Compound microbial preparation for degrading soil paclobutrazol to improve fruit quality and application thereof

Abstract

This invention belongs to the field of agricultural microbial technology, specifically relating to a compound microbial preparation for degrading paclobutrazol in soil and improving fruit quality, and its application. This invention provides a strain of Bacillus compostii (…). Bacillus stercoris KB97, a strain that promotes growth while also being able to grow using paclobutrazol as its sole carbon source, has been the subject of a groundbreaking study. This study found that combining KB97 with chitosan oligosaccharide and inositol, and applying it to the soil during the mango pit-hardening stage, significantly reduced soil paclobutrazol residues, alleviated the inhibitory effects of paclobutrazol on the tree, and improved fruit quality. Specifically, this resulted in reduced postharvest weight loss, inhibited fruit diseases, delayed peel color change, and improved fruit storage life. This invention establishes a complete chain from "soil pesticide reduction—plant growth promotion—fruit quality improvement," providing a green, integrated solution for the mango industry.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to a strain of Bacillus compostii (Bacillus). Bacillus stercoris KB97, a compound microbial preparation containing this strain with chitosan oligosaccharide and / or inositol, and its application in degrading paclobutrazol residues in soil, alleviating the inhibitory effect of paclobutrazol on mangoes, and / or improving the quality of mango fruits. Background Technology

[0002] Paclobutrazol is widely used in mango production, but years of use have resulted in high levels of its residues in the soil, which can easily lead to tree decline, reduced fruit quality, and especially reduced storage tolerance of mangoes. Currently, the use of microorganisms to degrade paclobutrazol is a research hotspot, with reports involving strains such as Pseudomonas, Micrococcus, and Streptomyces. However, these strains generally have limited functions, focusing primarily on degradation itself, lacking a comprehensive promotional effect on crop growth, and failing to improve postharvest storage performance or enhance the intrinsic quality of the fruit. Some degrading bacteria also pose biosafety risks. Therefore, screening a safe strain that can efficiently degrade paclobutrazol, possess multiple growth-promoting functions, and improve the postharvest storage performance of mangoes has always been a technical challenge in this field: because these three functions belong to different metabolic regulatory networks, their synergistic achievement in the same strain has a high degree of uncertainty.

[0003] Even after obtaining the target strain, further enhancing its application effect remains a hurdle. While biostimulants such as chitosan oligosaccharides and inositol can promote the growth of certain microorganisms or induce plant resistance, the compatibility between strains and stimulants is extremely strong, with different combinations showing vastly different effects. Although existing technologies include "Bacillus + chitosan oligosaccharides" combinations, these are mostly aimed at conventional growth promotion or disease resistance in crops such as cucumbers and tomatoes, with no research on paclobutrazol degradation and mango fruit quality improvement. The application of inositol in such combinations is even less reported. More importantly, current post-harvest preservation methods for mangoes mainly rely on post-harvest chemical soaking or low-temperature storage, which are "post-harvest remedial" measures and cannot fundamentally improve the fruit's storage resistance. The technical approach of achieving post-harvest preservation through pre-harvest soil application lacks research on application timing, strain colonization, and effect evaluation, remaining largely unexplored for a long time.

[0004] This invention provides a systematic solution to the three major challenges mentioned above: lack of multifunctional and safe bacterial strains, lack of efficient compound combinations, and lack of pre-harvest soil application and preservation methods. Summary of the Invention

[0005] To address the technical challenges of the existing technologies, this invention provides a Bacillus composting strain KB97, a ternary compound microbial preparation containing this strain, chitosan oligosaccharide, and inositol, and a method for applying this compound microbial preparation to the soil during the mango pit hardening stage. This technical solution can efficiently degrade paclobutrazol residues in the soil, significantly alleviate the inhibitory effect of paclobutrazol on mango growth, and greatly improve fruit quality—specifically by reducing postharvest weight loss, inhibiting fruit diseases, delaying peel color change, improving fruit appearance, and enhancing fruit storage resistance. It establishes a complete chain from "soil paclobutrazol reduction" to "plant growth promotion" and then to "fruit quality improvement," providing an integrated green solution for the mango industry.

[0006] To achieve the above objectives, the applicant selected a strain of Bacillus compostii (Bacillus subtilis). Bacillus stercoris KB97, its classification name is: Bacillus compostii KB97 Bacillus stercoris KB97, with accession number CCTCCNO: M 20261063, is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on May 25, 2026.

[0007] Secondly, the present invention provides a compound microbial preparation for degrading paclobutrazol in soil and improving fruit quality, comprising the aforementioned Bacillus composting KB97 and chitosan oligosaccharide; the mass concentration of the chitosan oligosaccharide is 50 mg / kg to 100 mg / kg, and the viable count of the Bacillus composting KB97 is 10. 6 ~10 8 CFU / g; the fruit quality specifically refers to the peel color, weight loss rate, and / or disease incidence rate of mango fruit.

[0008] Furthermore, the compound microbial preparation also contains inositol; the mass concentration of inositol is 50 mg / kg to 100 mg / kg.

[0009] Thirdly, the present invention provides the application of the aforementioned Bacillus compostingus KB97 in the preparation of a microbial preparation for degrading paclobutrazol residues in soil to alleviate the inhibitory effect of paclobutrazol on mangoes.

[0010] Fourthly, the present invention provides the application of the aforementioned compound microbial preparation in the preparation of a microbial preparation for degrading paclobutrazol residues in soil to alleviate the inhibitory effect of paclobutrazol on mangoes.

[0011] Fifthly, the present invention provides the application of the aforementioned compound microbial preparation in the preparation of a formulation for improving the postharvest storage and preservation effect of mangoes.

[0012] To further explain, the improvement of postharvest storage and preservation of mangoes includes: reducing the postharvest weight loss rate of mangoes, inhibiting the occurrence of postharvest diseases of mangoes, delaying the color change of mango peels, and / or improving the storage resistance of mango fruits.

[0013] Sixthly, the present invention provides a method for alleviating the inhibitory effect of paclobutrazol on mangoes, wherein the aforementioned Bacillus composting KB97 or the aforementioned compound microbial preparation is applied to the rhizosphere of the mango plant, wherein the amount of Bacillus composting KB97 applied is such that its final concentration in the rhizosphere soil reaches 10. 6 ~10 8 CFU / g soil.

[0014] In a seventh aspect, the present invention provides a method for improving the post-harvest storage and preservation effect of mangoes, wherein the compound microbial preparation is applied to the soil during the mango pit hardening period, and the application amount is 10-20L of diluted microbial agent solution per tree.

[0015] Eighthly, the present invention provides a compound microbial preparation product comprising the aforementioned Bacillus compostingus KB97, chitosan oligosaccharide and inositol, as well as agriculturally acceptable excipients.

[0016] The present invention has the following beneficial effects:

[0017] This invention provides a comprehensive composting Bacillus KB97 strain that can secrete IAA, fix nitrogen, solubilize phosphorus, and dissolve potassium, while also possessing multiple growth-promoting properties. Furthermore, it can efficiently degrade pesticide residues in soil using paclobutrazol as the sole carbon source. Building upon this, this invention is the first to combine KB97 with chitosan oligosaccharide and inositol in a ternary compound. Pot experiments have demonstrated that the combined use of these three (T6 group) is significantly superior to single strains or any binary combination in promoting mango plant height, stem diameter, leaf area, and biomass accumulation, as well as reducing paclobutrazol residues in the soil, producing a clear synergistic effect. Particularly noteworthy is this breakthrough application of the compound microbial preparation to the soil during the mango pit hardening stage (one month before harvest). This not only reduces the inhibitory effect of paclobutrazol on the plant at the source but also significantly improves the fruit quality. Field postharvest storage trials show that this treatment can significantly reduce postharvest weight loss, inhibit fruit diseases, delay peel yellowing, and improve fruit storage tolerance. Even more remarkably, this compound microbial agent also exhibits significant restorative capabilities for trees weakened by long-term paclobutrazol use, restoring tree vigor and providing a practical solution to the widespread tree decline problem in mango-producing areas. Overall, this invention establishes a complete chain from "reducing paclobutrazol in the soil" to "promoting plant growth" and then to "enhancing fruit quality," providing an integrated green technology solution for the mango industry. Attached Figure Description

[0018] Figure 1The growth of 127 bacterial strains in paclobutrazol-only carbon source medium (OD) 600 ).

[0019] Figure 2 This image shows the results of screening for the clear zone of paclobutrazol-degrading bacteria.

[0020] Figure 3 The growth-promoting characteristics and preferred strains of 21 paclobutrazol-degrading bacteria are shown in the figure.

[0021] Figure 4 This is a colony morphology diagram of Bacillus compostii KB97.

[0022] Figure 5 Phylogenetic tree of Bacillus compostii KB97 based on 16S rRNA gene sequence.

[0023] Figure 6 The results of different treatments on the fresh weight, dry weight, number of leaves, leaf area, stem diameter, plant height, and SPAD value of mango seedlings are shown in the figure.

[0024] Figure 7 The figure shows the effect of different treatments on the residual amount of paclobutrazol in the soil.

[0025] Figure 8 The graph shows the effects of different concentrations and different biostimulants on the growth of strain KB97.

[0026] Figure 9 The figure shows the effect of different treatments on the agronomic traits of mango seedlings in a pot experiment using compound microbial agents.

[0027] Figure 10 The figure shows the effect of different treatments on the residual amount of paclobutrazol in the soil during a pot experiment of compound microbial preparations.

[0028] Figure 11 These are photographs showing the effects of different treatments on the phenotype of mango fruit 30 days after harvest in a field trial.

[0029] Figure 12 This is a graph showing the changes in mango weight loss rate under different treatments during storage at 13℃ in a field trial.

[0030] Figure 13 This is a statistical chart showing the number of mango diseases (lesions) under different treatments during storage at 13℃ in a field trial.

[0031] Figure 14 This is a graph showing the changes in total color difference (ΔEab) of mangoes under different treatments during storage at 13℃ in a field trial.

[0032] Figure 15 This is a comparison chart of the color change levels of mangoes after 30 days of storage at 13℃ under different treatments in a field trial.

[0033] The applicant has named this strain Bacillus compostingus KB97, and its classification name is: Bacillus compostingus KB97 Bacillus stercoris KB97 was deposited on May 25, 2026, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO. M 20261063. Detailed Implementation

[0034] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0035] Unless otherwise stated, each feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features.

[0036] Example 1: Isolation, identification and growth-promoting function study of Bacillus compostii KB97

[0037] 1. Preliminary screening of bacterial strains:

[0038] Using 127 bacterial strains preserved in the laboratory as material, they were inoculated into liquid culture medium with paclobutrazol as the sole carbon source (paclobutrazol concentrations of 1 mg / L, 10 mg / L, and 100 mg / L, respectively), and cultured at 30℃ with shaking at 180 rpm. The OD600 value of the culture medium was measured. The results are as follows: Figure 1 As shown, 30 bacterial strains were screened out whose growth was significantly promoted in the presence of paclobutrazol.

[0039] 2. Secondary screening:

[0040] The paclobutrazol degradation ability of the above 30 bacterial strains was determined (clear zone method), and 21 strains of degrading bacteria that produced clear zones were obtained. Figure 2 Further testing was conducted on the 21 strains to determine their IAA production capacity, nitrogen fixation, organic phosphorus solubilization, inorganic phosphorus solubilization, potassium solubilization, and other growth-promoting indicators. Based on these comprehensive indicators, strains KB66, KB89, and KB97 were selected. Figure 3 ).

[0041] 3. Strain identification:

[0042] 16S rRNA gene sequencing and physiological and biochemical identification were performed on KB66, KB89, and KB97. The results showed that KB66 and KB89 are opportunistic pathogens posing a biosafety risk. KB97 was identified as *Bacillus compostii* (…). Bacillus stercoris Its colony morphology is as follows Figure 4 As shown (KB97 strain was inoculated onto TSA medium and incubated in a constant temperature incubator for 24 hours, the results are as follows). Figure 4 As shown, circular colonies appeared on the petri dish; they were pale yellow and opaque, with smooth surfaces and regular edges, consistent with the morphological characteristics of Bacillus compostii. The 16S rRNA gene phylogenetic tree is shown below. Figure 3 As shown (specific molecular identification analysis involved extracting genomic DNA from strain KB97, amplifying the 16S rRNA gene fragment using the universal bacterial primers 27F / 1492R, sequencing the fragment, and performing BLAST alignment with the NCBI database to construct a phylogenetic tree. Results are as follows...) Figure 5 As shown, strain KB97 and Bacillus stercoris Clustering into the same branch indicates that this strain belongs to [a specific taxonomic group]. Bacillus stercoris ).

[0043] The applicant has named the strain Bacillus compostii KB97 and deposited it at the China Center for Type Culture Collection (CCTCC) on May 25, 2026, at Wuhan University, Wuhan, China, with accession number CCTCC NO.M20261063.

[0044] Example 2: Pot experiment on the degradation of paclobutrazol and its growth-promoting effect by Bacillus composting KB97

[0045] 1. Experimental materials:

[0046] Test strain: Bacillus compostii KB97 screened in Example 1.

[0047] The test crop: Mango seeds.

[0048] Test soil: Natural soil taken from a mango orchard.

[0049] Mango seedling cultivation: Soak mango seeds for 24 hours beforehand and then disinfect them. Select seeds of uniform size and plant them in flowerpots filled with substrate. Once the mango seedlings have grown 2-3 true leaves, select seedlings of uniform growth and transplant them into natural soil collected from the mango orchard.

[0050] 2. Experimental Design:

[0051] The experiment consisted of 6 treatments, with 6 replicates for each treatment, as detailed below:

[0052] CK: Water control (no bacterial inoculation, no paclobutrazol).

[0053] H: Apply only KB97 bacterial suspension (final concentration 10). 7 CFU / g (soil)

[0054] P10: Paclobutrazol was applied only, and the final concentration of paclobutrazol in the soil was 10 mg / kg.

[0055] P10+H: Apply paclobutrazol (10 mg / kg) + KB97 bacterial suspension (10 mg / kg) 7 CFU / g (soil)

[0056] P20: Paclobutrazol was applied only, and the final concentration of paclobutrazol in the soil was 20 mg / kg.

[0057] P20+H: Apply paclobutrazol (20 mg / kg) + KB97 bacterial suspension (10 mg / kg). 7 CFU / g (soil).

[0058] 3. Implementation Methods

[0059] Paclobutrazol treatment: Prepare paclobutrazol solutions with concentrations of 100 mg / L and 200 mg / L respectively, and apply them evenly to the potting soil so that the final concentrations of paclobutrazol in the soil reach 10 mg / kg (corresponding to 100 mg / L solution) and 20 mg / kg (corresponding to 200 mg / L solution) respectively.

[0060] Inoculation treatment: One week after paclobutrazol treatment, KB97 bacterial solution was inoculated into the corresponding potted plants to achieve a final concentration of KB97 in the soil of 10. 7 CFU / g soil. A control treatment (CK) was also set up, which was not inoculated with bacteria, and its culture conditions were completely identical to other inoculation treatments.

[0061] 4. Cultivation conditions

[0062] All treated mango seedlings were cultured in a greenhouse with a natural temperature range of 25–30℃ and a humidity range of 50–70%RH. Each treatment was replicated six times, and all potted plants were arranged randomly.

[0063] 5. Measurement Indicators

[0064] Thirty days after transplanting, the agronomic traits of mango seedlings in each treatment (including plant height, stem diameter, number of leaves, chlorophyll content, etc.) were measured, and the residual amount of paclobutrazol in the soil was also measured.

[0065] Plant height: Measure the height from the base of the stem to the terminal bud using a ruler.

[0066] Stem diameter: Measure the stem diameter 1 cm above the cotyledon node using vernier calipers.

[0067] Leaf area: Scan all leaves and calculate the total leaf area using image analysis software.

[0068] Number of leaves: Record the total number of unfolded leaves for each plant.

[0069] Fresh weight and dry weight: Weigh the fresh weight of the above-ground and underground parts separately, then fix the green at 105℃ for 30 minutes, dry at 80℃ to constant weight, weigh the dry weight, and calculate the total fresh weight and total dry weight.

[0070] SPAD value: The relative chlorophyll content of leaves under each treatment function was determined using a handheld chlorophyll meter (SPAD-502).

[0071] Soil paclobutrazol residues: The residual concentration of paclobutrazol in rhizosphere soil was determined by high performance liquid chromatography (HPLC).

[0072] 6. Experimental Results:

[0073] The results are as follows Figure 6 , Figure 7 As shown, compared with the control (CK), the treatments with paclobutrazol alone (P10, P20) significantly inhibited the biomass, stem diameter, number of leaves, leaf area, and SPAD value of mango seedlings. However, compared with the treatments with paclobutrazol alone, the inoculation treatments (P10+H, P20+H) significantly improved all of the above-mentioned growth indicators of mango seedlings (including plant height, stem diameter, SPAD value, leaf area, number of leaves, and aboveground and underground dry and fresh weight), effectively alleviating the inhibitory effect of paclobutrazol on seedling growth and significantly reducing the residual amount of paclobutrazol in the soil. These results indicate that Bacillus compostii KB97 can effectively degrade paclobutrazol in the soil and reduce the inhibitory effect of paclobutrazol on the growth of mango seedlings.

[0074] Example 3: Screening of a combination of Bacillus composting KB97 and biostimulants

[0075] 1. Test materials

[0076] Test strain: Bacillus compostii KB97.

[0077] The tested biostimulants were: chitosan oligosaccharide, inositol, ectoin, fucoidan oligosaccharide, probiotic lipid, and γ-aminobutyric acid.

[0078] 2. Test Methods

[0079] First, a seed culture of Bacillus compostii KB97 was prepared. Six biostimulants were added to TSB liquid medium to achieve biostimulant concentrations of 0 (blank control), 0.005%, 0.01%, 0.02%, 0.05%, 0.1%, and 0.5% in each treatment group. Each concentration treatment was replicated in triplicate. After sterilizing the medium, the KB97 seed culture was inoculated at the same inoculum size and cultured. During culture, OD600 values ​​were measured periodically. Growth curves of the strain under different biostimulants and concentrations were plotted with culture time on the x-axis and OD600 value on the y-axis to compare the effects of each treatment on KB97 growth.

[0080] 3. Test Results

[0081] The results are as follows Figure 8As shown, chitosan oligosaccharide and inositol exhibited the most significant growth-promoting effects on strain KB97 at a concentration of 0.01%. Therefore, chitosan oligosaccharide and inositol at a concentration of 0.01% were selected as additives for subsequent compounding experiments.

[0082] Example 4: Pot Experiment of Bacillus composting KB97 Combined with Biostimulants

[0083] 1. Test materials

[0084] Test strain: Bacillus compostii KB97 obtained from screening in Example 1 ( Bacillus stercoris ).

[0085] The test crop: Mango seeds.

[0086] The tested growth-promoting substances were: chitosan oligosaccharide (molecular weight ≤3000 Da) and inositol (analytical grade).

[0087] The tested growth regulator was paclobutrazol (15% wettable powder).

[0088] Test soil: Natural soil taken from a mango orchard.

[0089] 2. Experimental Design:

[0090] There are a total of 7 treatments, with 6 replicates per treatment, as follows:

[0091] CK: Water control (no KB97 inoculation, no paclobutrazol, no added chitosan oligosaccharide and inositol).

[0092] CK1: Paclobutrazol was applied only, and the final concentration of paclobutrazol in the soil was 10 mg / kg;

[0093] T1: Apply paclobutrazol (10 mg / kg) + KB97 bacterial suspension (1 × 10⁻⁶). 7 CFU / g (soil)

[0094] T2: Apply paclobutrazol (10 mg / kg) + chitosan oligosaccharide (50 mg / kg).

[0095] T3: Administer paclobutrazol (10 mg / kg) + inositol (50 mg / kg).

[0096] T4: Apply paclobutrazol (10 mg / kg) + KB97 bacterial suspension (1 × 10⁻⁶). 7 CFU / g2 + chitosan oligosaccharide (50mg / kg2);

[0097] T5: Apply paclobutrazol (10 mg / kg) + KB97 bacterial suspension (1 × 10⁻⁶). 7 CFU / g±) + inositol (50mg / kg±);

[0098] T6: Apply paclobutrazol (10 mg / kg) + KB97 bacterial suspension (1 × 10⁻⁶). 7 CFU / g ±) + Chitosan oligosaccharide (50mg / kg ±) + Inositol (50mg / kg ±).

[0099] 3. Cultivation and treatment methods

[0100] Soak mango seeds for 24 hours beforehand, and then disinfect them by soaking in 75% ethanol for 30 seconds, disinfecting with 0.1% mercuric chloride for 5 minutes, and rinsing with sterile water 5 times. Select seeds of uniform size and sow them in flowerpots filled with seedling substrate. When the mango seedlings have grown 2-3 true leaves, select seedlings of uniform growth and transplant them into plastic pots (20cm in diameter and 18cm in height) filled with natural soil, one seedling per pot.

[0101] Paclobutrazol treatment: Prepare a 100 mg / L paclobutrazol solution and apply it evenly into the pot to make the final concentration of paclobutrazol in the soil 10 mg / kg.

[0102] Inoculation and growth-promoting substance treatment: 7 days after transplanting, inoculate with KB97 bacterial suspension (final concentration 1×10⁻⁶) according to the treatment requirements. 7 The treatment included CFU / g soil, along with chitosan oligosaccharide (50 mg / kg soil) and / or inositol (50 mg / kg soil). The control (CK) received no inoculation and no growth-promoting substances; CK1 received only paclobutrazol. All treatments were conducted in a greenhouse under natural light, with a temperature range of 25–30°C and relative humidity of 50%–70%. Routine water management was applied, with watering every 3 days to maintain soil moisture at 60%–70% of field capacity.

[0103] 4. Measurement Indicators and Methods

[0104] Thirty days after transplanting, six mango seedlings from each treatment were randomly selected for whole-plant harvesting. The above-ground and underground parts were separated, and the following indicators were measured:

[0105] Plant height: Measure the height from the base of the stem to the terminal bud using a ruler.

[0106] Stem diameter: Measure the stem diameter 1 cm above the cotyledon node using vernier calipers.

[0107] Leaf area: Scan all leaves and calculate the total leaf area using image analysis software.

[0108] Number of leaves: Record the total number of unfolded leaves for each plant.

[0109] Fresh weight and dry weight: Weigh the fresh weight of the above-ground and underground parts separately, then fix the green at 105℃ for 30 minutes, dry at 80℃ to constant weight, weigh the dry weight, and calculate the total fresh weight and total dry weight.

[0110] SPAD value: The relative chlorophyll content of leaves under each treatment function was determined using a handheld chlorophyll meter (SPAD-502).

[0111] Soil paclobutrazol residues: The residual concentration of paclobutrazol in rhizosphere soil was determined by high performance liquid chromatography (HPLC).

[0112] 5. Experimental Results:

[0113] The results are as follows Figure 9 , Figure 10 As shown. The effects of each treatment on mango seedling growth and soil paclobutrazol residues are as follows:

[0114] Compared with paclobutrazol alone (CK1), T1 (paclobutrazol + KB97), T2 (paclobutrazol + chitosan oligosaccharide), and T3 (paclobutrazol + inositol) all showed some growth-promoting effects, but the improvement was limited.

[0115] The growth-promoting effects of T4 (paclobutrazol + KB97 + chitosan oligosaccharide) and T5 (paclobutrazol + KB97 + inositol) were better than those of T1, T2, and T3, indicating that the combination of KB97 with chitosan oligosaccharide or inositol has a synergistic effect.

[0116] The T6 treatment (paclobutrazol + KB97 + chitosan oligosaccharide + inositol) showed the best performance among all treatments. It produced the highest plant height, stem diameter, SPAD value, leaf area, number of leaves, total dry weight, and total fresh weight for mango seedlings, and had the lowest paclobutrazol residue in the soil. Specific data are as follows (mean ± standard deviation, n=6): The T6 treatment had a plant height of 24.40 cm (CK1: 19.90 cm), stem diameter of 7.89 mm (CK1: 6.15 mm), SPAD value of 62.01 (CK1: 38.69), total leaf area of ​​608.66 cm² (CK1: 173.66 cm²), number of leaves of 13.60 (CK1: 7.60), total fresh weight of 30.40 g (CK1: 16.62 g), and total dry weight of 12.72 g (CK1: 7.56 g). Meanwhile, the paclobutrazol residue in the soil treated with T6 was only 0.21 mg / kg, significantly lower than that of CK1 (2.12 mg / kg) and other treatments (P<0.05). The paclobutrazol residue in the soil of each treatment group, from highest to lowest, was as follows: CK1 (2.12 mg / kg) > T3 (1.90 mg / kg) > T2 (1.82 mg / kg) > T1 (0.84 mg / kg) > T5 (0.35 mg / kg) > T4 (0.26 mg / kg) > T6 (0.21 mg / kg).

[0117] The above results indicate that the combination of Bacillus composting KB97 with chitosan oligosaccharide and inositol (T6) has a significantly better growth-promoting effect on mango seedlings than any single combination or application of either ingredient alone. Furthermore, it effectively reduces paclobutrazol residues in the soil, demonstrating a significant synergistic effect. This example verifies the excellent effect of the KB97 + chitosan oligosaccharide + inositol ternary combination in alleviating the growth inhibition of mangoes by paclobutrazol, and can be used to prepare microbial compound preparations that reduce paclobutrazol residues.

[0118] Example 5: Effects of the combination of Bacillus composting KB97 and biostimulants on mango fruit quality

[0119] 1. Test location: Shuijiao Mango Base, Sanya City.

[0120] 2. Experimental variety: Tainong mango ( Mangiferaindica L.cv.Tainong) is treated with paclobutrazol multiple times a year through soil or foliar application.

[0121] 3. Experimental Design:

[0122] Set up 5 processes: 10 trees per process

[0123] CK: Healthy trees (not affected by excessive use of paclobutrazol, the trees are basically normal), no treatment was given (control);

[0124] RF: Declining tree condition (caused by excessive paclobutrazol) + conventional fertilization;

[0125] RM: Declining tree body + conventional fertilization + T6 compound microbial preparation (T6 combination in Example 4);

[0126] HF: Healthy tree body + regular fertilization;

[0127] HM: Healthy tree body + conventional fertilization + T6 compound microbial preparation.

[0128] 4. Experimental Method: During the mango pit hardening stage (one month before harvest), T6 compound microbial preparation (containing Bacillus compostii KB97 1×10⁻⁶) was applied. 7 After diluting the ingredients (CFU / mL, chitosan oligosaccharide 0.01%, inositol 0.01%) according to the specified ratio, apply 20L per tree to the roots. Harvest the fruit after it matures, select fruits of uniform size and free from pests and diseases, and conduct a preservation test under storage conditions of 13℃ and 90% relative humidity.

[0129] 5. Experimental Results:

[0130] Fruit appearance quality (see) Figure 11After 30 days of storage, the fruits in the CK group suffered severe water loss and shriveling, and were severely affected by diseases; the fruits in the HM group were plump and bright in color, with the best appearance quality; the RM and HF groups showed performance in between.

[0131] Fruit weight loss / storage quality (see Figure 12 During storage, the weight loss rate of mangoes in all treatment groups increased continuously over time. The CK group showed the fastest increase, with a weight loss rate as high as 38.44% at 30 days; the HM group showed the slowest increase, with a weight loss rate of only 24.81% at 30 days; the weight loss rates of the RM and HF groups were 29.67% and 32.15%, respectively, while there was no significant difference between the RF and CK groups.

[0132] Fruit disease incidence / commercial quality (see Figure 13 After 30 days of storage, the average number of lesions was highest in the CK group, lowest in the HM group, followed by the RM and HF groups, while there was no significant difference between the RF and CK groups.

[0133] Peel color / appearance quality (see Figure 14 , Figure 15 During storage, the total color difference ΔEab of mangoes in all treatments increased continuously with prolonged storage time, but the rate of increase differed significantly. The HM group showed the slowest increase in ΔEab, reaching only 12.38 after 30 days of storage, maintaining a low level throughout and exhibiting the best color retention. The RM group followed, with ΔEab at 17.53 after 30 days, effectively delaying color change. The HF and RF groups showed moderate color retention, with ΔEab values ​​of 21.44 and 24.12 respectively after 30 days. The CK group showed the fastest increase in ΔEab, reaching 25.06 after 30 days, indicating the most significant color deterioration. Furthermore, the HM group exhibited the lowest color change level and the slowest rate of yellowing.

[0134] Comprehensive quality evaluation: The comprehensive quality of each treatment, from best to worst, is as follows: HM (healthy tree + T6) > RM (declining tree + T6) > HF (healthy tree + conventional fertilization) > RF (declining tree + conventional fertilization) > CK (blank control). The above results indicate that the compound microbial agent described in this invention can comprehensively improve the fruit quality of mangoes from multiple dimensions, including weight loss rate, disease control, and color maintenance. It is noteworthy that the comprehensive fruit quality of declining trees treated with the T6 compound microbial agent (RM treatment) was even better than that of healthy trees treated with conventional fertilization (HF treatment). This result fully demonstrates that the compound microbial agent described in this invention can not only effectively degrade paclobutrazol residues in the soil but also repair declining trees caused by long-term paclobutrazol use, restoring the post-harvest quality of their fruit to a level that even surpasses that of healthy trees treated with conventional fertilization. In other words, this invention achieves tree decline reversal "guided by quality restoration," rather than merely superficial "preservation treatment."

[0135] Therefore, it can be seen that the combination of Bacillus KB97 compost and biostimulants (compound microbial preparation) described in this invention can systematically improve the quality of mango fruit from the source by applying it to the root soil one month before harvest. Specifically, it includes: (1) reducing the rate of postharvest water loss and extending the storage period; (2) enhancing the disease resistance of the fruit and reducing postharvest diseases; (3) delaying the color change process of the peel and maintaining a good appearance; and (4) repairing the deteriorating tree and restoring its ability to produce high-quality fruit. Healthy trees treated with this compound microbial preparation (HM treatment) showed the best performance in all quality indicators, and deteriorating trees treated with this preparation (RM treatment) also showed excellent performance, which was significantly better than conventional fertilization and the blank control overall.

[0136] In summary, the applicant of this invention has independently screened and identified a Bacillus composting strain KB97 with good biosafety. Bacillus stercoris (KB97). Studies have confirmed that this strain not only possesses the ability to degrade the pesticide paclobutrazol, but also significantly alleviates the inhibitory effect of paclobutrazol on mango growth. Based on this, this invention successfully constructed a method for the synergistic application of Bacillus compostingus KB97 with chitosan oligosaccharide and inositol to degrade paclobutrazol, promote mango growth, and improve fruit quality through a complete technical path of "strain function verification—growth-promoting substance screening—synergistic compound optimization—multi-level effect evaluation". This method breaks through the core technical bottleneck of limited repair effects and unstable functions of single strains or single substances in existing technologies, and realizes the significant synergistic effect of the combination of Bacillus compostingus KB97, chitosan oligosaccharide, and inositol in promoting plant growth, degrading soil paclobutrazol residues, and comprehensively improving fruit quality. Indoor compound experiments confirmed the optimal growth-promoting concentrations of chitosan oligosaccharide and inositol for KB97 at the growth curve level; pot experiments verified the significant synergistic effect of the ternary compound treatment (T6) on agronomic traits, physiological indicators, and pesticide residue degradation at the plant level; field and postharvest experiments further verified the excellent performance of this combination at the fruit quality level. The applicant's research findings provide new technical solutions and important theoretical foundations for pesticide residue remediation, mango plant growth promotion, fruit quality improvement, and post-harvest preservation.

[0137] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the 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. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A strain of Bacillus compostii ( Bacillus stercoris KB97, with accession number CCTCC NO: M20261063, is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on May 25, 2026.

2. A compound microbial preparation for degrading paclobutrazol in soil and improving fruit quality, characterized in that, It comprises Bacillus compostingus KB97 as described in claim 1 and chitosan oligosaccharide; the mass concentration of the chitosan oligosaccharide is 50 mg / kg to 100 mg / kg, and the viable count of the Bacillus compostingus KB97 is 10. 6 ~10 8 CFU / g; the fruit quality specifically refers to the peel color, weight loss rate, and / or disease incidence rate of mango fruit.

3. The compound microbial preparation according to claim 2, characterized in that, It also contains inositol; the inositol concentration is 50 mg / kg to 100 mg / kg.

4. The use of Bacillus compostingus KB97 as described in claim 1 in the preparation of a microbial preparation for degrading paclobutrazol residues in soil to alleviate the inhibitory effect of paclobutrazol on mangoes.

5. The use of the compound microbial preparation according to claim 2 or 3 in the preparation of a microbial preparation for degrading paclobutrazol residues in soil to alleviate the inhibitory effect of paclobutrazol on mangoes.

6. The application of the compound microbial preparation according to claim 3 in the preparation of a preparation for improving the postharvest storage and preservation effect of mangoes.

7. The application according to claim 6, characterized in that, The improvements in postharvest storage and preservation of mangoes include: reducing postharvest weight loss, inhibiting postharvest diseases, delaying color change of mango peel, and / or improving the storage resistance of mangoes.

8. A method for alleviating the inhibitory effect of paclobutrazol on mango, characterized in that, Apply the Bacillus KB97 of claim 1 or the compound microbial preparation of claim 2 or 3 to the rhizosphere of mango plants, wherein the amount of Bacillus KB97 applied is such that its final concentration in the rhizosphere soil reaches 10. 6 ~10 8 CFU / g soil.

9. A method for improving the post-harvest storage and preservation effect of mangoes, characterized in that, During the mango pit hardening stage, the compound microbial preparation described in claim 3 is applied to the soil at a rate of 10-20 L of diluted microbial agent solution per tree.

10. A compound microbial preparation product, characterized in that, It comprises Bacillus KB97 of claim 1, chitosan oligosaccharide and inositol, as well as agriculturally acceptable adjuvants.

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