Application of Bacillus cereus SZ-4 or its wettable powder

Abstract

This invention discloses the application of *Bacillus cereus* SZ-4 or its wettable powder, belonging to the field of pesticide technology. The application described herein involves field treatment of crops with *Bacillus cereus* SZ-4 or a diluted solution of its wettable powder; the field treatment includes at least one of disease control, crop growth promotion, and rhizosphere soil improvement. The *Bacillus cereus* SZ-4 wettable powder of this invention, relying on the biological functional characteristics of the strain itself, simultaneously achieves multiple functions including crop disease control, plant growth regulation, and improvement of the microecological environment of the cultivation soil. It effectively overcomes the limitations of existing conventional biological agents, such as single function, insufficient efficacy stability, and the technical defects of chemical agents, such as residues, pathogen resistance, and soil degradation. This agent has a scientific mechanism of action, stable effects, and high application safety, making it suitable for ginseng green cultivation and various crop planting systems, possessing a good application foundation and promotional value.

Description

Technical Field

[0001] This invention belongs to the field of pesticide technology, specifically relating to the application of Bacillus cereus SZ-4 or its wettable powder. Background Technology

[0002] Ginseng ( Panax ginseng *Callicarpa meyer* is a perennial herbaceous medicinal plant belonging to the Araliaceae family. Its dried roots and rhizomes are core medicinal materials in the traditional Chinese medicine system, possessing extremely high medicinal and economic value. With the rapid development of the national health industry, the market demand for medicinal ginseng continues to rise, and large-scale artificial cultivation has become the mainstream development model for the ginseng industry. In the process of large-scale ginseng cultivation, foliar fungal diseases are the core factor restricting yield and quality improvement, among which *Botrytis cinerea* (*Callicarpa meyer*) is the most prevalent. Botrytis cinerea Gray mold and Alternaria (induced by) Alternaria spp . The most serious damage is caused by black spot disease induced by ginseng. Both types of diseases mainly infect the above-ground leaf tissues of ginseng. After infection, they accelerate the premature aging of leaves, inhibit the photosynthesis of plants, seriously hinder the accumulation of nutrients in ginseng, and ultimately cause a significant decrease in ginseng root yield and deterioration of medicinal quality, which greatly restricts the healthy development of large-scale and standardized ginseng planting industry.

[0003] Currently, the mainstream control methods for ginseng field diseases still rely on broad-spectrum chemical fungicides such as carbendazim and mancozeb. These agents are fast-acting and have significant short-term control effects, and are widely used in the field control of ginseng gray mold and black spot. However, the long-term large-scale and singular application of chemical pesticides in cultivation has exposed many unavoidable technical defects and industrial drawbacks, specifically in the following three aspects: First, pathogen resistance accumulates continuously, leading to a gradual decline in control effectiveness and rising planting costs. Long-term, single-use application of chemical fungicides induces intergenerational resistance in pathogens such as Botrytis cinerea and Alternaria alternata, resulting in a continuous increase in pathogen resistance levels in the field. To achieve basic disease prevention, growers can only continuously increase pesticide concentrations and application frequency, significantly increasing both labor and pesticide costs in the field. Furthermore, the disease recurrence rate remains high, and the long-term control stability of chemical pesticides is severely insufficient, creating a vicious cycle of "high input, low efficacy."

[0004] Secondly, the risk of excessive pesticide residues is prominent, hindering the quality and safety of medicinal materials and the internationalization of the industry. Ginseng, a unique traditional Chinese medicine with both medicinal and edible uses, has roots and rhizomes that can be used directly in medicine or processed for consumption, making it subject to extremely stringent requirements for pesticide residue levels. Frequent application of chemical pesticides easily leads to excessive pesticide residues in medicinal materials, seriously affecting the medicinal safety and commercial quality of ginseng, and failing to meet increasingly stringent pesticide residue limits in domestic and international markets. This significantly restricts the high-end, standardized, and international development of my country's ginseng industry.

[0005] Third, it disrupts the microecological balance of the rhizosphere soil, exacerbating continuous cropping obstacles and soil degradation. Chemical fungicides have broad-spectrum killing properties; while killing pathogenic fungi, they indiscriminately inhibit the activity of beneficial soil microorganisms and reduce soil enzyme activity. Long-term application directly leads to an imbalance in the soil microbial community structure, deterioration of soil physicochemical properties, and decline in soil fertility. Ginseng is a perennial rootstock crop with a strong dependence on the rhizosphere soil environment. Disruption of the soil microecology will significantly exacerbate continuous cropping obstacles in ginseng, resulting in weak plant growth, decreased disease resistance, and a vicious cycle of frequent diseases, soil degradation, and reduced yield.

[0006] In summary, the traditional pest control model relying solely on chemical pesticides cannot simultaneously address the three core needs of efficient disease control, crop quality and safety, and soil ecological protection. Existing technologies have seen some research attempting to use biological control strains and plant-derived active substances to target and inhibit ginseng gray mold and black spot, or to apply soil conditioners alone to improve degraded soils from continuous cropping. However, these existing solutions all have limitations, mostly addressing only single problems and lacking a systematic, integrated solution encompassing disease control, crop safety and quality improvement, and soil ecological restoration.

[0007] More importantly, among the currently available research and existing technologies, there is no mature technology system that can simultaneously meet the three core application conditions: first, it has a stable and efficient inhibitory effect on the two core foliar fungal diseases of ginseng, gray mold and black spot; second, the application concentration is safe and controllable, with no risk of plant damage, which can ensure the quality and safety of ginseng medicinal materials; and third, it can effectively repair the rhizosphere soil of ginseng, improve the key physicochemical properties of the soil, enhance soil enzyme activity, and optimize the soil micro-ecological environment.

[0008] Therefore, developing a safe, efficient, green and environmentally friendly microbial preparation that can simultaneously control major fungal diseases in ginseng, promote plant growth, and restore the rhizosphere soil microecology is a core technical challenge that urgently needs to be overcome in this field. It has important practical significance and application value for promoting green and ecological ginseng cultivation and breaking through the bottleneck of industrial development. Summary of the Invention

[0009] The purpose of this invention is to provide the application of Bacillus cereus SZ-4 and Bacillus cereus SZ-4 wettable powder to simultaneously control major fungal diseases of ginseng, promote plant growth, and repair rhizosphere soil.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention discloses the application of Bacillus cereus SZ-4 or Bacillus cereus SZ- wettable powder in crop cultivation, wherein a diluted solution of Bacillus cereus SZ-4 or Bacillus cereus SZ-4 wettable powder is used to treat crops in the field; the field treatment includes at least one of disease control treatment, crop growth promotion treatment, and rhizosphere soil improvement treatment.

[0011] The dead Bacillus glutathione described in this invention Bacillus vallismortis SZ-4, deposited at the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, deposited on September 25, 2013, accession number: CGMCC No. 8273. This invention relates to *Bacillus cereus*. Bacillus vallismortis SZ-4 is a biocontrol bacterium isolated by the inventors' research group in the early stage, which has good control effect on fungal diseases such as root rot. This strain is currently deposited at the China General Microbiological Culture Collection Center (CGMCC No. 8273) and has an invention patent entitled "A type of Bacillus cereus and its application", with authorization announcement number CN103952329B.

[0012] The Bacillus oryzae SZ-wettable powder described in this invention is the Bacillus oryzae SZ-wettable powder disclosed in publication number CN120615916 A.

[0013] In some embodiments of the present invention, the wettable powder of Bacillus cereus SZ-4 is diluted 100 to 1000 times.

[0014] In some embodiments of the present invention, the crop is any one or more of ginseng, rice, soybean, bok choy, wheat, rapeseed, and carrot.

[0015] In some embodiments of the present invention, the disease control treatment is for the prevention and control of ginseng gray mold and / or ginseng black spot disease; when carrying out the disease control treatment, a 100-fold diluted solution of Bacillus oryzae SZ-4 wettable powder is applied.

[0016] In some embodiments of the present invention, the crop growth-promoting treatment is a spraying or root irrigation treatment applied to the seed germination stage and / or seedling growth stage of the crop; when performing the crop growth-promoting treatment, a 100-fold diluted solution of the Bacillus oryzae SZ-4 wettable powder is applied.

[0017] In some embodiments of the present invention, the rhizosphere soil improvement treatment is carried out under conditions of infection with gray mold or black spot fungus; the rhizosphere soil improvement treatment includes reducing soil electrical conductivity, increasing soil organic matter content, increasing soil available nitrogen content, increasing soil available phosphorus content and / or increasing soil available potassium content.

[0018] In some embodiments of the present invention, for soils affected by gray mold, a 100-fold or 1000-fold diluted solution of Bacillus cereus SZ-4 wettable powder is applied; for soils affected by black spot disease, a 600-fold diluted solution of Bacillus cereus SZ-4 wettable powder is applied.

[0019] In some embodiments of the present invention, for soils affected by gray mold, a 100-fold dilution is used for treatment to regulate the activity of soil neutral phosphatase and sucrase; a 1000-fold dilution is used for treatment to improve soil organic matter.

[0020] In some embodiments of the present invention, the application concentration of the Bacillus cereus SZ-4 wettable powder is not less than 100 times the dilution concentration, so as to avoid inhibiting the seed germination or seedling growth of non-target crops.

[0021] In some embodiments of the present invention, the field treatment is applied by foliar spraying or soil irrigation.

[0022] Compared with the prior art, the present invention has the following beneficial effects: The Bacillus oryzae SZ-4 wettable powder of this invention, relying on the biological functional characteristics of the strain itself, simultaneously achieves multiple functions including crop disease control, plant growth regulation, and improvement of the microecology of cultivation soil. It effectively overcomes the limitations of existing conventional biological agents, such as single function, insufficient efficacy stability, and the technical shortcomings of chemical agents, including residues, pathogen resistance, and soil degradation. This agent has a scientific mechanism of action, stable effects, and high application safety, making it suitable for ginseng green cultivation and various crop planting systems, possessing a good foundation for application and promotional value.

[0023] The *Bacillus cereus* SZ-4 strain used in this invention possesses excellent complex physiological activities, naturally exhibiting nitrogen fixation, phosphorus solubilization, and potassium solubilization functions. It can activate inert nutrients in the soil that are difficult for plants to absorb and utilize, improving soil nutrient utilization efficiency. Simultaneously, it can secrete auxin IAA and synthesize siderophores, regulating crop growth and development through multiple physiological pathways such as nutrient activation, growth regulation, and micronutrient utilization, providing a stable biological regulatory basis for crop germination, root development, and plant growth. Based on the above-mentioned strain characteristics, appropriate concentrations of the inoculant treatment show good biosafety for various crops such as ginseng, rice, wheat, soybean, rapeseed, carrot, and bok choy. At conventional field application concentrations, it does not cause phytotoxicity to crops and can promote seed germination and normal seedling growth. Only ultra-high concentration treatments may cause slight growth inhibition in some crops. Within the range of conventional field application dosages, crop growth is safe and controllable, adaptable to various tillage and planting patterns, and meets the safety production requirements of ecological planting.

[0024] In terms of disease control, this wettable powder has a stable and highly effective control effect on gray mold and black spot, which are the main diseases occurring in ginseng production. Field pot trials showed that at the standard application concentration, the control effect on both target diseases could reach over 80%. Compared with existing Bacillus subtilis wettable powder and carbendazim chemical agents, the control efficacy can be improved by more than 47%, effectively making up for the shortcomings of weak control efficacy of conventional biological agents and the significant drawbacks of long-term use of chemical agents. Meanwhile, this microbial agent can significantly alleviate the growth inhibition and physiological damage of ginseng plants caused by disease stress, and improve the growth status of plants under disease conditions. The whole plant fresh weight, plant height, root dry weight, root length and root diameter of ginseng treated with the microbial agent are significantly increased, with increases of 44%~65%, 30%~70%, 21%~61%, more than 18% and more than 23%, respectively. By repairing and promoting the growth and development of plants, especially the root system, it enhances the plant's nutrient absorption capacity and stress resistance, effectively improves the crop growth under disease stress, and steadily improves the plant's biomass and growth quality.

[0025] At the soil improvement level, the application of the microbial agent of this invention can systematically optimize the physicochemical properties and biological activity of ginseng cultivation soil. After treatment with the microbial agent, the soil pH is slightly optimized and the electrical conductivity is slightly reduced, which is beneficial to improving the soil salinity accumulation and the imbalance of physicochemical properties. The soil organic matter content increases by 25.07% to 73.53%, and the contents of available nitrogen, available phosphorus, and available potassium are all increased to varying degrees, effectively improving the soil nutrient supply level. At the same time, the activities of key soil metabolic enzymes are significantly improved, with neutral phosphatase, sucrase, and urease activities increasing by more than 24.6%, more than 25%, and 50%, respectively. This can effectively enhance the soil material cycle and nutrient transformation capacity, continuously improve the soil microecological degradation caused by continuous cropping, and promote the restoration and healthy maintenance of soil fertility.

[0026] In summary, the wettable powder prepared by this invention using specific functional strains organically combines disease control, plant growth promotion, and soil improvement functions. The various functions work synergistically and complement each other, solving the problems of single function and limited effect of traditional plant protection products. It has high overall application safety and stable effect, which can meet the technical requirements of green and efficient ginseng cultivation. At the same time, it is applicable to the ecological production of a variety of crops and has good prospects for industrial application. Attached Figure Description

[0027] Appendix Figure 1 The images show the results of the growth-promoting function assay for Bacillus oryzae SZ-4 strain. A represents the morphology of a single colony of Bacillus oryzae SZ-4; B represents the nitrogen-fixing capacity assay for SZ-4 strain; C represents the phosphorus-solubilizing capacity assay for SZ-4 strain; D represents the potassium-solubilizing capacity assay for SZ-4 strain; E represents the siderophore production capacity assay for SZ-4 strain; and F represents the IAA production capacity assay for SZ-4 strain.

[0028] Appendix Figure 2 The following is a graph showing the effect of different WP treatments on the growth of ginseng seedlings in Example 2; where A is the CK (sterile water) treatment; B is the 500-fold dilution of 50% carbendazim WP; C is the 300-fold dilution of 50 billion / g Bacillus subtilis WP; D is the 100-fold dilution of SZ-4 WP; E is the 50-fold dilution of SZ-4 WP; and F is the 25-fold dilution of SZ-4 WP.

[0029] Appendix Figure 3 The following is a diagram showing the effects of different WP treatments on rice seedling growth in Example 2; where A is the CK (sterile water) treatment; B is the 500-fold dilution of 50% carbendazim WP; C is the 300-fold dilution of 50 billion / g Bacillus subtilis WP; D is the 100-fold dilution of SZ-4 WP; E is the 50-fold dilution of SZ-4 WP; and F is the 25-fold dilution of SZ-4 WP.

[0030] Appendix Figure 4 The following is a diagram showing the effects of different WP treatments on wheat seedling growth in Example 2; where A is the CK (sterile water) treatment; B is the 500-fold dilution of 50% carbendazim WP; C is the 300-fold dilution of 50 billion / g Bacillus subtilis WP; D is the 100-fold dilution of SZ-4 WP; E is the 50-fold dilution of SZ-4 WP; and F is the 25-fold dilution of SZ-4 WP.

[0031] Appendix Figure 5 The following is a graph showing the effects of different WP treatments on soybean seedling growth in Example 2; where A is the CK (sterile water) treatment; B is the 500-fold dilution of 50% carbendazim WP; C is the 300-fold dilution of 50 billion / g Bacillus subtilis WP; D is the 100-fold dilution of SZ-4 WP; E is the 50-fold dilution of SZ-4 WP; and F is the 25-fold dilution of SZ-4 WP.

[0032] Appendix Figure 6 The following is a diagram showing the effects of different WP treatments on the growth of rapeseed seedlings in Example 2; where A is the CK (sterile water) treatment; B is the 500-fold dilution of 50% carbendazim WP; C is the 300-fold dilution of 50 billion / g Bacillus subtilis WP; D is the 100-fold dilution of SZ-4 WP; E is the 50-fold dilution of SZ-4 WP; and F is the 25-fold dilution of SZ-4 WP.

[0033] Appendix Figure 7The following is a diagram showing the effects of different WP treatments on the growth of carrot seedlings in Example 2; where A is the CK (sterile water) treatment; B is the 500-fold dilution of 50% carbendazim WP; C is the 300-fold dilution of 50 billion / g Bacillus subtilis WP; D is the 100-fold dilution of SZ-4 WP; E is the 50-fold dilution of SZ-4 WP; and F is the 25-fold dilution of SZ-4 WP.

[0034] Appendix Figure 8 The following is a diagram showing the effects of different WP treatments on the growth of Chinese cabbage seedlings in Example 2; where A is the CK (sterile water) treatment; B is the 500-fold dilution of 50% carbendazim WP; C is the 300-fold dilution of 50 billion / g Bacillus subtilis WP; D is the 100-fold dilution of SZ-4 WP; E is the 50-fold dilution of SZ-4 WP; and F is the 25-fold dilution of SZ-4 WP.

[0035] Appendix Figure 9 The results show the effects of fungicide application on soil pH and electrical conductivity in ginseng under gray mold infection. The left figure shows the effect of pH, and the right figure shows the effect of electrical conductivity. CK represents the blank control group artificially inoculated with pathogens; A represents the SZ-4 WP 100-fold dilution treatment group; B represents the SZ-4 WP 600-fold dilution treatment group; C represents the SZ-4 WP 1000-fold dilution treatment group; D represents the Bacillus subtilis WP 300-fold dilution treatment group; and E represents the carbendazim WP 500-fold dilution treatment group.

[0036] Appendix Figure 10 The results show the effects of fungicide application on soil pH and electrical conductivity in ginseng infected with black spot disease; the left figure shows the effect of pH, and the right figure shows the effect of electrical conductivity; where CK represents the blank control group artificially inoculated with pathogens; A represents the SZ-4 WP 100-fold dilution treatment group; B represents the SZ-4 WP 600-fold dilution treatment group; C represents the SZ-4 WP 1000-fold dilution treatment group; D represents the Bacillus subtilis WP 300-fold dilution treatment group; and E represents the carbendazim WP 500-fold dilution treatment group.

[0037] Appendix Figure 11 The figures show the effects of different treatments on soil nutrient content under gray mold stress. The top left figure shows the effect on surface organic matter, the top right figure shows the effect on available nitrogen, the bottom left figure shows the effect on available phosphorus, and the bottom right figure shows the effect on available potassium. CK represents the blank control group artificially inoculated with pathogens; A represents the SZ-4 WP 100x dilution treatment group; B represents the SZ-4 WP 600x dilution treatment group; C represents the SZ-4 WP 1000x dilution treatment group; D represents the Bacillus subtilis WP 300x dilution treatment group; and E represents the carbendazim WP 500x dilution treatment group.

[0038] Appendix Figure 12 The figures show the effects of different treatments on soil nutrient content under black spot disease stress. The top left figure shows the effect on surface organic matter, the top right figure shows the effect on available nitrogen, the bottom left figure shows the effect on available phosphorus, and the bottom right figure shows the effect on available potassium. CK represents the blank control group artificially inoculated with pathogens; A represents the SZ-4 WP 100x dilution treatment group; B represents the SZ-4 WP 600x dilution treatment group; C represents the SZ-4 WP 1000x dilution treatment group; D represents the Bacillus subtilis WP 300x dilution treatment group; and E represents the carbendazim WP 500x dilution treatment group.

[0039] Appendix Figure 13 The figures show the effects of different treatments on ginseng soil enzyme activity under gray mold stress. The left figure shows the effect on neutral phosphatase, the middle figure shows the effect on urease, and the right figure shows the effect on sucrase. CK represents the blank control group artificially inoculated with pathogens; A represents the SZ-4 WP 100-fold dilution treatment group; B represents the SZ-4 WP 600-fold dilution treatment group; C represents the SZ-4 WP 1000-fold dilution treatment group; D represents the Bacillus subtilis WP 300-fold dilution treatment group; and E represents the carbendazim WP 500-fold dilution treatment group.

[0040] Appendix Figure 14 The figures show the effects of different treatments on ginseng soil enzyme activity under black spot disease stress. The left figure shows the effect on neutral phosphatase, the middle figure shows the effect on urease, and the right figure shows the effect on sucrase. CK represents the blank control group artificially inoculated with pathogens; A represents the SZ-4 WP 100-fold dilution treatment group; B represents the SZ-4 WP 600-fold dilution treatment group; C represents the SZ-4 WP 1000-fold dilution treatment group; D represents the Bacillus subtilis WP 300-fold dilution treatment group; and E represents the carbendazim WP 500-fold dilution treatment group. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0042] In the embodiments of this invention, strain activation was performed using NB medium; phosphorus solubilization capacity was tested using PKO (Pikovaskaia's) medium; potassium solubilization capacity was tested using potassium solubilization medium; siderophore production capacity was tested using CAS (Chrome Azurol S) medium; and nitrogen fixation capacity was tested using Ashby medium.

[0043] In this embodiment of the invention, the detection of indole-3-acetic acid (IAA) was performed using Salkowski's reagent: 10 mL of 0.5 mol / L FeCl3 solution and 500 mL of 35% HClO4. The mixture was shaken well before use and stored away from light. Disodium hydrogen phosphate, sodium tetraborate, potassium chloride, potassium dichromate, sulfuric acid, sodium hydroxide, ferrous sulfate, bromocresol green, methyl red, ethanol, hydrochloric acid, sodium tetraborate, sodium carbonate, phosphorus-free activated carbon, ammonium molybdate, potassium antimony tartrate, ascorbic acid, potassium dihydrogen phosphate, ammonium acetate, lithium chloride, potassium sodium tartrate, sucrose, toluene, glucose, citric acid, potassium hydroxide, phenol, methanol, acetone, sodium hypochlorite, urea, ammonium sulfate, disodium phenyl phosphate, aluminum sulfate, dibromo-p-benzoquinone imine chloride, and 3,5-dinitrosalicylic acid were all analytical grade reagents.

[0044] The 50% carbendazim wettable powder used in this embodiment of the invention was purchased from Sichuan Guoguang Agrochemical Co., Ltd., and a 500-fold dilution is the recommended field application dose; the 5×10¹⁰ CFU / g Bacillus subtilis wettable powder was purchased from Shandong Muyushi Biotechnology Co., Ltd., and a 300-fold dilution is the recommended field application dose.

[0045] In this embodiment of the invention, the ginseng seeds were split seeds from the previous year provided by Jilin Shenboshi Crop Health Technology Co., Ltd., with a splitting rate of 98%; the rice seeds were "Jingeng 616", purchased from Huai'an Jindi Seed Industry Co., Ltd.; the wheat seeds were "Xinxing 169", purchased from Shandong Xinxing Seed Industry Co., Ltd.; the soybean seeds were "Zhonghuang 13", purchased from Liangshan County Jindou Seed Industry Co., Ltd.; the rapeseed seeds were "Huayou 567", purchased from Henan Huayi Seed Industry Co., Ltd.; and the carrot and bok choy seeds were purchased from Cangzhou Jinkeli Seedling Co., Ltd.

[0046] The test soil in this embodiment of the invention was improved soil with a formula of seedling soil: perlite: vermiculite = 2:1:1. Its basic physicochemical properties were tested and found to be: pH 8.04, organic matter content 3.66%. Plate separation method was used to verify that *Botrytis cinerea* and *Alternaria alternata* were not detected. The three-year-old ginseng seedlings used in the test were provided by Jilin Shenboshi Crop Health Technology Co., Ltd.

[0047] The ginseng pathogens in this embodiment of the invention, namely Botrytis cinerea and Alternaria alternata, were provided by the Plant Pathology Laboratory of Jilin Agricultural University.

[0048] The Bacillus oryzae SZ-4 wettable powder used in this invention embodiment was prepared according to Example 1 of patent publication number CN120615916 A. The mass ratio of each raw material in this powder is as follows: 28% Bacillus oryzae SZ-4 fermentation centrifugation precipitate, 56% diatomaceous earth, 6.6% sodium butylnaphthalene sulfonate, 4.4% calcium lignosulfonate, 3% dextrin, and 2% sodium carboxymethyl cellulose (CMC-Na). The above raw material components were accurately weighed, mixed evenly, and then freeze-dried to obtain Bacillus oryzae SZ-4 wettable powder, abbreviated as SZ-4 WP.

[0049] The n-fold solution described in this embodiment of the invention is prepared by mixing 1 part by mass of Bacillus cereus SZ-4 wettable powder with (n-1) parts by mass of water. For example, SZ-4 WP 100-fold solution refers to mixing 1 part by mass of Bacillus cereus SZ-4 wettable powder with 99 parts by mass of water.

[0050] The preparation methods of ginseng pathogenic fungal suspensions, namely *Botrytis cinerea* suspension and *Alternaria alternata* suspension, in this embodiment of the invention are as follows: Activated *Botrytis cinerea* and *Alternaria alternata* suspensions are prepared separately: 2-3 mm from the edge of each colony is taken, and mycelial cakes are cut using an 8 mm diameter punch. Two mycelial cakes are inoculated into each 100 mL of potato dextrose broth (PDB). The mixture is then cultured at 28°C and 150 r / min on a shaker for 4 days. After culture, the mycelium is removed by filtration through sterile gauze, and the filtrate is collected. The spore concentration is determined under a microscope using a hemocytometer, and then diluted with sterile distilled water to 1 × 10⁻⁶. 5 Sporangia / mL, for later use.

[0051] Example 1: This example systematically tests the growth-promoting functional characteristics of the strain SZ-4 of the present invention. The specific experimental methods and results are as follows: 1. Experimental methods.

[0052] 1.1 Phosphorus solubility test.

[0053] The activated strain SZ-4 was inoculated into NB medium and cultured with shaking at 32℃ and 180 rpm for 24 h. 500 μL of the bacterial suspension was used to wet a circular sterile filter paper disc with a diameter of 8 mm. After being dried with sterile air, the filter paper disc was placed in the center of a PKO solid medium. Three culture dishes were set up as biological replicates. The dishes were inverted and placed in a 32℃ incubator for 3–7 days. The formation of phosphate-solubilizing zones on the medium was observed to determine the strain's phosphate-solubilizing ability.

[0054] 1.2 Potassium solubilization capacity test.

[0055] Take a sterile filter paper disc loaded with the above-mentioned strain SZ-4 and place it in the center of a silicate solid medium supplemented with BTB reagent. Set up three culture dishes as biological replicates. Invert the dishes and incubate them in a 32°C incubator for 3–7 days. Observe whether a bright yellow potassium-solubilizing zone appears on the medium to determine the strain's potassium-solubilizing ability.

[0056] 1.3 Testing of iron production capacity.

[0057] Sterile filter paper discs loaded with strain SZ-4 were inoculated into the center of CAS solid medium. Three culture dishes were set up as biological replicates and incubated at 32°C upside down for 3–7 days. The presence of a yellow halo around the medium was observed to determine the strain’s ability to produce siderophores.

[0058] 1.4 Nitrogen fixation capacity test.

[0059] The activated strain SZ-4 was streaked onto Ashby solid medium and cultured at 32°C for 3–7 days. The culture was repeated three times to observe whether strain SZ-4 could grow normally on nitrogen-free Ashby medium, thereby determining the strain's nitrogen-fixing ability.

[0060] 1.5 Detection of indoleacetic acid (IAA) production capacity.

[0061] The activated strain SZ-4 was inoculated into NA liquid medium supplemented with 50 mg / L tryptophan and cultured at 32℃ and 180 rpm for 36 h with shaking. 10 mL of the cultured bacterial suspension was centrifuged at 8000 rpm for 10 min, and the supernatant was collected. 0.5 mL of the supernatant was transferred to a sterile test tube, and 2 mL of Salkowski colorimetric solution was added. After mixing, the sample to be tested was prepared.

[0062] Two control groups were set up: a positive control consisting of a mixture of 0.5 mL of 50 mg / L IAA standard solution and 2 mL of Lalkowski colorimetric solution; and a negative control consisting of a mixture of 0.5 mL of blank NA liquid medium without inoculation of the strain and 2 mL of Lalkowski colorimetric solution. All samples were placed at room temperature and protected from light for 30 min, and the color of the samples was observed and recorded to determine whether the strain could produce indoleacetic acid.

[0063] 2. Experimental results and analysis.

[0064] Colony morphology observation results are as follows Figure 1 As shown in Figure A: The colonies of Bacillus oryzae SZ-4 are generally round with neat edges and regular shape. The colonies are raised and convex, and the surface is smooth and moist. When the strain is cultured to an older stage, irregular wrinkles will appear on the surface of the colonies, and the overall color of the colonies will be milky white.

[0065] Results of nitrogen fixation capacity test of strains as follows Figure 1 As shown in B: After streaking Bacillus oryzae SZ-4 onto Ashby's nitrogen-free medium and subculturing it three times, the strain could still grow normally and be stably passaged, indicating that Bacillus oryzae SZ-4 of the present invention has good nitrogen-fixing ability.

[0066] Results of phosphate solubility test of strains as follows Figure 1 As shown in C: After Bacillus oryzae SZ-4 was inoculated into PKO solid medium and cultured, a clear transparent phosphate-solubilizing zone could be formed around the bacterial colony, proving that the strain has a highly efficient phosphate-solubilizing function.

[0067] The results of the potassium-solubilizing ability test of the strain are as follows Figure 1 As shown in D: After Bacillus oryzae SZ-4 was inoculated into silicate solid medium with BTB chromogenic agent, irregular bright yellow color areas appeared around the bacterial cells, indicating that the strain can decompose silicate components in the medium and has significant potassium solubilization characteristics.

[0068] Results of the test for the siderophore production capacity of the strain are as follows Figure 1 As shown in E, Bacillus cereus SZ-4 can grow stably on CAS detection solid medium, and a distinct pale yellow halo can be produced around the colony, confirming that this strain can secrete siderophores and has iron-loving properties.

[0069] Qualitative test results of the strain's ability to produce indoleacetic acid (IAA) are as follows: Figure 1 As shown in F: According to the Salkowski colorimetric method, the test supernatant system inoculated with Bacillus oryzae SZ-4 showed a distinct light pink color reaction, which was significantly different from the negative control blank system, proving that this strain can synthesize and secrete indoleacetic acid (IAA) and has the ability to produce IAA.

[0070] In summary, Bacillus cereus SZ-4 not only has stable colony morphology, but also possesses multiple plant growth-promoting functions, including nitrogen fixation, phosphorus solubilization, potassium solubilization, iron carrier production, and secretion of indoleacetic acid (IAA). Its functional traits are comprehensive and stable, demonstrating good potential for agricultural growth-promoting applications.

[0071] Example 2: In this example, plant seeds were treated with Bacillus cereus SZ-4 wettable powder (SZ-4 WP) at different dilution ratios. A sterile water blank control, a biological agent control, and a chemical agent control were set up simultaneously to systematically explore the effects of this wettable powder on plant seed germination and seedling growth, and to clarify its growth-promoting effect and field application safety.

[0072] The experiment used seeds of seven crops: ginseng, rice, wheat, soybean, rapeseed, carrot, and bok choy. The specific experimental procedures for each type of seed are detailed below.

[0073] 1. Test treatment settings.

[0074] This experiment consisted of 6 treatment groups, with 5 biological replicates for each treatment group. The specific treatment methods are as follows: Treatment group 1 (1× concentration): SZ-4 WP 100 times dilution; Treatment group 2 (2× concentration): SZ-4 WP 50 times dilution; Treatment group 3 (4× concentration): SZ-4 WP 25 times dilution; Blank control group (CK): sterile water; Biological control group: 5 × 10⁶ effective viable bacteria 10 CFU / g Bacillus subtilis wettable powder 300 times dilution; Chemical control group: 50% carbendazim wettable powder, diluted 500 times.

[0075] 2. Seed pretreatment.

[0076] Select healthy plant seeds that are plump, uniform in size, undamaged, free from disease and pests, and exhibit consistent growth as test materials. First, soak them in 75% alcohol for 30 seconds, then soak them in 5% NaClO solution for 1 minute, and finally rinse them 5 times with sterile distilled water until there is no odor. Drain the water and set aside.

[0077] After the seeds were disinfected and drained, they were completely immersed in the corresponding treatment solutions of each group and soaked at room temperature for 24 hours. After soaking, the seeds were removed and allowed to drain naturally to remove any residual solution from the surface. This completed the seed pretreatment and was ready for subsequent germination and seedling emergence tests.

[0078] 3. Seed germination test.

[0079] Using 9 cm petri dishes, the bottom was lined with double layers of filter paper, and 5 mL of sterile water was added to moisten the bottom. The pretreated seeds from each group were evenly placed on the filter paper in their respective petri dishes, with 20 seeds evenly placed in each dish. All petri dishes were placed in an artificial climate incubator for constant temperature and light incubation. The incubation program was set as follows: 18℃ dark incubation for 16 h, followed by 24℃, 1000 Lx light incubation for 8 h, with a day-night cycle.

[0080] The day the seeds were placed in the incubator was designated as day 0. The number of germinated seeds in each group was recorded daily at 12:00 PM. The germination criterion was that the length of the taproot or shoot exceeded half of the seed's maximum length. Data was collected continuously for 7 days. After the experiment, the data from each group were summarized, and the seed germination rate, germination potential, and germination index were calculated. The formulas for each indicator are as follows: Germination rate (G) = Number of germinated seeds / Total number of seeds tested × 100%.

[0081] Germination potential (GP) = Total number of seeds that germinate within a specified time / Total number of seeds tested × 100%.

[0082] Germination Index (GI) = ∑ (number of germinated germinations at different times / corresponding number of germination days).

[0083] 4. Seed germination and seedling growth experiment.

[0084] The safety and growth-promoting effects of germination and seedling development were tested using an indoor pot cultivation method. The cultivation substrate was a 2:1 ratio of fresh forest soil to vermiculite, divided into 8 cm × 8 cm seedling pots (filled to a height of 5 cm). Ten pre-treated seeds were placed in each pot, initially watered with 15 mL of sterile water, and then covered with a 1 cm layer of cultivation substrate. All seedling pots were placed in an artificial climate incubator with the same parameters as the germination test, maintaining consistent environmental conditions.

[0085] After seed germination, each pot was thinned to 5 seedlings of uniform growth, with the day of thinning recorded as day 0. On day 7, according to the corresponding pesticide treatment method for each seed group, 15 mL of the corresponding treatment solution was applied as a supplementary watering. During this period, sterile water was sprayed regularly to keep the substrate moist. After 21 days, the growth status of the seedlings was observed and recorded, and the symptoms of pesticide damage were described (refer to Table 1). The degree of pesticide damage was determined according to the standards in Table 2. At the same time, plant height and root length were measured.

[0086] Table 1. Description of symptoms of pesticide damage.

[0087] .

[0088] Table 2. Determination of the degree of drug damage.

[0089] .

[0090] 5. Experimental Results and Analysis.

[0091] This experiment used ginseng, rice, wheat, soybean, rapeseed, carrot, and bok choy as test materials. Through seed germination tests and seedling pot growth tests, the safety and growth-promoting effects of SZ-4 WP on the growth of various plants were systematically evaluated. The specific experimental results are as follows: 5.1 Effects on ginseng seed germination and seedling growth.

[0092] The effects on ginseng are shown in Table 3 and Figure 2 As shown in Table 3, there are significant differences in the regulatory effects of different concentrations of SZ-4 WP on ginseng seed germination and seedling growth indicators. The germination rate of ginseng seeds under each concentration of SZ-4 WP treatment reached over 93%, indicating a good overall germination foundation. The germination rate of seeds treated with 100 times dilution of SZ-4 WP was consistent with the blank control group, but the germination potential and germination index were significantly better than all other treatments, demonstrating strong seed germination vitality and high seedling uniformity. It also effectively promoted seedling root growth, with a root-promoting effect superior to 300 times dilution of Bacillus subtilis, 500 times dilution of carbendazim, and high concentrations of SZ-4 WP. The 25 times dilution of SZ-4 WP treatment showed the lowest levels of germination indicators among the SZ-4 WP gradient treatments, exhibiting a slight inhibitory effect on ginseng seed germination and resulting in a reduced seed germination rate.

[0093] Table 3. Effects of different treatments on ginseng seed germination indices.

[0094] .

[0095] Note: In Table 3, A is the treatment with SZ-4 WP at 100 times dilution, B is the treatment with SZ-4 WP at 50 times dilution, C is the treatment with SZ-4 WP at 25 times dilution, D is the treatment with Bacillus subtilis WP at 300 times dilution, and E is the treatment with carbendazim WP at 500 times dilution. In Tables 4 to 9, the treatment groups A, B, C, D, and E are the same as in Table 3.

[0096] Depend on Figure 2 It was found that the leaf color and morphology of ginseng seedlings in all treatments were normal, and there were no visible symptoms of phytotoxicity. The results of the investigation 21 days after application showed that, except for the 25-fold dilution of SZ-4 WP, the root length of the other treatments was not significantly different from that of the control (CK), with a root length range of 36.11~43.99 mm. This indicates that low concentrations of SZ-4 WP (100-fold and 50-fold dilutions) did not affect the normal growth of ginseng roots, while high concentrations of SZ-4 WP (25-fold dilution) had a significant effect on root length. The plant height of the 50-fold dilution, 25-fold dilution, and 500-fold dilution treatments of carbendazim WP were significantly different from that of the control (P<0.05), indicating that the experimental dosage had a certain impact on the plant height growth of ginseng seedlings, but did not reach the standard of moderate phytotoxicity (refer to Table 2 for the determination of phytotoxicity level), and the overall application was safe.

[0097] 5.2 Effects on rice seed germination and seedling growth.

[0098] The effects on rice are shown in Table 4 and Figure 3As shown in Table 4, compared with the blank control group, treatments with 100-fold dilution of SZ-4 WP and 300-fold dilution of Bacillus subtilis had no significant effect on the germination rate of rice seeds. However, treatment with 100-fold dilution of SZ-4 WP effectively improved the germination potential and germination index of rice seeds, showing a significant promoting effect on seed germination. The 25-fold dilution treatment group had the lowest germination indices, with germination rate, germination potential, and germination index of 76.67%, 1.67%, and 9.30%, respectively, exhibiting a slight inhibitory effect on rice seed germination.

[0099] Table 4. Effects of different treatments on rice seed germination indices.

[0100]

[0101] Depend on Figure 3 It was observed that the number of tillers and leaf color of rice seedlings in each treatment group were normal, and no visible symptoms of phytotoxicity were observed. Results measured 21 days after application showed that the plant height of rice seedlings in each SZ-4 WP treatment group was not significantly different from the blank control group, indicating that SZ-4 WP did not affect the aboveground growth of rice plants. The root length of seedlings in different concentrations of SZ-4 WP treatment groups was significantly higher than that in the blank control group (P < 0.05), confirming that SZ-4 WP has a significant promoting effect on the root growth of rice seedlings.

[0102] 5.3 Effects on wheat seed germination and seedling growth.

[0103] The effects on wheat are shown in Table 5 and Figure 4 As shown in Table 5, compared with the blank control group, except for the SZ-4WP 25-fold dilution treatment group which showed a 4% decrease in wheat seed germination rate, all other treatment groups achieved a 100% germination rate, with no significant germination inhibition. The germination potential and germination index showed consistent trends across groups, with the SZ-4WP 100-fold dilution treatment group exhibiting the highest germination potential and germination index at 50% and 13.38 respectively, significantly superior to the blank control group (40% and 12.45), indicating that this concentration effectively improves the uniformity and speed of wheat seed germination. The SZ-4WP of this invention demonstrates outstanding performance in promoting wheat root elongation and early germination activity, outperforming both biological and chemical controls.

[0104] Table 5. Effects of different treatments on wheat seed germination indices.

[0105] .

[0106] Depend on Figure 4It was found that the wheat seedlings in all treatment groups were growing well, with no visible symptoms of herbicide damage. A survey 21 days after application showed that the seedling height of the 300-fold dilution of Bacillus subtilis and the 500-fold dilution of carbendazim treatment groups was not significantly different from the control group. The seedling height of the SZ-4 WP treatment groups was slightly lower than the control group, but none met the criteria for herbicide damage, indicating no substantial growth inhibition. Regarding root indicators, the root length of wheat seedlings in all SZ-4 WP treatment groups was superior to that in the control group. The 50-fold dilution treatment group had the longest root length, reaching 181.46 mm, an increase of approximately 30% compared to the control group, demonstrating a significant promoting effect on wheat root elongation (P < 0.05).

[0107] 5.4 Effects on soybean seed germination and seedling growth.

[0108] The effects on soybeans are shown in Table 6 and Figure 5 As shown in Table 6, among soybean seed germination rates, the SZ-4 WP 25-fold dilution treatment had the lowest germination rate at 83.33%, while the remaining treatments all exceeded 90%, close to the CK (96.67%). Germination potential and germination index showed consistent trends. The SZ-4 WP 100-fold dilution treatment had significantly higher germination potential and germination index (71.67% and 13.98, respectively) than the CK (63.33% and 12.70), demonstrating a germination-promoting effect. The Bacillus subtilis WP 300-fold dilution treatment had the lowest germination potential (28.33%), but did not affect the final germination rate.

[0109] Table 6. Effects of different treatments on soybean seed germination indices.

[0110]

[0111] Depend on Figure 5 It was found that the higher the concentration of SZ-4WP, the more pronounced the dwarfing trend in soybean seedlings. A survey conducted 21 days post-application showed no significant difference in root length between all treatments and the control (CK), indicating that the tested agent had no significant effect on soybean root growth. Regarding plant height, the 25-fold dilution of SZ-4WP (80.06 mm) was significantly lower than the CK (118.24 mm) (P<0.05), showing a slight inhibitory effect, but not reaching the phytotoxicity standard.

[0112] 5.5 Effects on rapeseed seed germination and seedling growth.

[0113] The effects on rapeseed are shown in Table 7 and Figure 6As shown in Table 7, the germination rate of rapeseed seeds decreased with increasing SZ-4 WP concentration: the germination rate of CK was 91.67%, slightly increased to 93.33% under 100-fold SZ-4 WP treatment, and decreased to 73.33% under 25-fold SZ-4 WP treatment, indicating that high concentrations of the inoculant had a slight inhibitory effect on germination. Germination potential and germination index showed the same trend; the 100-fold SZ-4 WP treatment (36.67%, 11.81) was significantly higher than CK (25.00%, 10.93), indicating that 100-fold SZ-4 WP could promote early seed germination growth.

[0114] Table 7. Effects of different treatments on rapeseed seed germination indices.

[0115] .

[0116] Depend on Figure 6 It was observed that only the SZ-4 WP 25x dilution treatment group showed slight uneven emergence; the seedlings in the other treatment groups grew uniformly and were in good condition, with no visible phytotoxicity symptoms. A survey 21 days after application showed that the root length of rapeseed seedlings in the 500x dilution carbendazim treatment group was 58.15 mm, significantly lower than the 69.11 mm in the control group (P < 0.05), indicating root suppression. The root length of seedlings in all SZ-4 WP treatment groups was significantly better than the control group (P < 0.05), with the 100x dilution treatment group having the longest root length at 87.52 mm, demonstrating a significant root-promoting effect. Regarding plant height, only the SZ-4 WP 25x dilution treatment group was significantly shorter than the control group; the other treatment groups showed no significant difference from the control group.

[0117] 5.6 Effects on carrot seed germination and seedling growth.

[0118] The effects on carrots are shown in Table 8 and Figure 7 As shown in Table 8, different treatments had varying effects on the germination indices of carrot seeds. The blank control group (CK) showed the best germination rate (96.67%), germination potential (13.33%), and germination index (10.55) among all treatment groups, indicating optimal seed vigor under this treatment. In contrast, the SZ-4WP 25-fold dilution treatment had the lowest germination rate (78.33%), germination potential (0.00), and germination index (8.08), indicating a certain degree of inhibitory effect at this concentration. Bacillus subtilis WP 300 and carbendazim WP 500 showed germination rates similar to CK (91.67% and 95.00%, respectively), but their germination potential and germination index were lower, indicating that these two agents slowed down the germination rate of carrot seeds.

[0119] Table 8. Effects of different treatments on carrot seed germination indices.

[0120] .

[0121] Depend on Figure 7 It was found that the carrot seedlings in all treatment groups showed good growth and no visible signs of pesticide damage. Results measured 21 days after application showed significant differences in root length and plant height among the groups (P < 0.05). The SZ-4 WP 50x dilution treatment group had the longest root length (57.09 mm), significantly better than the other treatment groups, effectively promoting root elongation. The Bacillus subtilis 300x dilution and carbendazim 500x dilution treatment groups had root lengths of 43.99 mm and 44.28 mm, respectively, showing a slight inhibitory effect on carrot root growth. Regarding plant height, only the SZ-4 WP 25x dilution treatment group had a plant height of 48.39 mm, significantly lower than the blank control group (57.28 mm); the other treatment groups showed no significant differences in plant height.

[0122] 5.7 Effects on seed germination and seedling growth of Chinese cabbage.

[0123] The effects on bok choy are shown in Table 9 and Figure 8 As shown in Table 9, the effects of different treatment groups on the germination of pak choi seeds varied significantly. Except for the SZ-4 WP 50-fold dilution (93.33%) and 25-fold dilution (78.33%) treatment groups, the germination rate of all other treatment groups reached 100%, consistent with the blank control group. This indicates that under appropriate concentrations of SZ-4 WP, treatment does not adversely affect the normal germination of pak choi seeds. The SZ-4 WP 100-fold dilution treatment group had the highest germination potential at 61.67%, significantly higher than the blank control group's 41.67% (P < 0.05), effectively improving the initial germination speed and uniformity of pak choi seeds, and enhancing seed germination vigor. With increasing SZ-4 WP concentration, the growth-promoting effect gradually weakened, and high concentrations showed a slight germination-inhibiting trend.

[0124] Table 9. Effects of different treatments on germination indices of Chinese cabbage seeds.

[0125] .

[0126] Depend on Figure 8It was found that only the SZ-4 WP 25x dilution treatment group showed slight growth abnormalities in the pakchoi seedlings, while the seedlings in the other treatment groups grew well with no visible phytotoxicity symptoms. Results 21 days after application showed significant differences in the effects of different treatments on root length and plant height of the pakchoi seedlings (P < 0.05). Among the root length indicators, the SZ-4 WP 100x dilution treatment group had the longest root length, reaching 84.23 mm, significantly better than the blank control group, demonstrating a prominent root-promoting effect. The Bacillus subtilis 300x dilution treatment group had the highest seedling height, at 98.95 mm, significantly higher than the blank control group (85.70 mm) and other treatment groups, promoting the growth of the above-ground parts of the pakchoi plant. The high-concentration SZ-4 WP (25x and 50x dilution) treatment groups showed a significant decrease in seedling height, indicating that SZ-4 WP has a certain inhibitory effect on the growth of pakchoi seedlings with increasing application concentration.

[0127] In summary, the results of this embodiment demonstrate that SZ-4 WP exhibits both safety and growth-promoting effects on ginseng and various plants at appropriate concentrations. Specifically, a 100-fold dilution significantly improved the germination potential or germination index of ginseng, rice, soybeans, and bok choy, and promoted root growth in wheat and rapeseed. No visible phytotoxicity symptoms were observed in any of the tested crops, indicating that SZ-4 poses extremely low risk to non-target crops and has high safety. However, when the concentration increased to 25 times the concentration, the germination rate and plant height of crops such as rice, carrots, and bok choy showed a slight decrease, suggesting that high concentrations of SZ-4 may have mild toxicity to seed germination and seedling growth. Therefore, in practical applications, the application concentration should be strictly controlled, and a 100-fold dilution is recommended as an appropriate dosage to avoid the adverse effects of high concentrations.

[0128] Example 3: This example systematically studies the outdoor potted plant control effect of the wettable powder SZ-4WP of the present invention.

[0129] 1. Test treatment settings.

[0130] This experiment included a total of 6 treatment groups: (1) Artificially inoculated pathogen blank control group (CK); (2) SZ-4 WP 100 times dilution; (3) SZ-4 WP 600 times dilution; (4) SZ-4 WP 1000 times dilution; (5) Bacillus subtilis WP 300 times dilution; (6) Carbendazim WP 500 times dilution.

[0131] 2. Experimental methods.

[0132] Three-year-old ginseng seedlings with uniform growth and free from pests and diseases were selected for the experiment and planted in PP plastic pots with a diameter of 30 cm and a height of 28 cm. The cultivation substrate ratio was seedling soil: perlite: vermiculite = 2:1:1. Three ginseng seedlings were planted in each pot, and the substrate weight of each pot was kept consistent. The potted plants were placed uniformly in an outdoor ginseng shed and maintained using conventional field cultivation management methods. After the ginseng entered the leaf expansion stage, healthy and disease-free plants with uniform growth were selected for efficacy testing.

[0133] Before the experiment, healthy ginseng leaves of uniform size and growth were selected. Using a sterile inoculation needle, wounds were gently made on the leaves, avoiding the veins, with five inoculation wounds on each leaf. The fungicide was applied by foliar spraying until the leaves of different treatments were evenly covered with the solution. 24 hours after application, 10 μL of pathogenic spore suspension was dripped into the leaf wounds. The fungicide was sprayed every 7 days during the experiment, for a total of 4 applications.

[0134] The experiment adopted a randomized group design, with 5 ginseng plants and 50 leaves in each treatment group, and 3 biological replicates. The routine field management measures such as water and fertilizer, temperature and humidity, and weeding were uniform throughout the experiment to ensure consistent experimental conditions and eliminate external interference.

[0135] Thirty days after pathogen inoculation, the disease incidence of ginseng plants in each treatment group was investigated and recorded. The disease was graded based on the percentage of lesion area on a single leaf of each palmate compound leaf relative to the total leaf area. The total number of leaves, the number of diseased leaves at each grade, and the disease index and control effect were calculated. The relevant calculation formulas are as follows: Disease index = ∑ (number of diseased leaves at each level × disease level value / total number of leaves × highest level value) × 100 Prevention efficacy (%) = (Disease index of control group - Disease index of treatment group / Disease index of control group) × 100 The severity of ginseng black spot disease is divided into 9 levels, as shown in Table 10.

[0136] Table 10 Grading standards for the severity of ginseng black spot disease.

[0137]

[0138] The severity of ginseng gray mold disease is divided into 9 levels, as shown in Table 11.

[0139] Table 11 Grading criteria for ginseng gray mold disease.

[0140]

[0141] 3. Experimental results.

[0142] 3.1 The control effects of different treatments on ginseng gray mold are shown in Table 12. The disease index of the control group (CK) was 65.93. Under different dilutions of Bacillus subtilis SZ-4 WP (treatments A, B, and C), the disease indices were 12.81, 37.92, and 48.89, respectively, with control effects of 80.56%, 42.48%, and 25.85%. Treatment A showed significantly improved control efficacy compared to Bacillus subtilis WP 300 times dilution (D) and carbendazim WP 500 times dilution (E). This indicates that Bacillus subtilis SZ-4 WP 100 and 600 times dilution can effectively control ginseng gray mold caused by Botrytis cinerea, and the control effect is significantly better than that of conventional biological agents Bacillus subtilis and conventional chemical agents carbendazim. P <0.05).

[0143] Table 12. Control effects of different treatments on gray mold in ginseng.

[0144]

[0145] Note: CK: Control group artificially inoculated with pathogens; A: SZ-4 WP 100x dilution; B: SZ-4 WP 600x dilution; C: SZ-4 WP 1000x dilution; D: Bacillus subtilis WP 300x dilution; E: Carbendazim WP 500x dilution. Data in the table are mean ± standard deviation, tested using the LSD method (P < 0.05 level).

[0146] 3.2 The control effects of different treatments on ginseng black spot disease are shown in Table 13. 30 days after inoculation with *Alternaria alternata*, the disease index of ginseng under the CK treatment was 71.85. Under different dilutions of *Bacillus subtilis* SZ-4 WP treatments (A, B, C), the disease indices were 13.70, 17.26, and 35.41, respectively. The control effects on ginseng black spot disease caused by *Alternaria alternata* were 80.93%, 75.98%, and 50.72%, respectively, all significantly higher than those treated with *Bacillus subtilis* WP 300x dilution (D) and carbendazim WP 500x dilution (E). P <0.05%, and the control efficacy increased by 12.68%, indicating that SZ-4 WP is significantly more effective than conventional biological agents Bacillus subtilis and conventional chemical agents carbendazim in controlling ginseng black spot disease. P <0.05). This indicates that at the tested concentration, SZ-4 WP showed better control of ginseng black spot disease caused by Alternaria alternata.

[0147] Table 13 shows the prevention and control of black spot disease in ginseng under different treatments.

[0148]

[0149] In Table 13, treatment groups A, B, C, D, and E are the same as those in Table 12.

[0150] The results of this embodiment show that the 100-fold dilution of SZ-4 WP has a control efficacy of over 80% against gray mold and black spot disease in ginseng, which is significantly better than that of conventional biocontrol agents such as Bacillus subtilis WP and carbendazim WP.

[0151] Example 4: This example focuses on the SZ-4 WP of the present invention and conducts a systematic experimental study on its effect on the growth performance of ginseng under pathogenic stress. The study verifies in detail the regulatory effect of SZ-4 WP at different dilutions on the growth indicators of ginseng plants under Botrytis cinerea and Alternaria infection stress. The specific experimental process and results are as follows.

[0152] 1. Test treatment settings.

[0153] This experiment included a total of 6 treatment groups: (1) Artificially inoculated pathogen blank control group (CK); (2) SZ-4 WP 100 times dilution; (3) SZ-4 WP 600 times dilution; (4) SZ-4 WP 1000 times dilution; (5) Bacillus subtilis WP 300 times dilution; (6) Carbendazim WP 500 times dilution.

[0154] 2. Experimental methods.

[0155] When the ginseng leaves expanded, each group of ginseng plants underwent root irrigation with 50 mL of pesticide solution per plant; the control group (CK) received an equal amount of 50 mL of sterile water per plant. Fifteen ginseng plants were used as replicates for each treatment group. Twenty-four hours after pesticide treatment, all plants were uniformly inoculated with 50 mL of pathogen spore suspension for artificial inoculation stress; subsequent irrigations were performed every 7 days for a total of four applications. Thirty days after pathogen inoculation, the field growth status and various growth indicators of each group of ginseng plants were systematically investigated, observed, and recorded.

[0156] After the experiment, soil samples from the rhizosphere of ginseng plants in each group were collected and sieved through a 20-mesh sieve to form two samples: one soil sample was stored in a -20 ℃ low-temperature environment for subsequent soil enzyme activity detection and determination (Example 5); the other soil sample was air-dried in a cool and ventilated place for basic soil physicochemical property detection and analysis.

[0157] Fresh, intact ginseng plant samples were collected from each group. Surface impurities were removed by rinsing with clean water, and residual moisture was absorbed using filter paper. Growth indicators were then measured: the fresh weight of the whole ginseng plant and the fresh weight of the roots were weighed and recorded. Plant height, taproot length, and taproot thickness were precisely measured and recorded using calipers. After completing these morphological measurements, the ginseng plants were dried in an oven at low temperature until constant weight, and the dry weight of the roots was weighed and recorded. Finally, the dried ginseng plants were pulverized, passed through a 65-mesh sieve, and sealed for later testing.

[0158] 3. Experimental results.

[0159] 3.1 Effects of SZ-4WP on ginseng growth performance under Botrytis cinerea stress.

[0160] Table 14 shows the effects of different pesticide treatments on various growth indicators of ginseng under the disease stress condition of artificial inoculation with Botrytis cinerea. As shown in Table 14, treatment C (SZ-4 WP 1000x dilution) had the highest whole plant fresh weight (15.22 g), which increased by 44% compared to the control (CK). There were no significant differences in whole plant fresh weight among treatments A (SZ-4 WP 100x dilution), B (SZ-4 WP 600x dilution), and E (carbendazim WP 500x dilution) (P>0.05). Treatment D (Bacillus subtilis WP 300x dilution) showed no significant difference in whole plant fresh weight compared to the control (CK). Ginseng plant height increased by more than 30% compared to the control (CK) in treatments A and B, showing a significant growth-promoting effect. The plant height of the other treatments (C, D, and E) was at the same level as the control (23-27 cm), with no significant improvement effect.

[0161] Results of ginseng root growth indicators showed that treatment C had the highest fresh and dry root weights among all groups, significantly increasing by 26% and 61% respectively compared to the blank control group (P<0.05), and was significantly superior to all other experimental treatment groups. Treatments A and B showed approximately 20% higher dry root weights than the control group (CK), indicating that SZ-4 WP can effectively promote ginseng root growth and development under gray mold stress. Treatments D and E showed no significant difference in root yield indicators compared to the control group (CK), and did not exhibit a significant growth-promoting effect.

[0162] Among the root morphology indicators, the ginseng root length of treatment B was the longest, at 21.73 cm, which was significantly better than that of the blank control group (16.96 cm). The ginseng root diameter of treatment C was the largest (15.69 mm), which was significantly higher than that of treatments CK and D. The root diameters of treatments A and B were 14.55 mm and 14.71 mm, respectively, both of which were significantly higher than that of treatment D (11.74 mm).

[0163] Table 14 shows the effects of different treatments on ginseng growth under gray mold stress.

[0164] .

[0165] Note: CK: Control group artificially inoculated with pathogens; A: SZ-4 WP 100x dilution; B: SZ-4 WP 600x dilution; C: SZ-4 WP 1000x dilution; D: Bacillus subtilis WP 300x dilution; E: Carbendazim WP 500x dilution. Data in the table are mean ± standard deviation, tested using the LSD method (P < 0.05 level).

[0166] In summary, the experimental results show that under Botrytis cinerea stress, 100-fold and 600-fold dilutions of SZ-4 WP effectively alleviated the inhibitory effect of Botrytis cinerea on ginseng plant growth, significantly improving core growth indicators such as whole plant fresh weight, plant height, root length, and root dry weight. A 1000-fold dilution of SZ-4 WP wettable powder significantly promoted root thickening and increased root weight, but had no significant effect on plant height. Conventional chemical fungicides such as carbendazim wettable powder and conventional biological control agents such as Bacillus subtilis wettable powder showed no significant improvement or growth-promoting effect on any of the growth indicators of ginseng under Botrytis cinerea stress.

[0167] 3.2 Effects of SZ-4WP on ginseng growth performance under Alternaria stellatus stress.

[0168] Table 15 shows the effects of different treatments on ginseng growth indicators under the stress of ginseng black spot disease induced by artificial inoculation with Alternaria alternata. Table 15 indicates that treatments A (SZ-4 WP 100x dilution) and B (SZ-4 WP 600x dilution) produced the best whole-plant fresh weight of ginseng, increasing by more than 65% compared to the blank control group, and significantly better than treatments C, D, and E (P<0.05). Treatment B resulted in a ginseng plant height of 42.60 cm, more than 70% higher than the blank control group's 24.81 cm, significantly higher than all other treatment groups. Treatments C, D, and E showed no significant difference in plant height and whole-plant fresh weight compared to the blank control group (P>0.05), indicating no significant improvement in growth.

[0169] There were no significant differences in the fresh weight of ginseng roots among the groups (P>0.05). Regarding root dry weight, treatment A had the highest value, increasing by 21% compared to the control group, a significant difference. The root dry weights of treatments B, C, D, and E ranged from 2.09 g to 2.17 g, all significantly higher than treatment E, but not significantly different from treatment A. In terms of root length, treatment A had the longest root length at 22.32 cm, increasing by more than 18% compared to the control group, significantly better than the control group and treatments D and E.

[0170] The results of root diameter analysis showed no significant differences in root diameter among treatment groups A, B, C, and D, and all were significantly higher than the blank control group and treatment group E. Among them, treatment B had the largest root diameter, at 15.74 mm, which was more than 27% higher than the blank control group. Treatment C had a root diameter of 15.25 mm, but its plant height was not significantly different from the blank control group, indicating that 1000-fold diluted SZ-4 WP can specifically promote root growth and thickening of ginseng under black spot disease stress.

[0171] Table 15 shows the effects of different treatments on ginseng growth under black spot disease stress.

[0172] .

[0173] In Table 15, treatment groups A, B, C, D, and E are the same as those in Table 14.

[0174] The results of the above experiments indicate that under the stress of Alternaria alternata-induced ginseng black spot disease, 100-fold and 600-fold dilutions of SZ-4 WP effectively alleviated the inhibitory effect of the disease on ginseng plant growth, and significantly promoted aboveground plant height, overall biomass, and root growth. A 1000-fold dilution of SZ-4 WP wettable powder primarily promoted thickening of ginseng roots. However, conventional chemical agents such as carbendazim wettable powder and biological control agents such as Bacillus subtilis wettable powder showed no significant improvement in any growth indicators of ginseng under black spot disease stress.

[0175] Pathogenic fungal infections can severely inhibit plant growth and development. The results of this example show that treatment with 100-fold and 600-fold dilutions of SZ-4 WP significantly alleviated the inhibitory effects of gray mold and black spot on ginseng growth, resulting in ginseng's whole plant fresh weight, plant height, and root dry weight being close to or even better than the blank control group. In contrast, Bacillus subtilis and carbendazim wettable powder did not show any growth-promoting effect. These results indicate that SZ-4 WP can achieve a synergistic effect of "disease prevention and growth promotion" in practical applications.

[0176] Example 5: Based on the experimental system of Example 4, this example collects soil samples from each group as the research object to further explore the regulatory effects of different dilutions of SZ-4 WP on the physicochemical properties, soil nutrients, and soil enzyme activities of ginseng-growing soil under Botrytis cinerea and Alternaria infection stress. It elucidates the mechanism of action of the present invention SZ-4 WP in alleviating soil-borne diseases of ginseng and improving the rhizosphere soil environment. The specific experimental methods and results are as follows.

[0177] 1. Test methods.

[0178] 1.1 Determination of soil physicochemical properties.

[0179] Soil physicochemical indicators were tested according to the standard methods in "Soil Agrochemical Analysis and Environmental Monitoring" (Yang Jianhong). Soil suspensions were prepared using the international standard soil-to-water ratio of 1:5, and soil pH and electrical conductivity (EC) were measured using an ion analyzer. Soil organic matter (OM) content was determined using the potassium dichromate volumetric method; soil available nitrogen (AHN) content was determined using the alkaline hydrolysis-diffusion method; soil available phosphorus (AP) content was determined using the sodium bicarbonate extraction colorimetric method; and soil available potassium (AK) content was determined using flame atomic absorption spectrophotometry.

[0180] 1.2 Determination of soil enzyme activity.

[0181] Soil enzyme activity was determined according to the standard methods in "Soil Enzymes and Their Research Methods" (Guan Songyin): Soil sucrase (S-SC) activity was determined by the 3,5-dinitrosalicylic acid colorimetric method; soil urease (S-UE) activity was determined by the indophenol colorimetric method; and soil neutral phosphatase (S-NP) activity was determined by the disodium phenyl phosphate method.

[0182] In this embodiment, all experimental treatment groups are uniformly as follows: CK is the blank control group artificially inoculated with pathogens; A is the SZ-4WP 100-fold dilution treatment group; B is the SZ-4 WP 600-fold dilution treatment group; C is the SZ-4 WP 1000-fold dilution treatment group; D is the Bacillus subtilis WP 300-fold dilution treatment group; and E is the carbendazim WP 500-fold dilution treatment group.

[0183] 2. Effects of different treatments on soil pH and electrical conductivity of ginseng under pathogen stress.

[0184] 2.1 Stress conditions for Botrytis cinerea (gray mold).

[0185] The effects of different treatments on soil pH in ginseng cultivation under Botrytis cinerea infection stress are as follows: Figure 9 As shown in the left figure, there are significant differences in pH values ​​among the different treatment groups. P <0.05. There was no significant difference between treatment A (7.85) and treatment E (7.84). P >0.05). Compared with CK (7.93), the soil pH values ​​of treatments A, B (7.87), C (7.89) and E were all slightly decreased. As the dilution factor of SZ-4 WP treatment increased from 100 times to 1000 times, the soil pH value showed a slight upward trend (from 7.85 to 7.89).

[0186] The effects of different treatments on soil electrical conductivity (EC) in ginseng cultivation, such as Figure 9The right figure shows the soil electrical conductivity (EC) of each treatment group, which ranged from 449.33 to 457.33 μS / cm. Compared with the control group (454.66 μS / cm), treatments A, B, C, and E significantly reduced soil electrical conductivity, with treatment A showing the lowest conductivity at 449.33 μS / cm. Treatments B and C showed no significant difference in conductivity, indicating that the regulatory effect of SZ-4 WP on soil soluble salt content tended to stabilize within this dilution range. Treatment D had a conductivity of 457.33 μS / cm, significantly higher than the control group.

[0187] 2.2 Stress conditions of Alternaria (black spot disease).

[0188] The effects of different treatments on soil pH in ginseng cultivation under Alternaria infection stress are as follows: Figure 10 As shown in the left figure, there were significant differences in soil pH values ​​among the treatment groups (P < 0.05). Treatment B showed the largest decrease in soil pH compared to the other treatment groups, dropping from 7.92 (CK) to 7.79. Compared to CK, SZ-4 WP (B, C), commercially available agents D, and E all reduced soil pH to some extent. The pH value of treatment A (7.96) was slightly higher than that of treatments CK and C. This indicates that different exogenous additive treatments can bring slightly alkaline soils closer to neutral or slightly acidic conditions.

[0189] The effects of different treatments on soil electrical conductivity are as follows: Figure 10 As shown in the right figure, the soil electrical conductivity values ​​in this experiment ranged from 447.00 to 456.33 μS / cm. Compared with the control (CK), the electrical conductivity of treatments B, D, and E all decreased significantly. P <0.05), with treatment B showing the lowest conductivity at 447.00 μS / cm. The conductivity values ​​of treatments A and C were similar, indicating that SZ-4 WP had a stable regulatory effect on the soil conductivity of ginseng under black spot stress within the dilution range of 100 to 1000 times.

[0190] 3. Effects of different treatments on soil nutrients in ginseng under pathogen stress.

[0191] Soil organic matter (OM) is a core nutrient indicator of soil, providing organic carbon sources for ginseng growth and improving soil structure. Available nitrogen (AHN), available phosphorus (AP), and available potassium (AK) are readily available nutrients that can be directly absorbed and utilized by ginseng, and are key indicators for evaluating soil fertility and function. This experiment compared soil nutrient indicators in the control group (CK), different dilutions of SZ-4 WP (A, B, C), Bacillus subtilis WP treatment group (D), and carbendazim WP treatment group (E) to clarify the regulatory effect of SZ-4 WP on soil fertility of ginseng under disease stress.

[0192] 3.1 Characteristics of soil nutrient changes under Botrytis cinerea stress.

[0193] Under Botrytis cinerea stress, the contents of OM, AHN, AP, and AK in the soil of each treatment group showed significant differences. Specific results are as follows: Figure 11 As shown.

[0194] Soil organic matter content results as follows Figure 11 As shown in the upper left figure: Treatment C had the highest soil organic matter content at 4.79%, a significant increase of 25.07% compared to the control group (3.83%), demonstrating that SZ-4 WP at a dilution of 1000 times can effectively promote soil organic matter accumulation under gray mold stress. Treatment E had an organic matter content of 2.48%, which was not significantly different from Treatment A (2.84%). It is speculated that chemical agents inhibit the decomposition and metabolism of soil microorganisms, and the application of high-concentration inoculants can easily disrupt the balance of the soil microbial community, thereby limiting organic matter accumulation.

[0195] Soil alkaline nitrogen content results as follows Figure 11 As shown in the upper right figure, treatment group B had the highest available nitrogen content at 24.69 mg / kg, a 39.26% increase compared to treatment group CK (17.73 mg / kg). This indicates that 600-fold dilution of SZ-4 WP effectively alleviated the inhibitory effect of Botrytis cinerea infection on soil nitrogen activation and significantly improved soil nitrogen supply capacity. The available nitrogen contents of treatment groups CK and A were 17.73 mg / kg and 17.59 mg / kg, respectively, with no significant difference between them and significantly lower than the other treatment groups. This suggests that high-concentration application of 100-fold dilution of SZ-4 WP could not improve soil nitrogen availability and may have synergistically inhibited the activity of soil nitrogen cycling microorganisms, thus reducing available nitrogen content.

[0196] Soil available phosphorus content results as follows Figure 11 As shown in the lower left figure, treatment group A had the highest available phosphorus content at 16.02 mg / kg, a slight increase of 6% compared to the control group (15.10 mg / kg), indicating only a mild phosphorus-promoting effect. Treatment groups C (14.37 mg / kg) and E (14.33 mg / kg) had the lowest available phosphorus content with no significant difference, indicating that SZ-4 WP 1000-fold dilution and carbendazim WP inhibited the activity of soil phosphorus-solubilizing microorganisms and failed to increase the available phosphorus content in the soil. Overall, the differences in available phosphorus content among the treatment groups were not significant (P>0.05), suggesting that the regulatory effects of each agent on available phosphorus in ginseng soil under gray mold stress were limited.

[0197] Soil available potassium content results as follows Figure 11As shown in the lower right figure, the available potassium content differed significantly among the treatment groups (P < 0.05). Treatment A had an available potassium content of 267.76 mg / kg, an increase of 18.62% compared to the control group (225.72 mg / kg), and was 1.36 times and 1.37 times higher than treatment D (197.12 mg / kg) and treatment E (196.00 mg / kg), respectively, demonstrating that a 100-fold dilution of SZ-4 WP can significantly increase the available potassium content in the soil. Treatments D and E had significantly lower available potassium contents than the control group and all SZ-4 WP treatment groups, confirming that commercially available biocontrol agents and chemical agents cannot improve soil potassium supply capacity and may even have negative effects, highlighting the significant advantages of the SZ-4 WP of this invention in regulating soil potassium fertility.

[0198] 3.2 Characteristics of soil nutrient changes under Alternaria stress.

[0199] Under Alternaria stress, the contents of OM, AHN, AP, and AK in the soil of each treatment group showed significant differences. Specific results are as follows: Figure 12 As shown.

[0200] Soil organic matter content results as follows Figure 12 As shown in the top left figure: the OM content in treatment B (5.31%) was significantly higher than all other treatment groups, increasing by 73.53% compared to the control (3.06%), indicating that SZ-4 WP at this concentration can significantly promote soil organic matter accumulation. Treatment E had the lowest OM content, only 2.35%, significantly lower than the control and all SZ-4 WP treatments (…). P <0.05), indicating that the application of carbendazim may disrupt the soil microbial community structure, inhibit the activity of microorganisms related to the decomposition and transformation of organic matter, and thus exacerbate the consumption of soil organic matter.

[0201] Soil alkaline nitrogen content results as follows Figure 12 As shown in the upper right figure, the AHN content under treatments D (24.13 mg / kg) and E (23.94 mg / kg) was significantly higher than that under control (17.83 mg / kg), indicating that these treatments were beneficial in mitigating the reduction in available nitrogen content in the soil under black spot infection. The AHN content under treatment B was 22.56 mg / kg, 26.53% higher than that under control, indicating that this concentration of SZ-4 WP could effectively alleviate the inhibitory effect of black spot pathogen infection on available nitrogen in the soil. Treatment A (17.73 mg / kg) had the lowest AHN content compared to control and showed no significant difference, suggesting that a 100-fold dilution of SZ-4 WP may have a certain inhibitory effect on soil nitrogen transformation-related microorganisms, leading to insufficient accumulation of available nitrogen.

[0202] Soil available phosphorus content results as follows Figure 12As shown in the lower left figure, the available phosphorus content differed significantly among the treatment groups (P < 0.05). Treatment group C had the highest available phosphorus content at 20.55 mg / kg, a 17.50% increase compared to the control group (17.49 mg / kg), demonstrating that 1000 times dilution of SZ-4 WP can effectively increase the available phosphorus content in the soil under black spot disease stress. The available phosphorus content in treatment groups B (15.63 mg / kg) and D (15.58 mg / kg) was significantly lower than that in the control group, indicating that 600 times dilution of SZ-4 WP and Bacillus subtilis WP had no effect on increasing available phosphorus in the soil and had a certain phosphorus-inhibiting effect.

[0203] Soil available potassium content results as follows Figure 12 As shown in the lower right figure, the available potassium content differed significantly among the treatment groups (P < 0.05). Treatment group C had the highest available potassium content at 239.16 mg / kg, a 28.22% increase compared to the control group (CK) (186.52 mg / kg), confirming that 1000x dilution of SZ-4 WP significantly activated soil potassium and increased available potassium content under black spot disease stress. Treatment groups CK and A had the lowest available potassium content, indicating that infection by Alternaria alternata and treatment with high concentrations of SZ-4 WP (100x dilution) inhibited soil potassium activation and reduced soil potassium supply capacity.

[0204] 4. Effects of different treatments on ginseng soil enzyme activity under pathogen stress.

[0205] 4.1 Changes in soil enzyme activity under Botrytis cinerea stress.

[0206] Soil enzyme activity results under Botrytis cinerea stress are as follows: Figure 13 As shown. Soil neutral phosphatase (S-NP) activity results are as follows. Figure 13 As shown in the left figure: Treatment A (3.42 U / g) showed higher S-NP activity, increasing by 27.6% compared to CK (2.68 U / g), but there was no significant difference compared to treatments D (2.88 U / g) and E (3.42 U / g). P >0.05). Treatments B (2.62 U / g) and C (2.29 U / g) showed no significant difference from the control (CK). In conclusion, with increasing dilution ratio, S-NP activity gradually decreased after applying SZ-4 WP, with 100-fold dilution of SZ-4 WP significantly improving soil neutral phosphatase activity.

[0207] Soil urease (S-UE) activity results as follows Figure 13As shown in the figure, the soil S-UE ranged from 4.50 to 6.51 U / g for each treatment, with treatment C (6.51 U / g) significantly higher than CK (4.70 U / g) and other treatments. There were no significant differences in S-UE activity among treatments A (4.50 U / g), B (4.76 U / g), D (4.53 U / g), and E (4.55 U / g). P >0.05). This indicates that a 1000-fold dilution of SZ-4WP can significantly regulate soil urease activity, while the other treatments have little effect on soil urease activity.

[0208] Soil sucrase (S-SC) activity results are as follows Figure 13 As shown in the right figure, the sucrase activity of each SZ-4 WP treatment group gradually decreased with increasing dilution factor. Treatment group A had the highest S-SC activity at 2.74 U / g, which was more than 80% higher than the CK group (1.45 U / g). Treatment groups B, D, and E showed no significant difference in S-SC activity (P > 0.05), and all were higher than the CK group. Treatment group C had the lowest S-SC activity at 1.19 U / g, significantly lower than all other treatment groups (P < 0.05).

[0209] In summary, under gray mold stress, the 100-fold dilution of SZ-4 WP of this invention can significantly enhance the activities of enzymes related to soil carbon conversion (sucrase) and phosphorus conversion (neutral phosphatase), but has no significant effect on nitrogen conversion enzyme activity; the 600-fold dilution of SZ-4 WP has no significant regulatory effect on the activities of the three types of soil enzymes; and the 1000-fold dilution of SZ-4 WP has a significant promoting effect on nitrogen conversion (urease).

[0210] 4.2 Changes in soil enzyme activity under Alternaria stress.

[0211] Soil enzyme activity results under Alternaria stress for each treatment are as follows: Figure 14 As shown. Soil neutral phosphatase (S-NP) activity results are as follows. Figure 14 As shown in the left figure, the S-NP activities of each treatment group ranged from 2.70 to 3.28 U / g, with no significant difference between groups (P > 0.05), indicating that the effects of all treatments on soil phosphorus invertase activity under black spot stress were relatively stable.

[0212] Soil urease (S-UE) activity results as follows Figure 14 As shown in the figure, the S-UE urease activity range for each treatment was 2.80–5.47 U / g, with no significant difference between treatments A (4.03 U / g) and D (3.52 U / g). P>0.05), indicating that SZ-4 WP 100-fold dilution and Bacillus subtilis WP have similar effects and no significant impact on soil urease activity. Treatment B (5.46 U / g) significantly promoted S-UE, increasing it by more than 50% compared to CK (3.62 U / g).

[0213] Soil sucrase (S-SC) activity results are as follows Figure 14 As shown in the right figure: the S-SC activity range for each treatment was 1.25~2.08 U / g, and there were significant differences among the treatments. P <0.05). Under the SZ-4 WP treatment conditions (A, B, C), the effect of promoting carbon metabolism gradually decreased with the increase of dilution factor. Among them, treatment A (2.08 U / g) showed the highest S-SC activity, which was significantly higher than CK and all other treatments.

[0214] In summary, under the stress of black spot disease, the application of SZ-4 WP at a dilution of 100 times has a promoting effect on the activity of enzymes related to soil carbon transformation (sucrase). SZ-4 WP at a dilution of 600 times has a significant promoting effect on nitrogen transformation (urease). SZ-4 WP at a dilution of 1000 times has no significant regulatory effect on the activities of soil neutral phosphatase, urease, and sucrase, and its soil improvement function is not prominent.

[0215] Studies have shown that the optimal soil pH for ginseng cultivation is 5.0–7.0. With increasing cultivation years, soil pH has continuously decreased, dropping from 5.55 to 4.75 in the main ginseng-producing area of ​​Jilin Province between 1963 and 2022. In this experiment, both gray mold and black spot infections led to a slight decrease in soil pH.

[0216] The results of this embodiment show that all microbial agent treatments significantly reduced soil electrical conductivity, with the 100-fold dilution (for gray mold) and the 600-fold dilution (for black spot) showing the most significant reductions. This indicates that SZ-4 WP can reduce soluble salt accumulation and improve salinization. While the chemical agent carbendazim also reduces electrical conductivity, it significantly inhibits organic matter accumulation, whereas SZ-4 WP has no such negative effect, demonstrating its application advantages. Carbendazim may poison beneficial microorganisms and inhibit organic matter transformation; SZ-4 WP, on the other hand, can promote microbial activity and accelerate organic matter accumulation, thus achieving both disease prevention and soil improvement.

[0217] Regarding organic matter: Under gray mold stress, a 1000-fold dilution of SZ-4 WP increased organic matter by 25% (compared to the control); under black spot stress, a 600-fold dilution increased it by 73%. However, a 100-fold dilution actually decreased organic matter, because high concentrations of the inoculant intensified carbon source competition or led to the dominance of r-strategy microorganisms that rapidly decompose organic matter. This indicates that the inoculant concentration needs to be optimized. Furthermore, SZ-4 WP treatment can simultaneously increase the content of available nitrogen, available phosphorus, and available potassium.

[0218] Soil enzyme regulation exhibits concentration-dependent effects. Under gray mold stress, a 100-fold dilution significantly increased the activities of neutral phosphatase and sucrase, a 600-fold dilution promoted urease activity, while a 1000-fold dilution had no significant effect. This is related to the rhizosphere colonization capacity and the impact on the microbial community of different concentrations of inoculants.

[0219] The above-described experimental examples merely illustrate specific embodiments of the present invention, and while the descriptions are detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. The application of *Bacillus cereus* SZ-4 or *Bacillus cereus* SZ- wettable powder in crop cultivation, characterized in that... The crop was treated in the field using Bacillus cereus SZ-4 or a diluted wettable powder of Bacillus cereus SZ-4; the field treatment included at least one of disease control treatment, crop growth promotion treatment, and rhizosphere soil improvement treatment.

2. The application according to claim 1, characterized in that, The wettable powder of Bacillus cereus SZ-4 is diluted 100 to 1000 times.

3. The application according to claim 1, characterized in that, The crops mentioned are any one or more of ginseng, rice, soybeans, bok choy, wheat, rapeseed, and carrots.

4. The application according to claim 1, characterized in that, The disease control treatment is for the prevention and control of ginseng gray mold and / or ginseng black spot disease; when carrying out the disease control treatment, apply a 100-fold dilution of Bacillus oryzae SZ-4 wettable powder.

5. The application according to claim 1, characterized in that, The crop growth-promoting treatment is a spraying or root irrigation treatment applied during the seed germination stage and / or seedling growth stage of the crop; when performing the crop growth-promoting treatment, a 100-fold dilution of the Bacillus oryzae SZ-4 wettable powder is applied.

6. The application according to claim 1, characterized in that, The rhizosphere soil improvement treatment is carried out under conditions of infection with gray mold or black spot fungus; the rhizosphere soil improvement treatment includes reducing soil electrical conductivity, increasing soil organic matter content, increasing soil available nitrogen content, increasing soil available phosphorus content and / or increasing soil available potassium content.

7. The application according to claim 6, characterized in that, For soils affected by gray mold, apply a 100-fold or 1000-fold dilution of Bacillus cereus SZ-4 wettable powder; for soils affected by black spot, apply a 600-fold dilution of Bacillus cereus SZ-4 wettable powder.

8. The application according to claim 7, characterized in that, For soils affected by gray mold, a 100-fold dilution was used to regulate the activity of neutral phosphatase and sucrase in the soil; a 1000-fold dilution was used to improve soil organic matter.

9. The application according to claim 1, characterized in that, The application concentration of the Bacillus cereus SZ-4 wettable powder solution should not be less than 100 times the dilution to avoid inhibiting the seed germination or seedling growth of non-target crops.

10. The application according to claim 1, characterized in that, The field treatment is applied by foliar spraying or soil irrigation.

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