Disease-preventing and growth-promoting compound microbial agent and application thereof

Abstract

The invention belongs to the field of microbial agents, and particularly relates to a compound microbial agent and application thereof.Activated bacillus subtilis SL-44 and klebsiella oxytoca Rs-5 are subjected to amplification culture, a bacillus subtilis SL-44 fermentation liquor and a klebsiella oxytoca Rs-5 fermentation liquor are obtained, the fermentation liquor is added into the fermentation liquor, the fermentation liquor is added into the fermentation liquor, the fermentation liquor is added into the fermentation liquor, and the fermentation liquor is added into the fermentation liquor to prepare the compound microbial agent. And carrying out mixed fermentation culture on the bacillus subtilis SL-44 fermentation liquor and the klebsiella oxytoca Rs-5 fermentation liquor to obtain the compound microbial agent. The compound microbial agent has the capabilities of producing siderophore, dissolving phosphorus, producing indoleacetic acid, producing 1-aminocyclopropane-1-carboxylic acid deaminase and producing a biological membrane, and can remarkably promote the germination of plant seedlings, increase the plant biomass, improve the stress resistance of plants and improve the appearance and internal quality of plant fruits.

Description

Technical Field

[0001] This invention belongs to the field of microbial agents, specifically relating to a compound microbial agent for disease prevention and growth promotion and its application. Background Technology

[0002] Microbial inoculants are active preparations made from plant rhizosphere growth-promoting bacteria (PGPR). They are environmentally friendly and economical biofertilizers that accumulate crop productivity by directly or indirectly promoting plant development, serving as a renewable source of plant nutrition and sustainable agronomy. Using microbial inoculants can not only address soil problems caused by continuous cropping and chemical fertilizer use, but also promote plant growth and increase fruit yield and quality. PGPR has a regulatory effect on plant growth and disease resistance. Its growth-promoting mechanisms include the secretion of auxin (IAA) to directly regulate plant growth, the conversion of insoluble phosphorus in the soil into a plant-absorbable form, and the fixation and reduction of atmospheric N2 to form NH4. + Microbial agents can promote plant growth by indirectly increasing endogenous auxins or decreasing ethylene levels. Their disease resistance mechanisms include secreting antimicrobial substances, competing with pathogens for ecological niches and nutrients, and inducing resistance. Furthermore, microbial agents can avoid environmental pollution caused by chemical fertilizers, making the preparation of microbial agents using PGPR (Potentially Generated Resin Producing Agents) of great significance.

[0003] Single microbial inoculants have limited effects, weak performance, and poor colonization ability. Combining different strains can combine the advantages of various microorganisms to work synergistically on plants, improving their growth-promoting or disease-preventing abilities. However, most currently available compound inoculants suffer from the problem of reduced efficacy due to resource competition or metabolic antagonism among the individual strains. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a compound microbial agent and its application. The compound microbial agent provided by this invention has the ability to produce iron phosphate, lyse phosphorus, produce indoleacetic acid, produce 1-aminocyclopropane-1-carboxylic acid deaminase, and produce biofilm. It can significantly promote seedling germination, increase plant biomass, enhance plant stress resistance, and improve the appearance and internal quality of plant fruits.

[0005] To achieve the above objectives, the specific technical solution of the present invention is as follows: The first aspect of this invention provides a composite microbial agent, which is prepared by the following steps: Activated Bacillus subtilis ( Bacillus subtilis SL-44 and Klebsiella acidogenic bacteria ( Klebsiella oxytoca Rs-5 were cultured separately to obtain Bacillus subtilis SL-44 fermentation broth and Klebsiella acidogenic Rs-5 fermentation broth; The compound microbial agent was obtained by mixing and fermenting the fermentation broth of Bacillus subtilis SL-44 and Klebsiella acidogenic bacteria Rs-5. The viable count of Bacillus subtilis SL-44 per milliliter of the aforementioned compound microbial agent is ≥2×10⁻⁶. 8 CFU, viable count of Klebsiella pneumoniae Rs-5 ≥ 4 × 10⁻⁶ 9 CFU.

[0006] Bacillus subtilis SL-44 and Klebsiella acidogenic bacteria Rs-5 exhibit good compatibility and can coexist well. Furthermore, Klebsiella acidogenic bacteria RS-5 has significant abilities to produce indoleacetic acid, phosphorus-soluble substances, and iron-producing substances. These secondary metabolites can be directly utilized by Bacillus subtilis SL-44, thereby increasing the biomass accumulation and metabolic activity of Bacillus subtilis SL-44. Conversely, Bacillus subtilis SL-44 has good biofilm production capabilities, which can provide physical protection and microaerobic / nutrient gradients for Klebsiella acidogenic bacteria RS-5, enhancing its colonization rate and survival ability under adverse conditions in the soil.

[0007] Furthermore, the fermentation culture temperature is 25℃~32℃, and the fermentation culture time is 40h~60h.

[0008] Furthermore, the fermentation culture temperature is 30°C, and the fermentation culture time is 48 hours.

[0009] Furthermore, the temperature for the expanded culture is 25℃~32℃, and the culture time is 40h~60h.

[0010] Furthermore, the temperature for the expanded culture is 28°C, and the culture time is 48 hours.

[0011] A second aspect of the present invention provides an application of the above-described composite microbial agent, wherein the application is any one or more of the following: a. Promotes plant growth; b. Improve plant stress resistance; c. Improve the quality of the plant's fruit.

[0012] Furthermore, promoting plant growth means increasing plant fresh weight, increasing plant dry weight, increasing plant root weight, increasing plant height, or promoting seedling germination.

[0013] Furthermore, the improvement of plant stress resistance is achieved by reducing plant malondialdehyde content, increasing plant proline content, increasing plant peroxidase content, increasing plant superoxide dismutase content, or increasing plant catalase content.

[0014] Furthermore, the improvement of plant fruit quality refers to increasing the fruit's weight, firmness, soluble sugar content, soluble solids content, soluble protein content, anthocyanin content, or vitamin C content.

[0015] Furthermore, the plant is rapeseed, grape, cucumber, or tomato; the plant fruit is grape, cucumber, or tomato.

[0016] Furthermore, the application method is to water the plant roots or spray the leaves with the compound microbial agent.

[0017] Furthermore, the application dosage of the compound microbial agent is 3L / mu to 12L / mu.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a compound microbial agent for disease prevention and growth promotion. The compound microbial agent for disease prevention and growth promotion provided by this invention is prepared by the following steps: activating Bacillus subtilis (… Bacillus subtilis SL-44 and Klebsiella acidogenic bacteria ( Klebsiella oxytoca Rs-5 were cultured separately to obtain Bacillus subtilis SL-44 fermentation broth and Klebsiella acidogenic Rs-5 fermentation broth. The Bacillus subtilis SL-44 and Klebsiella acidogenic Rs-5 fermentation broths were then mixed and fermented to obtain the composite microbial agent. Bacillus subtilis SL-44 and Klebsiella acidogenic Rs-5 exhibit good compatibility. Furthermore, Klebsiella acidogenic Rs-5 demonstrates significant IAA production, phosphorus-containing capacity, and heparin-producing capacity. These secondary metabolites can be directly utilized by Bacillus subtilis SL-44, enhancing its biomass accumulation and metabolic activity. Conversely, Bacillus subtilis SL-44 possesses good biofilm production capabilities, providing physical protection and a microaerobic / nutrient gradient for Klebsiella acidogenic Rs-5, thereby enhancing its colonization rate and survival ability under adverse conditions in the soil. Therefore, Bacillus subtilis SL-44 and Klebsiella pneumoniae Rs-5 work synergistically to give the prepared compound microbial agent strong stability and biological activity. The compound microbial agent provided by this invention has the ability to produce ironophiles, phosphate solubilizers, indoleacetic acid, 1-aminocyclopropane-1-carboxylic acid deaminase, and biofilm, which can significantly promote seedling germination, increase plant biomass, improve plant stress resistance, and improve the appearance and internal quality of plant fruits. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The results show the compatibility test results between Bacillus subtilis SL-44 strain and Klebsiella acidogenic strain Rs-5. The colony in the middle of the plate is Bacillus subtilis SL-44 strain.

[0021] Figure 2 Statistics on the iron-producing capacity of compound microbial agents.

[0022] Figure 3 Statistics on the phosphorus-solubility of compound microbial agents.

[0023] Figure 4 Statistics on the ability of compound microbial agents to produce 1-aminocyclopropane-1-carboxylic acid deaminase.

[0024] Figure 5 Statistics on the indoleacetic acid production capacity of compound microbial agents.

[0025] Figure 6 Statistics on the biofilm production capacity of compound microbial agents.

[0026] Figure 7 This is a statistical chart showing the effect of compound microbial inoculants on the growth of rapeseed seedlings. Detailed Implementation

[0027] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific 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. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0028] Bacillus subtilis in this invention ( Bacillus subtilis For strain information of SL-44, please refer to: Tao Jing. Screening of growth-promoting and biocontrol bacteria for processing tomatoes under drip irrigation conditions and a preliminary study on their antibacterial and growth-promoting mechanisms [D]. Shihezi University, 2006.; Klebsiella acidogenic bacteria in this invention ( Klebsiella oxytocaFor information on the Rs-5 strain, please refer to: Mo Wenping. Screening of salt-solubilizing and growth-promoting bacteria in cotton and a preliminary study on their salt-solubilizing and growth-promoting mechanisms [D]. Shihezi University, 2006.; Enterobacter cloacae in this invention ( Enterobacter cloacae For information on the strain of Rs-2, please refer to: Wang Xiaobo. Research on the phosphorus-solubilizing mechanism of Enterobacter cloacae Rs-2 and its application in biofertilizer [D]. Xi'an University of Technology, 2023.

[0029] Microbial inoculants are active preparations made from plant rhizosphere growth-promoting bacteria. They are environmentally friendly and economical biofertilizers that can accumulate crop productivity by directly or indirectly promoting plant development, and are a renewable source of plant nutrition and sustainable agronomy. Single microbial inoculants have relatively limited effects, weak performance, and poor colonization ability. Combining different strains can combine the advantages of various bacteria to work synergistically on plants, improving their growth-promoting or disease-preventing abilities. However, in most currently developed compound inoculants, the efficacy of the individual strains is easily reduced due to resource competition or metabolic antagonism, and they also suffer from difficulties in field colonization and poor environmental adaptability.

[0030] This invention provides a compound microbial agent and its application. The compound microbial agent provided by this invention has the ability to produce iron phosphate, phosphate solubilizer, indoleacetic acid, 1-aminocyclopropane-1-carboxylic acid deaminase, and biofilm. It can significantly promote the germination of plant seedlings, increase plant biomass, improve plant stress resistance, and improve the appearance and internal quality of plant fruits.

[0031] Example 1: A compound microbial agent, prepared by the following steps: Strain activation: Bacillus subtilis SL-44 and Klebsiella acidogenic strain Rs-5 were inoculated into LB solid medium by streak plating and incubated upside down in a 28°C incubator for 48 hours. Then, single colonies were picked and placed into LB liquid medium and incubated overnight at 28°C and 180 rpm to complete the activation.

[0032] Preparation of Bacillus subtilis SL-44 fermentation broth: Activated Bacillus subtilis SL-44 was inoculated into LB medium and cultured at 28℃ and 180 r / min for 2 days. The viable count was determined to be 10⁻⁶. 8 CFU / mL was used to obtain Bacillus subtilis SL-44 fermentation broth.

[0033] Preparation of fermentation broth for Klebsiella acidogenic bacteria Rs-5: Activated Klebsiella acidogenic bacteria Rs-5 were inoculated into LB medium and cultured at 28℃ and 180 r / min for 2 days. The viable count was determined to be 10⁻⁶. 8 CFU / mL was used to obtain the fermentation broth of Klebsiella acidogenic bacteria Rs-5.

[0034] 15 mL of Bacillus subtilis SL-44 fermentation broth and 15 mL of Klebsiella acidogenic Rs-5 fermentation broth were inoculated into 5 mL of LLB liquid medium and cultured. The culture parameters were set as follows: temperature 30℃, rotation speed 180 r / min, initial pH 7.0, dissolved oxygen (DO) 30 v / v%, and cultured for 2 days to obtain a compound microbial agent. The viable count of Bacillus subtilis SL-44 in this compound microbial agent was 2 × 10⁻⁶. 8 The viable count of Klebsiella pneumoniae Rs-5 was 4 × 10⁻⁶ CFU / mL. 9 CFU / mL, this invention labels this compound microbial agent as SL-44+Rs-5.

[0035] Comparative Example 1: A compound microbial inoculant, prepared by the following steps: Strain activation: Enterobacter cloacae Rs-2 strain was inoculated into LB solid medium by streak plating and incubated upside down in a 28°C incubator for 48 hours. Then, a single colony was picked and placed into LB liquid medium and incubated overnight at 28°C and 180 rpm to complete the activation.

[0036] Preparation of fermentation broth for Enterobacter cloacae Rs-2: Activated Enterobacter cloacae Rs-2 strain was inoculated into LB medium and cultured at 28℃ and 180 r / min for 2 days. The viable count was determined to be 10⁻⁶. 8 CFU / mL was used to obtain the fermentation broth of Klebsiella acidogenic bacteria Rs-2.

[0037] 15 mL of *Enterobacter cloacae* Rs-2 fermentation broth and 15 mL of *Bacillus subtilis* SL-44 fermentation broth prepared in Example 1 were inoculated into 5 L LB liquid medium and cultured. The culture parameters were set as follows: temperature 30℃, rotation speed 180 r / min, initial pH 7.0, DO 30 v / v%, and cultured for 2 days to obtain a composite microbial agent. The viable count of *Bacillus subtilis* SL-44 in this composite microbial agent was 2 × 10⁻⁶. 8 CFU / mL, viable count of Enterobacter cloacae Rs-2 was 3 × 10⁻⁶. 9 CFU / mL, this invention labels this compound microbial agent as SL-44+Rs-2.

[0038] Comparative Example 2: A compound microbial inoculant, prepared by the following steps: 15 mL of the Klebsiella acidogenic Rs-5 fermentation broth prepared in Example 1 and 15 mL of the Enterobacter cloacae Rs-2 fermentation broth prepared in Comparative Example 1 were inoculated into 5 L LB liquid medium and cultured. The culture parameters were set as follows: temperature 30℃, rotation speed 180 r / min, initial pH 7.0, DO 30 v / v%, and cultured for 2 days to obtain a compound microbial agent. The viable count of Klebsiella acidogenic Rs-5 in this compound microbial agent was 4 × 10⁻⁶. 9 CFU / mL, viable count of Enterobacter cloacae Rs-2 was 3 × 10⁻⁶. 9 CFU / mL, this invention labels this compound microbial agent as Rs-5+Rs-2.

[0039] Comparative Example 3: A compound microbial agent, prepared by the following steps: 10 mL of Bacillus subtilis SL-44 fermentation broth prepared in Example 1, 10 mL of Klebsiella acidogenic Rs-5 fermentation broth, and 10 mL of Enterobacter cloacae Rs-2 fermentation broth prepared in Comparative Example 1 were inoculated into 5 L LB liquid medium and cultured. The culture parameters were set as follows: temperature 30℃, rotation speed 180 r / min, initial pH 7.0, DO 30 v / v%, and cultured for 2 days to obtain a compound microbial agent. The viable count of Bacillus subtilis SL-44 in this compound microbial agent was 2 × 10⁻⁶. 8 The viable count of Klebsiella pneumoniae Rs-5 was 4 × 10⁻⁶ CFU / mL. 9 CFU / mL, viable count of Enterobacter cloacae Rs-2 was 3 × 10⁻⁶. 9 CFU / mL, this invention labels this compound microbial agent as SL-44+Rs-5+Rs-2.

[0040] Experimental Example 1: Performance Determination of Compound Microbial Inoculants The present invention first performs relevant performance tests on the composite microbial agents prepared in Example 1 and Comparative Examples 1-3, the Bacillus subtilis SL-44 fermentation broth and Klebsiella acidogenic bacteria Rs-5 fermentation broth in Example 1, and the Enterobacter cloacae Rs-2 fermentation broth in Comparative Example 1.

[0041] 1. Compatibility Compatibility test of Bacillus subtilis SL-44, Klebsiella pneumoniae Rs-5, and Enterobacter cloacae Rs-2: 100 μL of activated single bacterial suspension was spread onto LB solid medium. A sterilized circular filter paper (φ=0.5 cm) was placed in the center of each LB solid medium. Then, 10 μL of the other activated bacterial suspensions were inoculated onto the filter paper. The mixture was incubated at 30℃ for 48 h, and the presence of inhibition zones was observed. The presence of inhibition zones indicated an inhibitory effect between the strains in the compound microbial agent; otherwise, no inhibitory effect was observed.

[0042] The results are as follows Figure 1 As shown, Bacillus subtilis SL-44 and Klebsiella acidogenic bacteria Rs-5 exhibited good growth after co-culture, with intact colony morphology and no obvious growth defects or inhibition, indicating that Bacillus subtilis SL-44 and Klebsiella acidogenic bacteria Rs-5 have good compatibility.

[0043] 2. Determination of the ability to produce ferrophosphate Ferrophiles are chelating factors secreted by bacteria in low-iron environments to meet their own life needs. 3+ It has strong binding specificity.

[0044] Three fermentation broths and three compound microbial agents were centrifuged at 6600 rpm for 10 min. The resulting supernatant was mixed with an equal volume of chromium azurite S (CAS) detection solution and reacted in the dark for 0.5 h. The absorbance was measured at 630 nm to obtain the As value. Then, the total amount of ferrophile generated was calculated according to the following formula.

[0045] Total iron production (%) = [(Ar-As) / Ar]×100%; As: absorbance value (630 nm) of the test sample (i.e., supernatant of the fermentation broth of the strain) after reaction with CAS detection solution; Ar: absorbance value (630 nm) of blank culture medium (without inoculation) after reaction with CAS detection solution, as a reference.

[0046] like Figure 2 As shown, SL-44+Rs-5 can produce a large amount of siderophile, while SL-44+Rs-2, Rs-5+Rs-2, and SL-44+Rs-5+Rs-2 have less than ideal siderophile production capabilities. During the 0h~48h culture period, the siderophile content increased rapidly, reaching its maximum at 48h, which may be related to the rapid growth of the bacteria. The iron-producing contents of SL-44 fermentation broth, Rs-5 fermentation broth, Rs-2 fermentation broth, SL-44+Rs-5 compound microbial agent, Rs-5+Rs-2 compound microbial agent, and SL-44+Rs-2 compound microbial agent were 74.08%, 91.60%, 85.12%, 94.29%, 78.99%, 80.11%, and 74.15%, respectively. After 48 hours of cultivation, the rate of iron production by the agents also began to decrease, but the content remained stable. This may be due to the decrease in the number of viable bacteria. However, the iron-producing ability of SL-44+Rs-5 remained stable, indicating that SL-44+Rs-5 has a good synergistic effect.

[0047] 3. Phosphorus solubility test The inorganic phosphorus solubility of the strains was quantitatively determined using Pikovskaya (PVK) medium. Three fermentation broths and three compound microbial agents were inoculated into PVK medium and cultured continuously at 30℃ and 180 rpm for 60 h. Samples were taken at different incubation times, and the supernatant was obtained by centrifugation. The soluble inorganic phosphorus content was quantified by determining the soluble phosphate (available phosphorus) content of the fermentation broth using the molybdenum blue colorimetric method. The absorbance of the reaction solution at 700 nm was measured using ultraviolet spectroscopy. Measurements were taken every 6 h, and after a decrease, measurements were taken every 24 h, for a total of 7 days to continuously measure the phosphorus solubility of the fermentation broth or compound microbial agents.

[0048] like Figure 3 As shown, both the SL-44+Rs-5 composite microbial inoculant and the Rs-5 fermentation broth exhibited good solubility. Within 0-18 hours, the available phosphorus content generated from the dissolution of Ca3(PO4)2 by the SL-44+Rs-5 composite microbial inoculant and the Rs-5 inoculant rapidly increased, accompanied by a rapid decrease in ambient pH. The phosphorus solubility and the change in ambient pH showed a significant negative correlation. The available phosphorus content of the Rs-5 fermentation broth, Rs-2 fermentation broth, and the SL-44+Rs-5, SL-44+Rs-2, Rs-5+Rs-2, and SL-44+Rs-5+Rs-2 composite microbial inoculants reached their peak at 18 hours, at 65.39 mg / L, 60.78 mg / L, and 75.42 mg / L, respectively. The effective phosphorus content of SL-44 fermentation broth reached a peak of 19.69 mg / L at 24 hours, possibly due to the slower growth of SL-44. The SL-44+Rs-5 composite microbial agent exhibited the strongest phosphorus-containing capacity, increasing by 283.04% and 15.82% compared to SL-44 and Rs-5 fermentation broths, respectively. This is because under phosphorus stress, bacteria stimulate and induce the production of large amounts of organic acids or release H+ to meet their phosphorus requirements. + Substances that have good solubility for poorly soluble inorganic phosphates can lower the environmental pH. Furthermore, the available phosphorus content decreases rapidly between 18 and 72 hours, possibly because the bacteria consume the available phosphorus they produce to synthesize nucleic acids for their own growth and reproduction.

[0049] 4. Assay for the ability to produce 1-aminocyclopropane-1-carboxylic acid deaminase (ACC deaminase) Three fermentation broths and three compound microbial agents were cultured in nutrient agar liquid medium (NA medium) for 24 h. The resulting cultures were then centrifuged at 5000 rpm for 10 min at 4 °C to collect the cells. The cells were washed twice with DF liquid medium (ammonium sulfate-free) by centrifugation. Finally, the collected cells were resuspended in ADF medium and cultured at 28 °C and 200 rpm for 24 h to induce ACC deaminase production. After 24 h of culture, the cells were collected by centrifugation at 4 °C, washed twice with 0.1 mol / L Tris-HCl buffer (pH 7.6), and resuspended in 600 μL of 0.1 mol / L Tris-HCl buffer (pH 8.5). 30 μL of toluene was added and the mixture was rapidly shaken for 30 s to lyse the cells. 50 μL of 0.5 mol / L ACC was added to 400 μL of the toluene-containing cell extract and mixed thoroughly. Add 50 μL of 2 mmol / L FeSO₂ and 1.5 mL of 0.1 mol / L Tris-HCl, mix well, aerate thoroughly, seal, and incubate at 30 °C in a water bath for 30 min with shaking. Immediately add 0.5 mL of 4 mol / L HCl to terminate the reaction. Take 1 mL of the reaction solution, add 0.2 mL of 0.1% 2,4-dinitrophenylhydrazine and 2 mmol / L HCl solution, mix well, incubate at 30 °C for 15 min, and finally add 1 mL of 2 mol / L NaOH, mix well, and measure the absorbance at 540 nm. Perform three replicates for each group, simultaneously using redistilled water instead of the enzyme extraction buffer as a blank control. Repeat three times.

[0050] The unit enzyme activity of ACC deaminase is defined as the activity required to form 1 μmol of α-butanone per minute in the enzyme assay system. Protein concentration was determined using the Bradford colorimetric method, with bovine serum albumin as the protein standard. Specific activity (U / mg) was calculated by dividing enzyme activity by enzyme protein concentration. Enzyme activity measurements for each strain were calculated after subtracting spontaneous products from the control samples, and were repeated three times.

[0051] like Figure 4 As shown, after ACC induction, the ACC deaminase activities of SL-44 fermentation broth, Rs-5 fermentation broth, Rs-2 fermentation broth, SL-44+Rs-5 compound microbial agent, SL-44+Rs-2 compound microbial agent, Rs-5+Rs-2 compound microbial agent, and SL-44+Rs-5+Rs-2 compound microbial agent at 24 h were 3.09 U / mg, 5.18 U / mg, 4.89 U / mg, 4.98 U / mg, 4.62 U / mg, 4.85 U / mg, and 4.66 U / mg, respectively.

[0052] 5. Determination of ability to produce indoleacetic acid (IAA) IAA is an endogenous auxin that is ubiquitous in plants. Its biosynthetic precursor is L-tryptophan (L-Trp). It is mainly used to regulate the growth and division of plant cells, promote seed germination or root development, and can also induce plant disease resistance.

[0053] Three fermentation broths and three compound microbial agents were inoculated at a 1% inoculum volume ratio into LB liquid medium containing 100 mg / L L-tryptophan (L-Trp). The mixture was cultured continuously for 7 days, with samples taken every 24 hours. After centrifugation at 6500 rpm for 10 min, 2 mL of the supernatant was collected, and 4 mL of IAA colorimetric reagent was added. The mixture was then incubated in the dark at 40℃ for 30 min, and the OD530 was measured. The IAA content in the fermentation broth was calculated using a standard curve.

[0054] like Figure 5 As shown, SL-44 produces extremely low IAA content; while Rs-5, SL-44+Rs-5, and Rs-5+Rs-2 produce higher IAA content. IAA accumulation is also relatively slow within the 0h~48h range; from 48h~120h, IAA accumulates rapidly and reaches its peak. The IAA production of Rs-5, Rs-2, and SL-44+Rs-5, SL-44+Rs-2, Rs-5+Rs-2, and SL-44+Rs-5+Rs-2 are respectively... The IAA concentrations were 51.72 μg / mL, 33.75 μg / mL, 68.59 μg / mL, 30.69 μg / mL, 45.16 μg / mL, and 35.21 μg / mL, respectively. Among these, SL-44+Rs-5 consistently led other treatments in IAA production after 72 hours. Subsequently, the IAA concentration gradually decreased, and the viable bacterial count also declined rapidly, but the IAA concentration remained relatively stable. This may be because the bacteria continued some life activities during their decline phase, consuming some of the IAA. Therefore, the compound microbial agent (SL-44+Rs-5) provided in Example 1 has a strong IAA production capacity, and the production rate is much higher than its own consumption rate. In particular, IAA can effectively regulate plant growth even at very low concentrations.

[0055] 6. Measurement of biofilm production capacity Three fermentation broths and three compound microbial agents cultured to the stable phase were used to adjust OD under aseptic conditions. 600 The value was 0.5. Then, 1% of the inoculum was inoculated into 100 mL of LB. After thorough mixing, the mixture was aseptically transferred to a 24-well plate with 2 mL per well. The bacterial culture and culture medium were removed aseptically on days 1, 3, 5, and 7. The plates were then rinsed twice with sterile water, 1 mL of 1 w / v% crystal violet solution was added, and the plates were stained for 15 min. The staining solution was aspirated, and the plates were rinsed twice with sterile water. Then, 1 mL of anhydrous ethanol was added to decolorize the plates for 5 min. The absorbance was then measured at 595 nm.

[0056] from Figure 6 It can be seen that the SL-44 fermentation broth and the SL-44+Rs-5, SL-44+Rs-2, Rs-5+Rs-2, and SL-44+Rs-5+Rs-2 composite microbial agents all exhibit good biofilm-forming capabilities, while the Rs-5 fermentation broth, Rs-2 fermentation broth, and Rs-5+Rs-2 composite microbial agents do not possess biofilm-forming capabilities. At day 1, the biofilm is in its initial stage, where motile bacteria gradually differentiate into stationary cells and release signaling substances to attract other bacteria to aggregate, gradually increasing the biofilm thickness. At this point, SL-44's biofilm-forming capability is significantly higher than that of the composite microbial agents, possibly because Rs-5 has a certain inhibitory effect on the growth of SL-44 in the early stages. Subsequently, the biofilm biomass reaches its maximum at day 3, at which point the biofilm enters its mature stage. Mature biofilms exhibit the highest stability and the strongest resistance to changes in the external environment. At 7 days, as nutrients were gradually depleted, the biofilm gradually decomposed, and the biofilm cells redifferentiated from fixed cells into free cells and left the biofilm. At the same time, some bacteria began to lyse. However, at this time, the biofilm content of the compound microbial agent was higher than that of SL-44, indicating that Rs-5 had the effect of enhancing biofilm stability.

[0057] 7. Potted plant experiment The effects of inoculants on germination rate and biomass of plants were investigated through pot experiments with potted plants.

[0058] The pot experiment consisted of 7 treatments: CK: no compound microbial inoculant added; SL-44: SL-44 fermentation broth (SL-44 viable count was 2×10⁻⁶). 8 Treatment with CFU / mL; Rs-5: Rs-5 fermentation broth (Rs-5 viable count is 4×10⁻⁶). 9 Treatment with CFU / mL); Rs-2: Rs-2 fermentation broth (Rs-2 viable count is 4×10⁻⁶). 9 CFU / mL) treatment; SL-44+Rs-5: Example 1 compound microbial agent (SL-44 viable count 2×10⁻⁶) 8 The viable count of CFU / mL and Rs-5 was 4 × 10⁻⁶. 9 Treatment with CFU / mL; SL-44+Rs-2: Comparative Example 1 compound microbial agent (SL-44 viable count 2×10⁻⁶) 8 The viable count of CFU / mL and Rs-2 was 4 × 10⁻⁶. 9 Treatment with CFU / mL; Rs-5 + Rs-2: Comparative Example 2 compound microbial agent (Rs-5 viable count 4 × 10⁻⁶) 9 The viable count of CFU / mL and Rs-2 was 4 × 10⁻⁶. 9Treatment with CFU / mL; SL-44+Rs-5+Rs-2: Comparative Example 3 compound microbial agent (SL-44 viable count 2×10⁻⁶) 8 The viable count of CFU / mL and Rs-5 was 4 × 10⁻⁶. 9 The viable count of CFU / mL and Rs-2 was 4 × 10⁻⁶. 9 Treatment with CFU / mL.

[0059] Each seedling pot contained 1 kg of sterilized soil and sown with 6 plump, uniformly sized seeds. The soil used for the pot experiment was taken from the Lintong Campus of Xi'an University of Technology (34°21′N, 109°11′E). The original soil contained 16.22 g / kg of organic matter (SOM), 35.57 mg / kg of available nitrogen (SAN), 10.46 mg / kg of available phosphorus (SAP), and 170.10 mg / kg of available potassium (SAK), respectively, and had a pH value of 7.98 and an soluble salt content (EC value) of 480.23 µS / cm.

[0060] Seed disinfection: Soak seeds in 70 v / v% ethanol for 5 minutes, then soak in 0.1 w / v% HgCl2 for 10 minutes, and finally wash with sterile water.

[0061] The seedlings were grown in a constant temperature and light incubator at 28°C, with a light duration of 14 hours and a relative humidity of 50%. One week after seed germination, 4 mL of inoculum was applied to the roots of each seedling. In the control (CK) treatment, an equal volume of sterile water was applied to the roots of each seedling. Inoculation was repeated every two weeks. Plant biomass was measured seven weeks after seed germination.

[0062] Plant biomass includes fresh weight (FW), dry weight (DW), plant height (PH), and root weight (RL). Fresh plant samples are washed with pure water, blotted dry with paper, and their fresh weight is measured. They are then transferred to a drying oven and dried at 90°C until constant weight is achieved, at which point their dry weight is measured. Plant height is measured from the base of the stem to the growing point, and root weight is measured below the base of the stem. Plant diameter is measured using vernier calipers.

[0063] like Figure 7As shown, the application of SL-44 fermentation broth, Rs-5 fermentation broth, Rs-2 fermentation broth, and the compound microbial agents SL-44+Rs-5, SL-44+Rs-2, Rs-5+Rs-2, and SL-44+Rs-5+Rs-2 respectively did not have a very significant effect on the germination rate of rapeseed seeds. By day 2, the germination rate of all treatments had reached almost 100%, which may be due to the rapid germination of rapeseed seeds. Five days after the seedlings continued to grow, significant differences in seedling length were observed. The seedling lengths after treatment with SL-44 fermentation broth, Rs-5 fermentation broth, Rs-2 fermentation broth, SL-44+Rs-5 compound microbial agent, SL-44+Rs-2 compound microbial agent, Rs-5+Rs-2 compound microbial agent, and SL-44+Rs-5+Rs-2 compound microbial agent were 35.46cm, 45.63cm, 62.40cm, 50.76cm, 88.60cm, 56.12cm, 51.23cm, and 68.99cm, respectively. Compared to... In the control group (CK), the growth rate of rapeseed seedlings increased by 28.68%, 75.97%, 43.15%, 149.86%, 58.26%, 44.47%, and 94.56% respectively after applying SL-44 fermentation broth, Rs-5 fermentation broth, Rs-2 fermentation broth, SL-44+Rs-5 compound microbial agent, SL-44+Rs-2 compound microbial agent, Rs-5+Rs-2 compound microbial agent, and SL-44+Rs-5+Rs-2 compound microbial agent. The compound microbial agent of Example 1 had the most significant promoting effect on the germination and growth of the seedlings.

[0064] The application of SL-44 fermentation broth, Rs-5 fermentation broth, Rs-2 fermentation broth, SL-44+Rs-5 compound microbial agent, SL-44+Rs-2 compound microbial agent, Rs-5+Rs-2 compound microbial agent, and SL-44+Rs-5+Rs-2 compound microbial agent has a significant effect on rapeseed biomass.

[0065] Table 1 Effects of microbial inoculants on plant biomass Note: Different lowercase letters indicate that different treatment groups have significant differences in various indicators (dry weight, fresh weight, root weight, plant height).

[0066] As shown in Table 1, compared with the control group, the fresh weight of rapeseed seedlings treated with SL-44 fermentation broth, Rs-5 fermentation broth, Rs-2 fermentation broth, SL-44+Rs-5 compound microbial agent, SL-44+Rs-2 compound microbial agent, Rs-5+Rs-2 compound microbial agent, and SL-44+Rs-5+Rs-2 compound microbial agent increased by 54.94%, 109.82%, 77.10%, 265.83%, 93.24%, 87.36%, and 164.58%, respectively. The dry weight increased by 48.51%, 118.66%, 88.06%, 267.91%, 120.90%, 112.69%, and 135.82%, respectively; the plant height increased by 42.32%, 63.93%, 55.67%, 128.67%, 86.26%, 63.30%, and 91.98%, respectively; and the root weight increased by 56.10%, 126.83%, 107.32%, 287.80%, 143.90%, 129.27%, and 182.93%, respectively. This indicates that the rapeseed plants treated with the compound microbial inoculant showed significantly better fresh weight, dry weight, root weight, and plant height than those treated with other methods.

[0067] Experimental Example 2: Application of Compound Microbial Inoculants in Grape Cultivation I. Experimental Methods 1. Field Experiment Design The experiment consisted of five treatments, as shown in Table 2. Grapevines of similar growth (all varieties "Sunshine Rose") were selected as the test material, with 30 vines per column as one treatment. Each column was further divided into three equal plots. Except for fertilization, all other management practices were identical. Fertilizer was applied through the rhizosphere. The dosage of the microbial agent was as follows.

[0068] Table 2 Grape Field Experimental Treatment Groups 2. Sampling Method 2.1 Leaf sampling Leaf collection: Twenty fresh leaves of similar growth were collected from the four cardinal directions of the grapevine, with 100 leaves collected from each treatment group. All samples were sealed in polyethylene plastic bags and stored in an insulated box with ice packs.

[0069] 2.2 Fruit Sampling After the fruit matured, three bunches of normally developed fruit without obvious pests or diseases were randomly selected from each treatment group, sealed in polyethylene plastic bags, and stored at low temperature in an insulated box with ice packs.

[0070] 3. Leaf index measurement The contents of chlorophyll and carotenoids in grape leaves were determined by ethanol extraction spectrophotometry. Proline content was determined by the sulfosalicylic acid method. Malondialdehyde content was determined by the thiobarbituric acid method. Peroxidase (POD) activity was determined by spectrophotometric monitoring of guaiacol oxidation at 470 nm, catalase (CAT) activity was determined based on the decomposition rate of hydrogen peroxide at 240 nm, and superoxide dismutase (SOD) activity was assessed using the nitroblue tetrazolium (NBT) reduction inhibition method at 560 nm.

[0071] 4. Fruit index determination 4.1 Appearance quality The apparent quality of grapes includes: fresh weight, dry weight, firmness, longitudinal diameter, transverse diameter, and shape index of each grape. The fresh and dry weight of each grape was determined using an electronic balance. The firmness of the grapes was determined using a fruit firmness meter. The longitudinal and transverse diameters of the grapes were measured using vernier calipers, and the shape index is the ratio of the longitudinal to transverse diameters.

[0072] 4.2 Intrinsic Quality The intrinsic quality of grape berries includes: pH value, titratable acid content, soluble sugar content, sugar-acid ratio, soluble solids content, soluble protein content, vitamin C content, and anthocyanin content. The pH value of grape pulp filtrate was determined using a pH meter. Titratable acid was determined using an acid-base titration method. Soluble sugar content was determined using the sulfuric acid-phenol method. The soluble solids content of grape berries was determined using a handheld refractometer. Soluble protein was determined according to the Coomassie Brilliant Blue G-250 method. Vitamin C content was determined using a vitamin C colorimetric assay kit. Anthocyanin content was determined using a pH difference method.

[0073] II. Experimental Results 1. Leaf Indicators Table 3. Leaf Indicators of Grapes Note: Different lowercase letters in the table indicate significant differences between groups.

[0074] As shown in Table 3, the application of the compound microbial agent from Example 1 significantly affected the stress resistance of grapevines. Compared with the control group (CK), the levels of proline, malondialdehyde (MDA), chlorophyll, and antioxidant enzyme activity in grape leaves of groups T1, T2, T3, and T4 were significantly different. MDA content reflects the degree of stress damage suffered by plants; the MDA content in groups T1, T2, T3, and T4 decreased by 21.37%, 45.35%, 19.36%, and 17.01% respectively compared to CK. Proline content can be used as a physiological indicator for drought-resistant breeding. Compared with the control group (CK), the proline content of groups T2, T3, and T4 increased by 37.21%, 220.87%, 148.58%, and 77.29%, respectively; the chlorophyll a content of groups T1, T2, T3, and T4 increased by 12.56%, 72.44%, 71.43%, and 49.66%, respectively, compared with the control group; the optimal application rates for chlorophyll b and carotenoids were 6 L / mu, which were 1.87 mg / g and 3.08 mg / g, respectively. Antioxidant enzymes in plants can eliminate reactive oxygen species in cells, reduce membrane peroxidation, stabilize membrane permeability, improve photosynthesis, and promote healthy plant growth. In the T1, T2, T3, and T4 groups, POD increased by 29.46%, 68.19%, 74.28%, and 54.30%, respectively; SOD increased by 12.22%, 28.35%, 27.50%, and 21.56%, respectively; and CAT increased by 55.55%, 74.07%, 77.77%, 62.96%, and 11.11%, respectively.

[0075] 2. Fruit appearance quality Table 4. Apparent quality of grapes Note: Different lowercase letters in the table indicate significant differences between groups.

[0076] As shown in Table 4, the application of the compound microbial agent of Example 1 significantly improved the appearance quality of grapes. Compared with the control group (CK), the fresh weight, dry weight, and firmness of grapes in groups T1, T2, T3, and T4 were significantly different. Compared with the control group (CK), the fresh weight of grapes in groups T1, T2, T3, and T4 increased by 28.57%, 65.13%, 53.75%, and 49.95%, respectively; the dry weight increased by 1.11%, 29.37%, 28.48%, and 9.79%, respectively; and the firmness increased by 16.29%, 31.75%, 19.79%, and 16.70%, respectively. This may be because the compound microbial agent of Example 1 has the ability to produce ACC deaminase, which inhibits the production of abscisic acid and prevents fruit drop. The transverse diameter and longitudinal diameter were positively correlated with the fresh weight and dry weight. The fruit shape index of different treatments were close to 1, indicating that the shape was approximately spherical.

[0077] 3. Internal quality of the fruit Table 5. Internal quality of grapes Note: Different lowercase letters in the table indicate significant differences between groups.

[0078] As shown in Table 5, the application of the compound microbial agent of Example 1 significantly altered the titratable acid content, soluble sugar content, sugar-acid ratio, soluble solids content, and soluble protein content of grapes. Compared with the control group (CK), the titratable acidity of grapes in groups T1, T2, T3, and T4 decreased by 5.77%, 15.55%, 14.34%, and 13.71%, respectively; the soluble sugar increased by 4.66%, 28.15%, 21.80%, and 21.28%, respectively; the sugar-acid ratio increased by 11.22%, 51.84%, 42.29%, and 40.64%; the soluble solids increased by 10.43%, 34.62%, 31.87%, and 22.83%; the soluble protein increased by 75.08%, 148.00%, 165.40%, and 106.63%; the anthocyanin content increased by 4.80%, 37.47%, 42.54%, and 22.76%; and the vitamin C content increased by 43.70%, 104.12%, 106.48%, and 82.13%, respectively. Soluble solids, soluble protein, vitamin C and other indicators are important indicators for judging fruit quality. The above results show that applying the compound microbial agent provided by this invention to grapes can significantly improve fruit quality.

[0079] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0080] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A compound microbial agent, characterized in that, The composite microbial agent is prepared by the following steps: Activated Bacillus subtilis ( Bacillus subtilis SL-44 and Klebsiella acidogenic bacteria ( Klebsiella oxytoca Rs-5 were cultured separately to obtain Bacillus subtilis SL-44 fermentation broth and Klebsiella acidogenic Rs-5 fermentation broth; The compound microbial agent was obtained by mixing and fermenting the fermentation broth of Bacillus subtilis SL-44 and Klebsiella acidogenic bacteria Rs-5. The viable count of Bacillus subtilis SL-44 per milliliter of the aforementioned compound microbial agent is ≥2×10⁻⁶. 8 CFU, viable count of Klebsiella pneumoniae Rs-5 ≥ 4 × 10⁻⁶ 9 CFU.

2. The compound microbial agent according to claim 1, characterized in that, The fermentation culture temperature is 25℃~32℃, and the fermentation culture time is 40h~60h.

3. The compound microbial agent according to claim 1, characterized in that, The temperature for the expansion culture is 25℃~32℃, and the culture time is 40h~60h.

4. The application of the compound microbial agent according to claim 1, characterized in that, The application is any one or more of the following: a. Promotes plant growth; b. Improve plant stress resistance; c. Improve the quality of plant fruits.

5. The application according to claim 4, characterized in that, The promotion of plant growth refers to increasing the fresh weight of plants, increasing the dry weight of plants, increasing the root weight of plants, increasing the height of plants, or promoting the germination of plant seedlings.

6. The application according to claim 4, characterized in that, The improvement of plant fruit quality refers to increasing the fruit's weight, firmness, soluble sugar content, soluble solids content, soluble protein content, anthocyanin content, or vitamin C content.

7. The application according to claim 4, characterized in that, The plant is rapeseed, grape, cucumber or tomato; the fruit of the plant is grape, cucumber or tomato.

8. The application according to claim 4, characterized in that, The application method is to water the plant roots or spray the leaves with the compound microbial agent.

9. The application according to claim 8, characterized in that, The application dosage of the compound microbial agent is 3L / mu to 12L / mu.

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