Composting bacillus and application of combination of composting bacillus and chlorine dioxide in prevention and control of apple continuous cropping obstacles
By combining Bacillus DF1 compost with chlorine dioxide, the problems of increased soil chloride ions and decreased enzyme activity caused by chlorine dioxide treatment were solved, promoting the growth of apple seedlings and optimizing the soil microbial community structure, thus alleviating the problem of continuous cropping obstacles in apples.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANDONG AGRICULTURAL UNIVERSITY
- Filing Date
- 2026-01-07
- Publication Date
- 2026-05-01
AI Technical Summary
While existing methods of treating apple-growing soil with chlorine dioxide can effectively reduce the number of harmful Fusarium spores, they may also lead to increased soil chloride ion content, decreased enzyme activity, and microbial community imbalance, thus affecting apple growth and fruit quality.
The combined treatment of Bacillus stercoris DF1 and chlorine dioxide was carried out by treating the soil with chlorine dioxide solution before transplanting fruit trees and applying liquid Bacillus stercoris fertilizer after planting. This synergistic effect improved root vitality and soil enzyme activity and reduced soil chloride ion content.
It significantly improves the growth and root vitality of apple seedlings, optimizes the soil microbial community structure, improves soil enzyme activity, reduces continuous cropping obstacles in apples, and enhances the health and yield of fruit trees.
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Abstract
Description
A strain of Bacillus compost and its application in combination with chlorine dioxide in controlling apple continuous cropping obstacles Technical Field
[0001] This invention relates to the field of agricultural microbial technology, specifically to a strain of Bacillus compostii and its application in combination with chlorine dioxide in controlling continuous cropping obstacles in apples. Background Technology
[0002] Apple continuous cropping obstacle refers to the phenomenon that occurs when apple trees are cultivated on the same land or in the same area for a long period of time, resulting in frequent outbreaks of pests and diseases, slow growth and development of young trees, weak plants, fragile root systems, short tree lifespan, or deterioration in fruit quality and reduced productivity. The occurrence of apple continuous cropping obstacle greatly restricts the healthy growth of apple seedlings. Therefore, finding effective methods to alleviate apple continuous cropping obstacle is of great significance to the healthy and sustainable development of the apple industry.
[0003] There are many factors contributing to continuous cropping obstacles in apple orchards, including deterioration of soil physicochemical properties, imbalance of soil microbial community structure due to increased harmful fungi, and accumulation of autotoxic substances. Among these, harmful fungi are the most significant factor influencing continuous cropping obstacles. Soil disinfection aims to control the number of harmful bacteria, optimize the rhizosphere microecological environment, and ultimately reduce the harm of continuous cropping obstacles to crops. Currently, chemical disinfection of soil widely uses fumigants such as dazomet, methylphenidate, and chloropicrin, as well as strong oxidants such as potassium permanganate, peracetic acid, and sodium hypochlorite, and various disinfectants and soil conditioners. Methyl bromide is currently recognized as the most effective soil fumigant, but due to its severe ozone-depleting effects, its use has been banned since the 20th century under environmental protection policies in various countries. Finding alternatives has become a key research focus in the field of soil disinfection.
[0004] Strong oxidants can sterilize and disinfect soil through their strong oxidizing properties, degrade autotoxic substances, and are environmentally friendly and harmless to humans, giving them a unique competitive advantage in controlling continuous cropping obstacles in apple orchards. The inventors' team previously investigated the impact of chlorine dioxide treatment on the microecological environment of continuously cropped soil and its effectiveness in controlling continuous cropping obstacles in apple orchards. Studies showed that chlorine dioxide treatment of continuously cropped apple soil significantly reduced the number of harmful Fusarium spores in the soil, optimized the soil microbial community structure, and effectively controlled continuous cropping obstacles. However, chlorine dioxide can decompose into chloride ions. While chloride ions are an essential micronutrient for plants, excessive chloride can be toxic, hindering growth and reducing yield and quality. Furthermore, chlorine dioxide soil disinfection can reduce the activity of sucrase, catalase, urease, and neutral phosphatase in continuously cropped soil, potentially causing an imbalance in the soil's biological ecosystem. Therefore, addressing these drawbacks of chlorine dioxide treatment for continuously cropped soil is a pressing technical problem that needs to be solved. Summary of the Invention
[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide a strain of Bacillus compostii and its application in combination with chlorine dioxide in the control of continuous cropping obstacles in apples.
[0006] Specifically, the present invention relates to the following technical solution: In the first aspect, the present invention provides a strain of Bacillus stercoris DF1, which was deposited on October 23, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with the biological accession number CGMCC No. 36317 and the classification name Bacillus stercoris.
[0007] Compared with existing composting Bacillus, the Bacillus stercoris DF1 of the present invention has the following characteristics: (1) It has antibacterial activity against a variety of pathogens that cause continuous cropping obstacles in apples, such as Fusarium oxysporum, Fusarium rotundum, Fusarium moniliforme, and Fusarium moniliforme.
[0008] (2) It has the properties of being resistant to salt, alkali and chlorine dioxide, and also has the ability to promote growth.
[0009] (3) Combining Bacillus stercoris DF1 with chlorine dioxide can synergistically improve the root vigor of apples, improve the soil enzyme activity reduction caused by chlorine dioxide treatment alone, and reduce the increase in soil chloride ion content caused by chlorine dioxide treatment.
[0010] In a second aspect, the present invention provides a liquid microbial fertilizer containing the aforementioned Bacillus stercoris DF1.
[0011] Preferably, in the liquid microbial fertilizer, Bacillus stercoris DF1 exists in the form of cultured live bacteria, bacterial suspension, or fermentation broth.
[0012] Preferably, the liquid microbial fertilizer is prepared by the following method: Bacillus stercoris DF1 is inoculated into LB liquid medium and incubated at 32°C and 180 rpm. -1 Cultured on a shaker for 2 days.
[0013] In a third aspect, the present invention provides the use of the above-mentioned Bacillus stercoris DF1 or liquid microbial fertilizer in at least one of the following (1)-(4): (1) inhibiting the growth of plant pathogens; (2) preparing a product for inhibiting plant pathogens; (3) preventing and controlling diseases caused by plant pathogens; (4) preparing a product for preventing and controlling diseases caused by plant pathogens.
[0014] In the above applications, the plant pathogens are Fusarium oxysporum, Fusarium solani, Fusarium latifolium, or Fusarium moniliforme.
[0015] In a fourth aspect of the present invention, the above-mentioned Bacillus stercoris DF1 or liquid microbial fertilizer is provided for use in the following (1) or (2): (1) alleviating continuous cropping obstacles in apple trees; (2) preparing a biocontrol agent to alleviate continuous cropping obstacles in apple trees.
[0016] The fifth aspect of the present invention provides a method for controlling apple continuous cropping obstacles using chlorine dioxide combined with Bacillus stercoris DF1, comprising the following steps: (1) treating the continuous cropping soil with chlorine dioxide solution before transplanting the fruit trees; (2) watering and drying the continuous cropping soil treated with chlorine dioxide solution in sequence, and then planting the fruit trees in the dried continuous cropping soil, and applying Bacillus stercoris DF1 liquid fertilizer after planting.
[0017] Preferably, in step (1), the soil from continuous cropping is treated with chlorine dioxide solution 7 days before the fruit trees are transplanted.
[0018] Preferably, in step (1), the concentration of the chlorine dioxide solution is 600 mg·L⁻¹. -1 The mass ratio of chlorine dioxide solution to the treated continuously cropped soil was 1:10.
[0019] This invention does not require applying chlorine dioxide to the entire orchard. It only requires selecting a planting ditch and sterilizing within that ditch area, thus minimizing the cost of soil treatment while ensuring the sterilization effect of chlorine dioxide.
[0020] Preferably, in step (2), the drying treatment time is 7 days. Through drying treatment, the added chlorine dioxide can fully exert its function and decompose, reducing or avoiding damage to the root system of apple seedlings caused by chlorine dioxide, and maximizing the growth of the plants.
[0021] Preferably, in step (2), the amount of liquid microbial fertilizer of Bacillus stercoris DF1 applied is 1% of the volume of the continuously cropped soil.
[0022] The beneficial effects of the present invention are as follows: (1) The present invention screened and isolated a strain of Bacillus stercoris DF1, which has salt and alkali resistance, nitrogen fixation, phosphorus solubilization, potassium solubilization and IAA production; it has an inhibitory effect on pathogens such as Fusarium oxysporum, Fusarium rotundum, Fusarium flocculation, and Fusarium moniliforme that cause continuous cropping obstacles in apples, and can be used for the prevention and control of continuous cropping obstacles in apples.
[0023] (2) The combined application of Bacillus stercoris DF1 and chlorine dioxide to control apple continuous cropping obstacles has a synergistic effect; moreover, Bacillus stercoris DF1 can effectively reduce the increase in soil chloride ion content caused by chlorine dioxide disinfection, and improve the reduction in soil sucrase, catalase, urease and neutral phosphatase activities caused by chlorine dioxide disinfection. Attached Figure Description
[0024] Figure 1 shows the observation of a single colony of strain DF1 and Gram staining; Figure 2 shows the phylogenetic tree of strain DF1 based on 16S rRNA sequence analysis; Figure 3 shows the antagonistic effect of Bacillus compostii DF1 against four Fusarium species; Figure 4 shows the salt and alkali tolerance of Bacillus compostii DF1; Figure 5 shows the growth-promoting function of Bacillus compostii DF1; Figure 6 shows the chlorine dioxide tolerance of Bacillus compostii DF1; Figure 7 shows the effect of different treatments on seedling growth under potted conditions; Figure 8 shows the effect of different treatments on seedling root vigor under potted conditions; Figure 9 shows the effect of different treatments on soil enzyme activity under potted conditions; Figure 10 shows the effect of different treatments on soil chloride ion content under potted conditions. Detailed Implementation
[0025] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.
[0026] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments. If the specific experimental conditions are not specified in the embodiments, they are generally performed under conventional conditions or according to the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following embodiments, unless otherwise specified, can be obtained commercially. Specifically: The composition of the culture medium used in this invention is as follows: LB medium: 10 g trypsinized peptone, 5 g yeast extract, 10 g NaCl, shake the container until the solute dissolves, adjust the pH to 7.0 with 5 mol / L NaOH. Make up to 1 L with distilled water, incubate at 121℃ for 20 min.
[0027] PDA medium: 200 g peeled potato, 20 g glucose, 20 g agar, distilled water to a final volume of 1 L, incubate at 121 °C for 20 min.
[0028] Nitrogen-fixing medium: 10g glucose, 0.5g KH2PO4, 0.1g K2HPO4, 0.1g NaCl, 0.2g MgSO4, 5g CaCO3, 0.1g K2SO4, 10g agar, distilled water to a final volume of 1L, 121℃, 15 min.
[0029] Phosphate-solubilizing medium: 10g glucose, 0.5g (NH4)2SO4, 0.3g NaCl, 0.3g MgSO4·7H2O, 0.03g MnSO4·H2O, 0.3g KCl, 0.03g FeSO4·7H2O, 15g agar. Adjust the pH to 7.0, bring the volume to 1L with distilled water, and incubate at 121℃ for 15 min.
[0030] Amylase-degrading medium: 20 g starch, 0.5 g KCl, 2 g NaNO3, 1 g K3PO4, 0.5 g MgSO4•7H2O, 5 g NaCl, 18 g agar, and distilled water to a final volume of 1 L. Incubate at 121℃ for 20 min. After the strain has cultured successfully, add iodine solution to the medium for observation.
[0031] Protease medium: 1% agar, 5% skim milk powder, 115℃, 30 min, mix and pour into a plate.
[0032] IAA production and testing medium: Inoculate the strain with 1.0 g•L -1 Tryptophan was cultured in PDB medium at 25±0.1℃ for 7 days. After filtration through Whatman filter paper, 1 mL of the filtrate was mixed with 2 mL of Salkowski reagent (2% 0.5M FeCl3 + 35% perchloric acid). The mixture was incubated at room temperature for 20 min, and the color change was observed.
[0033] Example 1: Isolation and screening of strains In October 2024, at Donghuashan Manor in Dongwangjiazhuang Village, Dawangzhuang Town, Laiwu District, Jinan City, Shandong Province, three different healthy and robust pear trees were selected, and rhizosphere soil was collected and stored in a 4°C refrigerator for the isolation and screening of strains.
[0034] 1. Isolation of bacterial strains: Thoroughly mix the collected soil sample, weigh 10 g, and add it to a 250 mL Erlenmeyer flask containing 90 mL of sterile water. Incubate at 180 rpm. -1 Shaking on a shaker for 30 minutes yields 10 -1Dilute the bacterial suspension, take 10 of the above-mentioned amount. -1 1 mL of bacterial suspension was added to a test tube containing 9 mL of sterile water to obtain 10. -2 Dilute the bacterial suspension, and so on, until the bacterial suspension is diluted to 10. -3 10 -4 10 -5 Take 100 μL of bacterial suspension for each concentration gradient and add it to LB medium. Spread it evenly using a sterile spreader, invert the plate and incubate at 32°C. After 1 day, pick the bacteria from the plate and streak them onto a new LB plate to cultivate single colonies.
[0035] 2. Strain Screening: The isolated bacteria were inoculated with four Fusarium species (Fusarium solani, Fusarium moniliforme, Fusarium flocculatione, and Fusarium oxysporum) onto PDA medium for plate confrontation experiments to screen for the strain with the best antagonistic effect against the four Fusarium species. After preliminary isolation and screening, a strain with the best antagonistic effect against the four Fusarium species was obtained and named DF1.
[0036] Example 2: Identification of strain DF1 1. Morphological identification: After culturing strain DF1 in LB medium for 2-3 days, the morphology of single colonies is shown in Figure 1-A. The colonies are generally white, with a smooth surface and regular edges. Gram staining is positive, as shown in Figure 1-B.
[0037]
[0038] A phylogenetic tree was constructed based on the sequencing results, as shown in Figure 2. Since strain DF1 and Bacillus stercoris are from the same line, the tested strain DF1 is indeed Bacillus stercoris.
[0039] Based on morphological and molecular biological identification results, strain DF1 was named *Bacillus stercoris* DF1. The strain was then bio-preserved, and the preservation information is as follows: Biological material: DF1; Classification and nomenclature: *Bacillus stercoris*; Preservation institution: China General Microbiological Culture Collection Center (CGMCC); Abbreviation: CGMCC; Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Preservation date: October 23, 2025; Registration number: CGMCC No. 36317.
[0040] Example 3: Performance evaluation of Bacillus composting DF1 1. Antibacterial performance evaluation: Fusarium oxysporum, Fusarium solani, Fusarium moniliforme, and Fusarium moniliforme were inoculated onto PDA medium. The antagonistic effect of Bacillus composting DF1 on the above pathogens was carried out. After incubation at 28 ℃ for 7 days, the bacteria were taken out and the colony diameter was measured using the cross-cross method to determine its antibacterial effect.
[0041] Inhibition rate (%) = [(control colony diameter - treated colony diameter) / control colony diameter] × 100%.
[0042] The results are shown in Figure 3 and Table 1. Figure 3 shows that Bacillus composting DF1 has an inhibitory effect on Fusarium oxysporum (Figure 3-A), Fusarium solani (Figure 3-B), Fusarium moniliforme (Figure 3-C), and Fusarium moniliforme (Figure 3-D).
[0043] Table 1. Inhibitory effect of Bacillus composting DF1 on pathogenic fungi. Note: Different lowercase letters indicate significant differences between different treatments (P<0.05).
[0044] Table 1 shows that Bacillus composting DF1 has inhibitory effects on all four Fusarium species: Bacillus composting DF1 showed the best inhibitory effect on Fusarium moniliforme, with an inhibition rate of 68.5%, followed by Fusarium oxysporum, Fusarium latifolium, and Fusarium solani. The inhibition rate against all three pathogens exceeded 45%, indicating that Bacillus composting DF1 has a good inhibitory effect on the main pathogenic fungi causing continuous cropping obstacles in apples.
[0045] 2. Salt and alkali tolerance test: (1) Salt tolerance test: LB liquid medium was used as the base medium. NaCl was added to the base medium to make the NaCl content in the base medium 5%, 7%, 9%, 11% and 13% respectively; the same amount of Bacillus composting DF1 was inoculated into the base medium with different NaCl contents, the pH was maintained at 7.0-7.5, and the culture was carried out for 48 h. The A of the culture medium was measured. 600 value.
[0046] (2) Alkali tolerance test: LB liquid medium was used as the basal medium, with the NaCl content maintained at 5%, and the pH of the medium controlled at 8.0, 9.0, 10.0, 11.0, and 12.0. The same amount of Bacillus composting DF1 was inoculated into the basal medium at different pH values, and cultured for 48 h. The alkali tolerance of the culture solution was then measured. 600 value.
[0047] The results of the salt and alkali tolerance tests are shown in Figure 4. DF1 stopped growing when the NaCl concentration reached 13% and when the pH reached 10. This indicates that strain DF1 has a certain degree of salt and alkali tolerance.
[0048] 3. Growth-promoting performance evaluation: The strain DF1 was inoculated onto nitrogen-fixing medium, phosphorus-solubilizing medium, amylase-solubilizing medium, protease-solubilizing medium and IAA production and testing medium, respectively, and the relevant growth-promoting functions of Bacillus compostingus DF1 were qualitatively analyzed.
[0049] The results showed that Bacillus composting DF1 possesses nitrogen-fixing ability (Figure 5-A), can convert insoluble phosphate (Figure 5-B), and exhibits strong amylase (Figure 5-C) and protease (Figure 5-D) activities. After the addition of Salkowski's reagent, the Bacillus composting bacterial solution turned red, indicating the production of IAA during its growth (Figure 5-E).
[0050] 4. Chlorine dioxide tolerance test: LB liquid medium was used as the basal medium. Chlorine dioxide solution was added to the basal medium to make the concentration of chlorine dioxide in the medium 100 mg·L⁻¹. -1 200 mg·L -1 300mg·L -1 400 mg·L -1 500mg·L -1 600mg·L -1 The same amount of Bacillus composting DF1 was inoculated into basal culture media with different chlorine dioxide concentrations, cultured for 48 h, and the A content of the culture medium was measured. 600 value.
[0051] The test results are shown in Figure 6. The results indicate that Bacillus composting DF1 has a certain tolerance to chlorine dioxide.
[0052] Example 4: Pot Experiment 1. Experimental Design The pot experiment was conducted from May to October 2025 at the National Apple Engineering Experimental Center and the Apple Replanting and Microbiology Laboratory on the South Campus of Shandong Agricultural University. The experimental soil was taken from an old apple orchard in Jianzishan Scenic Area, Xintai City, Shandong Province.
[0053] Preparation of liquid microbial fertilizer using Bacillus stercoris DF1: Bacillus stercoris DF1 was inoculated into LB liquid medium and incubated at 32℃ and 180 rpm. -1 The bacteria were cultured on a shaker for 2 days. The entire system (including the culture medium) after cultivation was completed was a liquid Bacillus composting DF1 microbial fertilizer, with a viable count of 2.3 × 10⁻⁶ bacteria. 9 CFU·mL -1 .
[0054] The experiment consisted of five treatments: continuous cropping (CK1), methyl bromide (CK2), chlorine dioxide (T1), Bacillus DF1 liquid fertilizer (T2), and chlorine dioxide combined with Bacillus DF1 liquid fertilizer (T3). Each treatment had 10 replicates.
[0055] Continuous cropping treatment (CK1): The experimental soil was not treated, and 2 seedlings were planted in each pot; Methyl bromide treatment (CK2): The experimental soil was fumigated with methyl bromide 7 days before planting, and 2 seedlings were planted in each pot; Chlorine dioxide treatment (T1): Chlorine dioxide powder was prepared to a concentration of 600 mg·L⁻¹. -1 Solution (prepared and used immediately): Water the soil thoroughly with chlorine dioxide solution 7 days before planting (chlorine dioxide solution to soil mass ratio 1:10), planting 2 seedlings per pot; Treatment with Bacillus DF1 liquid compost fertilizer (T2): The test soil is untreated, 2 seedlings are planted per pot, and one week after planting, each pot is watered with 1% (by volume) of Bacillus DF1 liquid compost fertilizer; Treatment with chlorine dioxide combined with Bacillus DF1 liquid compost fertilizer (T3): Chlorine dioxide powder is prepared to a concentration of 600 mg·L⁻¹. -1 Prepare the solution (use immediately). Water the soil thoroughly with chlorine dioxide solution 7 days before planting (chlorine dioxide solution to soil mass ratio is 1:10). Plant 2 seedlings in each pot. One week after planting, water each pot with 1% of the test soil volume of Bacillus DF1 compost liquid fertilizer.
[0056] All treatments were subject to unified field management, including timely thinning, sampling, and measurement of relevant indicators.
[0057] 2. Determination of plant biomass: Three seedlings of similar size and growth from each treatment were selected, and their plant height and root diameter at the base of the plant were measured. Fresh weight of the Pingyi sweet tea seedlings, collected from broken pots, was measured using an electronic balance. After blanching and drying, their dry weight was determined.
[0058] Determination of soil microbial quantity: The number of soil bacteria, fungi, and actinomycetes was determined by microbial plate count.
[0059] Determination of soil enzyme activity: The activities of four soil enzymes (urease, sucrase, catalase, and neutral phosphatase) were measured.
[0060] Determination of seedling root activity: Fresh, tender white roots were collected and root activity was determined using the triphenyltetrazolium chloride (TTC) method.
[0061] Determination of soil chloride ion content: Soil chloride ions were determined using the Mohr method as specified in industry standard NY / T 1378-2007.
[0062] Determination of real-time quantitative analysis of harmful soil fungi: Soil DNA was extracted using the Soil Genomic DNA Kit. The copy numbers of four Fusarium genes in soil DNA from different treatments were analyzed in real-time using a CFX96 TMThermal Cycler (Bio-Rad).
[0063] 3. Experimental Results: (1) Effects of different treatments on the growth of Pingyi sweet tea seedlings Figure 7 shows the effects of different treatments on the growth of Pingyi sweet tea seedlings. As shown in Table 2, the treatment with chlorine dioxide combined with Bacillus DF1 liquid fertilizer significantly promoted the growth of Pingyi sweet tea seedlings. In August, compared with the continuous cropping control, the plant height and diameter at birth of the chlorine dioxide treatment (T1) increased by 16.64% and 47.9%, respectively; the plant height and diameter at birth of the Bacillus DF1 liquid fertilizer (T2) increased by 14.44% and 35.29%, respectively; and the plant height and diameter at birth of the chlorine dioxide combined with Bacillus DF1 liquid fertilizer (T3) increased by 31.78% and 70.09%, respectively.
[0064] Compared with the continuous cropping control (CK1), the fresh weight and dry weight of chlorine dioxide treatment (T1) increased by 218.62% and 371.90%, respectively; the fresh weight and dry weight of Bacillus DF1 liquid microbial fertilizer (T2) increased by 189.94% and 290.95%, respectively; and the fresh weight and dry weight of chlorine dioxide combined with Bacillus DF1 liquid microbial fertilizer (T3) increased by 310.80% and 431.40%, respectively, significantly promoting the plant growth and biomass increase of Pingyi sweet tea seedlings. Compared with continuously cropped soil, the use of a high concentration of chlorine dioxide solution (i.e., 600 mg·L⁻¹) significantly improved the growth and biomass of Pingyi sweet tea seedlings. -1After chlorine dioxide disinfection of the soil, no problem of high concentration damage to seedlings was observed. The treatment of chlorine dioxide combined with Bacillus subtilis DF1 liquid microbial fertilizer (T3) significantly promoted plant growth. After soil disinfection, it further promoted plant growth and effectively alleviated the problem of continuous cropping of apples.
[0065] Table 2 Effects of different treatments on the biomass of Pingyi sweet tea Note: CK1, soil control for continuous cropping; CK2, methyl bromide treatment; T1, chlorine dioxide treatment; T2, Bacillus DF1 liquid fertilizer for composting; T3, chlorine dioxide combined with Bacillus DF1 liquid fertilizer for composting. Different lowercase letters for the same sampling time indicate significant differences between treatments (P<0.05), the same applies below.
[0066] (2) Effects of different treatments on root vigor: Figure 8 shows the effects of different treatments on the root vigor of Pingyi sweet tea seedlings. As shown in the figure, the methyl bromide treatment (CK2) showed the best root vigor, which increased by 78.87% compared with the continuous cropping treatment (CK1); the chlorine dioxide treatment (T1) increased by 25.63%; the Bacillus DF1 liquid fertilizer (T2) increased by 16.51%; and the chlorine dioxide combined with Bacillus DF1 liquid fertilizer (T3) increased by 45.80%. The results show that the root vigor of Pingyi sweet tea seedlings was significantly improved after the combined treatment of chlorine dioxide and Bacillus DF1 liquid fertilizer, which has a synergistic effect.
[0067] (3) Effects of different treatments on soil microbial numbers. As shown in Table 3, compared with the continuous cropping control (CK1), the number of soil bacteria, fungi, and actinomycetes was significantly reduced in the chlorine dioxide treatment (T1) and the methyl bromide treatment (CK2). The number of fungi decreased and the number of bacteria increased in the Bacillus compost DF1 liquid fertilizer treatment (T2). The chlorine dioxide combined with Bacillus compost DF1 liquid fertilizer treatment (T3) significantly changed the soil microbial community structure, and there were obvious differences among the treatments. Among them, the methyl bromide fumigation treatment had the fewest fungi, bacteria, and actinomycetes.
[0068] Compared with the continuous cropping control (CK1), methyl bromide fumigation treatment (CK2) reduced soil fungal counts by 84.98%, actinomycete counts by 51.39%, and bacterial counts by 52.19%. Chlorine dioxide treatment (T1) reduced soil fungal counts by 56.15%, actinomycete counts by 52.63%, and bacterial counts by 57.73%. Bacillus spores DF1 liquid fertilizer treatment (T2) reduced soil fungal counts by 25.91%, increased actinomycete counts by 14.11%, and increased bacterial counts by 53.34%. Chlorine dioxide combined with Bacillus spores DF1 liquid fertilizer treatment (T3) reduced soil fungal counts by 42.30%, decreased actinomycete counts by 1.16%, and increased bacterial counts by 46.80%.
[0069] Table 3. Effects of different treatments on soil microorganisms (4) Effects of different treatments on soil enzyme activity The quantity and activity of soil microorganisms can affect the activity of soil enzymes. In the treatments in July and August 2025, the soil enzyme activity measurement results showed that chlorine dioxide treatment (T1) reduced the activities of urease, sucrase, phosphatase and catalase. The treatment of Bacillus compost DF1 liquid microbial fertilizer (T2) and the treatment of chlorine dioxide combined with Bacillus compost DF1 liquid microbial fertilizer (T3) increased the activities of urease, sucrase, phosphatase and catalase. Among them, the treatment of chlorine dioxide combined with Bacillus compost DF1 liquid microbial fertilizer (T3) had a more significant effect (Figure 9).
[0070] In July and August, the sucrase activity in the chlorine dioxide treatment (T1) decreased by 13.32% and 12.73% compared to the continuous cropping control (CK1), respectively. The activity in the Bacillus DF1 liquid microbial fertilizer treatment (T2) increased by 7.6% and 23.58% compared to the continuous cropping control (CK1), respectively. The activity in the chlorine dioxide combined with Bacillus DF1 liquid microbial fertilizer treatment (T3) increased by 66.37% and 72.64% compared to the continuous cropping control (CK1), respectively.
[0071] In July and August, the neutral phosphatase activity in the chlorine dioxide treatment (T1) decreased by 13.17% and 19.31% respectively compared to the continuous cropping control (CK1). The activity in the Bacillus DF1 liquid fertilizer treatment (T2) increased by 15.81% and 7.65% respectively compared to the continuous cropping control (CK1). The activity in the chlorine dioxide combined with Bacillus DF1 liquid fertilizer treatment (T3) increased by 38.68% and 35.77% respectively compared to the continuous cropping control (CK1).
[0072] In July and August, the urease activity in the chlorine dioxide treatment (T1) decreased by 21.66% and 33.75% compared to the continuous cropping control (CK1), respectively. The activity in the Bacillus DF1 liquid fertilizer treatment (T2) increased by 21.31% and 10% compared to the continuous cropping control (CK1), respectively. The activity in the chlorine dioxide combined with the Bacillus DF1 liquid fertilizer treatment (T3) increased by 83.33% and 62.5% compared to the continuous cropping control (CK1), respectively.
[0073] In July and August, the catalase activity in the chlorine dioxide treatment (T1) decreased by 27.78% and 12.38% compared to the continuous cropping control (CK1), respectively. The catalase activity in the Bacillus DF1 liquid fertilizer treatment (T2) increased by 8.30% and 12.38% compared to the continuous cropping control (CK1), respectively. The chlorine dioxide combined with Bacillus DF1 liquid fertilizer treatment (T3) increased by 45.83% and 51.42% compared to the continuous cropping control (CK1), respectively. The application of chlorine dioxide combined with Bacillus DF1 liquid fertilizer in the soil can effectively improve the soil microbial community structure and significantly increase soil enzyme activity.
[0074] (5) Effects of different treatments on soil chloride ion content: Chloride ion residue is easily observed after soil disinfection with chlorine dioxide (Figure 10), which may lead to the soil chloride ion content exceeding the national standard. Measurements showed that the soil chloride ion content after chlorine dioxide treatment (T1) did not show a significant increase, being 1.6 times that of continuously cropped soil (CK1), with an overall level of 53 mg·kg⁻¹. -1 Less than 100 mg / kg -1 The concentration was within the safe range for soil. Then, the addition of Bacillus DF1 liquid compost fertilizer slightly reduced the chloride ion concentration in the soil, bringing the overall concentration to 48 mg / kg. -1 The results indicate that the Bacillus DF1 liquid compost fertilizer promoted soil vitality to a certain extent.
[0075] (6) Effects of different treatments on the number of harmful fungi in the soil. The data analysis in Table 4 shows that chlorine dioxide disinfection of continuously cropped soil can greatly limit the growth of four Fusarium fungi and reduce their abundance in the soil. The gene copy number of pathogenic Fusarium fungi in the chlorine dioxide treatment (T1) was significantly lower than that in the continuously cropped control treatment (CK1), with the gene copy numbers of Fusarium oxysporum, Fusarium solani, Fusarium solani, and Fusarium moniliforme decreasing by 40.44%, 25.06%, 66.03%, and 44.95%, respectively. Compared with the continuously cropped control (CK1), the chlorine dioxide combined with Bacillus compost DF1 liquid fertilizer (T3) treatment of the soil reduced the gene copy numbers of Fusarium oxysporum, Fusarium solani, Fusarium solani, and Fusarium moniliforme by 69.28%, 52.39%, 68.89%, and 52.29%, respectively, showing a significant inhibitory effect.
[0076] Table 4. Real-time fluorescence quantitative analysis of different treatments on harmful fungi In conclusion, treatment of continuously cropped soil with chlorine dioxide combined with Bacillus subtilis DF1 liquid fertilizer significantly promoted apple seedling growth compared to the continuous cropping control. Biomass factors such as plant height and stem diameter were also significantly superior to the continuous cropping treatment. Furthermore, prior treatment with chlorine dioxide significantly reduced the abundance of pathogenic Fusarium spores in the soil, while subsequent application of Bacillus subtilis DF1 liquid fertilizer optimized the soil microbial community structure. Therefore, treatment of continuously cropped soil with chlorine dioxide combined with Bacillus subtilis DF1 liquid fertilizer has a significant effect on alleviating continuous cropping obstacles in apple orchards.
[0077] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.
Claims
1. A strain of Bacillus stercoris DF1, with the biological accession number CGMCC No.36317.
2. A liquid microbial fertilizer containing Bacillus stercoris DF1 as claimed in claim 1.
3. The liquid microbial fertilizer according to claim 2, characterized in that, In the liquid microbial fertilizer, Bacillus stercoris DF1 exists in the form of cultured live bacteria, bacterial suspension, or fermentation broth.
4. The microbial agent according to claim 3, characterized in that, The liquid microbial fertilizer was prepared by the following method: Bacillus stercoris DF1 was inoculated into LB liquid medium and incubated at 32°C and 180 rpm. -1 Cultured on a shaker for 2 days.
5. The use of Bacillus stercoris DF1 as described in claim 1 or the liquid microbial fertilizer as described in any one of claims 2-4 in at least one of the following (1)-(4): (1) inhibiting the growth of plant pathogens; (2) preparing a product for inhibiting plant pathogens; (3) preventing and controlling diseases caused by plant pathogens; (4) preparing a product for preventing and controlling diseases caused by plant pathogens; preferably, the plant pathogen is Fusarium oxysporum, Fusarium solani, Fusarium moniliforme, or Fusarium moniliforme.
6. The application of Bacillus stercoris DF1 as described in claim 1 or the liquid microbial fertilizer as described in any one of claims 2-4 in the following (1) or (2): (1) alleviating the continuous cropping obstacles of apple trees; (2) preparing a biocontrol agent to alleviate the continuous cropping obstacles of apple trees.
7. A method for controlling apple continuous cropping obstacles using chlorine dioxide combined with Bacillus stercoris DF1, characterized in that, Includes the following steps: (1) Treat the continuous cropping soil with chlorine dioxide solution before transplanting the fruit trees; (2) Water and dry the continuous cropping soil after chlorine dioxide solution treatment, and then plant the fruit trees in the dried continuous cropping soil. After planting, apply liquid bacterial fertilizer of Bacillus stercoris DF1.
8. The method according to claim 7, characterized in that, In step (1), the soil from continuous cropping is treated with chlorine dioxide solution 7 days before the fruit trees are transplanted.
9. The method according to claim 7, characterized in that, In step (1), the concentration of the chlorine dioxide solution is 600 mg·L⁻¹. -1 The mass ratio of chlorine dioxide solution to the treated continuously cropped soil was 1:
10.
10. The method according to claim 7, characterized in that, In step (2), the amount of liquid microbial fertilizer of Bacillus stercoris DF1 applied is 1% of the volume of soil in continuous cropping.