Method for reducing the damage of pests and pesticide residues on wolfberry by using plant probiotics

Abstract

The application relates to the field of agricultural planting technology, and particularly discloses a method for reducing disease and insect pests and pesticide residues of wolfberry by using plant probiotics, which comprises the following steps: adopting a high-yield beta-glucanase strain I and a high-yield chitinase strain II to prepare Sb microbial agents through high-density fermentation technology and then spray drying, diluting the Sb microbial agents with water and spraying the Sb microbial agents on the surface of wolfberry plants during the growth period of the wolfberry, or granulating the Sb microbial agents into fermentation agents, mixing the fermentation agents into a substrate to prepare Sb biological fertilizers, and applying the Sb biological fertilizers during the dormancy period of the wolfberry to reduce diseases and insect pests; and adopting a high-yield esterase strain III to prepare a compound microbial agent through high-density liquid fermentation, diluting the compound microbial agent with water and spraying the compound microbial agent on the surface of wolfberry plants during the growth period of the wolfberry to reduce organic phosphorus pesticide residues. The method has the advantages of reducing the diseases and insect pests and pesticide residues of wolfberry, and improving the yield and quality of wolfberry.

Description

Technical Field

[0001] This application relates to the field of agricultural planting technology, and more specifically, it relates to a method for reducing pests and diseases and pesticide residues in wolfberry using plant probiotics. Background Technology

[0002] Due to its unique plateau basin climate, excellent latitude, and suitable sandy loam alkaline soil, Qaidam wolfberries are characterized by large seeds and high content of active ingredients such as flavonoids and polysaccharides, resulting in rich nutritional value and unique health benefits. Compared to other producing areas, the polysaccharide content of Qaidam organic wolfberries is moderate, making them suitable for drying and processing into concentrated juice, wolfberry powder, and wolfberry polysaccharide products. Under the special natural environment, wolfberries grown in the Qaidam Basin possess unparalleled advantages, with high-quality, large fruits accounting for over 70%.

[0003] With the development and expansion of the Qaidam Basin goji berry industry, significant problems urgently need to be addressed, most notably the increase in pests and diseases. Goji berry plants flower and bear fruit multiple times a year, resulting in multiple generations of major pests and diseases annually. The simultaneous occurrence of pests and diseases at the same time and overlapping life stages is extremely common, making control extremely difficult. Surveys have identified hundreds of types of goji berry pests and diseases, including dozens of major ones, many of which are unique to goji berries, causing reduced yields and, in severe cases, even total crop failure.

[0004] In addition, existing methods for controlling diseases and pests in wolfberry include agricultural control, chemical control, and biological control. Due to the numerous diseases and pests affecting wolfberry, the application of chemical pesticides has become an important control measure. Among these, organophosphate pesticides are commonly used for insect and acar control. Chlorpyrifos, as a highly effective, broad-spectrum, and moderately toxic organophosphate insecticide, has good contact, stomach poison, and fumigation effects and is widely used to control pests such as stem borers, armyworms, scale insects, aphids, bollworms, thrips, leafhoppers, and mites on various crops. With the banning of highly effective and toxic pesticides (such as methamidophos and methamidophos), chlorpyrifos has gradually replaced them, leading to its wider application and larger dosage. It has become an important pesticide for controlling diseases and pests in wolfberry, but it has also caused problems such as declining wolfberry quality, environmental pollution, and excessive pesticide residues. Therefore, reducing the use of chlorpyrifos and eliminating its residues in wolfberry products is of great significance for increasing wolfberry yield, improving wolfberry quality, and ensuring food safety. Summary of the Invention

[0005] In order to reduce pests and diseases and pesticide residues in wolfberry and improve wolfberry yield and quality, this application provides a method for reducing pests and diseases and pesticide residues in wolfberry using plant probiotics.

[0006] This application provides a method for reducing pests, diseases, and pesticide residues in wolfberry using plant probiotics, employing the following technical solution:

[0007] A method for reducing diseases, pests, and pesticide residues in wolfberry using plant probiotics includes: using strain I, which produces high levels of β-glucanase, and strain II, which produces high levels of chitinase, through high-density fermentation technology and then spray-drying to produce an Sb inoculum. This inoculum is diluted with water and sprayed onto the surface of wolfberry plants during the wolfberry growing season, or granulated into a fermentation agent and mixed into a substrate to produce an Sb bio-fertilizer, which is applied during the wolfberry dormancy period to reduce diseases and pests; and using strain III, which produces high levels of esterase, through high-density liquid fermentation to produce a compound inoculum, which is diluted with water and sprayed onto the surface of wolfberry plants during the wolfberry growing season to reduce organophosphorus pesticide residues.

[0008] Preferably, strain I is Bacillus subtilis that produces high levels of β-glucanase, strain II is Bacillus subtilis that produces high levels of chitinase, and strain III is Lactobacillus plantarum zju-005 that produces high levels of esterase.

[0009] Preferably, the Sb microbial agent and the compound microbial agent are diluted with water at a weight ratio of 1:100 and sprayed on the surface of the wolfberry plants at a rate of 1000 mL per plant, once a week for 4 consecutive weeks; the Sb bio-fertilizer is applied to each wolfberry plant at a rate of 1 kg.

[0010] Preferably, the preparation method of the Sb inoculant includes the following steps:

[0011] S101, Liquid Culture: After mixing strain I and strain II, inoculate the mixture into liquid culture medium at an inoculation rate of 2%, and incubate at 36°C with shaking for 22 hours until the bacterial density exceeds 1×10⁻⁶. 9 Sb bacterial suspension was obtained by mixing cfu / mL; the liquid culture medium formula was: glucose 11 g / L, soybean meal 20 g / L, shrimp meal 1 g / L, with the remainder being water, and the pH was adjusted to 6.5. The culture was then sterilized at 121℃ for 20 min.

[0012] S102. Fermentation Culture: The Sb bacterial solution was then inoculated into the optimized fermentation medium and cultured at 37℃ and 180rpm for 36 hours. The fermentation was repeated 5 times to obtain a high-density Sb bacterial solution. The fermentation medium formula was: soybean meal 20g / L, wood ash 10g / L, sucrose 5g / L, starch 10g / L, and inducer 5g / L. The inducer was a mixture of β-glucan and chitin.

[0013] S103. Preparation of Sb bacterial culture in microporous corn flour: Corn flour is mixed with water to form corn milk with a concentration of 50%. Raw amylase composed of 5 U / L α-amylase and 500 U / L saccharifying enzyme is added. The mixture is shaken and cultured at 37°C for 24 hours. The starch in the corn flour particles is partially hydrolyzed to form pores. After centrifugation, the mixture is dried at 120°C to obtain microporous corn flour. Then, microporous corn flour is added to the high-density Sb bacterial culture to a concentration of 5% (W / V). The mixture is shaken and embedded at 20°C for 1 hour to obtain microporous corn flour Sb bacterial culture.

[0014] S104. Spray drying: Wood ash powder and soybean powder are used as drying aids, glutinous rice flour is used as wall material, jasmine leaf powder and hyaluronic acid are added to form a slurry solid. The slurry solid is then mixed with microporous corn flour Sb bacterial solution in a ratio of 3:20, stirred evenly, and finally spray dried to produce Sb bacterial agent. The inlet temperature during spray drying is 170-190℃, and the outlet temperature is 80-100℃.

[0015] Preferably, in step S104, the prepared slurry contains 10 parts of microporous corn flour Sb bacterial solution, 1 part of glutinous rice flour, 0.1 parts of jasmine leaf powder, 0.1 parts of hyaluronic acid, 0.2 parts of wood ash, and 0.1 parts of soybean flour.

[0016] Preferably, in step S102, the fermentation is carried out in a fermenter, and the fermentation conditions are as follows: fermentation time 0-10 h, stirring speed 100 rpm, and aeration rate 10 m³ / h. 3 / h; fermentation time 10-20h, stirring speed 150rpm, aeration rate 15m³ / h. 3 / h; fermentation time 20-36h, stirring speed 150rpm, aeration rate 18m³ / h. 3 / h; temperature throughout: 37±1℃, pressure: 0.03-0.04Mpa, pH: natural.

[0017] Preferably, the preparation method of the Sb bio-fertilizer is as follows:

[0018] S201, Solid-state fermentation: The prepared Sb inoculum is inoculated into a solid-state fermentation medium at a ratio of 0.1% and fermented at 37°C for 24 hours to obtain solid-state fermented product; wherein, the formula of the solid-state fermentation medium is 2 parts corn flour, 2 parts soybean meal, 1 part wood ash, 1 part wheat bran, and 3-4 parts water.

[0019] S202, Preparation of fermentation agent: Mix wood ash, soybean powder and solid fermentation material in a ratio of 5:1:5, and granulate in a granulator at 85℃ to obtain granulated mixed fermentation agent of strain I and strain II.

[0020] S203. Preparation of bio-fertilizer: Mix straw powder, soybean meal, wood ash, wheat bran, shrimp powder and mixed fermentation agent in a ratio of 30:3:3:3:1:1, add appropriate water, and let it ferment naturally for 5-10 days to obtain Sb bio-fertilizer.

[0021] Preferably, the preparation method of the compound microbial agent is as follows:

[0022] S301. Activation of strains: Under aseptic conditions, strain III, Saccharomyces cerevisiae and Acetobacter acetic acid were inoculated onto MRS broth medium, YPD medium and AC medium respectively, and cultured in shake flasks at 37℃ and 180r / min for 24h. The samples were then collected for later use.

[0023] S302. Preparation of bacterial culture medium: The activated bacterial strain III, Saccharomyces cerevisiae, and Acetic Acid Bacillus were inoculated again into their respective culture media and fermented for 10-12 hours. Then, they were transferred to their respective solid culture media and cultured for 48 hours to obtain bacterial culture medium. The bacterial density was controlled at 10⁻⁶. 9 Approximately CFU / mL;

[0024] S303. Preparation of microbial agent: The culture medium of the strain was transferred to black tea medium. The inoculation amounts of strain III, Saccharomyces cerevisiae and Acetobacter acetic acid were 6%, 2% and 2% respectively, with a total inoculation amount of 10% (v / v). The medium was then placed in a shake flask at 37℃ and 180r / min for 24h and the compound microbial agent was collected.

[0025] Preferably, the preparation method of the black tea culture medium is as follows: 1% loose tea leaves are placed in boiling double-distilled water, stirred thoroughly and soaked for 10 minutes, then filtered twice with 8 layers of gauze, 7% sucrose is added, and after high-temperature sterilization, the mixture is cooled to 25-30°C and sealed for later use.

[0026] Preferably, the sterilization conditions for the black tea culture medium are: 121℃ for 15 min.

[0027] In summary, this application has the following beneficial effects:

[0028] 1. Since strains I and II used in this application continuously produce β-glucanase and chitinase respectively during their growth, and can coexist and reproduce during mixed fermentation, while simultaneously producing high levels of both β-glucanase and chitinase, the inoculants of strains I and II can be mixed and fermented to prepare an Sb inoculant containing both strains I and II. When used, this inoculant can be sprayed onto the surface of the wolfberry plants, or granulated into a fermentation agent and mixed into the substrate to produce Sb bio-fertilizer, reducing diseases and pests. Field trials have shown that the Sb inoculant and Sb bio-fertilizer prepared using strains I and II can effectively inhibit the occurrence of wolfberry diseases and pests (aphids, psyllids, powdery mildew) and improve wolfberry production performance.

[0029] 2. The β-glucanase-producing strain I in this application exhibits significant antagonistic and inhibitory effects on pathological fungi of Lycium barbarum (black fruit disease fungi, powdery mildew fungi, and root rot fungi). This is because β-glucanase can strongly disrupt the cell walls of pathological fungi, thus significantly inhibiting and killing them. The chitinase-producing strain II, after entering Lycium barbarum insect pest larvae, significantly inhibits digestive enzymes, thereby killing the larvae.

[0030] 3. After screening strain III, this application blended strain III with acetic acid bacteria and brewer's yeast in an appropriate ratio to produce a microecologically stable mixed bacterial solution. Field trials showed that the bacterial solution of strain III had a significant reduction effect on pesticide residues, and the microecologically stable mixed bacterial solution containing strain III had a significant reduction effect on chlorpyrifos and imidacloprid residues. Furthermore, acetic acid bacteria and yeast can also produce abundant hydrolytic enzymes and metabolites, thus providing a synergistic effect on the pesticide residue reduction of strain III.

[0031] 4. *Lactobacillus plantarum* and *Bacillus subtilis* have relatively good abilities to degrade chlorpyrifos, and probiotic strains play a positive role in chlorpyrifos degradation. Furthermore, *Lactobacillus plantarum* is a food-grade safe microorganism that can widely colonize plants and has probiotic and disease-resistant effects. The abundant hydrolytic enzymes produced by lactic acid bacteria, including esterases, will play an important role in the effective degradation of pesticide residues. Attached Figure Description

[0032] Figure 1 This is a plate antagonistic diagram of strain I of this application against the black fruit disease fungus;

[0033] Figure 2 This is a growth curve of strain I of this application and a graph showing the changes in its β-glucanase activity;

[0034] Figure 3 This is a growth curve of strain II of this application and a graph showing the changes in its chitinase activity;

[0035] Figure 4 This is a growth curve and a graph showing the changes in the activities of β-glucanase and chitinase when strain I and strain II of this application are cultured together;

[0036] Figure 5 Standard curves of chlorpyrifos, dichlorvos, and dimethoate under the action of different organic solvents;

[0037] Figure 6 This is a graph showing the changes in chlorpyrifos concentration and degradation rate over time under the action of different probiotics in this application;

[0038] Figure 7 This is a graph showing the changes in dichlorvos concentration and degradation rate over time under the action of different probiotics in this application;

[0039] Figure 8 This is a graph showing the changes in dimethoate concentration and degradation rate over time under the action of different probiotics in this application;

[0040] Figure 9 This is a graph showing the comparison of pesticide degradation rates of different wolfberries under the action of probiotics in this application (96 h);

[0041] Figure 10 This is a dynamic graph showing the reduction of chlorpyrifos residue in wolfberry fruit, as described in this application;

[0042] Figure 11 This is a dynamic graph showing the reduction of chlorpyrifos and imidacloprid residues in wolfberry fruit. Detailed Implementation

[0043] The present application will be further described in detail below with reference to the accompanying drawings and embodiments.

[0044] Example

[0045] A method for reducing diseases, pests, and pesticide residues in wolfberry using plant probiotics includes: using strain I, which produces high levels of β-glucanase, and strain II, which produces high levels of chitinase, through high-density fermentation technology and then spray-drying to produce an Sb inoculum. This inoculum is diluted with water and sprayed onto the surface of wolfberry plants during the wolfberry growing season, or granulated into a fermentation agent and mixed into a substrate to produce an Sb bio-fertilizer, which is applied during the wolfberry dormancy period to reduce diseases and pests; and using strain III, which produces high levels of esterase, through high-density liquid fermentation to produce an inoculum, which is diluted with water and sprayed onto the surface of wolfberry plants during the wolfberry growing season to reduce organophosphorus pesticide residues.

[0046] The Sb microbial agent and compound microbial agent are diluted with water at a weight ratio of 1:100 and sprayed on the surface of the wolfberry plants at a rate of 1000 mL per plant, once a week for 4 consecutive weeks; the Sb bio-fertilizer is applied to each wolfberry plant at a rate of 1 kg.

[0047] The strain I is Bacillus subtilis that produces high levels of β-glucanase, strain II is Bacillus subtilis that produces high levels of chitinase, and strain III is Lactobacillus plantarum zju-005 that produces high levels of esterase.

[0048] Preparation of Sb inoculant

[0049] The preparation method of Sb inoculant includes the following steps:

[0050] S101, Liquid culture: The initial densities of both strain I and strain II exceeded 1×10⁻⁶. 6 The bacterial count was 2% (cFU / mL). After mixing the two, the mixture was inoculated into liquid culture medium and cultured at 36°C with shaking for 22 hours until the bacterial density exceeded 1×10⁻⁶. 9 Sb bacterial suspension was obtained by mixing cfu / mL; the liquid culture medium formula was: glucose 11 g / L, soybean meal 20 g / L, shrimp meal 1 g / L, with the remainder being water, and the pH was adjusted to 6.5. The culture was then sterilized at 121℃ for 20 min.

[0051] S102. Fermentation Culture: The Sb bacterial solution was then inoculated into the optimized fermentation medium and cultured at 37℃ and 180rpm for 36 hours. The fermentation was repeated 5 times to obtain a high-density Sb bacterial solution. The fermentation medium formula was: soybean meal 20g / L, wood ash 10g / L, sucrose 5g / L, starch 10g / L, and inducer 5g / L. The inducer was a mixture of β-glucan and chitin.

[0052] In step S102, the fermentation is carried out in a fermenter, and the fermentation conditions are as follows: fermentation time 0-10 h, stirring speed 100 rpm, and aeration rate 10 m³ / h. 3 / h; fermentation time 10-20h, stirring speed 150rpm, aeration rate 15m³ / h. 3 / h; fermentation time 20-36h, stirring speed 150rpm, aeration rate 18m³ / h. 3 / h; temperature throughout: 37±1℃, pressure: 0.03-0.04Mpa, pH: natural.

[0053] S103. Preparation of Sb bacterial culture in microporous corn flour: Corn flour is mixed with water to form corn milk with a concentration of 50%. Raw amylase composed of 5 U / L α-amylase and 500 U / L saccharifying enzyme is added. The mixture is shaken and cultured at 37°C for 24 hours. The starch in the corn flour particles is partially hydrolyzed to form pores. After centrifugation, the mixture is dried at 120°C to obtain microporous corn flour. Then, microporous corn flour is added to the high-density Sb bacterial culture to a concentration of 5% (W / V). The mixture is shaken and embedded at 20°C for 1 hour to obtain microporous corn flour Sb bacterial culture.

[0054] S104. Spray drying: Wood ash powder and soybean powder are used as drying aids, glutinous rice flour is used as wall material, jasmine leaf powder and hyaluronic acid are added to form a slurry solid. The slurry solid is then mixed with microporous corn flour Sb bacterial solution at a ratio of 3:20, stirred evenly, and finally spray dried to produce Sb bacterial agent. The inlet temperature during spray drying is 170-190℃, and the outlet temperature is 80-100℃.

[0055] In step S104, the prepared slurry contains 10 parts of microporous corn flour Sb bacterial solution, 1 part of glutinous rice flour, 0.1 part of jasmine leaf powder, 0.1 part of hyaluronic acid, 0.2 parts of wood ash, and 0.1 parts of soybean flour.

[0056] The preparation method of Sb bio-fertilizer is as follows:

[0057] S201, Solid-state fermentation: The prepared Sb inoculum is inoculated into a solid-state fermentation medium at a ratio of 0.1% and fermented at 37°C for 24 hours to obtain solid-state fermented product; wherein, the formula of the solid-state fermentation medium is 2 parts corn flour, 2 parts soybean meal, 1 part wood ash, 1 part wheat bran, and 3-4 parts water.

[0058] S202, Preparation of fermentation agent: Mix wood ash, soybean powder and solid fermentation material in a ratio of 5:1:5, and granulate in a granulator at 85℃ to obtain granulated mixed fermentation agent of strain I and strain II; use wood ash powder and soybean powder as drying aids, the former has a growth-promoting effect on Bacillus subtilis, and the latter contains insect digestive enzyme inhibitors.

[0059] S203. Preparation of bio-fertilizer: Mix straw powder, soybean meal, wood ash, wheat bran, shrimp powder and mixed fermentation agent in a ratio of 30:3:3:3:1:1, add appropriate water, and let it ferment naturally for 5-10 days to obtain Sb bio-fertilizer.

[0060] The preparation method of the compound microbial agent is as follows:

[0061] S301. Activation of strains: Under aseptic conditions, strain III, Saccharomyces cerevisiae and Acetobacter acetic acid were inoculated onto MRS broth medium, YPD medium and AC medium respectively, and cultured in shake flasks at 37℃ and 180r / min for 24h. The samples were then collected for later use.

[0062] S302. Preparation of bacterial culture medium: The activated strain III, Saccharomyces cerevisiae, and Acetic Acid Bacillus were inoculated again into MRS broth medium, YPD medium, and AC medium, respectively, and fermented for 10-12 hours. Then, they were transferred to MRS solid broth medium, YPD solid medium, and AC solid medium, respectively, and cultured for 48 hours to obtain bacterial culture medium. The bacterial density was controlled at 10-1. 9 Approximately CFU / mL;

[0063] S303. Preparation of microbial agent: The culture medium of the strain was transferred to black tea medium. The inoculation amounts of strain III, Saccharomyces cerevisiae and Acetobacter acetic acid were 6%, 2% and 2% respectively, with a total inoculation amount of 10% (v / v). The medium was then placed in a shake flask at 37℃ and 180r / min for 24h and the compound microbial agent was collected.

[0064] In this application, the preparation methods for each culture medium are as follows:

[0065] MRS medium: 10.0g peptone, 10.0g beef extract, 5.0g yeast extract, 2.0g diammonium citrate, 20.0g glucose, 5.0g sodium acetate, 2.0g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 0.2g manganese sulfate, 1.0mL Tween 80, and water to a final volume of 1000mL; adjust pH to 6.0.

[0066] MRS solid medium: 10.0g peptone, 10.0g beef extract, 5.0g yeast extract, 2.0g diammonium citrate, 20.0g glucose, 5.0g sodium acetate, 2.0g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 0.2g manganese sulfate, 1.0mL Tween 80, 2% agar powder; pH adjusted to 6.0.

[0067] YPD medium (w / v): 1% yeast extract, 2% tryptone, 2% glucose.

[0068] YPD solid medium (w / v): 1% yeast extract, 2% tryptone, 2% glucose, 2% agar powder.

[0069] AC medium (w / v): 1% yeast extract, 2% tryptone, 2% calcium carbonate; after high-temperature sterilization and cooling to room temperature, add 3.5% 95% ethanol (v / v).

[0070] AC solid medium (w / v): 1% yeast extract, 2% tryptone, 2% calcium carbonate, 2% agar powder; after high-temperature sterilization and cooling to 40 °C, 3.5% 95% ethanol (v / v) is added.

[0071] Black tea culture medium: Place 1% loose tea leaves in boiling double-distilled water, stir thoroughly and soak for 10 minutes, then filter twice through 8 layers of gauze, add 7% sucrose, sterilize at high temperature, cool to 25-30℃, and seal for later use. The sterilization conditions for black tea culture medium are: 121℃, 15 minutes.

[0072] Screening of strains I, II and III

[0073] 1. The screening method for strain I is as follows:

[0074] (1) Culture medium

[0075] Plate screening medium: 250 mL 2% β-glucan, 0.5 g Congo red, 0.7 g K2HPO4, 0.3 g KH2PO4, 0.5 g MgSO4, 0.02 g FeSO4·H2O, 5 g NaCl, 20 g agar, and distilled water to a final volume of 1000 mL, pH 7.0–7.2.

[0076] Initial fermentation medium: 500 mL 2% β-glucan, 10 g peptone, 0.7 g K2HPO4, 0.3 g KH2PO4, 0.5 g MgSO4, 0.02 g FeSO4·H2O, 5 g NaCl, and distilled water to a final volume of 1000 mL, pH 7.0–7.2.

[0077] (2) Screening of glucanase-producing strain I

[0078] Primary screening: Soil samples were collected from bamboo forests. 10g of soil was added to 90mL of sterile physiological saline containing small glass beads. After thorough shaking, the sample was serially diluted with the initial fermentation medium. The samples were incubated at 28℃ for 4–6 hours, then treated at 75℃ for 15 minutes. 0.1mL of each appropriate dilution was then pipetted onto selection plates and incubated at 30℃ for 1–3 days. The presence of a clear zone around the colony was observed, and the H / C ratio (diameter of the clear zone / colony diameter) was recorded. Single colonies with high H / C ratios were selected and streaked onto plates for isolation and purification.

[0079] Secondary screening: The strains obtained from the initial screening were inoculated into the initial fermentation medium (25 mL / 100 mL). After culturing at 30 °C and 200 rpm for 12 h, the strains were inoculated into the initial fermentation medium (50 mL / 250 mL) at a volume fraction of 1%. The culture was continued at 30 °C and 200 rpm. The β-glucanase activity in the fermentation supernatant was measured periodically.

[0080] (3) Determination of glucanase activity

[0081] The DNS method (3,5-dinitrosalicylic acid colorimetric method) was used, with glucose as the standard, to construct a standard curve. The absorbance of the test samples and sample blanks was measured separately. Glucose solutions of different concentrations were prepared, and 1 mL was accurately transferred, incubated in a 40℃ water bath for 60 min, 1 mL of DNS was added, and the solution was boiled for color development for 5 min. The solution was then cooled to room temperature in a water bath, and the absorbance was measured at 540 nm to construct the glucose standard curve.

[0082] Centrifuge the fermentation broth at 4℃ and 5000×g for 10 min. Collect 0.5 mL of the supernatant in a test tube, add 0.5 mL of 1% β-glucan, incubate at 40℃ for 60 min, add 1 mL of DNS and boil for 5 min for color development. For the blank setting: add 0.5 mL of 1% colloidal chitin, incubate at 40℃ for 60 min, add 1 mL of DNS and boil for 5 min, then add the supernatant and for color development. After color development, centrifuge at 4℃ and 5000×g for 3 min, collect the supernatant and measure the absorbance at 540 nm. Calculate the reducing sugar content based on the standard curve. Define the amount of enzyme required to produce the equivalent of 1 μmol of glucose per minute as one unit of enzyme activity (U).

[0083] (4) Molecular biological identification of strains

[0084] The 16S rDNA sequence was amplified from the bacterial genomic DNA using universal primers 27F (A-GAGTTTGATCMTGGCTCAG) and 1492R (TACGGY-TACCTTGTTACGACTT) (PCR amplification conditions: 94℃ for 5 min; 94℃ for 30 s, 48℃ for 30 s, 72℃ for 90 s, 30 cycles; 72℃ for 10 min). The PCR-amplified DNA fragments were separated by electrophoresis on a 1% agarose gel, and the purified DNA was recovered using a kit and directly sent for sequencing. Sequencing results were analyzed for sequence homology using NCBI to determine the taxonomic species.

[0085] (5) Results

[0086] A strain of Bacillus subtilis (strain I) was screened. This bacterium produces a high level of β-glucanase, which has an inhibitory or bactericidal effect on pathogenic fungi because the fungal cell wall is rich in glucan. Figure 1 As shown in Table 1, the study found that β-glucanase is closely related to the antifungal effect, and strain I showed a significant inhibitory and killing effect on the pathogenic fungus of Lycium barbarum. Figure 1 As shown, in bidirectional culture, the black fruit disease fungus *Colletotrichum gloeosporioides* Penz could only grow towards the side without strain I. The colonies of *Colletotrichum gloeosporioides* Penz spread out in a fan shape, with a relatively clear antagonistic zone. This indicates that strain I has a significant inhibitory effect on the black fruit disease fungus *Colletotrichum gloeosporioides* Penz.

[0087] Table 1. Inhibitory and bactericidal effects of bacterial suspension of strain I and its derived bio-fertilizer on the pathogenic fungus of Lycium barbarum.

[0088]

[0089] Note: The inhibition band refers to the difference between the inhibition radius and the radius of the colony to be tested. -: indicates no inhibition band; +: indicates the inhibition band is <5mm; ++: indicates the inhibition band is >5mm.

[0090] 2. The screening method for strain II is as follows:

[0091] (1) Preparation of colloidal chitin

[0092] 30g of chitin powder (Sigma-Aldrich, USA) was slowly added to 300mL of pre-cooled concentrated hydrochloric acid. The mixture was magnetically stirred for 2 hours and then allowed to stand at 4°C for 24 hours. The mixture was then slowly added to 2L of pre-cooled distilled water and stirred until homogeneous. The mixture was centrifuged at 5000×g for 10 minutes to collect the white colloidal chitin precipitate, which was repeatedly washed until the pH reached approximately 6.5. Finally, the precipitate was resuspended in 500mL of distilled water to obtain a 6% colloidal chitin stock solution.

[0093] (2) Culture medium

[0094] Plate screening medium: 250 mL of 2% colloidal chitin, 0.5 g of Congo red, 0.7 g of K2HPO4, 0.3 g of KH2PO4, 0.5 g of MgSO4, 0.02 g of FeSO4·H2O, 5 g of NaCl, 20 g of agar, and distilled water to a final volume of 1000 mL, pH 7.0–7.2.

[0095] Initial fermentation medium: 500 mL of 2% colloidal chitin, 10 g of peptone, 0.7 g of K2HPO4, 0.3 g of KH2PO4, 0.5 g of MgSO4, 0.02 g of FeSO4·H2O, 5 g of NaCl, and distilled water to a final volume of 1000 mL, pH 7.0–7.2.

[0096] (3) Screening of chitinase-producing Bacillus

[0097] Initial screening: Collect coastal near-tidal flat soil. Add 10g of soil sample to 90mL of sterile physiological saline containing small glass beads. After thorough shaking, perform serial dilutions with the initial fermentation medium. Incubate at 28℃ for 4-6 hours, then treat at 75℃ for 15 minutes. Use a pipette to transfer 0.1mL of the appropriate dilution to screening plates and incubate at 30℃ for 1-3 days. Observe whether a clear zone appears around the colony and record the H / C value (diameter of the clear zone / colony diameter). Select single colonies with high H / C values ​​and streak them onto plates for isolation and purification.

[0098] Secondary screening: The strains obtained from the initial screening were inoculated into the initial fermentation medium (25 mL / 100 mL). After culturing at 30°C and 200 rpm for 12 h, the strains were inoculated into the initial fermentation medium (50 mL / 250 mL) at a volume fraction of 1%. The strains were then cultured at 30°C and 200 rpm. Chitinase activity in the fermentation supernatant was measured periodically.

[0099] (4) Determination of chitinase activity

[0100] The DNS method (3,5-dinitrosalicylic acid colorimetric method) was used, with N-acetyl-β-D-glucosamine as the standard. A standard curve was plotted, and the absorbance of the test samples and sample blanks was measured separately. N-acetyl-D-glucosamine solutions of different concentrations were prepared. 1 mL of each solution was accurately transferred, incubated in a 40°C water bath for 60 min, 1 mL of DNS was added, and the solution was boiled for color development for 5 min. The solution was then cooled to room temperature in a water bath, and the absorbance was measured at 540 nm to plot the N-acetyl-D-glucosamine standard curve.

[0101] Centrifuge the fermentation broth at 4℃ and 5000×g for 10 min. Collect 0.5 mL of the supernatant in a test tube, add 0.5 mL of 1% colloidal chitin, incubate at 40℃ for 60 min, add 1 mL of DNS and boil for 5 min for color development. For the blank setting: add 0.5 mL of 1% colloidal chitin, incubate at 40℃ for 60 min, add 1 mL of DNS and boil for 5 min, then add the supernatant and for color development. After color development, centrifuge at 4℃ and 5000×g for 3 min, collect the supernatant and measure the absorbance at 540 nm. Calculate the reducing sugar content based on the standard curve. Define the amount of enzyme required to produce the equivalent of 1 μmol of β-DN-acetylglucosamine reducing sugar per minute as one enzyme activity unit (U).

[0102] (5) Determination of the inhibition rate (%) of insect digestive enzymes

[0103] The midgut and hemolymph of 4-year-old caterpillars were collected to measure the activities of protease, amylase, and esterase.

[0104] Digestive enzyme inhibition rate (%) = 1 - (specific enzyme activity of control group - specific enzyme activity of treatment group) / specific enzyme activity of control group.

[0105] (6) Results

[0106] A strain of Bacillus subtilis (strain II) was screened. This bacterium produces a high amount of chitinase, which significantly inhibits digestive enzymes after entering the larvae of wolfberry pests, thus killing the larvae of wolfberry pests.

[0107] Table 2. Inhibitory effects of bacterial suspension of strain II and bio-fertilizer derived from it on digestive enzymes of Lycium barbarum insect larvae.

[0108]

[0109] Table 2 shows that the bacterial solution of strain II and the bio-fertilizer made from its compost have chitinase activity, which significantly inhibits the digestive enzymes of wolfberry insect larvae.

[0110] 3. Growth curves of strain I and strain II and changes in their enzyme activity.

[0111] Fermentation medium: soybean meal, wood ash, sucrose, starch, inducers (β-glucan and chitin).

[0112] Table 3. Growth curves of strain I and changes in its β-glucanase activity.

[0113]

[0114] Table 4. Growth curves of strain II and changes in chitinase activity.

[0115]

[0116] Table 5. Growth curves and changes in the activities of β-glucanase and chitinase when strains I and II were co-cultured.

[0117]

[0118] Summary: Based on Tables 3-5 and Figures 2-4 When the culture medium contained the inducing components β-glucan and chitin, strains I and II continuously produced β-glucanase and chitinase during their growth, respectively, showing a significant correlation between growth and enzyme production. This means that for strains I and II, increased cell density equates to increased enzyme production. When strains I and II were co-fermented, although the growth status of strains I and II could not be directly distinguished, the activities of β-glucanase and chitinase in the fermentation system were significantly positively correlated with cell density, indirectly indicating that both strains I and II were growing well. This means that high production can be achieved simultaneously through co-fermentation of strains I and II.

[0119] 4. The screening method for strain III is as follows:

[0120] This application selects laboratory-preserved probiotics, including *Lactobacillus plantarum*, *Lactobacillus rhamnosus*, *Lactococcus lactis*, and *Bacillus subtilis*, as well as commercially available probiotic lactic acid bacteria yogurt starter cultures, as screening targets for probiotics. Chlorpyrifos, dichlorvos, and dimethoate are selected as representative varieties of commonly used chemical pesticides in wolfberry, and probiotics with pesticide degradation capabilities are screened. Petroleum ether, xylene, and ethyl acetate are selected as extraction solvents to study their pesticide extraction capabilities and to screen for the optimal organic solvent. High-performance liquid chromatography (HPLC) is used to determine and calculate the pesticide content and degradation rate at different times under the action of different pre-screened probiotics, and to study and compare the pesticide degradation capabilities of different pre-screened probiotics.

[0121] 4.1 Experimental Materials and Methods

[0122] (1) Experimental strains

[0123] Lactobacillus plantarum zju-005, Lactobacillus rhamnosus zju-012, Lactococcus lactis zju-006, Bacillus subtilis LJH25, Bacillus natto zju-025, Bacillus natto CICC 20640, Bacillus natto CICC 20641, and Bacillus natto CICC 20642 were all laboratory-preserved strains. The lactic acid bacteria yogurt starter (Lactobacillus bulgaricus and Streptococcus thermophilus) was purchased from Beijing Chuanxiu Technology Co., Ltd.

[0124] (2) Main reagents

[0125] Chlorpyrifos, dichlorvos, and dimethoate standards (all purchased from Beijing Century Aoke Biotechnology Co., Ltd., purity > 99%); MRS broth medium and MRS solid medium (both purchased from Beijing Aoboxing Biotechnology Co., Ltd.); PBS buffer (pH 7.4 ± 0.2); soybean sprouts (commercially available); yeast extract, tryptone, NaCl, sucrose, agar powder, petroleum ether, xylene, ethyl acetate, methanol, formic acid, anhydrous Na2SO4, and double-distilled water (all reagents were of chromatographic or analytical grade).

[0126] (3) Solution preparation

[0127] 100 mg / mL Chlorpyrifos solution: Dissolve 0.25 g of chlorpyrifos standard in 2.5 mL of methanol and store at 4 °C.

[0128] 100 mg / mL dichlorvos solution: Dissolve 0.25 g of dichlorvos standard in 2.5 mL of methanol and store at 4 °C.

[0129] 100 mg / mL dimethoate solution: Dissolve 0.25 g of dimethoate standard in 2.5 mL of methanol and store at 4 °C.

[0130] Formic acid solution (v / v): 0.1% formic acid (chromatographic grade) is placed in double-distilled water and mixed thoroughly.

[0131] (4) Preparation of culture medium

[0132] LB medium (w / v): 1% yeast extract, 2% tryptone, 1% NaCl.

[0133] Soybean juice fermentation medium (w / v): soybean sprout juice (20% soybean sprouts placed in double-distilled water, boiled and concentrated to 50%, filtered), 2% sucrose.

[0134] Soybean sprout solid culture medium (w / v): soybean sprout juice (20% soybean sprouts placed in double-distilled water, boiled and concentrated to 50%, then filtered), 2% sucrose, 2% agar powder. All the above culture media were sterilized at 121℃ for 15 min.

[0135] (5) Activation of strains

[0136] Under aseptic conditions, *Lactobacillus plantarum* zju-005, *Lactobacillus rhamnosus* zju-012, *Lactococcus lactis* zju-006, and lactic acid bacteria yogurt starter were inoculated onto MRS broth medium and cultured in shake flasks at 37°C and 180 rpm for 24 h. *Bacillus subtilis* LJH25, *Bacillus natto* zju-025, *Bacillus natto* CICC20640, *Bacillus natto* CICC20641, and *Bacillus natto* CICC20642 were inoculated onto LB medium and cultured in shake flasks at 37°C and 180 rpm for 24 h, and then collected for later use.

[0137] (6) Fermentation culture

[0138] The activated strain was transferred to the corresponding soybean juice fermentation medium (50 mL fermentation medium / 250 mL Erlenmeyer flask) at an inoculation amount of 6% (v / v), and fermented at 37℃ and 180 r / min for 24 h. Then, a second transfer fermentation was performed, and the samples were collected for later use.

[0139] (7) Enrichment culture

[0140] After the temperature of the soybean juice fermentation medium drops to room temperature, add pesticides to the corresponding soybean juice fermentation medium to a concentration of 100 mg / L, mix thoroughly, and then transfer the bacterial culture to the corresponding soybean juice fermentation medium at an inoculation amount of 10% (v / v). Incubate at 37℃ and 180 r / min in shake flasks. Transfer the culture every so often, keeping the inoculation amount constant, and gradually increase the pesticide concentration by 100 mg / L each time, until the concentration reaches 500 mg / L. Allow the culture to acclimate for more than 2 weeks, and then collect the culture for later use.

[0141] After diluting the strain by the appropriate factor, inoculate it onto the corresponding soybean juice solid culture medium containing 500 mg / L pesticide. After 72 hours, observe the growth of the strain, screen out the strains with better growth, and number and store them.

[0142] (8) Comparison of pesticide extraction from wolfberry using different organic solvents

[0143] Petroleum ether, xylene, and ethyl acetate were selected as extraction solvents. 100 mg / mL solutions of chlorpyrifos, dichlorvos, and dimethoate were added to MRS broth medium (50 mL medium / 250 mL Erlenmeyer flask) at concentration gradients of 62.5, 125, 250, 500, and 1000 mg / L, respectively. The mixtures were shaken at 180 rpm for 0.5 h at room temperature. 10 mL of the pesticide-containing culture medium was transferred to a test tube, and an equal volume of extraction solvent was added. The mixture was thoroughly shaken and allowed to stand until complete separation. The organic phase was collected, and an equal volume of extraction solvent was added for a second extraction. The organic phases were combined, and anhydrous Na2SO4 was added to absorb water. The mixture was then concentrated to 1–2 mL by rotation, and finally brought to a final volume of 10 mL with methanol. The collected solution was then stored for later use.

[0144] (9) Determination of pesticide content by high performance liquid chromatography (HPLC)

[0145] Different pesticide samples were filtered through a 0.22 μm microporous membrane, and the filtrates were collected for analysis. The analysis was performed using a Synergi4uHydro-RP 80A reversed-phase C18 column (250 × 4.6 mm), with mobile phase V... (甲酸溶液) ∶V (甲醇) The ratio was 15:85, the flow rate was 1.0 mL / min, the column temperature was 35℃, and the injection volume was 20 μL. The detection wavelength was the corresponding characteristic absorption peak. The characteristic absorption peaks of chlorpyrifos, dichlorvos, and dimethoate were 292, 211, and 210 nm, respectively.

[0146] Collect the corresponding peak diagrams of the samples to be tested and calculate the content of different pesticide samples.

[0147] (10) Establishment of standard curve and determination of recovery rate

[0148] For the extracted samples, a standard curve was established with concentration gradients of 62.5, 125, 250, 500 and 1000 mg / L, with concentration as the x-axis and peak area as the y-axis, and the regression equation was obtained.

[0149] The 100 mg / mL pesticide solution was successively diluted to 62.5, 125, 250, 500 and 1000 mg / L, and used as a blank control. The recovery rate and coefficient of variation of pesticides with different concentrations were determined. Three sets of parallel experiments were conducted to compare the extraction effects of three organic solvents, ethyl acetate, xylene and petroleum ether, on the corresponding pesticides.

[0150] The recovery rate (AR) is calculated using the formula: Recovery Rate % = C1 / C0 × 100%

[0151] Wherein, C1 is the mass concentration (mg / L) of pesticide in the treated sample, and C0 is the mass concentration (mg / L) of pesticide in the control group.

[0152] The formula for calculating the coefficient of variation (CV) is: Coefficient of variation % = Standard deviation / Mean × 100%

[0153] (11) Preparation of different probiotic systems for reducing pesticide use in wolfberry

[0154] After the temperature of the soybean juice fermentation medium dropped to room temperature, pesticides were added to the corresponding soybean juice fermentation medium to a concentration of 250 mg / L. After thorough mixing, the culture solution of the strain after initial screening was transferred to the corresponding soybean juice fermentation medium at an inoculation amount of 10% (v / v). The culture was then placed in a shake flask at 37°C and 180 r / min. Bacterial culture samples were collected at 0, 24, 48, 72 and 96 h for later use.

[0155] Take 10 mL of the pesticide-containing culture medium and extract with ethyl acetate. Use the sample without bacterial culture as a blank control, and perform the same procedures as the treated sample.

[0156] (12) Determination of pesticide content and degradation rate by high performance liquid chromatography (HPLC)

[0157] A culture medium containing chlorpyrifos without inoculation of the bacterial strain was used as a blank control, and the resulting solution was extracted and used as the standard test solution. An appropriate amount of the test sample was taken and subjected to HPLC pretreatment.

[0158] Collect the corresponding peak diagrams of the samples to be tested, calculate the pesticide content of wolfberry at different times and the degradation rate of wolfberry pesticide by probiotics, conduct three sets of parallel experiments, compare the degradation ability of different probiotics on wolfberry pesticide, and the degradation trend over time.

[0159] The formula for calculating the degradation rate is: X = (Cck - Cx) / Cck

[0160] In the formula: X is the degradation rate, Cck is the pesticide concentration (mg / L) in the control sample, and Cx is the pesticide concentration (mg / L) in the treated sample.

[0161] 4.2 Experimental Results and Discussion

[0162] (1) Initial screening of probiotics with the ability to reduce pesticide residues in wolfberries

[0163] Eight laboratory-preserved bacterial strains and nine commercially available lactic acid bacteria yogurt starter cultures or mixtures were selected. After enrichment culture and plate growth, the statistical results are shown in Table 6.

[0164] Table 6 shows that under the action of three kinds of wolfberry pesticides, the four strains of Bacillus natto diluted to 10 6 At that time, growth was not obvious. Only Bacillus natto 20640 showed some growth, with colony counts between 11 and 27; and Bacillus natto 20641 showed slight growth under the action of dichlorvos. The remaining 5 probiotic strains or mixtures were diluted to 10 times.7 At the same time, all showed varying degrees of good growth. Under the action of chlorpyrifos, the number of colonies growing, arranged in descending order, was Lactobacillus plantarum, Bacillus subtilis, Lactobacillus rhamnosus, lactic acid bacteria yogurt starter, and Lactococcus lactis, with Lactobacillus plantarum reaching a growth rate of 2.6 × 10⁻⁶. 9 cfu / mL; the growth trend of the strain was similar to that of chlorpyrifos, but the overall inhibitory effect was more significant, with the best-growing *Lactobacillus plantarum* reaching 1.0 × 10⁻⁶ cfu / mL. 9 CFU / mL; the order of activity under the action of dichlorvos was Bacillus subtilis, Lactobacillus rhamnosus, Lactobacillus plantarum, lactic acid bacteria yogurt starter, and Lactococcus lactis. The inhibitory effect on these strains was also stronger than that of chlorpyrifos. Bacillus subtilis showed the best growth, reaching 1.2 × 10⁻⁶ CFU / mL. 9 cfu / mL.

[0165] Taking all factors into consideration, Lactobacillus plantarum, Bacillus subtilis, Lactobacillus rhamnosus, lactic acid bacteria yogurt starter, and Lactococcus lactis all exhibit good growth capabilities in pesticide systems. Therefore, these five probiotic strains or mixtures will be initially screened for further research.

[0166] Table 6. Growth of different probiotics on plates under different pesticide treatments on wolfberries.

[0167]

[0168] Note: " / " indicates no growth.

[0169] (2) Comparison of pesticide extraction capabilities of different organic solvents for wolfberry

[0170] The extraction of organophosphorus pesticides is often carried out in liquid-liquid systems. Xylene, petroleum ether, and ethyl acetate were selected for the extraction of three commonly used pesticides from wolfberry: chlorpyrifos, dichlorvos, and dimethoate. The relationship between pesticide concentration and peak area in wolfberry was established, and the corresponding recoveries were calculated. The results are as follows: Figure 5 As shown in Table 7.

[0171] exist Figure 5 In the control groups of chlorpyrifos, dichlorvos, and dimethoate, the standard curve regression equations are as follows:

[0172] y = 5263x + 711194 (R2=0.992),

[0173] y = 3450x + 488981 (R2=0.993),

[0174] y = 3878x + 541065 (R2 = 0.991).

[0175] The relative extraction efficiencies of the three organic solvents for chlorpyrifos, in descending order, were petroleum ether, ethyl acetate, and xylene, with petroleum ether and ethyl acetate being relatively close. In dichlorvos extraction, ethyl acetate was more efficient than both petroleum ether and xylene. In dimethoate extraction, xylene was significantly less efficient than both petroleum ether and ethyl acetate. Due to the different properties of pesticides and extraction solvents, there are instances where increasing pesticide concentration leads to a decrease in solvent extraction efficiency.

[0176] Table 7 Comparison of the extraction effects of different organic solvents on pesticides in wolfberry

[0177]

[0178] Note: AR refers to average recovery rate; CV refers to coefficient of variation.

[0179] Table 7 shows that the average recovery rates of chlorpyrifos extracted with organic solvents ranged from 64.6% to 91.0%, with coefficients of variation ranging from 3.2% to 3.8%; for dichlorvos, the average recovery rates ranged from 75.4% to 89.1%, with coefficients of variation ranging from 3.2% to 4.5%; and for dimethoate, the average recovery rates ranged from 58.8% to 83.9%, with coefficients of variation ranging from 3.6% to 4.9%. The low coefficients of variation ensured the accuracy of the extraction operation.

[0180] The extraction capabilities of petroleum ether, xylene, and ethyl acetate for three pesticides found in wolfberry were selected and studied. The average recoveries of petroleum ether and ethyl acetate for chlorpyrifos were 91.0% and 89.5%, respectively, which were quite close and higher than those of other extraction solvents or pesticides. The average recoveries of ethyl acetate for dichlorvos and dimethoate were 89.1% and 83.9%, respectively, representing the highest recoveries among the corresponding pesticides.

[0181] Taking all factors into consideration, xylene had the lowest extraction efficiency among the three wolfberry pesticides, while petroleum ether and ethyl acetate had similar extraction rates. Petroleum ether had a relatively large coefficient of variation, which, corresponding to its volatile nature, led to a relatively high experimental error due to its instability. Therefore, based on extraction efficiency and solvent stability, ethyl acetate was chosen as the extraction solvent for wolfberry pesticides and for subsequent experimental studies.

[0182] 4.3 Determination of the ability of different probiotics to reduce pesticide residues in wolfberry

[0183] (1) Effects of different probiotics on pesticide degradation in wolfberry

[0184] Five probiotic strains or mixtures obtained from the initial screening were used in degradation experiments of three types of pesticides on wolfberry. Ethyl acetate was used as the extraction solvent, and relationship curves between time, concentration, and degradation rate were established. The results are as follows: Figure 6-8 As shown.

[0185] Depend on Figure 6It can be seen that under the action of probiotic strains, the concentration of chlorpyrifos decreased continuously over time, while the degradation rate showed an increasing trend. After 96 hours of cultivation, the concentration of chlorpyrifos in the control group decreased from 248.5 mg / L to 217.7 mg / L, indicating that chlorpyrifos undergoes a certain degree of self-degradation. In the groups of lactic acid bacteria yogurt starter, Lactococcus lactis, Lactobacillus rhamnosus, Bacillus subtilis, and Lactobacillus plantarum, the concentration of chlorpyrifos pesticide decreased to 191.6, 194.3, 169.9, 152.4, and 140.8 mg / L, respectively, with corresponding degradation rates of 23.2%, 23.1%, 33.2%, 39.6%, and 45.1%. In the first 24 hours of cultivation, only Lactobacillus plantarum showed a relatively fast growth rate and strong adaptability; after 48 hours of cultivation, the changing trends of chlorpyrifos concentration and degradation rate gradually slowed down.

[0186] Taking all factors into consideration, Lactobacillus plantarum and Bacillus subtilis have relatively good ability to degrade chlorpyrifos, and probiotic strains have a positive effect on the degradation of chlorpyrifos. The degradation rate increases rapidly at first and then slowly within a certain time range.

[0187] Depend on Figure 7 It can be seen that under the action of probiotic strains, the concentration of dichlorvos decreased continuously over time, while the degradation rate showed an increasing trend, eventually stabilizing. After 96 hours of cultivation, the concentration of dichlorvos pesticide in the lactic acid bacteria yogurt starter, *Lactococcus lactis*, *Lactobacillus rhamnosus*, *Bacillus subtilis*, and *Lactobacillus plantarum* groups decreased from approximately 250 mg / L to 188.2, 200.3, 164.8, 159.4, and 170.8 mg / L, respectively, with corresponding degradation rates of 25.3%, 20.2%, 32.0%, 36.8%, and 31.0%. Considering all factors, *Bacillus subtilis* exhibits relatively good dichlorvos degradation ability.

[0188] Depend on Figure 8 It can be seen that under the action of probiotic strains, the concentration of dimethoate continuously decreased over time, while the degradation rate showed an increasing trend, eventually stabilizing. After 96 hours of cultivation, the concentrations of dimethoate pesticide in the lactic acid bacteria yogurt starter, *Lactococcus lactis*, *Lactobacillus rhamnosus*, *Bacillus subtilis*, and *Lactobacillus plantarum* groups decreased from approximately 250 mg / L to 198.8, 210.1, 188.7, 187.4, and 174.1 mg / L, respectively, with corresponding degradation rates of 21.1%, 16.2%, 23.7%, 25.7%, and 28.8%. Considering all factors, *Lactobacillus plantarum* exhibits relatively good ability to degrade dimethoate.

[0189] (2) Comparison of pesticide degradation in different wolfberries under the action of probiotics

[0190] A cross-sectional comparison was conducted on the degradation differences of different pesticides on wolfberry by probiotic strains. The pesticide degradation rates of wolfberry after 96 hours of growth of different probiotics were selected as data support. The results are as follows: Figure 9 As shown.

[0191] Depend on Figure 9 It was found that after 96 hours of cultivation, apart from the lactic acid bacteria yogurt starter, the degradation abilities of the other four probiotic strains for the three pesticides on wolfberry, ranked in descending order, were chlorpyrifos, dichlorvos, and dimethoate. Overall, chlorpyrifos was more easily degraded, while dimethoate was the most difficult to degrade. Among the same pesticides, *Lactobacillus plantarum* showed the best degradation ability for chlorpyrifos, while *Bacillus subtilis* showed better degradation performance for dichlorvos.

[0192] In summary, HPLC analysis of the degradation concentrations and rates of pesticides under the action of different probiotic strains showed that the five initially screened strains exhibited varying degrees of degradation activity against three commonly used pesticides in wolfberry: chlorpyrifos, dichlorvos, and dimethoate. Furthermore, the degradation rate increased rapidly at first and then slowed down over a certain time period. Among them, *Lactobacillus plantarum* and *Bacillus subtilis* showed relatively good degradation abilities against chlorpyrifos, with degradation rates of 45.1% and 39.6% respectively after 96 hours. *Bacillus subtilis* showed relatively good degradation abilities against dichlorvos, with a degradation rate of 36.8% after 96 hours. *Lactobacillus plantarum* showed relatively good degradation abilities against dimethoate, with a degradation rate of 28.8% after 96 hours. A comparative analysis revealed that chlorpyrifos showed greater degradation activity compared to dichlorvos and dimethoate among the different probiotic strains. Since chlorpyrifos is currently the most widely used and consumed organophosphorus pesticide, further research will be conducted using chlorpyrifos, the selected probiotic strains, and the extraction solvent.

[0193] Test methods

[0194] I. Field trial of Bacillus subtilis inoculum solution and bio-fertilizer with high yields of glucanase and chitinase for the control of diseases and pests (aphids, psyllids, powdery mildew) in wolfberry.

[0195] 1.1 Test time and location

[0196] Test location: Dagele Township, Golmud City.

[0197] Test period: July 20 - August 4, 2016.

[0198] 1.2 Materials and Methods

[0199] Experimental materials: Bacillus subtilis strains (strain I and strain II) that produce high levels of glucanase and chitinase.

[0200] Test method:

[0201] (1) High-density culture of strain I and strain II

[0202] The initial densities of both strain I and strain II exceeded 1 × 10⁻⁶. 6The concentration of the bacteria was 10⁶ / mL. After mixing the two, the inoculum was added to the liquid culture medium at an inoculum size of 2%, and cultured at 36°C with shaking for 22 hours until the bacterial density exceeded 1×10⁶. 9 Sb bacterial suspension was obtained by mixing cfu / mL; the liquid culture medium formula was: glucose 11 g / L, soybean meal 20 g / L, shrimp meal 1 g / L, with the remainder being water, and the pH was adjusted to 6.5. The culture was then sterilized at 121℃ for 20 min.

[0203] (2) Preparation of strain I and strain II inoculants (Sb inoculant)

[0204] Corn flour was mixed with water to make corn milk with a concentration of 50%. Raw amylase consisting of 5 U / L α-amylase and 500 U / L saccharifying enzyme was added, and the mixture was incubated at 37°C with shaking for 24 hours. The starch in the corn flour particles was partially hydrolyzed to form pores. After centrifugation, the mixture was dried at 120°C to obtain microporous corn flour. Then, microporous corn flour was added to the Sb bacterial culture to a concentration of 5% (W / V), and the mixture was incubated at 20°C with shaking for 1 hour to obtain microporous corn flour Sb bacterial culture.

[0205] Using plant ash powder and soybean flour as drying aids, glutinous rice flour as wall material, jasmine leaf powder and hyaluronic acid are added to form a slurry solid. The slurry solid is then mixed with microporous corn flour Sb bacterial solution in a ratio of 3:20, stirred evenly, and finally spray-dried to produce Sb bacterial agent. The inlet temperature during spray drying is 170℃ and the outlet temperature is 80℃.

[0206] The prepared slurry contains 10 parts microporous corn flour Sb bacterial solution, 1 part glutinous rice flour, 0.1 part jasmine leaf powder, 0.1 part hyaluronic acid, 0.2 parts wood ash, and 0.1 part soybean flour.

[0207] (3) Preparation of bio-fertilizer (Sb bio-fertilizer) by strain I and strain II

[0208] The prepared Sb inoculant was inoculated into a solid fermentation medium at a ratio of 0.1% and fermented at 37℃ for 24 hours to obtain a solid fermentation product. Then, wood ash, soybean meal, and solid fermentation product were mixed in a ratio of 5:1:5 and granulated at 85℃ using a granulator to obtain granular mixed fermentation agent of strains I and II. Finally, the compost components (60 parts straw, 6 parts soybean meal, 6 parts wood ash, 6 parts wheat bran, and 2 parts shrimp meal) were mixed evenly and sprayed with the prepared mixed fermentation agent (2 parts) at a rate of 1% (Sb inoculant volume L / compost weight Kg). The compost was kept away from rain and direct sunlight and composted for 6 days to obtain Sb bio-fertilizer.

[0209] (4) Preparation of control agent

[0210] Corn flour was mixed with water to make corn milk with a concentration of 50%. Raw amylase composed of α-amylase (EC.3.2.1.1) and saccharifying enzyme (EC.3.2.1.3) (enzyme dosages of 5 U / L and 500 U / L, respectively) was added. The mixture was cultured at 37°C with shaking for 24 hours. The starch in the corn flour particles was partially hydrolyzed to form pores. After centrifugation, the mixture was dried at 120°C to obtain microporous corn flour.

[0211] The relevant components (10 parts microporous corn flour, 1 part glutinous rice flour, 0.1 part jasmine leaf powder, 0.1 part hyaluronic acid, and 20 parts water) were mixed into a slurry and then spray-dried (inlet temperature 170℃, outlet temperature 80℃) to prepare control agent-I.

[0212] (5) Preparation of control fertilizer

[0213] Mix the compost components (60 parts straw, 6 parts soybean meal, 6 parts wood ash, 6 parts wheat bran, and 2 parts shrimp powder) evenly, avoid rain and direct sunlight, and compost for 8 days to obtain non-Sb bio-fertilizer.

[0214] (6) Determination of Sb density

[0215] The test was conducted in accordance with "GB / T 26428-2010 Detection of Bacillus subtilis in feed microbial preparations".

[0216] (7) Chitinase activity assay

[0217] Take 5 mL of Lsb bacterial culture and centrifuge at 5000 rpm for 10 min. Take 0.5 mL of the supernatant, add 1 mL of Lmcllvaine buffer and 0.5 mL of 3% colloidal chitin, incubate at 37°C for 30 min, then incubate in boiling water for 10 min. Take 1 mL of the supernatant, centrifuge at 5000 rpm for 5 min, and add 0.5 mL of DNS reagent to terminate the reaction. After boiling in water for 5 min, immediately cool, add 4 mL of deionized water, and measure the OD540 nm value. Plot a standard curve with N-acetylglucosamine content on the x-axis and OD540 nm value on the y-axis, with the blank tube as the zero point. Under the conditions of 37°C and pH 7.0, the amount of enzyme required to produce 1 μmol of N-acetylglucosamine reducing sugar per minute is defined as 1 enzyme activity unit (U).

[0218] (8) β-glucanase activity assay

[0219] The reducing sugar assay method is used. β-glucanase can degrade β-glucan into oligosaccharides and monosaccharides. Its reducing groups can react with DNS reagent under boiling water bath conditions to produce a colorimetric reaction. The intensity of the color is directly proportional to the amount of reducing sugar produced, which in turn is directly proportional to the β-glucanase activity of the reaction solution. Therefore, the amount of reducing sugar produced can be calculated by colorimetric determination of the absorbance of the reaction solution, thus obtaining the β-glucanase activity. The definition of β-glucanase activity unit (IU): Under the assay conditions, the amount of enzyme required to hydrolyze β-glucan to produce 1 μmol of reducing sugar (calculated as glucose) per minute is defined as one enzyme activity unit (IU).

[0220] (9) Use of Sb inoculant and Sb bio-fertilizer

[0221] When used to control aphids and psyllids, dilute the Sb inoculant with water at a weight ratio of 1:100 and spray it onto the surface of the wolfberry plants. The amount sprayed (based on the weight of the inoculant) is 1000 mL per wolfberry plant, sprayed once a week for 4 consecutive weeks. Apply Sb bio-fertilizer at a rate of 1 kg per wolfberry plant.

[0222] When used to suppress powdery mildew, spray Sb fungicide continuously for 5 days.

[0223] (10) The use of control agents and control fertilizers is the same as the use of Sb inoculants and Sb bio-fertilizers.

[0224] 1.3 Insect Infestation Assessment and Control Effectiveness

[0225] (1) Evaluation of psyllid infestation in wolfberry

[0226] Psyllid nymphs cause more damage than adults, and adults migrate frequently, making them difficult to count. Therefore, psyllid infestation is evaluated by counting nymphs. In a wolfberry forest, each implementation example or control example had 8 plots (4 wolfberry plants per plot), and an additional 8 plots were assumed as blank controls, for a total of 48 plots. Five branches from each wolfberry plant were randomly selected from five directions (east, south, west, north, and center). The number of psyllid nymphs on each branch was recorded before the experiment and 3 days after the experiment. Infestation was evaluated using the following formula:

[0227] Prevention and control effect = (1 - CK0 × PT1 / CK1 × PT0) × 100%

[0228] CK0: Number of live insects in the blank control area before the experiment.

[0229] CK1: Number of live insects in the blank control area after the experiment.

[0230] PT0: Number of live insects before the test in the example or control case.

[0231] PT1: Number of live insects after the test in the example or control case.

[0232] Goji berry yield per plant determination: In the goji berry forest, each example or control example was divided into 8 plots (4 goji berry plants per plot). After the fresh fruit was harvested, the yield of each plot was weighed separately.

[0233] The number of samples for detecting psyllid infestation and yield per plant in each example or control was 8.

[0234] (2) Evaluation of the aphid infestation on wolfberry

[0235] In the wolfberry forest, each example or control case consisted of 8 plots (4 wolfberry plants per plot), and 8 plots were assumed as blank controls, for a total of 48 plots. Five branches from each wolfberry plant were randomly selected from five directions (east, south, west, north, and center), and the aphid count on each branch was recorded before the experiment and 3 days after the experiment. The insect infestation was evaluated using the following formula.

[0236] Prevention and control effect = (1 - CK0 × PT1 / CK1 × PT0) × 100%

[0237] CK0: Number of live insects in the blank control area before the experiment.

[0238] CK1: Number of live insects in the blank control area after the experiment.

[0239] PT0: Number of live insects before the test in the example or control case.

[0240] PT1: Number of live insects after the test in the example or control case.

[0241] Goji berry yield per plant determination: In the goji berry forest, each example or control example was divided into 8 plots (4 goji berry plants per plot). After the fresh fruit was harvested, the yield of each plot was weighed separately.

[0242] The number of samples for detecting aphid infestation and yield per plant in each example or control was 8.

[0243] (3) Evaluation of the effectiveness of powdery mildew control

[0244] When powdery mildew occurs, the initial disease incidence is assessed before spraying with Sb fungicide. Sb fungicide is then sprayed continuously for 5 days, and the control efficacy is assessed twice, at 7 and 14 days after spraying. A fixed-point, fixed-plant, fixed-shoot survey is conducted, with 10 new shoots surveyed per plot, and 10 leaves surveyed from top to bottom on each shoot, for a total of 100 leaves surveyed per plot. Each leaf is graded according to the percentage of diseased area relative to the total leaf area, and the total number of leaves surveyed and the number of leaves at each grade are recorded. The grading criteria are as follows.

[0245] 0: No lesions;

[0246] l: The area of ​​lesions accounts for less than 5% of the total leaf area;

[0247] 3: The area of ​​lesions accounts for 6-10% of the total leaf area:

[0248] 5: The area of ​​lesions accounts for 11-20% of the total leaf area;

[0249] 7: The area of ​​lesions accounts for 21-40% of the total leaf area:

[0250] 9: The area of ​​diseased spots accounts for more than 41% of the total leaf area.

[0251] Calculation of protective efficacy:

[0252] Disease index = ∑(number of diseased leaves at each level × relative grade value) / (total number of leaves surveyed × 9) × 100

[0253] Efficacy (%) = [1 - (Disease index before treatment × Disease index after treatment) / (Disease index after treatment × Disease index before treatment)] × 100

[0254] 1.4 Experimental Results and Analysis

[0255] As shown in Tables 8, 9, and 10, the bacterial solution in the examples exhibited high chitinase activity, the bacterial agent contained high Sb density, and the prepared Sb bio-fertilizer also showed high chitinase activity and high Sb density. In contrast, the bacterial agent and bio-fertilizer in the control examples showed virtually no Sb or chitinase activity, indicating a close correlation between high Sb density and high chitinase activity. The psyllid and aphid infestations in the examples were significantly lower than in the control examples, while the yield per plant was significantly higher, demonstrating that the Sb-rich bacterial agent and bio-fertilizer effectively reduced psyllid and aphid infestations and improved goji berry production performance. Furthermore, the Sb bacterial agent also showed good control of powdery mildew on goji berries.

[0256] Table 8. Control effects of Sb inoculant and Sb biofertilizer on wolfberry psyllids.

[0257]

[0258] Table 9. Control effects of Sb inoculant and Sb biofertilizer on wolfberry aphids.

[0259]

[0260] Table 10. Control efficacy of Sb inoculant against powdery mildew of wolfberry.

[0261]

[0262] II. Field trials of strain III and its inoculant for reducing pesticide residues in wolfberry

[0263] Organophosphorus pesticides have advantages such as a wide variety, high efficacy, broad application range, and low cost. They are the most widely used pesticides in my country, accounting for 80-90% of total pesticide usage. Chlorpyrifos is a highly effective, broad-spectrum, and moderately toxic organophosphorus insecticide with good contact, stomach poison, and fumigation effects. It is widely used to control pests such as stem borers, armyworms, scale insects, aphids, bollworms, thrips, leafhoppers, and mites on various crops. With the banning of highly effective and toxic pesticides (such as methamidophos and methamidophos), chlorpyrifos has gradually replaced them, leading to its increasingly wider application and larger dosage. It has become an important pesticide for controlling pests and diseases in wolfberry products. Therefore, reducing chlorpyrifos residues in wolfberry products is of great significance for improving wolfberry quality and ensuring food safety.

[0264] This application involves preparing a bacterial solution of *Lactobacillus plantarum* (strain III) capable of degrading chlorpyrifos, and observing its degradation effect on chlorpyrifos in the reagent production environment of wolfberry through field trials.

[0265] 2.1 Experimental Materials and Methods

[0266] Test strains: Lactobacillus plantarum zju-005 (i.e., strain III) and inoculum (strain III mixed with acetic acid)

[0267] (Obtained by co-culturing of bacteria and yeast) for laboratory preservation.

[0268] Main reagent: Chlorpyrifos EC (Jiangsu Huifeng Agrochemical Co., Ltd., 40% active ingredient).

[0269] 2.2 Main Instruments and Equipment

[0270] Instrument and model: Agilent 6820 gas chromatograph; Manufacturer: Agilent Technologies.

[0271] 2.3 Detection and Analysis Methods

[0272] (1) Culture of strain III

[0273] The initial density of Lactobacillus plantarum is 10 to 10⁻⁶. 7 The bacterial suspension was inoculated into MRS medium at a concentration of 1% (cfu / mL) and cultured at 37°C with shaking for 24 hours to obtain a bacterial suspension with a density of 1×10⁻⁶. 9 cfu / mL.

[0274] The activated strain III, Saccharomyces cerevisiae CICC 33068, and Acetic Acid Bacillus CICC 20441 were then fermented in their respective culture media for a certain period of time, and then transferred to their respective solid culture media and cultured for 48 hours, with the strain density controlled at 102. 9 Approximately CFU / mL.

[0275] (2) Preparation of microbial agents

[0276] The bacterial culture was then transferred to black tea culture medium. The inoculation amounts of strain III, Saccharomyces cerevisiae, and Acetobacter acetic acid were 6%, 2%, and 2%, respectively, with a total inoculation amount of 10% (v / v). The culture was then placed in a shake flask at 37 ℃ and 180 r / min for 24 h and collected for later use to obtain the compound bacterial agent.

[0277] (3) Determination of chlorpyrifos residue in wolfberry poisoning

[0278] Gas chromatography was used, and the determination of multiple residues of organophosphorus, organochlorine, pyrethroid and carbamate pesticides in vegetables and fruits was carried out by a testing company in accordance with the standard NY / T 761-2008.

[0279] 2.4 Field Trials

[0280] Location: Dagele Township, Golmud City, Qinghai Province

[0281] (1) Field trial of strain III

[0282] Ten wolfberry trees were divided into a control group (Group C) and an experimental group (Group E). Chlorpyrifos was applied using a backpack sprayer via conventional spraying. In the experimental group, 5 kg of bacterial solution of strain III was sprayed per wolfberry tree at 4 h (E1), 7 d (E2), 14 d (E3), and 21 d (E4) after chlorpyrifos application. In the control group, 5 kg of water was sprayed per wolfberry tree at 4 h (C1), 7 d (C2), 14 d (C3), and 21 d (C4) after chlorpyrifos application. Fresh fruit was collected one day after the bacterial solution application in the experimental group and one day after the water application in the control group to test for chlorpyrifos residues.

[0283] (2) Field trials of microbial agents

[0284] The experiment consisted of 20 wolfberry trees each, divided into a control group (Group C) and an experimental group (Group E). Chlorpyrifos and imidacloprid were applied using a backpack sprayer via conventional spraying. In the experimental group, 5 kg of inoculant solution was sprayed per wolfberry tree at 4 h (E1), 7 d (E2), 14 d (E3), and 21 d (E4) after pesticide application. In the control group, 5 kg of water was sprayed per wolfberry tree at 4 h (C1), 7 d (C2), 14 d (C3), and 21 d (C4) after pesticide application. One day after spraying the inoculant solution in the experimental group and one day after spraying the water in the control group, fresh fruit was collected to test for chlorpyrifos residue.

[0285] 2.5 Results and Discussion

[0286] (1) Strain III reduces chlorpyrifos residue

[0287] The decline dynamics of chlorpyrifos residues in wolfberry fruit are shown in Table 11 and Figure 10 As shown.

[0288] Table 11 Reduction dynamics of chlorpyrifos residues in wolfberry fruit

[0289]

[0290] The results showed that the application of the inoculant containing strain III significantly reduced the residues of chlorpyrifos and imidacloprid in wolfberry poisoning, reaching the safety standard.

[0291] (2) Inoculants containing strain III can reduce the residues of chlorpyrifos and imidacloprid.

[0292] The decline dynamics of chlorpyrifos and imidacloprid residues in wolfberry fruit are shown in Table 12 and Figure 11 As shown.

[0293] Table 12 Reduction dynamics of chlorpyrifos and imidacloprid residues in wolfberry fruit

[0294]

[0295] The results showed that the application of the inoculant containing strain III significantly reduced the residues of chlorpyrifos and imidacloprid in wolfberry poisoning, reaching the safety standard.

[0296] 2.6 Summary

[0297] The bacterial solution prepared from *Lactobacillus plantarum* zju-005 (i.e., strain III), which was screened in the laboratory and has the ability to degrade chlorpyrifos, showed its ability to reduce pesticide residues in wolfberries and meet safety standards in field trials. Furthermore, the microecologically stable compound microbial agent obtained by co-culturing strain III with acetic acid bacteria and yeast was even more effective in reducing pesticide residues in wolfberries, and due to its microecological stability, it was more suitable for field application.

[0298] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for reducing pests, diseases, and pesticide residues in wolfberry using plant probiotics, characterized in that, This includes using strain I, which produces high levels of β-glucanase, and strain II, which produces high levels of chitinase, to produce Sb microbial agents through high-density fermentation and then spray-drying. These agents are diluted with water and sprayed onto the surface of goji berry plants during their growth period, or granulated into a fermentation agent and mixed into a substrate to produce Sb bio-fertilizer, which is applied during the dormant period of goji berries to reduce diseases and pests. Additionally, strain III, which produces high levels of esterase, is used to produce a compound microbial agent through high-density liquid fermentation. This agent is diluted with water and sprayed onto the surface of goji berry plants during their growth period to reduce organophosphorus pesticide residues.

2. The method for reducing pests and diseases and pesticide residues in wolfberry using plant probiotics according to claim 1, characterized in that: The strain I is Bacillus subtilis that produces high levels of β-glucanase, strain II is Bacillus subtilis that produces high levels of chitinase, and strain III is Lactobacillus plantarum zju-005 that produces high levels of esterase.

3. The method for reducing pests and diseases and pesticide residues in wolfberry using plant probiotics according to claim 1, characterized in that: The Sb microbial agent and compound microbial agent are diluted with water at a weight ratio of 1:100 and sprayed on the surface of the wolfberry plants at a rate of 1000 mL per plant, once a week for 4 consecutive weeks; the Sb bio-fertilizer is applied to each wolfberry plant at a rate of 1 kg.

4. The method for reducing pests and diseases and pesticide residues in wolfberry using plant probiotics according to claim 1, characterized in that: The preparation method of the Sb inoculant includes the following steps: S101, Liquid Culture: After mixing strain I and strain II, inoculate the mixture into liquid culture medium at an inoculation rate of 2%, and incubate at 36°C with shaking for 22 hours until the bacterial density exceeds 1×10⁻⁶. 9 cfu / mL, to obtain Sb bacterial culture; S102, Fermentation culture: The Sb bacterial solution was then inoculated into the optimized fermentation medium and cultured at 37℃ and 180rpm for 36h. The fermentation was repeated 5 times to obtain a high-density Sb bacterial solution. S103. Preparation of Sb bacterial culture in microporous corn flour: Corn flour is mixed with water to form corn milk with a concentration of 50%. Raw amylase composed of 5 U / L α-amylase and 500 U / L saccharifying enzyme is added. The mixture is shaken and cultured at 37°C for 24 hours. The starch in the corn flour particles is partially hydrolyzed to form pores. After centrifugation, the mixture is dried at 120°C to obtain microporous corn flour. Then, microporous corn flour is added to the high-density Sb bacterial culture to a concentration of 5% (W / V). The mixture is shaken and embedded at 20°C for 1 hour to obtain microporous corn flour Sb bacterial culture. S104. Spray drying: Wood ash powder and soybean powder are used as drying aids, glutinous rice flour is used as wall material, jasmine leaf powder and hyaluronic acid are added to form a slurry solid. The slurry solid is then mixed with microporous corn flour Sb bacterial solution at a ratio of 3:20, stirred evenly, and finally spray dried to produce Sb bacterial agent. The inlet temperature during spray drying is 170-190℃, and the outlet temperature is 80-100℃.

5. The method for reducing pests and pesticide residues in wolfberry using plant probiotics according to claim 4, characterized in that: In step S104, the prepared slurry contains 10 parts of microporous corn flour Sb bacterial solution, 1 part of glutinous rice flour, 0.1 part of jasmine leaf powder, 0.1 part of hyaluronic acid, 0.2 parts of wood ash, and 0.1 parts of soybean flour.

6. The method for reducing pests and pesticide residues in wolfberry using plant probiotics according to claim 4, characterized in that: In step S102, the fermentation is carried out in a fermenter, and the fermentation conditions are as follows: fermentation time 0-10 h, stirring speed 100 rpm, and aeration rate 10 m³ / h. 3 / h; fermentation time 10-20h, stirring speed 150rpm, aeration rate 15m³ / h. 3 / h; fermentation time 20-36h, stirring speed 150rpm, aeration rate 18m³ / h. 3 / h; temperature throughout: 37±1℃; pressure: 0.03-0.04 MPa; pH: natural.

7. The method for reducing pests and pesticide residues in wolfberry using plant probiotics according to claim 4, characterized in that: The preparation method of the Sb bio-fertilizer is as follows: S201, Solid-state fermentation: The prepared Sb inoculum is inoculated into a solid-state fermentation medium at a ratio of 0.1%, and fermented at 37°C for 24 hours to obtain solid-state fermentation product; S202, Preparation of fermentation agent: Mix wood ash, soybean powder and solid fermentation material in a ratio of 5:1:5, and granulate in a granulator at 85℃ to obtain granulated mixed fermentation agent of strain I and strain II. S203. Preparation of bio-fertilizer: Mix straw powder, soybean meal, wood ash, wheat bran, shrimp powder and mixed fermentation agent in a ratio of 30:3:3:3:1:1, add appropriate water, and let it ferment naturally for 5-10 days to obtain Sb bio-fertilizer.

8. The method for reducing pests and diseases and pesticide residues in wolfberry using plant probiotics according to claim 1, characterized in that: The preparation method of the compound microbial agent is as follows: S301. Activation of strains: Under aseptic conditions, strain III, Saccharomyces cerevisiae and Acetobacter acetic acid were inoculated onto MRS broth medium, YPD medium and AC medium respectively, and cultured in shake flasks at 37℃ and 180r / min for 24h. The samples were then collected for later use. S302. Preparation of bacterial culture medium: The activated bacterial strain III, Saccharomyces cerevisiae, and Acetic Acid Bacillus were inoculated again into their respective culture media and fermented for 10-12 hours. Then, they were transferred to their respective solid culture media and cultured for 48 hours to obtain bacterial culture medium. The bacterial density was controlled at 10⁻⁶. 9 Approximately CFU / mL; S303. Preparation of microbial agent: The culture medium of the strain was transferred to black tea medium. The inoculation amounts of strain III, Saccharomyces cerevisiae and Acetobacter acetic acid were 6%, 2% and 2% respectively, with a total inoculation amount of 10% (v / v). The medium was then placed in a shake flask at 37℃ and 180r / min for 24h and the compound microbial agent was collected.

9. The method for reducing pests and diseases and pesticide residues in wolfberry using plant probiotics according to claim 1, characterized in that: The preparation method of the black tea culture medium is as follows: 1% loose tea leaves are placed in boiling double-distilled water, stirred thoroughly and soaked for 10 minutes, then filtered twice with 8 layers of gauze, then 7% sucrose is added, and after high-temperature sterilization, the mixture is cooled to 25-30℃ and sealed for later use.

10. A method for reducing pests and diseases and pesticide residues in wolfberry using plant probiotics according to claim 9, characterized in that: The sterilization conditions for the black tea culture medium were all 121℃ for 15 min.

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