Microbial seed coating agent for resisting paris polyphylla root rot as well as preparation method and application of microbial seed coating agent

Abstract

The invention provides a paris polyphylla root rot resistant microbial seed coating as well as a preparation method and application thereof. Specifically, through a sodium alginate-polyethylene glycol-glycerol hydrogel system and assisted by bacillus velezensis TLD6 and aspergillus awamori TPD6, the microbial seed coating agent is developed, the resistance of paris polyphylla to root rot caused by fusarium oxysporum is improved, and the yield of paris polyphylla is increased. By measuring the indexes such as agronomic traits, mixed liquid storage stability, embedding rate, antibacterial property, disease index, prevention and treatment effect, superoxide dismutase, peroxidase and catalase activity, the conclusion that the microbial seed coating agent prepared from the sodium alginate-polyethylene glycol-glycerol hydrogel has a relatively good action effect on root rot resistance of paris polyphylla is obtained.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial agents and plant protection, specifically a microbial seed dressing agent for resisting root rot of Paris polyphylla, its preparation method and application. Background Technology

[0002] Paris polyphylla root rot is a typical soil-borne fungal disease caused by Fusarium oxysporum. It can infect the entire growth cycle of Paris polyphylla, and its main symptom is that the affected rhizomes gradually rot from the tail end, exhibiting a wet, soft rot or a cottony soft rot. The epidermis of the infected rhizomes turns dark brown, and in severe cases, it can lead to the death of the Paris polyphylla. This disease often breaks out and spreads in hot and humid environments, posing a significant threat to the cultivation of Paris polyphylla.

[0003] Currently, the control methods for Paris polyphylla root rot mainly include agricultural regulation, chemical pesticide use, and biological control. However, agricultural control measures are complex to implement and difficult to scale up; while the use of chemical pesticides can achieve short-term effects, it easily induces pesticide resistance in pathogens and causes environmental pollution and pesticide residue safety issues. In contrast, biological control, as a green and environmentally friendly disease prevention strategy, has good application prospects.

[0004] In recent years, various microbial resources have been developed for the prevention and control of plant diseases. For example, *Aspergillus pumilus* (… Aspergillus awamori Bacillus belye (B) possesses a rich enzyme system and metabolites, showing broad application potential in industrial and agricultural fields. Some studies have demonstrated its inhibitory activity against pathogens. Bacillus velezensis It also exhibits good antifungal activity and is widely used in the prevention and control of diseases such as root rot and damping-off in various crops.

[0005] Microbial seed coating agents, as a novel formulation, encapsulate seed surfaces with a mixture of biocontrol microorganisms and adhesive materials. This not only enhances seed protection against pathogens but also promotes growth in the early stages of plant development, thereby increasing crop yield and quality. The key lies in the selection of the carrier material and the efficiency of microbial encapsulation and release. Sodium alginate is a commonly used natural biodegradable polymer that can react with Ca... 2+ Sodium alginate forms a gel network structure with divalent cations, which is widely used in microcapsule preparation and cell immobilization. However, the gel structure formed by sodium alginate alone is relatively fragile and prone to microbial leakage or rapid release, limiting its persistence in microbial preparations.

[0006] To improve the mechanical properties and controlled-release capability of sodium alginate gel, we explored compounding it with materials such as polyethylene glycol (PEG) and glycerol (Gly). PEG exhibits excellent biocompatibility and membrane-forming properties, while glycerol, as a plasticizer, effectively improves the gel's flexibility and stability, enhancing its practical application as a carrier material. Although existing studies have reported the application of PEG-sodium alginate blends in medical and environmental fields, research on the sodium alginate-polyethylene glycol-glycerol (SA-PEG-Gly) ternary composite system as a carrier for microbial seed coating agents remains relatively scarce.

[0007] Based on this, this invention screened and optimized the sodium alginate-polyethylene glycol-glycerol compound ratio, using it as a hydrogel carrier. Combined with *Bacillus vesiculosus* TLD6 and *Aspergillus auris* TPD6, which have biocontrol potential, a microbial seed coating system was constructed. A stable coating material was formed through ionic cross-linking, and its physicochemical properties, biocontrol activity maintenance ability, disease resistance, and growth-promoting effect were systematically evaluated. This research provides a new approach for the development of efficient, green, and safe microbial seed coating agents, and is expected to provide technical support for the sustainable control of Paris polyphylla root rot. Summary of the Invention

[0008] This invention provides a microbial seed coating agent for controlling root rot in Paris polyphylla, its preparation method, and its application. Specifically, it is a composite hydrogel based on sodium alginate-polyethylene glycol-glycerol (SA-PEG-Gly) matrix, used to encapsulate two biocontrol strains, Bacillus vesiculus TLD6 and Aspergillus auris TPD6, to construct a stable and efficient microbial seed coating agent suitable for the prevention and control of root rot in Paris polyphylla and for promoting the growth of Paris polyphylla. This invention is achieved through the following technical solution: According to the first objective of this invention, the present invention provides a microbial seed coating agent for resisting root rot of Paris polyphylla, wherein the seed coating agent uses sodium alginate-polyethylene glycol-glycerol hydrogel as a carrier to encapsulate biocontrol bacteria; the biocontrol bacteria are Bacillus belyssus TLD6 and / or Aspergillus pumilus TPD6; the concentration of sodium alginate in the hydrogel carrier is 0.5%-1.5% w / v, the concentration of polyethylene glycol is 0.5%-1.5% w / v, and the concentration of glycerol is 0.5%-1% w / v.

[0009] As a preferred technical solution, the number of biocontrol bacteria in each 1 mL of seed coating agent is 10. 8 CFUs.

[0010] As a preferred technical solution, the hydrogel carrier exhibits pH-dependent swelling behavior, meaning that the swelling rate of the hydrogel increases as the pH value of the solution increases.

[0011] As a preferred technical solution, the seed coating agent has a viable bacterial count of not less than 6 log CFU / mL and an encapsulation rate of not less than 83% after 6 months of storage.

[0012] According to a second objective of the present invention, the present invention provides a method for preparing a microbial seed dressing agent for resisting root rot of Paris polyphylla, comprising the following steps: (1) Screening the optimal formulation of sodium alginate-polyethylene glycol-glycerol hydrogel system: Weigh sodium alginate, polyethylene glycol and glycerol, dissolve them in sterile water after autoclaving, stir evenly, cool to room temperature and then add biocontrol bacterial suspension and mix well, so that the concentrations of sodium alginate and polyethylene glycol are 0.5% w / v, 1% w / v or 1.5% w / v, respectively, and the concentrations of glycerol are 0.5% w / v, 0.75% w / v or 1% w / v, respectively; (2) Determination of the compatibility between hydrogel and biocontrol bacteria: After incubation at 27℃ for 48 hours, the number of viable bacteria was calculated using the dilution plating method. Sterile water was used as a control to calculate the survival rate. Concentrations with good biocompatibility were selected for subsequent experiments. The experiment was repeated at least 5 times. The formula for calculating the survival rate of bacteria is: Bacterial survival rate (%) = (C / V) × 100% In the formula, C is the number of viable bacteria after 48 hours of incubation, and V is the number of viable bacteria before incubation; (3) Preparation of sodium alginate-polyethylene glycol-glycerol hydrogel: Sodium alginate, polyethylene glycol and glycerol were dissolved in deionized water to prepare SA-PEG-Gly blend solution, and 2% w / v CaCl2 solution was added for crosslinking for 1 hour to form a gel; (4) Determination of swelling ratio and water content of hydrogel: The prepared hydrogel was air-dried at room temperature and then weighed (W). d The dried hydrogel was placed in solutions with pH values ​​of 4.1, 5.2, 6.8, and 8.0. At regular intervals, the hydrogel was removed, its surface moisture was wiped off, and it was weighed. It was then immersed back into the same solution to obtain the weight (W) of the hydrogel at different time points. t The experiment was repeated at least three times, and the swelling rate was calculated: Swelling rate % = (W t - W d ) / W d ×100; The water content was determined by gravimetric method. Sodium alginate-polyethylene glycol-glycerol hydrogel and sodium alginate hydrogel were prepared respectively. After the hydrogel was completely dried, it was weighed (Mo). Then the dried hydrogel was swollen in deionized water until it reached constant weight. The hydrogel was then dried at room temperature. It was weighed every 12 h (Mt). The experiment was repeated at least 5 times. The water content (%) was calculated using the following formula: Water content (%) = Mt–Mo / Mt×100. (5) Preparation of sodium alginate-polyethylene glycol-glycerol-biocontrol bacteria blend: After sterilizing the SA-PEG-Gly blend, add TLD6 and / or TPD6 bacterial suspension to prepare SA-PEG-Gly-TLD6, SA-PEG-Gly-TPD6 or composite blend; (6) Determine the storage stability, encapsulation rate, antibacterial properties and survival of biocontrol bacteria in soil of the blend: Store at 4℃ and 27±2℃ for 6 months and test viable bacteria count and pH value regularly; calculate encapsulation rate by phosphate buffer gel breaking method; determine antibacterial properties by plate confrontation method and calculate inhibition rate; evaluate biocontrol bacteria survival by soil release experiment; (7) Treat Paris polyphylla seedlings with blended solution: After disinfection, apply sterile hydrogel microparticles, TLD6 gel microparticles, TPD6 gel microparticles, TLD6+TPD6 gel microparticles or corresponding bacterial suspension to the roots for irrigation. (8) Determine the agronomic traits and disease resistance indicators of Paris polyphylla: including plant height, leaf length, leaf width, disease index, control effect and antioxidant enzyme activity.

[0013] As a preferred technical solution, in step (6), 1 mL of the hydrogel prepared by the blend is added to 9 mL of phosphate buffer solution, and shaken in a shaker at 3°C ​​and 100 r / min for 30 min to break the hydrogel and obtain a broken mixture. 1 mL of the broken mixture is taken, diluted with sterile water, and spread on a culture medium. After incubation for 48 h, the viable count is calculated. At the same time, the bacterial suspension before embedding is taken, diluted, spread on plates, incubated, and counted to calculate the viable count. Each experiment is repeated at least 3 times to calculate the embedding rate. Encapsulation efficiency = (Number of viable bacteria after encapsulation / Number of viable bacteria before encapsulation) × 100%; The antimicrobial properties of the biocontrol bacteria in the hydrogel were evaluated by placing hydrogel and 6 mm diameter pathogen blocks on both sides of a plate confrontation experiment. Plates inoculated only with pathogens served as a control. The plates were incubated at 25°C for 15 days, and the hyphal length of the pathogens was measured to calculate the inhibition rate. Inhibition rate (%) = (Control colony diameter - Treated colony diameter) / Control colony diameter × 100%; The soil was sieved and sterilized in a high-pressure steam autoclave at 121°C for two hours to prepare sterile soil. Following the ionogelation method described above, the biocontrol bacteria were encapsulated to prepare three types of gel microparticles: TLD6 gel microparticles, TPD6 gel microparticles, and TLD6+TPD6 composite gel microparticles. A total of 100 mg of gel microparticles was then added to 100 g of sterile soil. Sterile water was added to the sterile soil to maintain soil moisture at approximately 50%. The soil was placed in a sterile container and left at room temperature for 90 days. The soil was mixed every other day. On days 1, 7, 15, 30, 60, and 90 after the application of gel microparticles, 1 gram of soil was sampled to calculate the viable bacterial count. The samples were continuously diluted and spread onto culture media, and the colony counts on the culture media were recorded. Each experiment was repeated at least five times.

[0014] According to a third objective of the present invention, the present invention provides an application of a microbial seed dressing agent for preventing and controlling root rot in Paris polyphylla and promoting its growth. The application includes applying the seed dressing agent in the form of gel microparticles or bacterial suspension to Paris polyphylla seedlings to improve the plant's resistance to root rot caused by Fusarium oxysporum and to enhance the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT).

[0015] As a preferred technical solution, the application can reduce the disease index of Paris polyphylla root rot by at least 30% and improve the prevention and control effect by no less than 50%.

[0016] As a preferred technical solution, the application can promote the growth of Paris polyphylla plant height, leaf length, leaf width, root length and biomass, with the growth rate being no less than 15% compared with the control group.

[0017] The beneficial effects of this invention are as follows: This invention screened and optimized the ratio of sodium alginate-polyethylene glycol-glycerol compound and used it as a hydrogel carrier. It combined this compound with *Bacillus vesiculosus* TLD6 and *Aspergillus auris* TPD6, which have biocontrol potential, to construct a microbial seed coating system. A stable coating material was formed through ionic cross-linking, and this material was used to treat *Paris polyphylla* seedlings. A comprehensive evaluation was conducted, measuring the compatibility of the hydrogel with the biocontrol bacteria, the swelling rate and water content of the sodium alginate-polyethylene glycol-glycerol hydrogel, the storage stability of the sodium alginate-polyethylene glycol-glycerol-biocontrol bacteria mixture, the encapsulation rate, antibacterial properties, the survival of the biocontrol bacteria in the soil, plant height, leaf length, leaf width, aboveground fresh weight, aboveground dry weight, disease index, control effect, and the activities of superoxide dismutase, peroxidase, and catalase. The results showed that treating *Paris polyphylla* seedlings with sodium alginate-polyethylene glycol-glycerol combined with biocontrol potential microorganisms significantly improves the plant's resistance to root rot. The preparation process is simple and easy to operate. Attached Figure Description

[0018] Figure 1 This is a flowchart of the present invention.

[0019] Figure 2 The viability of the strains after incubation in different concentrations of film-forming materials for 48 hours, among which, Figure 2 A represents the percentage of viable Bacillus belyssus TLD6. Figure 2 B represents the percentage of viable bacteria in Aspergillus foetida TPD6.

[0020] Figure 3 Swelling rate of sodium alginate-polyethylene glycol-glycerin coated film in solutions of different pH values.

[0021] Figure 4 Changes in water content over time for sodium alginate hydrogel and sodium alginate-polyethylene glycol-glycerol hydrogel.

[0022] Figure 5 Storage stability of Bacillus vesiculosus TLD6 and Aspergillus aurae TPD6 in SA-PEG-Gly blends and encapsulation efficiency of hydrogels for biocontrol bacteria.

[0023] Figure 6 The antibacterial effects of single and combined strains of Fusarium oxysporum in hydrogels, among which, Figure 6 A shows the antibacterial effects of single and combined strains against Fusarium oxysporum. Figure 6 B represents the colony diameter and inhibition rate of single and combined strains against Fusarium oxysporum.

[0024] Figure 7 Release of Bacillus vesiculosus TLD6 and Aspergillus aurae TPD6 from coating films and their activity in soil.

[0025] Figure 8 The effects of microbial hydrogel particles on the growth of Paris polyphylla seedlings, among which, Figure 8 A represents the effect of microbial hydrogel particles on the plant height of Paris polyphylla. Figure 8 B represents the effect of microbial hydrogel particles on the leaf length of Paris polyphylla. Figure 8 C represents the effect of microbial hydrogel particles on the leaf width of Paris polyphylla. Figure 8 D represents the effect of microbial hydrogel particles on the root length of Paris polyphylla. Figure 8 E represents the effect of microbial hydrogel particles on the fresh weight of Paris polyphylla. Figure 8 F represents the effect of microbial hydrogel particles on the dry weight of Paris polyphylla. Figure 8 G is the morphological diagram of Paris polyphylla after processing.

[0026] Figure 9 The effect of microbial hydrogel microparticles on the prevention and control of root rot in Paris polyphylla, among which, Figure 9 A represents the disease index and prevention effect after treatment with microbial hydrogel particles. Figure 9B shows the morphological diagrams of Paris polyphylla after each treatment.

[0027] Figure 10 The effects of microbial hydrogel microparticles on the activities of SOD, CAT, and POD in the roots of Paris polyphylla, among which, Figure 10 A represents the effect of microbial hydrogel microparticles on SOD activity in the roots of Paris polyphylla. Figure 10 B represents the effect of microbial hydrogel microparticles on CAT activity in the roots of Paris polyphylla. Figure 10 C represents the effect of microbial hydrogel microparticles on the POD activity of Paris polyphylla roots. Detailed Implementation

[0028] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Example 1

[0030] like Figures 1-10 As shown, A microbial seed dressing agent for resisting root rot of Paris polyphylla, its preparation method and application, comprising the following steps Step 1: Screening for the optimal formulation of the sodium alginate-polyethylene glycol-glycerol hydrogel system. First, weigh out a certain amount of sodium alginate, polyethylene glycol, and glycerin, autoclave them, dissolve them in sterile water, stir evenly, and cool to room temperature. Then add the bacterial suspension and mix well so that the final concentrations of sodium alginate and polyethylene glycol in the final solution are (0.5 w / v, 1 w / v, 1.5% w / v), and the final concentrations of glycerin are (0.5 w / v, 0.75 w / v, 1% w / v).

[0031] Step 2: Determine the compatibility between the hydrogel and the biocontrol bacteria. The cells were incubated at 27°C for 48 hours. Viable bacteria were counted at 0 and 48 hours using the dilution plating method. Sterile water was used as a control instead of the film-forming material. The survival rate of the bacteria was calculated. A concentration with good biocompatibility was selected for subsequent experiments. The experiment was repeated 5 times.

[0032] Bacterial survival rate (%) = (C / V) × 100% C: Viable bacteria count after 48 hours of incubation; V: Viable bacteria count before incubation.

[0033] The compatibility of hydrogels with biocontrol bacteria, such as Figure 2As shown, the biocontrol bacteria TPD6 and TLD6 of *Paris polyphylla* retained 91.74% and 91.04% of their viability, respectively, in sterile water (CK). The number of viable bacteria retained by both strains gradually increased with increasing SA and Gly concentrations, with the highest number of viable bacteria retained in SA (1.5% w:v) and Gly (1% w:v). With increasing PEG concentration, the number of viable bacteria in both strains significantly decreased, with the highest number of viable bacteria retained in PEG (0.5% w:v).

[0034] Step 3, Preparation of sodium alginate-polyethylene glycol-glycerol hydrogel Sodium alginate, polyethylene glycol, and glycerol were completely dissolved in deionized water to prepare an SA-PEG-Gly blend. 2 g of anhydrous calcium chloride was dissolved in 100 mL of deionized water to prepare a 2% w:v CaCl2 solution. The blend was added to the 2% w:v CaCl2 solution, and after crosslinking for 1 h, the mixture was then placed in a Ca... 2+ Under the influence of [the agent], the blend will become a gel state; Step 4: Determination of swelling ratio and water content of sodium alginate-polyethylene glycol-glycerol hydrogel. Based on the optimal coating formulation selected in the above experiments, sodium alginate, polyethylene glycol, and glycerin were dissolved in distilled water to prepare a sodium alginate-polyethylene glycol-glycerin blend. After crosslinking with 2% w:v CaCl2 solution for 1 h, the prepared hydrogel was air-dried at room temperature and weighed (W). d The dried hydrogel was placed in solutions with different pH values ​​(4.1, 5.2, 6.8, and 8.0). At regular intervals, the hydrogel was removed, its surface moisture was wiped off, and it was weighed. It was then immersed back into the same solution to obtain the weight (W) of the hydrogel at different time points. t The experiment was repeated three times. The swelling ratio was calculated using the following formula: Swelling rate % = (W t - W d ) / W d ×100 The water content of sodium alginate-polyethylene glycol-glycerol hydrogel was determined by gravimetric method. 2 Sodium alginate-polyethylene glycol-glycerol hydrogels and sodium alginate hydrogels were prepared separately. After complete drying, the hydrogels were weighed (Mo). The dried hydrogels were then swollen in deionized water until a constant weight was reached. The hydrogels were then dried at room temperature, and weighed every 12 hours (Mt). The experiment was repeated 5 times. The following formula was used for calculation: Moisture content (%) = Mt – Mo / Mt ×100 Where M0 refers to the weight of the dry hydrogel, and Mt refers to the weight of the hydrogel after time t. The swelling ratio of SA-PEG-Gly hydrogel at different pH values ​​is as follows: Figure 3 As shown, the swelling ratios at pH 4.1, 5.2, 6.8, and 8.0 were 62.04%, 65.21%, 91.58%, and 148.66%, respectively. It was observed that the swelling ratio of the hydrogel increased with increasing solution pH. Under alkaline conditions, the swelling ratio of the hydrogel was relatively high, while under acidic conditions it was relatively low, demonstrating that the swelling of the hydrogel is pH-dependent. The water content of the SA-PEG-Gly hydrogel is as follows... Figure 4 As shown, the water content of both SA hydrogel and SA-PEG-Gly hydrogel gradually decreased over time. Within 60 hours, the water content of SA-PEG-Gly hydrogel decreased from 85% to 20%, and the water content of SA hydrogel decreased from 81% to 9%. However, the water content of SA-PEG-Gly hydrogel remained higher than that of SA hydrogel, indicating that the composite SA-PEG-Gly hydrogel has a stronger water retention capacity than the single SA hydrogel.

[0035] Step 5, Preparation of sodium alginate-polyethylene glycol-glycerol-biocontrol bacteria blend A certain amount of sodium alginate, polyethylene glycol, and glycerol were weighed and dissolved in distilled water to prepare a sodium alginate-polyethylene glycol-glycerol blend. The blend was sterilized at 121℃ for 20 min. Suspensions of Bacillus vesiculus TLD6 and Aspergillus aparine TPD6 were poured into the cooled SA-PEG-Gly blend and stirred until homogeneous, thus preparing sodium alginate-polyethylene glycol-glycerol-Bacillus vesiculus TLD6 (SA-PEG-Gly-TLD6), sodium alginate-polyethylene glycol-glycerol-Aspergillus aparine (SA-PEG-Gly-TPD6), and a composite gel sodium alginate-polyethylene glycol-glycerol-Bacillus vesiculus-Aspergillus aparine (SA-PEG-Gly-TLD6+TPD6). The blend contains 1% w:v sodium alginate, 0.5% w:v polyethylene glycol, and 1% w:v glycerol. Each mL of the blend contains 10 biocontrol bacteria. 8 CFUs. Preparation of composite gel: It is prepared by ion-gelling of equal proportions of SA-PEG-Gly-TLD6 blend and SA-PEG-Gly-TPD6 blend.

[0036] Step 6: Determine the storage stability, encapsulation rate, antibacterial properties, and survival of the biocontrol bacteria in the soil of the sodium alginate-polyethylene glycol-glycerol-biocontrol bacteria blend. The bacterial blends were stored in HDPE bottles at 4℃ and 27±2℃ for 6 months, with a sterile blend serving as a control. Every 30 days, 1 mL of the blend was serially diluted with sterile physiological saline, spread onto culture medium, and incubated for 48 hours before counting the viable cells. Each experiment was performed in triplicate. The pH of the blends was measured every 30 days for 6 months. Before measurement, the mixture was gently shaken to ensure uniform dispersion of the bacteria, and the pH value was recorded.

[0037] Encapsulation efficiency determination: 1 mL of the hydrogel prepared by the blend was added to 9 mL of phosphate buffer solution and shaken at 3°C ​​and 100 r / min for 30 min in a shaker to break the hydrogel and obtain a broken mixture. 1 mL of this broken mixture was diluted with sterile water, spread on a culture medium, and incubated for 48 h before counting to calculate the viable count. Simultaneously, the bacterial suspension before encapsulation was diluted, spread on plates, incubated, and counted to calculate the viable count. Each experiment was performed in triplicate.

[0038] Encapsulation efficiency = (Number of viable bacteria after encapsulation / Number of viable bacteria before encapsulation) × 100% The antimicrobial properties of biocontrol bacteria in the hydrogel were evaluated by placing hydrogel and 6 mm diameter pathogen blocks on both sides of a plate confrontation experiment. Plates inoculated only with pathogens served as a control. The plates were incubated at 25°C for 15 days, and the hyphal length of the pathogens was measured, and the inhibition rate was calculated.

[0039] Inhibition rate (%) = (Control colony diameter - Treated colony diameter) / Control colony diameter × 100% Determination of the release of biocontrol bacteria in the coating film and their survival in soil: Soil was sieved and sterilized in an autoclave at 121°C for two hours to prepare sterile soil. Following the ionogelation method described above, the biocontrol bacteria were encapsulated to prepare three types of gel microparticles (TLD6 gel microparticles, TPD6 gel microparticles, and TLD6+TPD6 composite gel microparticles). A total of 100 mg of gel microparticles was then added to 100 g of sterile soil. Sterile water was added to maintain soil moisture at approximately 50%. The soil was placed in a sterile container and left at room temperature for 90 days. The soil was mixed every other day. On days 1, 7, 15, 30, 60, and 90 after gel microparticle application, 1 gram of soil was collected to count the viable bacteria. Samples were serially diluted and spread onto culture media, and the colony counts were recorded. Each experiment was repeated five times. The storage stability and encapsulation efficiency of the sodium alginate-polyethylene glycol-glycerol-biocontrol bacteria blend were as follows: Figure 5As shown, the two blends were stored at 4℃ and 27±2℃ for 6 months, respectively. It was found that the viable bacterial counts in both blends showed a slow decreasing trend. During the six-month storage period, both blends maintained a high bacterial content, with viable bacterial counts greater than 6 log CFU / mL. Furthermore, compared to 30℃, both blends retained more viable bacterial counts at 4℃. Simultaneously, the encapsulation efficiency of the hydrogel for both strains reached over 83%. The antibacterial properties of the hydrogel are as follows: Figure 6 As shown, single strains and combined strains of TLD6 and TPD6 exhibited good inhibitory effects against Fusarium oxysporum. The release of biocontrol bacteria from the coating film and their survival in the soil are as follows... Figure 7 As shown, both single and mixed strains can be slowly released from the SA-PEG-Gly hydrogel and maintain high activity in the soil.

[0040] Step 7: Treat Paris polyphylla seedlings with a sodium alginate-polyethylene glycol-glycerol-biocontrol bacteria mixture. Seedlings were transplanted in June 2024. Before transplanting, the underground parts of the Paris polyphylla and the flowerpots were disinfected with 3% sodium hypochlorite. Two weeks after establishment, the seedlings underwent eight treatments: sterile water (CK), sterile hydrogel microparticles, TLD6 hydrogel microparticles, TPD6 hydrogel microparticles, TLD6+TPD6 hydrogel microparticles, TLD6 spore suspension root drenching, TPD6 bacterial suspension root drenching, and TLD6+TPD6 bacterial suspension root drenching. 20 g (containing 10⁸ CFU / g) was buried in the soil around the seedlings. The biocontrol bacterial suspension and spore suspension were inoculated into the Paris polyphylla seedlings via root drenching, with each seedling inoculated with 20 mL (containing 10⁸ CFU / g). 8 (CFU / mL). One week later, the treatment was repeated in the same manner, with 15 plants per treatment, maintaining five replicates. Seedling pots were randomly placed in the greenhouse, and each pot was injected with the same amount of sterile water twice a week to maintain soil moisture.

[0041] Step 8: Measure the plant height, leaf length, leaf width, fresh weight of the above-ground parts, dry weight of the upper parts, disease index, control effect, and activities of superoxide dismutase, peroxidase, and catalase of Paris polyphylla.

[0042] After 30 days of co-cultivation, three plants were randomly harvested from each replicate. Agronomic traits of *Paris polyphylla*, including plant height, leaf length, leaf width, root length, fresh weight of the underground part, and dry weight of the underground part, were measured using a ruler (accurate to 0.1 cm). A pot experiment was conducted in a greenhouse to evaluate the biocontrol effect of microbial gel particles on root rot of *Paris polyphylla*. Fifteen days after application of the gel particles and biocontrol bacterial solution, a suspension of *Fusarium oxysporum* (F) (1×10⁻⁶) was used. 5The root irrigation solution (CFU / mL) was applied to each Paris polyphylla plant at a rate of 20 mL. After the disease developed, the incidence rate and disease index were measured. Ten treatment groups were set up in this experiment: sterile gel microparticles + F, TPD6 gel microparticles + F, TLD6 gel microparticles + F, TPD6 + TLD6 gel microparticles + F, TPD6 bacterial solution + F, TLD6 bacterial solution + F, carbendazim + F, F treatment alone, and sterile distilled water treatment as a blank control (CK). Data were recorded after the disease developed. Five plants were randomly selected from each treatment and the treatment was repeated three times. The disease index and relative control efficacy were calculated. The grading standard for root rot of Paris polyphylla is based on the grading standard for root rot of Panax notoginseng, and is divided into 6 levels: Level 0: Healthy and asymptomatic; Level 1: Rot only occurs on the surface of the tuber, and 0% < rotten area ≤ 10%, or rot has spread to the interior and 5% < rotten area ≤ 10%; Level 2: Rot has spread to the interior, and 10% < rotten area ≤ 40%, with no rotten branches; Level 3: Rot has spread to the interior, and 40% < rotten area ≤ 50%, with rotten branches; Level 4: Rot has spread to the interior, and 50% < rotten area ≤ 70%, with rotten branches falling off; Level 5: Rot has spread to the interior, and the rotten area > 70% until the entire tuber is completely rotten, with rotten branches.

[0043] The formulas for calculating the disease index and relative efficacy are as follows: Disease index = ∑ (number of diseased leaves at each level × representative value for each level) / (highest disease level × total number of leaves surveyed) × 100; Prevention and control effect (%) = (Control disease index - Treatment disease index) / Control disease index × 100 Fresh Paris polyphylla tubers were collected on day 7 after inoculation with Fusarium oxysporum. The tubers were washed clean of soil, immediately frozen in liquid nitrogen, and the activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) in the tubers were measured using a kit from Suzhou Greens Biotechnology Co., Ltd. The experiments were performed in triplicate.

[0044] The effects of microbial hydrogel particles on Paris polyphylla seedlings, such as Figure 8 As shown, the results indicate that microbial hydrogel particles significantly promote the growth of Paris polyphylla seedlings. Different microbial hydrogel particles exhibited good promoting effects on plant height, leaf length, leaf width, root length, fresh weight of underground parts, and dry weight of underground parts. The control effect of microbial hydrogel particles on root rot of Paris polyphylla is shown in the figure. Figure 9 As shown, all treatments with microbial hydrogel particles significantly improved the control of root rot in Paris polyphylla. The effect of microbial hydrogel particles on the antioxidant enzyme activity of Paris polyphylla is as follows: Figure 10 As shown, microbial hydrogel particles significantly increased the activity of antioxidant enzymes such as SOD, POD and CAT in the roots of Paris polyphylla.

[0045] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A microbial seed dressing agent for resisting root rot of Paris polyphylla, characterized in that, The seed coating agent uses sodium alginate-polyethylene glycol-glycerol hydrogel as a carrier to encapsulate biocontrol bacteria; the biocontrol bacteria are Bacillus belyssus TLD6 and / or Aspergillus pumilus TPD6; the concentration of sodium alginate in the hydrogel carrier is 0.5%-1.5% w / v, the concentration of polyethylene glycol is 0.5%-1.5% w / v, and the concentration of glycerol is 0.5%-1% w / v.

2. The microbial seed dressing agent for resisting root rot of Paris polyphylla as described in claim 1, characterized in that, The number of biocontrol bacteria per 1 mL of seed dressing agent is 10. 8 CFUs.

3. The microbial seed dressing agent for resisting root rot of Paris polyphylla as described in claim 1, characterized in that, The hydrogel carrier exhibits pH-dependent swelling behavior, with the swelling rate of the hydrogel increasing as the pH of the solution increases.

4. The anti-root rot microbial seed dressing agent for Paris polyphylla as described in claim 1, characterized in that, The seed coating agent has a viable bacterial count of no less than 6 log CFU / mL and an encapsulation rate of no less than 83% after 6 months of storage.

5. The method for preparing a microbial seed dressing agent for resisting root rot of Paris polyphylla as described in any one of claims 1 to 4, characterized in that, Includes the following steps: (1) Screening the optimal formulation of sodium alginate-polyethylene glycol-glycerol hydrogel system: Weigh sodium alginate, polyethylene glycol and glycerol, dissolve them in sterile water after autoclaving, stir evenly, cool to room temperature and then add biocontrol bacterial suspension and mix well, so that the concentrations of sodium alginate and polyethylene glycol are 0.5% w / v, 1% w / v or 1.5% w / v, respectively, and the concentrations of glycerol are 0.5% w / v, 0.75% w / v or 1% w / v, respectively; (2) Determination of the compatibility between hydrogel and biocontrol bacteria: After incubation at 27℃ for 48 hours, the number of viable bacteria was calculated using the dilution plating method. Sterile water was used as a control to calculate the survival rate. Concentrations with good biocompatibility were selected for subsequent experiments. The experiment was repeated at least 5 times. The formula for calculating the survival rate of bacteria is: Bacterial survival rate (%) = (C / V) × 100% In the formula, C is the number of viable bacteria after 48 hours of incubation, and V is the number of viable bacteria before incubation; (3) Preparation of sodium alginate-polyethylene glycol-glycerol hydrogel: Sodium alginate, polyethylene glycol and glycerol were dissolved in deionized water to prepare SA-PEG-Gly blend solution, and 2% w / v CaCl2 solution was added for crosslinking for 1 hour to form a gel; (4) Determination of swelling ratio and water content of hydrogel: The prepared hydrogel was air-dried at room temperature and then weighed (W). d The dried hydrogel was placed in solutions with pH values ​​of 4.1, 5.2, 6.8, and 8.

0. At regular intervals, the hydrogel was removed, its surface moisture was wiped off, and it was weighed. It was then immersed back into the same solution to obtain the weight (W) of the hydrogel at different time points. t The experiment was repeated at least three times, and the swelling rate was calculated: Swelling rate % = (W t - W d ) / W d ×100; The water content was determined by gravimetric method. Sodium alginate-polyethylene glycol-glycerol hydrogel and sodium alginate hydrogel were prepared respectively. After the hydrogel was completely dried, it was weighed (Mo). Then the dried hydrogel was swollen in deionized water until it reached constant weight. The hydrogel was then dried at room temperature. It was weighed every 12 h (Mt). The experiment was repeated at least 5 times. The water content (%) was calculated using the following formula: Water content (%) = Mt–Mo / Mt×100. (5) Preparation of sodium alginate-polyethylene glycol-glycerol-biocontrol bacteria blend: After sterilizing the SA-PEG-Gly blend, add TLD6 and / or TPD6 bacterial suspension to prepare SA-PEG-Gly-TLD6, SA-PEG-Gly-TPD6 or composite blend; (6) Determine the storage stability, encapsulation rate, antibacterial properties and survival of biocontrol bacteria in soil of the blend: Store at 4℃ and 27±2℃ for 6 months and test viable bacteria count and pH value regularly; calculate encapsulation rate by phosphate buffer gel breaking method; determine antibacterial properties by plate confrontation method and calculate inhibition rate; evaluate biocontrol bacteria survival by soil release experiment; (7) Treat Paris polyphylla seedlings with blended solution: After disinfection, apply sterile hydrogel microparticles, TLD6 gel microparticles, TPD6 gel microparticles, TLD6+TPD6 gel microparticles or corresponding bacterial suspension to the roots for irrigation. (8) Determine the agronomic traits and disease resistance indicators of Paris polyphylla: including plant height, leaf length, leaf width, disease index, control effect and antioxidant enzyme activity.

6. The preparation method according to claim 5, characterized in that, In step (6), 1 mL of the hydrogel prepared by the blend was added to 9 mL of phosphate buffer solution and shaken in a shaker at 3°C ​​and 100 r / min for 30 min to break the hydrogel and obtain a broken mixture. 1 mL of the broken mixture was taken, diluted with sterile water, and spread on a culture medium. After incubation for 48 h, the viable count was calculated. At the same time, the bacterial suspension before embedding was taken, diluted, spread on plates, incubated, and counted to calculate the viable count. Each experiment was repeated at least 3 times to calculate the embedding rate. Encapsulation efficiency = (Number of viable bacteria after encapsulation / Number of viable bacteria before encapsulation) × 100%; The antimicrobial properties of the biocontrol bacteria in the hydrogel were evaluated by placing hydrogel and 6 mm diameter pathogen blocks on both sides of a plate confrontation experiment. Plates inoculated only with pathogens served as a control. The plates were incubated at 25°C for 15 days, and the hyphal length of the pathogens was measured to calculate the inhibition rate. Inhibition rate (%) = (Control colony diameter - Treated colony diameter) / Control colony diameter × 100%; The soil was sieved and sterilized in a high-pressure steam autoclave at 121°C for two hours to prepare sterile soil. Following the ionogelation method described above, the biocontrol bacteria were encapsulated to prepare three types of gel microparticles: TLD6 gel microparticles, TPD6 gel microparticles, and TLD6+TPD6 composite gel microparticles. A total of 100 mg of gel microparticles was then added to 100 g of sterile soil. Sterile water was added to the sterile soil to maintain soil moisture at approximately 50%. The soil was placed in a sterile container and left at room temperature for 90 days. The soil was mixed every other day. On days 1, 7, 15, 30, 60, and 90 after the application of gel microparticles, 1 gram of soil was sampled to calculate the viable bacterial count. The samples were continuously diluted and spread onto culture media, and the colony counts on the culture media were recorded. Each experiment was repeated at least five times.

7. The application of the anti-root rot microbial seed dressing agent according to any one of claims 1 to 4 in the prevention and control of root rot in Paris polyphylla and the promotion of Paris polyphylla growth, characterized in that, The application includes applying seed dressing agents in the form of gel microparticles or bacterial suspensions to Paris polyphylla seedlings to improve the plant's resistance to root rot caused by Fusarium oxysporum and to enhance the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT).

8. The application according to claim 7, characterized in that, The application can reduce the disease index of Paris polyphylla root rot by at least 30% and improve the prevention and control effect by no less than 50%.

9. The application according to claim 7, characterized in that, The application can promote the growth of Paris polyphylla plant height, leaf length, leaf width, root length and biomass, with the growth rate being no less than 15% compared with the control group.

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