A rhizosphere growth-promoting anti-epidemic mould composite live bacteria suspension agent and a preparation process thereof

Abstract

The application discloses a rhizosphere growth-promoting and epidemic-preventing mould composite active bacteria suspension agent and a preparation process thereof, and belongs to the technical field of pesticides. The suspension agent comprises Trichoderma harzianum and Bacillus subtilis composite functional bacteria, a microenvironment steady-state protection system (bacteria microcapsule protective agent, a pH buffer stabilizer, an oxidation-reduction potential regulator and a low-temperature protective agent), a rhizosphere targeted delivery carrier (citric acid modified rice husk biochar and chitosan-iron ion coordination polymer) and a suspension stabilizing system. Sequential symbiotic fermentation technology is adopted to make the two bacteria form metabolic synergy, and the total number of active bacteria reaches 1.0*10 10 2.0*10 10 cfu / mL, which is 40%-60% higher than that of mixing after separate fermentation. The microenvironment steady-state protection makes the shelf life at 25 DEG C reach 12 months, and the active bacteria attenuation rate is less than or equal to 15%. The rhizosphere targeted delivery makes the rhizosphere colonization amount increase by 2.4-3.6 times. The three technical links promote each other, the field effect on tomato root-knot nematode disease reaches 82.5%, the growth-promoting rate is 45%-68%, the persistence period is 45-60 days, and the requirements of green agricultural development are met.

Description

Technical Field

[0001] This invention relates to the field of pesticide technology, and more specifically, to a rhizosphere growth-promoting and disease-preventing compound live bacteria suspension and its preparation process. Background Technology

[0002] Rhizosphere growth-promoting bacteria and Trichoderma are two important agricultural microbial resources. Bacillus subtilis, a typical PGPR strain, can promote plant growth by producing active substances such as indoleacetic acid (IAA), siderophores, and extracellular polysaccharides, while simultaneously secreting lipopeptide antibiotics (such as surfactants and iturobrine) to inhibit pathogens. Trichoderma harzianum is one of the most widely used biocontrol fungi, controlling various soil-borne diseases through hyperparasitism, antibiotic activity, and the induction of systemic resistance (ISR).

[0003] Theoretically, combining PGPR bacteria with Trichoderma can achieve a dual function of "growth promotion + disease prevention". However, existing technologies have the following technical shortcomings in the development of compound microbial agents: (1) Defects in strain compatibility and fermentation process: Most existing compound microbial agents are prepared by fermenting fungi and bacteria separately and then simply mixing them physically. Chinese patent CN111088191A discloses a method for co-culturing Bacillus and Trichoderma, which adopts a segmented inoculation strategy, first culturing Trichoderma to the chlamydospore formation stage and then inoculating Bacillus for co-culturing. However, this method still has problems such as a long fermentation cycle (total cycle exceeding 120 hours), mutual inhibition of metabolites between the two bacteria, and difficulty in accurately controlling the proportion of live bacteria in the final product. In addition, the existing technology does not fully consider the difference in suspension stability between Trichoderma conidia and Bacillus spores in the suspension system, resulting in stratification, precipitation and uneven distribution of bacteria during product storage.

[0004] (2) Viable Bacterial Survival and Shelf Life Defects: The core technical indicator of viable bacterial formulations is the number of viable bacteria within the shelf life. While existing Trichoderma spore aqueous suspensions (such as CN104904752A) and Bacillus subtilis oil suspensions (such as CN200910166429) have solved the stability problem of single-species formulations, they lack a systematic protection strategy for complex viable bacterial systems. Viable bacteria in suspensions face multiple stresses such as pH fluctuations, oxidative stress, osmotic pressure changes, and temperature fluctuations, resulting in a shelf life (25℃) of typically only 3-6 months and a viable bacterial count decay rate exceeding 50%, severely restricting the commercialization of the products.

[0005] (3) Functional Synergy and Rhizosphere Colonization Deficiencies: Existing compound microbial agents simply superimpose growth-promoting and disease-preventing functions without designing a functional synergy mechanism from the perspective of rhizosphere microecology. The growth-promoting effect of PGPR requires effective colonization of the strain in the rhizosphere, while the disease-preventing effect of Trichoderma depends on its competitive advantage in the rhizosphere. Existing technologies have not established a "metabolic interaction-spatial complementarity-temporal synergy" system between the two functional bacteria, resulting in actual field effects that are far lower than theoretical expectations. In addition, existing suspensions lack rhizosphere-targeted delivery capabilities, and a large number of live bacteria are lost to non-rhizosphere areas after application to the soil, resulting in low rhizosphere colonization efficiency.

[0006] In summary, the existing technology has the following technical problems that need to be solved: (1) lacks precise control technology for fungal-bacterial sequential symbiotic fermentation, and the proportion of live bacteria and metabolite composition in compound microbial agents are uncontrollable; (2) lacks a microenvironmental stability protection system for compound live bacteria suspension system, resulting in short shelf life and rapid decay of live bacteria; (3) lacks rhizosphere targeted delivery and functional synergistic design, resulting in unstable field growth promotion and disease prevention effects. Summary of the Invention

[0007] The technical problem to be solved by this invention is to provide a rhizosphere growth-promoting and disease-preventing fungal compound live bacteria suspension and its preparation process. This suspension achieves metabolic synergy between fungi and bacteria through sequential symbiotic fermentation technology, extends the shelf life of the compound live bacteria through a microcapsule steady-state protection system, and improves the rhizosphere colonization efficiency through a rhizosphere-targeted delivery carrier. This makes the effects of growth promotion and disease prevention complementary and mutually reinforcing, overcoming the technical defects of the existing compound microbial agent fermentation process, poor live bacteria stability, and unstable field effects.

[0008] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows: A rhizosphere growth-promoting and disease-preventing compound live bacteria suspension, comprising the following components by weight percentage: (a) Multifunctional bacterial cells: Trichoderma harzianum conidia and hyphal fragments, with an effective viable count ≥2.0 × 10⁻⁶. 8 cfu / mL; Bacillus subtilis spores and vegetative cells, with an effective viable count ≥ 5.0 × 10⁻⁶. 8 cfu / mL; The ratio of the effective viable counts of the two strains was (1:2.5) to (1:4), and the total effective viable count was ≥8.0×10⁻⁶. 8 cfu / mL; (b) Microenvironmental stability protection system: The microbial cell protectant is 3%~6%, which is composed of trehalose, skim milk powder and maltodextrin in a mass ratio of (3~5):(2~3):(1~2), and is spray-dried to form porous microspheres with a particle size of 10~50μm; A pH buffer stabilizer of 0.5% to 1.5% is used, wherein the pH buffer stabilizer is a dipotassium hydrogen phosphate-citric acid buffer pair with a buffering range of pH 6.0 to 7.5; The redox potential regulator is 0.3%~1.0%, wherein the redox potential regulator is a compound of L-cysteine ​​hydrochloride and ascorbic acid in a mass ratio of (1~2):1; The cryoprotectant is 2% to 4%, wherein the cryoprotectant is a compound of glycerol and dimethyl sulfoxide in a volume ratio of (4 to 6): 1; (c) Rhizosphere targeted delivery vector: 1%~3% of modified biochar-based rhizosphere targeting carrier, wherein the modified biochar-based rhizosphere targeting carrier is rice husk biochar modified with citric acid, with a particle size of 50~200μm, a specific surface area of ​​200~500m² / g, and a surface carboxyl content ≥1.5mmol / g; The rhizosphere exudate-responsive release regulator is 0.5%~1.5%, which is a chitosan oligosaccharide-iron ion coordination polymer with a particle size of 100~500nm. It undergoes coordination dissociation in the presence of rhizosphere exudates (organic acids, sugars). (d) Suspension-stabilized system: The suspending agent is 2% to 4%, which is a compound of magnesium aluminum silicate and xanthan gum in a mass ratio of (2 to 3): 1; The wetting and dispersing agent is 1%~2%, wherein the wetting and dispersing agent is a compound of sodium lignosulfonate and alkyl naphthalene sulfonate formaldehyde condensate in a mass ratio of (1~1.5):1; Defoamer 0.1%~0.3%, preservative 0.1%~0.3% and the remainder sterile water.

[0009] The composite functional microbial cell is prepared using a sequential symbiotic fermentation process, specifically including the following steps: F1: Preparation of Trichoderma seed culture: Trichoderma harzianum inoculated onto PDA slant medium and cultured at 28℃ for 72-96 h until sporulation; spores were washed off with sterile water and prepared to a concentration of 1.0×10⁻⁶. 7 ~5.0×10 7 A spore suspension of spores / mL was inoculated into Trichoderma liquid seed culture medium at an inoculation rate of 5%~10% (v / v) and cultured at 28℃ and 180rpm for 24~36h to obtain Trichoderma seed liquid; F2: Trichoderma fermentation culture: Inoculate Trichoderma seed culture at an inoculum volume of 8%~12% (v / v) into Trichoderma fermentation medium, filling the medium to 30% capacity. Ferment at 28℃, with an aeration rate of 1:0.5~1:1 (v / v / min) and a stirring speed of 150~200 rpm for 48~60 h, until the conidial concentration in the culture medium reaches 2.0×10⁻⁶. 8~5.0×10 8 spores / mL, reducing sugar concentration dropped to below 5 g / L; F3: Preparation of Bacillus seed culture: Inoculate Bacillus subtilis strain onto LB slant medium and incubate at 37℃ for 18-24 h; wash off the bacterial cells with sterile water, and inoculate with Bacillus liquid seed culture medium at an inoculation rate of 3%-5% (v / v), and incubate at 37℃ with shaking at 200 rpm for 12-16 h until OD reaches 100%. 600 When the concentration reaches 1.5~2.0, Bacillus seed culture is obtained; F4: Sequential co-existing fermentation: When the Trichoderma fermentation culture reaches 48-60 hours, Bacillus seed culture is inoculated into the Trichoderma fermentation broth at an inoculation rate of 5%-8% (v / v). The fermentation conditions are adjusted to a temperature of 30-32℃, an aeration rate of 1:0.8-1:1.2 (v / v / min), and a stirring speed of 200-250 rpm. Fermentation culture is continued for 24-36 hours. During the fermentation process, the pH is maintained at 6.5-7.5 by adding food-grade sodium hydroxide solution. F5: Fermentation Termination and Cell Collection: When the Bacillus subtilis spore formation rate in the fermentation broth is ≥90% and the total effective viable cell count is ≥1.0×10⁻⁶. 10 Fermentation was terminated when cfu / mL was reached; the fermentation broth was centrifuged at 8000-10000 rpm for 10-15 min using a disc centrifuge, and the cell precipitate was collected; the cell precipitate was washed twice with sterile physiological saline and then resuspended in sterile water containing a cryoprotectant to obtain a concentrated multifunctional cell solution. The Trichoderma fermentation medium consists of: wheat bran 20-30 g / L, corn flour 15-25 g / L, soybean meal 10-15 g / L, KH2PO4 2-3 g / L, MgSO4·7H2O 0.5-1.0 g / L, and an initial pH of 6.0-6.5. The composition of the Bacillus liquid seed culture medium is as follows: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and an initial pH of 7.0-7.2.

[0010] The preparation method of the composite live bacteria suspension includes the following steps: P1: Microencapsulation of bacterial cells: The compound functional bacterial cell concentrate obtained in step F5 is mixed with the bacterial cell microcapsule protectant solution at a volume ratio of 1:(1~2). The bacterial cell microcapsule protectant solution is a solution with a total concentration of 15%~25% (w / v) prepared by dissolving trehalose, skim milk powder and maltodextrin in sterile water at a mass ratio of (3~5):(2~3):(1~2). The mixture is homogenized under high pressure (pressure 30~50MPa, 2~3 cycles) to make the protectant uniformly coat the bacterial cell surface. Then, it is spray-dried (inlet air temperature 120~140℃, outlet air temperature 60~70℃, atomizer speed 15000~20000rpm) to obtain bacterial cell microcapsule powder. P2: Microcapsule rehydration and activation: The bacterial microcapsule powder obtained in step P1 is added to a rehydration solution containing a pH buffer stabilizer and a redox potential regulator at a mass ratio of 1:(5~10). The pH of the rehydration solution is 6.5~7.0 and the redox potential is -150~-250mV. The microcapsule is rehydrated and activated by shaking in a water bath at 30℃ (100rpm) for 30~60min to obtain an activated bacterial suspension. P3: Rhizosphere-targeting carrier loading: The modified biochar-based rhizosphere-targeting carrier and the rhizosphere exudate-responsive release regulator were mixed at a mass ratio of (2~3):1 and ultrasonically dispersed (power 200W, frequency 40kHz) for 20~30min, so that the release regulator was loaded onto the surface and pores of the biochar through electrostatic adsorption and hydrogen bonding; then the mixture was added to the activated bacterial suspension obtained in step P2 at a mass ratio of 1:(3~5), and the mixture was shaken and adsorbed at 30℃ and 150rpm for 2~4h, so that the bacterial microcapsules were loaded onto the surface of the modified biochar carrier through van der Waals forces and electrostatic interactions, resulting in a rhizosphere-targeting bacterial complex suspension; P4: Preparation of the suspension stabilization system: Add suspending agent, wetting and dispersing agent, defoamer and preservative to the suspension obtained in step P3, and make up to the required volume with sterile water. Grind with a colloid mill (3000~5000 rpm) for 15~30 min to make the system uniformly dispersed. P5: Quality Inspection and Packaging: The effective viable count, pH, suspension rate, particle size distribution and contamination rate of the suspension are tested. After passing the test, the suspension is aseptically packaged to obtain the rhizosphere growth-promoting and disease-preventing fungal compound live bacteria suspension.

[0011] In the sequential symbiotic fermentation process described in this invention, Trichoderma and Bacillus form a symbiotic system of metabolic complementarity, spatial stratification, and temporal synergy: In the early stage of fermentation (0-48h), Trichoderma rapidly grows and produces spores using the carbon and nitrogen sources in the culture medium, secreting extracellular enzymes such as cellulase and xylanase to degrade complex organic matter, while simultaneously producing pyranone antibiotics to inhibit other bacteria; In the middle and late stages of fermentation (48-84h), after inoculation, Bacillus rapidly multiplies using oligosaccharides and amino acids produced by Trichoderma metabolism as a nutrient source, while simultaneously secreting lipopeptides such as surfactantin to further inhibit other bacteria, and producing growth-promoting substances such as IAA and siderophores; The metabolites of the two bacteria form a complementary effect between antibiotics and growth promoters, and Trichoderma hyphal fragments provide attachment sites for Bacillus, while the biosurfactants produced by Bacillus promote the dispersion of Trichoderma spores, achieving spatial stratification and complementary colonization.

[0012] The microenvironmental homeostasis protection system of this invention maintains the long-term survival of the composite live bacteria through a triple mechanism: the microcapsule protectant forms a glassy protective film on the surface of the bacteria, reducing water activity and blocking oxygen molecule penetration; the pH buffer stabilizer maintains the system pH in the optimal range of 6.0~7.5, avoiding acid and alkali stress; and the redox potential regulator maintains the weakly reducing environment of the system, inhibiting the oxidative damage of reactive oxygen to the bacterial membrane, and ensuring that the effective live bacteria count of the suspension decreases by ≤20% within 12 months in the temperature range of 4~25℃.

[0013] The rhizosphere targeted delivery carrier of this invention improves rhizosphere colonization efficiency through rhizosphere chemotaxis, carrier loading, and a response release mechanism: the abundant carboxyl groups and porous structure on the surface of modified biochar give it a strong adsorption capacity for water and nutrients in the rhizosphere soil, thus passively enriching it in the high-nutrient region of the rhizosphere; the rhizosphere exudate responsive release regulator undergoes coordination dissociation upon contact with organic acids and sugars secreted by the rhizosphere, resulting in a change in the surface charge of the carrier and promoting the release of microcapsules from the carrier surface; the released microcapsules are rehydrated and activated in the rhizosphere microenvironment, Trichoderma conidia germinate on the root surface to form invasive hyphae, and Bacillus subtilis spores germinate into vegetative bodies to secrete growth-promoting substances, forming a fungal-bacterial complementary microecological community in the rhizosphere.

[0014] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention achieves metabolic synergy and functional complementarity between *Trichoderma harzianum* and *Bacillus subtilis* through a sequential symbiotic fermentation process. Compared with existing techniques that involve separate fermentation followed by simple mixing, sequential symbiotic fermentation enables the metabolites of the two bacteria to form a synergistic network. *Trichoderma* secretes extracellular enzymes that degrade complex organic matter in the culture medium, providing usable small-molecule nutrients for *Bacillus subtilis*. The biosurfactants produced by *Bacillus subtilis* reduce the surface tension of the culture medium, promoting the dispersion of *Trichoderma* spores and oxygen transfer. The fermentation cycle is shortened from 150-180 hours for separate fermentation to 72-96 hours, and energy consumption is reduced by more than 35%. The total effective viable count in the final fermentation broth reaches 1.0 × 10⁻⁶. 10 ~2.0×10 10 The cfu / mL concentration was increased by 40% to 60% compared to the product obtained by mixing the products after separate fermentation.

[0015] (2) This invention constructs a quadruple microenvironmental homeostasis protection system, including microencapsulation, pH buffering, redox regulation, and low-temperature protection, which significantly extends the shelf life of the compound live bacteria suspension. Compared with existing live bacteria preparations that only add a single protective agent (such as glycerol or trehalose), the suspension of this invention, after being stored at 25°C for 12 months, maintains a viable count of ≥85% for Trichoderma harzianum and ≥90% for Bacillus subtilis, with a total viable count decline rate of ≤15%; while existing products typically have a viable count decline rate exceeding 50% under the same conditions. Under refrigeration conditions at 4°C, the effective viable count of the suspension of this invention remains at ≥80% within 24 months, extending the shelf life by 2 to 3 times.

[0016] (3) The high concentration and high activity of the complex microbial cells produced by sequential symbiotic fermentation provide high-quality contents for the microenvironment homeostasis protection, enabling the protection system to play its full role. The microenvironment homeostasis protection system extends the shelf life of live bacteria and provides positive feedback for targeted delivery to the rhizosphere, ensuring that the live bacteria are in the best physiological state when applied in the field. Field trials show that the suspension of this invention has a control effect of 82.5% on tomato root-knot nematode disease and 88.3% on cucumber wilt disease. At the same time, the growth-promoting effect on tomato plant height, root length and fresh weight is 1.45 times, 1.62 times and 1.58 times that of the control, respectively. Under the same effective live bacteria count and dosage, the field growth-promoting effect of the product of this invention is 35%~50% higher than that of existing complex microbial agents, the disease prevention effect is 25%~40% higher, and the effective period is extended to 45~60 days.

[0017] (4) The present invention uses food-grade or agricultural-grade raw materials as protective agents and carriers. The modified biochar is derived from rice husks, an agricultural waste, which is environmentally friendly and low-cost. The suspension contains no chemical pesticide components, is safe for non-target organisms, and is in line with the development direction of green agriculture. Attached Figure Description

[0018] Figure 1This is a flow chart of the sequential symbiotic fermentation process described in this invention; Figure 2 This is a flow chart of the preparation process of the composite live bacteria suspension of the present invention; Figure 3 This is a graph showing the change in viable cell count during the sequential symbiotic fermentation process of Example 1 and Comparative Examples 1-3 of the present invention; Figure 4 This is a comparison chart of the decline in viable bacteria count during shelf life between Example 1 and Comparative Examples 1-3 of the present invention; Figure 5 This is a comparison chart of the field control effects of Example 1 of the present invention and Comparative Examples 1-3 on tomato root-knot nematode disease; Figure 6 The figures show the field test results of the growth-promoting effect of Example 1 and Comparative Examples 1-3 on tomatoes. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0020] Example 1 (1) Sequential symbiotic fermentation F1: Preparation of Trichoderma seed culture: Trichoderma harzianum was inoculated onto PDA slant medium and cultured at 28℃ for 84 h until abundant spore production. Spores were washed off with 10 mL of sterile water containing 0.05% (v / v) Tween-80, counted using a hemocytometer, and prepared to a concentration of 3.0 × 10⁻⁶. 7 A spore suspension with spores / mL was prepared. Trichoderma liquid seed culture medium (wheat bran 25 g / L, corn flour 20 g / L, soybean meal 12 g / L, KH2PO4 2.5 g / L, MgSO4·7H2O 0.8 g / L, initial pH 6.2) was inoculated at an inoculum volume of 8% (v / v) in 100 mL Erlenmeyer flasks and cultured at 28℃ with shaking at 180 rpm for 30 h to obtain the Trichoderma seed culture.

[0021] F2: Trichoderma fermentation culture: Trichoderma seed culture was inoculated at 10% (v / v) into Trichoderma fermentation medium (composition same as seed medium) in a 3L / 10L fermenter. Fermentation was carried out at 28℃, with an aeration rate of 1:0.8 (v / v / min) and a stirring speed of 180 rpm for 54 hours. Conidia and reducing sugar concentrations were measured every 6 hours during fermentation. By the 54th hour, the conidia concentration reached 3.5 × 10⁻⁶. 8 The spores / mL and the reducing sugar concentration decreased to 3.2 g / L.

[0022] F3: Preparation of Bacillus seed culture: Bacillus subtilis strain was inoculated onto LB slant medium and cultured at 37℃ for 20 h. The bacterial cells were washed off with sterile physiological saline and inoculated into Bacillus liquid seed culture medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, initial pH 7.0) at an inoculation rate of 4% (v / v). The medium was filled into 100 mL / 500 mL Erlenmeyer flasks and cultured at 37℃ with shaking at 200 rpm for 14 h until OD reached. 600 When the concentration reaches 1.8, Bacillus seed culture is obtained.

[0023] F4: Sequential symbiotic fermentation: At 54 hours of Trichoderma fermentation, Bacillus seed culture was inoculated into the Trichoderma fermentation broth at an inoculation rate of 6% (v / v). The fermentation conditions were adjusted to a temperature of 31℃, an aeration rate of 1:1.0 (v / v / min), and a stirring speed of 220 rpm, and fermentation was continued for 30 hours. During fermentation, the pH was maintained at 7.0±0.3 by adding 2 mol / L food-grade sodium hydroxide solution.

[0024] F5: Fermentation Termination and Cell Collection: After 84 hours of fermentation, samples were taken for testing. The spore formation rate of Bacillus subtilis was 92.5%, and the conidia concentration of Trichoderma harzianum was 3.2 × 10⁻⁶. 8 The number of viable Bacillus subtilis cells was 8.5 × 10⁻⁶ spores / mL. 8 CFU / mL, total viable count was 1.17 × 10⁻⁶. 10 CFU / mL. The fermentation broth was centrifuged at 9000 rpm for 12 min using a disc centrifuge, and the bacterial cell precipitate was collected. The bacterial cell precipitate was washed twice with sterile physiological saline and then resuspended in sterile water containing a cryoprotectant (glycerol: dimethyl sulfoxide = 5:1, v / v, final concentration 3%) to obtain a multifunctional bacterial cell concentrate with a concentration factor of 5 times.

[0025] (2) Preparation of compound live bacteria suspension P1: Microencapsulation of Bacterial Cells: The concentrated multifunctional bacterial cell solution obtained in step F5 was mixed with the microencapsulation protectant solution at a volume ratio of 1:1.5. The microencapsulation protectant solution was prepared by dissolving 40g of trehalose, 25g of skim milk powder, and 15g of maltodextrin in 200mL of sterile water to prepare a solution with a total concentration of 20% (w / v). The mixture was homogenized three times at 40MPa using a high-pressure homogenizer, and then spray-dried (inlet air temperature 130℃, outlet air temperature 65℃, atomizer speed 18000rpm) to obtain microencapsulation powder. The microencapsulation powder was found to have a moisture content of 3.2% and an encapsulation efficiency of 88.5%.

[0026] P2: Microcapsule rehydration and activation: 80g of the bacterial microcapsule powder obtained in step P1 was added to 400mL of a rehydration solution containing a pH buffer stabilizer (dipotassium hydrogen phosphate-citric acid buffer pair, final concentration 1.0%) and a redox potential modifier (0.4% L-cysteine ​​hydrochloride and 0.3% ascorbic acid). The pH of the rehydration solution was 6.8 and the redox potential was -200mV. The microcapsule was rehydrated and activated by shaking in a water bath at 30℃ (100rpm) for 45min to obtain an activated bacterial suspension.

[0027] P3: Rhizosphere Targeted Carrier Loading: 20g of citric acid-modified rice husk biochar (particle size 80~150μm, specific surface area 350m² / g, surface carboxyl content 2.1mmol / g) was mixed with 8g of chitosan oligosaccharide-iron ion coordination polymer (particle size 200nm), added to 200mL of sterile water, and ultrasonically dispersed (power 200W, frequency 40kHz) for 25min. Then, this mixture was added to the activated bacterial suspension obtained in step P2, and adsorbed by shaking at 30℃ and 150rpm for 3h to obtain a rhizosphere targeted bacterial complex suspension.

[0028] P4: Preparation of the suspension stabilization system: Add 3.0 g of magnesium aluminum silicate, 1.2 g of xanthan gum, 1.5 g of sodium lignosulfonate, 1.0 g of alkyl naphthalene sulfonate formaldehyde condensate, 0.2 g of defoamer (polydimethylsiloxane emulsion), and 0.2 g of preservative (potassium sorbate) to the suspension obtained in step P3, and bring the volume to 1000 mL with sterile water. Grind the mixture in a colloid mill (4000 rpm) for 20 min to ensure uniform dispersion of the system.

[0029] P5: Quality Inspection and Packaging: Detection of the effective viable count of the suspension (Trichoderma harzianum 2.8 × 10⁻⁶). 8 CFU / mL, Bacillus subtilis 7.2 × 10⁻⁶ 8 cfu / mL, total viable count 1.0 × 10⁻⁶ 9 The results were measured for cfu / mL, pH (6.8), suspension rate (92.5%, determined according to CIPAC MT15.1 method), average particle size (8.5μm), and contamination rate (≤1.0%). After passing the test, the sample was aseptically dispensed into brown glass bottles to obtain the rhizosphere growth-promoting and disease-preventing fungal compound live bacteria suspension.

[0030] Example 2 The composite live bacteria suspension was prepared according to the method of Example 1, with the only difference being: in step F4, the inoculation amount of Bacillus seed liquid was 5% (v / v), and the sequential co-fermentation time was 36h; in step P1, the composition of the microcapsule protectant solution was 35g of trehalose, 20g of skim milk powder, and 12g of maltodextrin, with a total concentration of 18% (w / v); in step P3, the amount of citric acid modified rice husk biochar was 15g, and the amount of chitosan oligosaccharide-iron ion coordination polymer was 6g.

[0031] The number of viable Trichoderma harzianum bacteria in the suspension obtained in this embodiment was found to be 2.5 × 10⁻⁶. 8 CFU / mL, Bacillus subtilis viable count was 6.8 × 10⁻⁶. 8 CFU / mL, total viable count 9.3 × 10⁻⁶ 8 cfu / mL, suspension rate 90.2%.

[0032] Example 3 The composite live bacteria suspension was prepared according to the method of Example 1, with the only difference being: in step F4, the inoculation amount of Bacillus seed liquid was 8% (v / v), and the sequential co-fermentation time was 24h; in step P1, the composition of the microcapsule protectant solution was 50g of trehalose, 30g of skim milk powder, and 18g of maltodextrin, with a total concentration of 25% (w / v); in step P3, the amount of citric acid modified rice husk biochar was 25g, and the amount of chitosan oligosaccharide-iron ion coordination polymer was 10g.

[0033] The number of viable Trichoderma harzianum bacteria in the suspension obtained in this embodiment was 3.2 × 10⁻⁶. 8 CFU / mL, Bacillus subtilis viable count was 8.5 × 10⁻⁶. 8 cfu / mL, total viable count 1.17×10 9 cfu / mL, suspension rate 94.6%.

[0034] Example 4 The composite live bacteria suspension was prepared according to the method in Example 1, with the only difference being that the rhizosphere targeting carrier loading in step P3 adopted method two, that is, the chitosan oligosaccharide-iron ion coordination polymer was first mixed with the activated bacterial suspension and adsorbed for 1 hour, and then the modified biochar carrier was added to continue adsorption for 2 hours, so that the release regulator was mainly loaded on the surface of the bacterial microcapsules rather than the surface of the biochar.

[0035] Testing showed that the rhizosphere response release sensitivity of the suspension obtained in this embodiment was improved by approximately 25% compared to that in Example 1.

[0036] Example 5 The composite live bacteria suspension was prepared according to the method of Example 1, with the only difference being that the spray drying in step P1 adopted method three, that is, the inlet air temperature was reduced to 110°C, the outlet air temperature was reduced to 55°C, and the atomizer speed was increased to 22000 rpm, so as to reduce the impact of heat stress on bacterial activity.

[0037] Testing showed that the survival rate of live bacteria in the microcapsule powder obtained in this embodiment was about 12% higher than that in Example 1.

[0038] Comparative Example 1 (Pre-existing technology: simple mixing after separate fermentation) Following the method of Example 1 of Chinese Patent CN111088191A, Trichoderma harzianum and Bacillus subtilis were fermented independently. The fermentation period for Trichoderma harzianum was 72 hours, and the fermentation period for Bacillus subtilis was 36 hours. After fermentation, the cells were collected by centrifugation and physically mixed at a live cell ratio of 1:3. Then, a suspension was prepared according to steps P2 to P5 of Example 1.

[0039] The total number of viable bacteria in the suspension obtained in this comparative example was 6.5 × 10⁻⁶. 8 cfu / mL, suspension rate 85.3%.

[0040] Comparative Example 2 (Prior Technology: No Microenvironment Steady-State Protection System) The composite live bacteria suspension was prepared according to the method of Example 1, with the only difference being that: in step P1, the bacterial cell microcapsule protectant was not used, and the bacterial cell concentrate was directly mixed with the reconstitution solution; in step P2, the pH buffer stabilizer and the redox potential regulator were not used.

[0041] The initial total viable count of the suspension obtained in this comparative example was 9.8 × 10⁻⁶. 8 cfu / mL, suspension rate 88.5%.

[0042] Comparative Example 3 (Prior Technology: Rhizosphere-Free Targeted Delivery Vector) The composite live bacteria suspension was prepared according to the method of Example 1, except that: in step P3, the modified biochar-based rhizosphere targeting carrier and rhizosphere exudate responsive release regulator were not used, and the activated bacterial suspension was directly introduced into step P4.

[0043] The total number of viable bacteria in the suspension obtained in this comparative example was 1.05 × 10⁻⁶. 9 cfu / mL, suspension rate 91.2%.

[0044] Comparative Example 4 (no Bacillus, only Trichoderma single agent) The suspension was prepared according to the method of Example 1, except that sequential symbiotic fermentation was not performed, Trichoderma harzianum was fermented and cultured for 72 hours, and Bacillus subtilis was not inoculated.

[0045] Comparative Example 5 (no Trichoderma, only Bacillus single agent) The suspension was prepared according to the method of Example 1, except that sequential symbiotic fermentation was not performed, and only Bacillus subtilis was cultured for 36 hours without inoculating Trichoderma harzianum.

[0046] Experimental Example 1: Monitoring Changes in Viable Cell Count and Metabolites During Sequential Symbiotic Fermentation Fermentation was carried out according to the methods of Example 1 and Comparative Example 1, and samples were taken every 12 hours to test the following indicators: (1) Conidial concentration of Trichoderma harzianum: The hemocytometer counting method was used; (2) Viable count of Bacillus subtilis: Plate count method (LB medium, 37℃ for 24h); (3) Total viable count: the sum of the above two; (4) Reducing sugar concentration: DNS method; (5) IAA yield: Salkowski colorimetric method; (6) Surfactin yield: acid precipitation-methanol extraction-HPLC method; (7) Cellulase activity: DNS method, expressed as filter paper enzyme activity (FPA).

[0047] The results are shown in Table 1 and Figure 3 As shown.

[0048] Table 1. Changes in key indicators during sequential symbiotic fermentation. The results showed that the total viable cell count of the sequential symbiotic fermentation in Example 1 reached 11.7 × 10⁻⁶ after 84 hours. 8 CFU / mL, compared with Comparative Example 1, after separate fermentation and mixing (8.2 × 10⁻⁶). 8 The IAA yield in Example 1 (32.5 mg / L) was increased by 108.3% compared to Comparative Example 1 (15.6 mg / L), and the surfactant peptide yield (45.8 mg / L) was increased by 105.4% compared to Comparative Example 1 (22.3 mg / L), demonstrating that sequential symbiotic fermentation significantly promoted the synergistic accumulation of metabolites from both bacteria. Simultaneously, the reducing sugar consumption in Example 1 was more complete (0.8 g / L vs 1.5 g / L), and the culture medium utilization rate was increased by 46.7%.

[0049] Experimental Example 2: Shelf-life viable count decay test The suspensions of Examples 1-3 and Comparative Examples 1-3 were stored at 4°C, 15°C and 25°C, respectively. Samples were taken every 30 days to detect the viable counts of Trichoderma harzianum and Bacillus subtilis, and the viable count retention rate was calculated.

[0050] The results are shown in Table 2 and Figure 4 As shown.

[0051] Table 2. Viable cell count decline in each treatment group under 25℃ storage conditions. The results showed that the total viable cell count retention rate of Example 1 was 85.6% after 12 months of storage at 25°C, significantly higher than that of Comparative Example 1 (38.5%) and Comparative Example 2 (18.2%). Comparative Example 2 (without a microenvironmental homeostasis protection system) showed the fastest decline, demonstrating the synergistic protective effect of the microencapsulated protectant, pH buffer stabilizer, and redox potential regulator. Under storage conditions at 4°C, the retention rate of Example 1 was 96.5% after 12 months and 83.2% after 24 months.

[0052] Experimental Example 3: Determination of Rhizosphere Colonization Efficiency Pot experiments were conducted. Tomato seedlings (variety "Zhongshu No. 4", 3-leaf-1-heart stage) were transplanted into plastic pots filled with sterilized substrate (peat moss:vermiculite:perlite = 3:1:1). The suspensions from Examples 1, 3, 4, and 5 were prepared at an effective ingredient ratio of 5.0 × 10⁻⁶. 7 The dosage of CFU / g soil was applied to the rhizosphere soil. Rhizosphere soil samples (0-5 mm from the root surface) were collected at 3, 7, 14, 21, and 30 days after application. The rhizosphere colonization of Trichoderma harzianum and Bacillus subtilis was determined using the selective culture medium plate counting method.

[0053] The results are shown in Table 3.

[0054] Table 3. Results of rhizosphere colonization in each treatment group (×10) 8 cfu / g rhizosphere soil) The results showed that the colonization rates of *Trichoderma harzianum* and *Bacillus subtilis* in the rhizosphere of Example 1 were 2.4–3.5 times and 2.6–3.6 times higher, respectively, than those of Comparative Example 3 (without the rhizosphere-targeted delivery vector). Example 1 reached its peak colonization rate at 14 days (*Trichoderma harzianum* 35.2 × 10⁻⁶). 8 CFU / g, Bacillus subtilis 55.8 × 10⁻⁶ 8 The high colonization rate (cfu / g) and the sustained release effect of the rhizosphere-targeted delivery vector at 30 days demonstrate its effectiveness.

[0055] Experiment Example 4: Field Trial for Controlling Tomato Root-Knot Nematode Disease Experiment location: A vegetable planting base.

[0056] The test crop was tomato, variety "Zhongshu No. 4".

[0057] Experimental design: A randomized block design was used, with 3 replicates per treatment and a plot size of 30m² per replicate. 2 Apply the pesticide by root irrigation at the time of tomato transplanting, using a dosage of 5.0 × 10⁻⁶. 7CFU / g soil. Water treatment served as a blank control, and 10% thiazophos granules (recommended dose) served as a chemical control. Disease index and control efficacy were assessed at 30 and 60 days after application, and root knot index and control efficacy were calculated.

[0058] The results are shown in Table 4 and Figure 5 As shown.

[0059] Table 4. Field control efficacy of each treatment group against tomato root-knot nematode disease. The results showed that Example 1 had a control efficacy of 82.5% at 30 days, which was close to that of the chemical agent thiazophos (88.2%); and a control efficacy of 78.3% at 60 days, which was significantly higher than thiazophos's 70.2%, proving that the effective period of the product of the present invention was superior to that of chemical agents. The control efficacy of Comparative Example 4 (Trichoderma only) and Comparative Example 5 (Bacillus only) was significantly lower than that of Example 1, proving the synergistic disease prevention effect of the compound microbial agent.

[0060] Experiment Example 5: Field Trial of Growth-Promoting Effect The experimental site was the same as in Experiment 4. The pesticide was applied by root irrigation at the time of tomato transplanting, and the dosage was the same as in Experiment 4. Tomato plant height, root length, above-ground fresh weight, and root fresh weight were measured 30 days and 60 days after application, and the growth promotion rate was calculated.

[0061] The results are shown in Table 5 and Figure 6 As shown.

[0062] Table 5. Field growth-promoting effects of each treatment group on tomatoes (60 days) The results showed that Example 1 promoted the growth of tomato plant height, root length, aboveground fresh weight, and root fresh weight by 45.2%, 62.5%, 58.6%, and 68.5%, respectively, which were significantly higher than those of Comparative Examples 1-3 and 4-5. The root length promotion rate (62.5%) and root fresh weight promotion rate (68.5%) of Example 1 were particularly outstanding, demonstrating that the rhizosphere-targeted delivery vector significantly improved the colonization efficiency of the strain in the rhizosphere, thereby enhancing the growth-promoting effect.

[0063] Test Example 6: Safety Evaluation (1) Safety to non-target organisms: The acute LD50 of the suspension concentrate in Example 1 to silkworms was determined in accordance with the "Guidelines for Environmental Testing of Pesticide Registration". 50 (96h) >5000mg / L, acute LC50 toxicity to zebrafish 50 (96h) >1000mg / L, acute toxicity LC50 to earthworms 50 (14d) >1000mg / kg soil, all of which are classified as low toxicity.

[0064] (2) Safety to crops: Tomato, cucumber, pepper and eggplant seedlings were treated with the suspension from Example 1 at 1, 2 and 5 times the recommended dose, respectively, and the presence of phytotoxicity symptoms was observed within 7 days. The results showed that no phytotoxicity symptoms were observed in any of the dose treatments, indicating that the crops were safe.

[0065] (3) Impact on soil microbial diversity: 16S rRNA high-throughput sequencing was used to analyze changes in the bacterial community structure in the soil after application. The results showed that the Shannon index and Chao1 index of soil bacteria after treatment in Example 1 were not significantly different from those of the water control (P>0.05), proving that the suspension agent of the present invention has no significant negative impact on soil microbial diversity.

[0066] The above test results fully demonstrate that the rhizosphere growth-promoting and disease-preventing fungal compound live bacteria suspension of the present invention has achieved significant technical progress in three aspects: sequential symbiotic fermentation live bacteria yield, microenvironmental homeostasis protection shelf life, and rhizosphere targeted delivery and colonization efficiency. Moreover, the three technical aspects promote each other and synergistically enhance each other's effects, while also possessing good environmental safety, which meets the requirements of green agricultural development.

[0067] The above description is merely a preferred embodiment of the present invention; however, the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and its improved concepts, should be covered within the scope of protection of the present invention.

Claims

1. A rhizosphere growth-promoting and disease-preventing fungal compound live bacteria suspension and its preparation process, characterized in that, By weight percentage, it contains the following components: (a) Multifunctional bacterial cells: Trichoderma harzianum conidia and hyphal fragments, with an effective viable count ≥2.0 × 10⁻⁶. 8 cfu / mL; Bacillus subtilis spores and vegetative cells, with an effective viable count ≥ 5.0 × 10⁻⁶. 8 cfu / mL; (b) Microenvironmental stability protection system: The microbial cell protectant is 3%~6%, which is composed of trehalose, skim milk powder and maltodextrin in a mass ratio of (3~5):(2~3):(1~2), and is spray-dried to form porous microspheres with a particle size of 10~50μm; A pH buffer stabilizer of 0.5% to 1.5% is used, wherein the pH buffer stabilizer is a dipotassium hydrogen phosphate-citric acid buffer pair with a buffering range of pH 6.0 to 7.5; The redox potential regulator is 0.3%~1.0%, wherein the redox potential regulator is a compound of L-cysteine ​​hydrochloride and ascorbic acid in a mass ratio of (1~2):1; The cryoprotectant is 2% to 4%, wherein the cryoprotectant is a compound of glycerol and dimethyl sulfoxide in a volume ratio of (4 to 6): 1; (c) Rhizosphere targeted delivery vector: 1%~3% of modified biochar-based rhizosphere targeting carrier, wherein the modified biochar-based rhizosphere targeting carrier is rice husk biochar modified with citric acid, with a particle size of 50~200μm, a specific surface area of ​​200~500m² / g, and a surface carboxyl content ≥1.5mmol / g; The rhizosphere exudate-responsive release regulator is 0.5%~1.5%, wherein the rhizosphere exudate-responsive release regulator is a chitosan oligosaccharide-iron ion coordination polymer with a particle size of 100~500nm; (d) Suspension-stabilized system: The suspending agent is 2% to 4%, which is a compound of magnesium aluminum silicate and xanthan gum in a mass ratio of (2 to 3): 1; The wetting and dispersing agent is 1%~2%, wherein the wetting and dispersing agent is a compound of sodium lignosulfonate and alkyl naphthalene sulfonate formaldehyde condensate in a mass ratio of (1~1.5):1; Defoamer 0.1%~0.3%, preservative 0.1%~0.3% and the remainder sterile water.

2. The rhizosphere growth-promoting and disease-preventing fungal compound live bacteria suspension and its preparation process according to claim 1, characterized in that, The ratio of the effective viable counts of the two strains was (1:2.5) to (1:4), and the total effective viable count was ≥8.0×10⁻⁶. 8 cfu / mL.

3. The rhizosphere growth-promoting and disease-preventing fungal compound live bacteria suspension and its preparation process according to claim 1 or 2, characterized in that, The composite functional microbial cell was prepared using a sequential symbiotic fermentation process, which includes the following steps: F1: Preparation of Trichoderma seed culture: Trichoderma harzianum inoculated onto PDA slant medium and cultured at 28℃ for 72-96 h until sporulation; spores were washed off with sterile water and prepared to a concentration of 1.0×10⁻⁶. 7 ~5.0×10 7 A spore suspension of spores / mL was inoculated into Trichoderma liquid seed culture medium at an inoculation rate of 5%~10% (v / v) and cultured at 28℃ and 180rpm for 24~36h to obtain Trichoderma seed liquid; F2: Trichoderma fermentation culture: Inoculate Trichoderma seed culture at an inoculum volume of 8%~12% (v / v) into Trichoderma fermentation medium, filling the medium to 30% capacity. Ferment at 28℃, with an aeration rate of 1:0.5~1:1 (v / v / min) and a stirring speed of 150~200 rpm for 48~60 h, until the conidial concentration in the culture medium reaches 2.0×10⁻⁶. 8 ~5.0×10 8 spores / mL, reducing sugar concentration dropped to below 5 g / L; F3: Preparation of Bacillus seed culture: Inoculate Bacillus subtilis strain onto LB slant medium and incubate at 37℃ for 18-24 h; wash off the bacterial cells with sterile water, and inoculate with Bacillus liquid seed culture medium at an inoculation rate of 3%-5% (v / v), and incubate at 37℃ with shaking at 200 rpm for 12-16 h until OD reaches 100%. 600 When the concentration reaches 1.5~2.0, Bacillus seed culture is obtained; F4: Sequential co-existing fermentation: When the Trichoderma fermentation culture reaches 48-60 hours, Bacillus seed culture is inoculated into the Trichoderma fermentation broth at an inoculation rate of 5%-8% (v / v). The fermentation conditions are adjusted to a temperature of 30-32℃, an aeration rate of 1:0.8-1:1.2 (v / v / min), and a stirring speed of 200-250 rpm. Fermentation culture is continued for 24-36 hours. During the fermentation process, the pH is maintained at 6.5-7.5 by adding food-grade sodium hydroxide solution. F5: Fermentation Termination and Cell Collection: When the Bacillus subtilis spore formation rate in the fermentation broth is ≥90% and the total effective viable cell count is ≥1.0×10⁻⁶. 10 Fermentation was terminated when cfu / mL was reached; the fermentation broth was centrifuged at 8000-10000 rpm for 10-15 min using a disc centrifuge, and the bacterial precipitate was collected; the bacterial precipitate was washed twice with sterile physiological saline and then resuspended in sterile water containing a cryoprotectant to obtain a concentrated multifunctional bacterial solution.

4. The rhizosphere growth-promoting and disease-preventing fungal compound live bacteria suspension and its preparation process according to claim 3, characterized in that, The Trichoderma fermentation medium consists of: wheat bran 20-30 g / L, corn flour 15-25 g / L, soybean meal 10-15 g / L, KH2PO4 2-3 g / L, MgSO4·7H2O 0.5-1.0 g / L, and an initial pH of 6.0-6.5; the Bacillus liquid seed culture medium consists of: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, and an initial pH of 7.0-7.

2.

5. The rhizosphere growth-promoting and disease-preventing fungal compound live bacteria suspension and its preparation process according to claim 1 or 2, characterized in that, The preparation method of the composite live bacteria suspension includes the following steps: P1: Microencapsulation of bacterial cells: A compound functional bacterial cell concentrate and a bacterial cell microcapsule protectant solution are mixed at a volume ratio of 1:(1~2). The bacterial cell microcapsule protectant solution is a solution with a total concentration of 15%~25% (w / v) prepared by dissolving trehalose, skim milk powder and maltodextrin in sterile water at a mass ratio of (3~5):(2~3):(1~2). The mixture is homogenized under high pressure at 30~50MPa and circulated 2~3 times, and then spray-dried to obtain bacterial cell microcapsule powder. P2: Microcapsule rehydration and activation: Add the bacterial microcapsule powder to a rehydration solution containing pH buffer stabilizer and redox potential regulator at a mass ratio of 1:(5~10), and rehydrate and activate in a 30℃ water bath for 30~60 min to obtain an activated bacterial suspension; P3: Rhizosphere-targeting carrier loading: The modified biochar-based rhizosphere-targeting carrier and the rhizosphere exudate-responsive release regulator were mixed at a mass ratio of (2~3):1 and ultrasonically dispersed at a power of 200W and a frequency of 40kHz for 20~30min; then the mixture was added to the activated bacterial suspension at a mass ratio of 1:(3~5) and adsorbed by shaking at 30℃ and 150rpm for 2~4h to obtain a rhizosphere-targeting bacterial complex suspension; P4: Preparation of suspension stabilization system: Add suspending agent, wetting and dispersing agent, defoamer and preservative to the suspension, and make up to the required volume with sterile water. Grind with a colloid mill at a speed of 3000~5000 rpm for 15~30 min. P5: Quality Inspection and Packaging: The effective viable count, pH, suspension rate, particle size distribution and contamination rate of the suspension are tested. After passing the test, the suspension is aseptically packaged to obtain the rhizosphere growth-promoting and disease-preventing fungal compound live bacteria suspension.

6. The rhizosphere growth-promoting and disease-preventing fungal compound live bacteria suspension and its preparation process according to claim 5, characterized in that, In step P1, the inlet air temperature for spray drying is 120~140℃, the outlet air temperature is 60~70℃, and the atomizer speed is 15000~20000rpm.

7. The rhizosphere growth-promoting and disease-preventing fungal compound live bacteria suspension and its preparation process according to claim 5, characterized in that, The pH of the reconstituted solution in step P2 is 6.5~7.0, and the redox potential is -150~-250mV.

8. The rhizosphere growth-promoting and disease-preventing fungal compound live bacteria suspension and its preparation process according to claim 5, characterized in that, In step P3, the loading method of the rhizosphere exudate-responsive release regulator is as follows: the chitosan oligosaccharide-iron ion coordination polymer and the modified biochar-based rhizosphere targeting carrier are first mixed and ultrasonically dispersed, and then adsorbed by oscillation with the activated bacterial suspension.

9. A rhizosphere growth-promoting and disease-preventing fungal compound live bacteria suspension and its preparation process according to claim 1 or 2, characterized in that, The suspension is used to prevent and control root-knot nematode disease in tomatoes, wilt disease in cucumbers, blight in peppers, and verticillium wilt in eggplants, as well as to promote rhizosphere growth in solanaceous vegetables, cucurbits, and legumes.

10. A rhizosphere growth-promoting and disease-preventing fungal compound live bacteria suspension and its preparation process according to claim 1 or 2, characterized in that, The application methods are root drenching, drip irrigation, or seed coating, with an effective viable bacteria count of 1.0 × 10⁻⁶. 7 ~1.0×10 8 cfu / g soil.

Images