highest spore viability (3.01 × 10^9^ CFU/g), significantly surpassing the other treatments. Prioritizing suspension stability and spore viability, which are critical for the product’s shelf life and efficacy, the optimal total adjuvant dosage was determined to be 18%.

3.10. Screening of High-Temperature Stabilizers

As shown in Table 8, compared to the control without a stabilizer after 14 d of heat storage, the addition of stabilizers maintained a better wetting time, suspensibility, and spore viability of the WP. The addition of 2% CaCO_3_ resulted in the optimal wetting time and suspensibility (33.0 s and 73.4%, respectively). Spore viability decreased from 2.84 × 10^9^ CFU/g before storage to 1.78 × 10^9^ CFU/g, with a heat storage loss rate of 37.32%. Therefore, 2% CaCO_3_ was selected as the stabilizer for the B. atrophaeus YL84 wettable powder.

3.11. UV Protectant Screening

As shown in Table 9, the addition of various types and concentrations of UV protectants improved the spore viability of the WP compared with the control without a UV protectant. When 0.5% vitamin C (VC) was used as the UV protectant for B. atrophaeus YL84, the spore viability was the highest (1.49 × 10^9^ CFU/g) after 12 h of exposure to a 254 nm UV lamp (20 W, distance 60 cm). This was followed by 1% vitamin C, with no significant difference between the two treatments. Considering the overall balance of suspensibility, wetting time, and spore viability, 1% vitamin C was selected as the preferred UV protectant under the experimental conditions.

3.12. Quality Assessment of the Bacillus atrophaeus YL84 WP

The B. atrophaeus YL84 WP was prepared by mixing the YL84 fermentation broth with the screened carrier, wetting agent, dispersant, high-temperature stabilizer, and UV protectant at the optimized ratios. The quality of the formulated wettable powder was subsequently evaluated (Table 10).

3.13. Inhibition of Cytospora pyri Mycelial Growth by the YL84 WP

In the plate confrontation assay, both 50-fold and 100-fold dilutions of the YL84 WP significantly inhibited the mycelial growth of C. pyri. The 50-fold dilution exhibited the most potent inhibitory effect, with an inhibition rate of 75.26%, while the 100-fold dilution showed an inhibition rate of 68.13% (Table 11, Figures S3 and S4). These results suggest that the YL84 WP has promising application prospects.

3.14. Control Efficacy of the YL84 WP on Korla Pear Valsa Canker

The results of the detached-branch assay (Table 12) demonstrated that the 50-fold dilution of YL84 WP effectively suppressed lesion expansion caused by C. pyri. The blank control inoculated only with YL84 WP exhibited no disease symptoms. In contrast, branches inoculated solely with the pathogen developed distinct lesions with a mean length of 7.26 cm. The protective treatment, where YL84 WP was applied prior to pathogen inoculation, resulted in a mean lesion length of 1.97 cm, corresponding to the highest control efficacy of 72.83%. The therapeutic treatment, where the pathogen was inoculated before the application of the 50-fold YL84 WP, resulted in a mean lesion length of 2.28 cm and a control efficacy of 68.59%. These findings indicate that the 50-fold dilution of YL84 WP provides substantial protective and curative activity against C. pyri, effectively limiting lesion enlargement.

4. Discussion

As medium composition and fermentation conditions directly determine microbial proliferation rates and viable cell counts, their systematic optimization is a critical prerequisite for the efficient development and industrialization of microbial formulations. In this study, a combination of single-factor experiments and RSM was employed to systematically optimize the fermentation process for B. atrophaeus YL84, determining the optimal medium composition as follows: sucrose 13.9 g·L^−1^, tryptone 11.8 g·L^−1^, and MgSO_4_ 5.9 g·L^−1^. Although studies on the optimization of fermentation conditions for Bacillus species have increased in recent years, optimal parameters often vary significantly among strains due to differences in genetic background, physiological characteristics, and environmental adaptation [28]. Furthermore, the distinct local adaptation of microorganisms means that strains isolated from different ecological environments often exhibit specific fermentation requirements [29]. Therefore, during process optimization and scale-up, targeted parameter screening must be conducted based on the genetic traits, physiological state, and isolation source of the target strain to ensure process stability and reproducibility. Single-factor experiments in this study further clarified the optimal physical conditions for YL84 as pH 7.0, 32 °C, and 210 r·min^−1^. These conditions are highly consistent with the optimal parameters reported for B. atrophaeus XW2 isolated from healthy poplar leaves by Zhu [30] and B. atrophaeus E20303 isolated from Qarhan Salt Lake mud by Qiao et al. [31], suggesting that YL84 shares highly similar fundamental growth habits with B. atrophaeus strains from other sources.

WPs typically consist of carriers, wetting agents, suspending agents, dispersants, stabilizers, and protectants. During product development and process optimization, suspensibility, wetting time, and spore viability are commonly used as quality control indices [32]. Among these components, the carrier usually accounts for the largest proportion of the WP; thus, evaluating the biocompatibility between the carrier and the strain is particularly important [33]. Significant differences were observed among carrier materials in spore adsorption capacity; this variation is primarily attributable to inherent physicochemical properties of the carriers, including specific surface area, pore structure, surface charge, and chemical functional groups [31]. For example, carriers with high specific surface area and abundant mesoporous structure (e.g., attapulgite and precipitated silica) provide numerous attachment sites for spores and therefore exhibit higher adsorption capacities, whereas carriers with compact structures or smooth surfaces (e.g., talcum powder) tend to have lower adsorption capacity [34,35]. For instance, while some studies indicate that kaolin improves spore survival [36,37,38,39], others report poor compatibility between kaolin and certain biocontrol strains [40]. This demonstrates that although most carriers are inert materials, the adaptability of different strains to the same carrier varies significantly. Therefore, systematic compatibility assessments must be conducted for specific strains. In this study, using the fermentation broth as the active ingredient source, the carrier screening process primarily focused on the carrier’s adsorption capacity for the broth and its impact on key physicochemical indices such as wetting time and suspensibility. The results showed that attapulgite possessed high adsorption capacity, a short wetting time, and high suspensibility; consequently, attapulgite was selected as the carrier for the B. atrophaeus YL84 WP. This finding is consistent with the conclusions of Wang et al. [41].

In addition to the carrier, the selection of adjuvants significantly influences WP quality; superior wetting and dispersing agents can enhance the dispersibility and stability of the formulation [42]. In this study, wetting time, suspensibility, and inhibition rate served as the primary evaluation criteria to screen several candidate adjuvants. The candidate wetting agents included sodium alkyl naphthalene sulfonate, sodium dodecyl sulfate (SDS), and tween-60; candidate dispersants included sodium lignin sulfonate, polyethylene glycol 6000, and sodium tripolyphosphate. Comparison of different adjuvant combinations revealed that, with respect to wetting time, the combination of sodium lignin sulfonate and SDS yielded the shortest wetting time, a result similar to that of Zhang et al. [43]. However, when suspensibility and spore viability were prioritized, the combination of polyethylene glycol 6000 and sodium alkyl naphthalene sulfonate achieved the highest values, with suspensibility levels approaching those reported for a B. marinus WP by Liu et al. [44]. Based on this comprehensive comparison, sodium alkyl naphthalene sulfonate and polyethylene glycol 6000 were selected as the wetting agent and dispersant, respectively, for the B. atrophaeus YL84 WP. Subsequent optimization of their ratio and total dosage indicated that a wetting agent-to-dispersant ratio of 3:7 with a total dosage of 18% produced a formulation with a short wetting time, maximum suspensibility, and optimal spore viability.

The Xinjiang region is characterized by abundant sunshine, intense UV radiation, and high midday temperatures. Given this natural environment, the addition of UV protectants and high-temperature stabilizers has been proven to effectively improve the photostability of formulations, delay degradation caused by UV radiation, and reduce moisture-induced agglomeration under high temperatures, thereby mitigating the adverse effects of UV and heat on powder performance [45]. The results of this study showed that the addition of 2% (w/w) calcium carbonate (CaCO_3_) effectively alleviated the decline in spore viability of the B. atrophaeus YL84 WP at (54 ± 2 °C), while maintaining high wettability and suspensibility, consistent with the findings of Yang et al. [46]. Furthermore, the use of 1% vitamin C (VC) as a UV protectant significantly reduced the impact of UV radiation on spore viability. Notably, after 12 h of exposure to a 254 nm UV lamp (20 W, distance 60 cm), the spore viability of the B. atrophaeus YL84 WP remained at 1.43 × 10^9^ CFU/g. This aligns with the results of Luo et al. [47], further validating the positive role of UV protectants in enhancing the UV resistance of the formulation. Additionally, in plate confrontation and detached-branch assays, the 50-fold dilution of the B. atrophaeus YL84 WP exhibited significant disease control capability, with an inhibition rate of 75.26% against the mycelial growth of C. pyri. In detached-branch assays, the WP demonstrated protective and therapeutic efficacy of 72.83% and 68.59%, respectively. Similar levels of disease control have been reported for other Bacillus-based formulations, such as B. subtilis S1-0210, which achieved approximately 70% control efficacy against strawberry gray mold (Botrytis cinerea) [48] and B. velezensis F0b, which showed a maximum control efficacy of 73.14% against gray mold [26]. Although differences in host plants, target pathogens, and experimental systems limit direct quantitative comparisons, these results collectively suggest that the WP formulation of B. atrophaeus YL84 exhibits a biocontrol potential comparable to that of previously reported Bacillus-based formulations. In addition to the formulation-related factors discussed above, the antagonistic activity of Bacillus spp. is often associated with the production of nonribosomal peptide (NRP) lipopeptides and/or the induction of host resistance. Genome mining studies have suggested that some Bacillus atrophaeus strains harbor multiple biosynthetic gene clusters (e.g., NRPS/PKS-related clusters) potentially involved in the biosynthesis of antimicrobial secondary metabolites, including fengycin-like and other lipopeptide compounds [49]. These genomic features provide a plausible molecular basis for the biocontrol potential of B. atrophaeus and raise the possibility that strain YL84 may possess similar secondary metabolic capacity.

The improved spore viability and stability of the YL84 wettable powder can be attributed to the synergistic effects of the selected carrier and adjuvant system operating through multiple mechanisms [50]. First, the attapulgite carrier, owing to its distinctive morphology, high specific surface area, and abundant mesoporous structure, provides an excellent physical matrix for spore adsorption [33]. This structure not only facilitates the immobilization of large numbers of spores but also provides mechanical protection, thereby mitigating the effects of temperature and humidity fluctuations on spore viability. The adjuvant system plays a key role in dispersion and stabilization [51]. The combination of wetting agents and dispersants ensures rapid and uniform dispersion and effectively prevents particle agglomeration, thus ensuring homogeneous distribution and bioavailability of spores during application [52]. Meanwhile, stabilizers (e.g., CaCO_3_) help mitigate moisture-induced agglomeration and thermal stress during storage and transport [53]. In addition, the inclusion of a UV protectant (e.g., vitamin C) effectively reduces UV-induced damage to spores, further preserving biological activity [54]. Notably, the spore viability achieved in this study (2.63 × 10^9^ CFU/g) not only exceeds the threshold stipulated by the Chinese national standard (NY/T 2293.1-2012 [54], 2.0 × 10^8^ CFU/g) but is also comparable to the high viability reported for other effective Bacillus-based WPs, such as the B. velezensis SH 1471 formulation described by Shen et al. [25]. In summary, the physical protective function of attapulgite and the dispersive/stabilizing functions of the adjuvants act complementarily to reduce physicochemical stress on spore viability markedly. This multi-layered protective strategy constitutes the mechanistic basis for the high spore viability and sustained efficacy of the YL84 wettable powder.

From a horticultural practice perspective, the present data provide an initial reference for application of the YL84 WP, although field-oriented parameters still require optimization. Based on the detached-branch assays, the 50-fold dilution that showed consistent protection and curative activity can be considered a preliminary working concentration for follow-up greenhouse/field trials; however, definitive dosage recommendations should be established through dose–response tests under orchard conditions. In practice, application timing is expected to be critical, and treatments should be aligned with periods of high infection risk and/or pruning wound formation, when canker pathogens are more likely to invade. In addition, compatibility with common orchard inputs (e.g., copper-based bactericides/fungicides, mineral oils, or adjuvants) and tank-mix stability should be systematically evaluated, because pH, ionic strength, and co-formulants may affect spore viability and dispersibility. Therefore, future trials will prioritize (1) field dose optimization and (2) seasonality and spray scheduling under local climate conditions.

It should be noted that the bioefficacy of the WP formulation was evaluated exclusively under in vitro conditions and using detached branches under controlled laboratory conditions. Although these assays provide valuable preliminary insights into the antagonistic potential of the formulation against C. pyri, they do not fully replicate the complexity of field environments. In natural orchard systems, multiple factors such as environmental variability, plant physiological status, microbial community interactions, and application conditions may significantly influence biocontrol performance. Therefore, the results obtained in this study should be interpreted as indicative rather than fully predictive of field efficacy. Further evaluations under greenhouse and field conditions will be necessary to validate the practical applicability of the WP formulation. Although accelerated stress tests (UV and high-temperature exposure) indicated improved short-term robustness of the WP, the present study did not determine shelf-life stability under realistic storage conditions (e.g., months-long storage at different temperatures and humidity levels). Consequently, the duration over which key quality attributes (spore viability, wetting time, suspensibility, and dispersibility) remain within acceptable ranges is still unknown. To address this gap and to support translation toward commercialization, future work will include systematic storage stability programs (e.g., 4 °C /25 °C/40 °C and controlled humidity conditions) with periodic monitoring of the above indices and bioefficacy. Moreover, commercialization of microbial biopreparations typically faces practical challenges, including batch-to-batch consistency during scale-up fermentation, contamination control, downstream processing robustness, and compliance with regulatory registration requirements and phytosanitary standards. These considerations underscore that additional validation and standardization are required before the YL84 WP can be positioned as an orchard-ready product.

5. Conclusions

This study focused on B. atrophaeus YL84, where optimized fermentation conditions enhanced its antagonistic activity and facilitated its development into a WP formulation. This work provides a scientific foundation for the eco-friendly management of Korla pear valsa canker. Furthermore, our previous research has identified that B. atrophaeus YL84 also possesses notable plant growth-promoting traits and saline–alkali tolerance, underscoring its considerable application potential. Future studies will involve pot and field experiments to systematically evaluate the strain’s efficacy in promoting plant growth and enhancing stress tolerance. These investigations will pave the way for its broad application in agriculture, aiming to achieve integrated benefits of disease control, yield improvement, and environmental sustainability. Nevertheless, several limitations should be acknowledged. First, bioefficacy was evaluated only in vitro and on detached branches, and field performance under orchard conditions remains to be verified. Second, the mechanism underlying antagonism (e.g., lipopeptide production and/or host resistance induction) was not experimentally confirmed in this study. Third, although the WP showed robustness in accelerated UV/high-temperature tests, the shelf-life stability and property retention during long-term storage have not yet been determined. Nevertheless, potential field limitations—such as UV stress that can inactivate unprotected spores, uneven deposition on the complex surface of tree bark, and constrained humidity conditions that may hinder spore germination—may compromise efficacy and should be considered in practical application. Consequently, systematic field validation is necessary to bridge the lab-to-field gap, specifically to optimize application dosage, evaluate environmental persistence under field conditions, and thoroughly assess potential phytotoxicity to host trees. In addition, storage stability testing under realistic conditions and compatibility assessments with common orchard inputs will be conducted to support practical deployment within IPM programs. Finally, scale-up production, quality-control standardization, and compliance with regulatory registration and phytosanitary requirements will be addressed as key steps toward commercialization. Overall, this study provides a WP technology-oriented framework for developing Bacillus-based biopesticides with high spore viability and promising bioefficacy, while delineating clear priorities for future field and mechanistic research.