A compound microbial agent, a compound liquid microbial agent and application thereof

Abstract

The application belongs to the technical field of microbial inoculants, and particularly relates to a compound microbial inoculant, a compound liquid microbial inoculant and application.The application provides a compound microbial inoculant which is composed of Bacillus mojavensis (W1), Bacillus halodurans (L21) and Bacillus subtilis (M4). Bacillus mojavensis Bacillus halotolerans Bacillus subtilis The Bacillus mojavensis (W1), the Bacillus halodurans (L21) and the Bacillus subtilis (M4) in the compound microbial inoculant interact with each other, can activate the defense mechanism of plants, improve the superoxide dismutase (SOD) activity, the peroxidase (POD) activity and the catalase (CAT) activity, and reduce the content of malondialdehyde (MDA), so that the prevention and treatment effect of the root rot of Pterygioglobose in the compound microbial inoculant is improved.​

Description

Technical Field

[0001] This invention belongs to the field of microbial inoculant technology, specifically relating to a compound inoculant, a compound liquid inoculant, and their applications. Background Technology

[0002] Axe-winged sand mustard ( Pugionium dolabratum *Maxim.* is a perennial herbaceous plant belonging to the genus *Maxim.* of the Brassicaceae family, mainly distributed in arid and semi-arid sandy areas of Northwest China (such as Inner Mongolia, Ningxia, and Gansu). It has a well-developed root system and is drought-resistant and wind-blown sand-resistant, making it an important species for ecological restoration and economic crops in sandy areas. However, under artificial cultivation or environmental stress, it is susceptible to root rot, affecting its growth and yield.

[0003] The pathogens of root rot are mainly fungi: common pathogens include Fusarium spp. ( Fusarium spp. ), Pythium genus ( Pythium spp. Phytophthora ( ) Phytophthora spp. Of these, root rot caused by Fusarium is the most common, and it can produce a red or pink mold layer.

[0004] The main control methods for root rot in *Sinocyclocheilus serrata* are agricultural control, chemical control, and biological control. Agricultural control may require proper crop rotation, selection of well-drained soil to avoid waterlogging, soil disinfection before planting, and selection of healthy seedlings—a rather complex process. Chemical control may involve using fungicides such as carbendazim and thiophanate-methyl, applied as a root drench in the early stages of disease, but resistance is easily developed. Biological control utilizes beneficial microorganisms to inhibit pathogens.

[0005] Compound microbial agents are preparations containing a variety of beneficial microorganisms. There are no reports in the existing technology of using a combination of Bacillus mojavei, Bacillus halophilus, and Bacillus subtilis to control root rot of Acer buergerianum. Summary of the Invention

[0006] The purpose of this invention is to provide a compound microbial agent, a compound liquid microbial agent, and its application. The compound microbial agent provided by this invention has a good control effect on root rot of *Strombus haematomarginatus*.

[0007] To address the aforementioned technical problems, the present invention proposes the following technical solution:

[0008] This invention provides a compound microbial agent, wherein the compound microbial agent is composed of Bacillus mojaveniformis (… Bacillus mojavensis W1, Salt-resistant Bacillus ( Bacillus halotolerans L21 and Bacillus subtilis ( Bacillus subtilis) M4 composition; the preservation number of the Bacillus mojavensis W1 is CGMCC No. 35479, the preservation number of the Bacillus halodurans L21 is CGMCC No. 35478, and the preservation number of the Bacillus subtilis M4 is CGMCC No. 35477.

[0009] As a preferred solution, the viable cell number of the Bacillus mojavensis W1 in the complex microbial agent is ≥ 1 × 10 8 CFU / g or ≥ 1 × 10 8 CFU / mL; the viable cell number of the Bacillus halodurans L21 in the complex microbial agent is ≥ 1 × 10 8 CFU / g or ≥ 1 × 10 8 CFU / mL; and the viable cell number of the Bacillus subtilis M4 in the complex microbial agent is ≥ 1 × 10 8 CFU / g or ≥ 1 × 10 8 CFU / mL.

[0010] The application also provides a complex liquid microbial agent, which is a fermentation broth obtained by fermentation culture of the complex microbial agent according to the above solution.

[0011] As a preferred solution, the preparation method of the fermentation broth comprises: mixing a Bacillus mojavensis W1 fermentation broth, a Bacillus halodurans L21 fermentation broth and a Bacillus subtilis M4 fermentation broth to obtain the fermentation broth.

[0012] As a preferred solution, the volume ratio of the Bacillus mojavensis W1 fermentation broth, the Bacillus halodurans L21 fermentation broth and the Bacillus subtilis M4 fermentation broth during the mixing is (1~2):(1~2):(1~2).

[0013] As a preferred solution, the preparation method of the Bacillus mojavensis W1 fermentation broth comprises: inoculating the Bacillus mojavensis W1 into a fermentation medium to perform fermentation culture;

[0014] The preparation method of the Bacillus halodurans L21 fermentation broth comprises: inoculating the Bacillus halodurans L21 into a fermentation medium to perform fermentation culture;

[0015] The preparation method of the Bacillus subtilis M4 fermentation broth comprises: inoculating the Bacillus subtilis M4 into a fermentation medium to perform fermentation culture.

[0016] As a preferred solution, the fermentation culture is performed for 32~38h, the rotation speed is 160~190rpm, and the temperature is 25~32℃.

[0017] As a preferred solution, the culture medium composition applied in the fermentation culture includes: 9-11 g / L sucrose, 9-11 g / L yeast powder and 9-11 g / L K2HPO4; the initial pH of the culture medium is 6-7; and the liquid loading amount of the culture medium is 16%-22% of the culture container.

[0018] The application also provides the application of the composite microbial agent or the composite liquid microbial agent in the prevention and treatment of plant diseases.

[0019] As a preferred solution, the plant includes Arabis hallerstainti; and the disease includes root rot.

[0020] The application has the beneficial effect that the composite microbial agent provided by the application is prepared by the mutual interaction of Bacillus mojavensis W1, Bacillus halodurans L21 and Bacillus subtilis M4, can activate the defense mechanism of the plant, improve the superoxide dismutase SOD activity, peroxidase POD activity and catalase CAT activity, and reduce the content of malondialdehyde MDA, thereby improving the prevention and treatment effect of Arabis hallerstainti root rot. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 W1 phylogenetic tree constructed based on 16S rDNA sequences;

[0022] Figure 2 M4 phylogenetic tree constructed based on 16S rDNA sequences;

[0023] Figure 3 L21 phylogenetic tree constructed based on 16S rDNA sequences;

[0024] Figure 4 Strain compatibility determination diagram;

[0025] Figure 5 Antagonistic activity of mixed antagonistic bacteria on Fusarium oxysporum;

[0026] Figure 6 Influence diagram of carbon source types on the growth of composite bacteria;

[0027] Figure 7 Influence diagram of sucrose concentration on the growth of composite bacteria;

[0028] Figure 8 Influence diagram of nitrogen source types on the growth of composite bacteria;

[0029] Figure 9 Influence diagram of yeast powder concentration on the growth of composite bacteria;

[0030] Figure 10 Influence diagram of inorganic salt types on the growth of composite bacteria;

[0031] Figure 11 The effect of potassium dihydrogen phosphate concentration on the growth of the complex bacteria (Figure).

[0032] Figure 12 Curve of bacterial concentration change during the growth process of the compound bacteria;

[0033] Figure 13 Figure showing the effect of different liquid volumes on the growth of the compound bacteria;

[0034] Figure 14 The graph shows the effect of different initial pH values ​​of the culture medium on the fermentation of the complex bacteria.

[0035] Figure 15 The diagram shows the biocontrol effect of the compound bacteria on root rot of *Sinocyclocheilus fasciatus*.

[0036] Figure 16 Figure showing the effect of different treatments on SOD enzyme activity in potted *Strombax ceiba* leaves;

[0037] Figure 17 Figure showing the effect of different treatments on POD enzyme activity in potted *Strombus haematomarginatus* leaves;

[0038] Figure 18 Figure showing the effect of different treatments on CAT enzyme activity in potted *Strombus haematomarginatus* leaves;

[0039] Figure 19 Figure showing the effect of different treatments on malondialdehyde content in the leaves of potted *Strombax ceiba*.

[0040] Biological Preservation Instructions

[0041] Strain W1, classified as Bacillus mojavei ( Bacillus mojavensis It was deposited on July 31, 2025 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35479.

[0042] Strain L21, classified as halophilic Bacillus ( Bacillus halotolerans It was deposited on July 31, 2025 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35478.

[0043] Strain M4, classified as Bacillus subtilis ( Bacillus subtilis It was deposited on July 31, 2025 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35477. Detailed Implementation

[0044] This invention provides a compound microbial agent, wherein the compound microbial agent is composed of Bacillus mojaveniformis (… Bacillus mojavensis W1, Salt-resistant Bacillus ( Bacillus halotolerans L21 and Bacillus subtilis ( Bacillus subtilis The composition is M4; the preservation number of the Mojavectomyces W1 is CGMCC No. 35479, the preservation number of the halophilic Bacillus L21 is CGMCC No. 354798, and the preservation number of the Bacillus subtilis M4 is CGMCC No. 354797.

[0045] The combined application of Bacillus mojaveii W1, halophilic Bacillus L21, and Bacillus subtilis M4 in this invention further improves the control effect on root rot of Acer buergerianum.

[0046] As an optional implementation, the viable count of Bacillus mojaverifolium W1 in the compound bacterial agent of the present invention is ≥1×10⁻⁶. 8 CFU / g or ≥1×10 8 CFU / mL; the viable count of halophilic Bacillus L21 in the compound bacterial agent is ≥1×10⁻⁶. 8 CFU / g or ≥1×10 8 CFU / mL; the viable count of Bacillus subtilis M4 in the compound bacterial agent is ≥1×10⁻⁶. 8 CFU / g or ≥1×10 8 CFU / mL.

[0047] This invention provides a compound liquid microbial agent, which is a fermentation broth obtained by fermenting and culturing the compound microbial agent described in the above-mentioned technical solution.

[0048] As an optional implementation, the method for preparing the fermentation broth includes: mixing Bacillus mojaveii W1 fermentation broth, Bacillus halophilus L21 fermentation broth, and Bacillus subtilis M4 fermentation broth.

[0049] As an optional implementation, the volume ratio of the Bacillus mojaveii W1 fermentation broth, the halophilic Bacillus L21 fermentation broth, and the Bacillus subtilis M4 fermentation broth during mixing is (1~2):(1~2):(1~2), more preferably 1:1:1.

[0050] As an optional embodiment, the preparation method of the Bacillus mojavensis W1 fermentation liquor of the present application comprises inoculating the Bacillus mojavensis W1 into a fermentation medium for fermentation culture. As an optional embodiment, the inoculation form of the Bacillus mojavensis W1 comprises a single colony or a seed liquid. The preparation method of the single colony or the seed liquid is not particularly limited in the present application, and a conventional method can be used. The inoculation method is not particularly limited in the present application, and a conventional method can be used.

[0051] As an optional embodiment, the fermentation culture time of the present application is 32-38h, further preferably 33-36h, and more preferably 35h. The fermentation culture temperature of the present application is preferably 25-32℃, further preferably 26-30℃, and more preferably 28℃. The rotation speed of the fermentation culture is 160-190rpm, further preferably 170-185rpm, and more preferably 180rpm. As an optional embodiment, the medium used in the fermentation culture of the present application comprises 9-11g / L sucrose, which can also be 9.5-10.5g / L, and more preferably 10g / L; the medium of the present application comprises 9-11g of yeast powder, which can also be 9.8-10.5g / L, and more preferably 10g / L; the medium of the present application comprises 9-11g / L K2HPO4, which can also be 9.4-10.2g / L, and more preferably 10g / L. The sucrose, yeast powder and K2HPO4 in the medium of the present application can promote the growth of Bacillus mojavensis W1, Bacillus halodurans L21 and Bacillus subtilis M4, and increase the growth density of the complex microorganisms. The initial pH of the medium of the present application is 6-7, which can also be 6.2-6.6, and more preferably 6.4; the volume of the medium accounts for 16%-22% of the volume of the culture container, which can also be 17%-21%, and more preferably 20%. In the specific embodiments of the present application, 50mL of medium is added to each 250mL conical flask. The fermentation culture temperature, time, rotation speed, initial pH of the medium, and volume of the medium of the present application are all for promoting the growth of Bacillus mojavensis W1, Bacillus halodurans L21 and Bacillus subtilis M4.

[0052] The preparation method of the Bacillus halodurans L21 fermentation liquor of the present application comprises inoculating the Bacillus halodurans L21 into a fermentation medium for fermentation culture. The fermentation culture temperature, time, rotation speed, medium, volume ratio of the medium, and initial pH of the medium have been discussed above and will not be repeated here.

[0053] The preparation method of the Bacillus subtilis M4 fermentation liquor comprises inoculating the Bacillus subtilis M4 into a fermentation medium for fermentation culture.

[0054] The application also provides application of the composite microbial agent or the composite liquid microbial agent in the prevention and treatment of plant diseases.

[0055] In the application, the plant preferably comprises P. saxifraga.

[0056] In the application, the disease comprises root rot.

[0057] In the application, the application mode is preferably root irrigation.

[0058] In the application, the composite microbial agent is applied in a field or a seedling tray.

[0059] The composite microbial agent is applied in the four-leaf-one-heart stage, and the application frequency is 1-3 times, more preferably 2 times, and the interval time of 2 times is 7 days. The main root of P. saxifraga grown to the four-leaf-one-heart stage has been elongated, and the lateral root has begun to germinate. At this time, application of the microbial agent can avoid the problem of too short root system in the cotyledon stage and the defect of high lignification degree of the root system in the adult stage. At the same time, the individual difference of P. saxifraga in the four-leaf-one-heart stage is small, which can guarantee the repeatability and reliability of the experimental results.

[0060] In order to further illustrate the application, the technical solutions provided by the application are described in detail below in combination with the drawings and examples, but they should not be understood as limiting the scope of protection of the application.

[0061] Example 1

[0062] 1. Isolation and screening of antagonistic bacteria

[0063] The collected soil is subjected to dilution coating to isolate antagonistic strains, and the isolated bacteria are further purified until single colonies are obtained. Twelve antagonistic strains having a control effect on the Fusarium oxysporum causing root rot of P. saxifraga are obtained by plate confrontation, and the 12 bacterial strains are named as N35, M3, L12, N33, W3, N3O, W2, L7, N23, M4, L21 and W1, respectively. The control CK is treated with sterile water, and the inhibition rates of the CK and the antagonistic strains are shown in Table 1. As shown in Table 1, the 12 biocontrol strains show different inhibition effects on the root rot pathogen, and the strains W1, M4 and L21 have better inhibition effects, so the three strains are selected as the composite microbial agent for subsequent research. The calculation formula of the inhibition rate is as follows:

[0064] .

[0065] Table 1. Antibacterial rate of antagonistic strains

[0066]

[0067] Note: Different lowercase letters indicate significant differences between data points in the same column. p <0.05, the same applies below.

[0068] 2. Identification of antagonistic bacteria

[0069] (1) Morphological characteristics of antagonistic bacteria

[0070] Antagonistic strains W1, M4, and L21 were streaked onto three zones of LB agar plates and incubated at 29°C. Colony morphology was observed. Strains of strain W1 were round, milky white, with translucent edges, a moist surface, irregular shape, and slightly raised, uneven edges. Gram staining of strain W1 indicated that it was a Gram-positive strain.

[0071] The colony morphology of strain M4 is that of small, opaque, milky-white colonies with neat edges, dry, and difficult to pick up. Gram staining results of strain M4 indicate that it is a Gram-positive strain.

[0072] Colonies of strain L21 are opaque, milky white, with wrinkled edges and a moist surface. Gram staining results indicate that strain L21 is a Gram-positive strain.

[0073] (2) Molecular biological identification

[0074] Gene sequence alignment was performed using BLAST from NCBI, and a phylogenetic tree was constructed using MEGA. Strains W1 and... Bacillus mojavensis They share high gene homology and belong to the same branch, see [link / reference]. Figure 1 Based on morphological and physiological-biochemical characteristics, strain W1 was identified as Bacillus mojaveniformis (Mojaveniformis). Bacillus mojavensis ).

[0075] Strain M4 shares high genetic homology with Bacillus subtilis, belonging to the same clade (see [link]). Figure 2 Based on morphological and physiological-biochemical characteristics, strain M4 was identified as Bacillus subtilis (Bacillus subtilis). Bacillus subtilis ).

[0076] strain L21 and Bacillus halotolerans They share high gene homology and belong to the same branch, see [link / reference]. Figure 3 Based on morphological and physiological-biochemical characteristics, strain L21 was identified as a salt-tolerant Bacillus (Bacillus). Bacillus halotolerans ).

[0077] 3. Compatibility test results

[0078] The compatibility between the biocontrol strains was evaluated using the flat plate cross streak method: strains W1, M4, and L21 were inoculated in a triangular layout in three directions on an LB medium plate, and the cross region was observed after 3 days of incubation at 30°C. As shown in FIG. 1, there was no obvious growth inhibition region between strains W1, M4, and L21, indicating that they could be mixed and cultured. Figure 4

[0079] The compatibility between the strains was determined by the presence or absence of an inhibition zone. The presence of an obvious inhibition zone at the cross point indicates that the strains are antagonistic and should not be mixed and cultured. If there is no inhibition, it indicates that the strains are compatible and can be used for subsequent composite microbial agent development experiments.

[0080] 4. Antibacterial test of the composite bacteria

[0081] If the above compatibility identification results can be mixed and cultured, the selected mixed bacteria are subjected to antagonistic test using the flat plate confrontation method. The specific method is as follows:

[0082] M4 was inoculated into LB liquid medium for culture to obtain M4 bacterial liquid; L21 was inoculated into LB liquid medium for culture to obtain L21 bacterial liquid; and W1 was inoculated into LB liquid medium for culture to obtain W1 bacterial liquid. The culture conditions were 24°C, 280 rpm, 24 h, and a liquid volume of 100 mL / 250 mL conical flask.

[0083] The preparation method of L421 composite bacterial liquid was as follows: M4 bacterial liquid and L21 bacterial liquid were mixed at a volume ratio of 1:1 to obtain L421 bacterial liquid.

[0084] The preparation method of L121 composite bacterial liquid was as follows: W1 bacterial liquid and L21 bacterial liquid were mixed at a volume ratio of 1:1 to obtain L121 bacterial liquid.

[0085] The preparation method of L14 composite bacterial liquid was as follows: W1 bacterial liquid and M4 bacterial liquid were mixed at a volume ratio of 1:1 to obtain L14 bacterial liquid.

[0086] The preparation method of H1 composite bacterial liquid was as follows: W1 bacterial liquid, M4 bacterial liquid, and L21 bacterial liquid were mixed at a volume ratio of 1:1:1 to obtain H1 composite bacterial liquid.

[0087] Biocontrol bacteria treatment 1: (1) pathogenic bacteria pretreatment: a 5-day-old pathogenic bacteria plate was used, a sterile puncher was used to cut a 6-mm-diameter bacterial cake at the edge of the colony, and the cake was inoculated in the center of a fresh PDA plate. (2) Biocontrol bacteria treatment: 20 μL of the tested L421 bacterial liquid was inoculated at four orthogonal positions (cross-shaped distribution) with a radius of 2.5 cm from the center of the plate; 28°C, dark culture for 7 days; three parallel experiments were set.

[0088] ​Biocontrol treatment 2: same as biocontrol treatment 1, the only difference is that L121 bacterial liquid is inoculated, and 3 parallel experiments are set.

[0089] Biocontrol treatment 3: same as biocontrol treatment 1, the only difference is that L14 bacterial liquid is inoculated, and 3 parallel experiments are set.

[0090] Biocontrol treatment 4: same as biocontrol treatment 1, the only difference is that H1 bacterial liquid is inoculated, and 3 parallel experiments are set.

[0091] Control group (CK): set up a blank control inoculated with only pathogenic bacteria, each treatment is repeated 3 times, and cultured at 28℃ in the dark for 7 days.

[0092] After each treatment culture is completed, the antibacterial activity of the biocontrol bacteria is evaluated by measuring the diameter of the inhibition zone or the colony growth inhibition rate, and the phenotypic characteristics such as the morphological changes of the pathogenic bacteria colonies are recorded.

[0093] The antagonistic effect is as shown in Figure 5 The mixed bacteria L421, L121, L14 and H1 have control effect on the root rot pathogen of Arabis hallerstainii, and the inhibition rates of strains L421, L121, L14 and H1 on Fusarium oxysporum are 59.47%, 60.85%, 62.67% and 75.41%, respectively.

[0094] Example 2 Fermentation optimization of compound bacteria

[0095] The composition of LB liquid medium is: 10g peptone, 5g yeast powder, 10g sodium chloride and 1L distilled water, pH 7.0.

[0096] 1. Optimization of fermentation medium of compound bacteria

[0097] (1) Carbon source optimization: using LB liquid medium as the basis, 5 kinds of carbon source replacement medium were designed: using 10g of sucrose, maltose, xylose, glucose and fructose to replace an equal amount of peptone in LB liquid medium, respectively, and the rest of the ingredients remain unchanged.

[0098] The experiment adopts a completely randomized block design, and 3 biological replicates are set for each carbon source treatment.

[0099] Before inoculation, inoculate 100mL / 250 conical flask with 1% (v / v) of H1 compound bacterial liquid (the preparation method of H1 compound bacterial liquid is the same as that of Example 1), 100mL / 250 conical flask contains 100mL medium in 250mL conical flask, and cultivate at 28℃ constant temperature shaking bed with 180rpm rotation speed for 24h. After cultivation, the absorbance value of the culture solution obtained in each treatment group at 600nm wavelength was measured. The results are shown in Figure 6 It can be seen from the results that the OD 600The values from left to right are 1.57, 1.30, 1.49, 1.37, 1.32, sucrose is the most conducive to the growth of H1 complex bacteria, OD 600 The highest. Select sucrose configuration medium, the formula of the medium is: 10 g sucrose, 5 g yeast powder, 10 g sodium chloride and 1 L distilled water, recorded as medium 1.

[0100] Optimization of sucrose concentration: only adjust the concentration of sucrose on the basis of medium 1, the concentration of sucrose is set to 0.5%, 1%, 2%, 3%, 4% and 5%, inoculate H1 complex bacteria liquid at 28℃, 180 rpm shaking culture for 24h, by measuring OD 600 Value to evaluate the growth of complex bacteria, so as to determine the optimum concentration of nutrients. Each concentration is set to 3 repeats, and the results are shown in Figure 7 According to Figure 7 It can be seen that the OD 600 Value from left to right is 1.33, 1.51, 1.63, 1.54, 1.52, 1.47, when the concentration of sucrose is 2%, the concentration of strain fermentation liquid is the highest, OD 600 Value is 1.63.

[0101] (2) nitrogen source optimization: using LB liquid medium as the basis, respectively design 10 g / L of 7 kinds of nitrogen source yeast powder control, beef extract, protein peptone, glutamic acid, ammonium sulfate, ammonium chloride and potassium nitrate instead of the same amount of yeast powder component in the basic formula, keep other medium components unchanged.

[0102] The experiment adopts complete random block design, each carbon source treatment is set to 3 biological repeats. Before inoculation, H1 complex bacteria liquid (the preparation method of H1 complex bacteria liquid is the same as example 1) is inoculated into 100 mL / 250 conical flask at 2% (v / v) inoculation amount, 100 mL / 250 conical flask contains 100 mL medium in 250 mL conical flask, and the shaking culture is carried out at 28℃ constant temperature shaking table with 180 rpm rotation speed for 24h. After culture, the absorbance value of the culture liquid obtained in each treatment group at 600nm wavelength is measured. The results are shown in Figure 8 It can be seen that the OD 600 Value from left to right is 1.29, 0.07, 0.05, 0.61, 0.57, 0.16, 0.12, yeast powder is the most conducive to the growth of H1 complex bacteria, OD 600 The highest is 1.29. The optimized medium is: 10 g protein peptone, 5 g yeast powder, 10 g sodium chloride and 1 L distilled water, recorded as medium 2.

[0103] Optimization of yeast powder concentration: On the basis of medium 2, only the concentration of yeast powder was adjusted, and the concentration of yeast powder was set to 0.5%, 1%, 2%, 3%, 4% and 5% respectively. After inoculation with H1 complex bacterial liquid, it was cultured at 28°C and 180 rpm for 24h. The growth of the complex bacteria was evaluated by measuring the OD 600 value, and the optimal concentration of nutritional ingredients was determined. Each concentration was set in triplicate, and the results are shown in Figure 9 . According to Figure 9 , it can be seen that the OD 600 values of the fermentation broth from left to right are 1.15, 1.36, 1.23, 1.23, 1.23, 0.98, and the concentration of the strain fermentation broth is the highest when the concentration of yeast powder is 1%, and the OD 600 value is 1.36.

[0104] (3) Optimization of inorganic salts: Taking standard LB liquid medium as the benchmark, 7 representative inorganic salts such as K2HPO4, KH2PO4, FeSO4, ZnSO4, MgSO4, NaCl (control) and KCl were selected to replace the NaCl component in the basic formula in equal amounts, and other components remained constant.

[0105] According to the difference in the demand of microorganisms for inorganic elements, different concentrations were set: the addition amount of K2HPO4, KH2PO4, NaCl and KCl was 0.5% (w / v), and the addition amount of FeSO4, ZnSO4 and MgSO4 was 0.05% (w / v). H1 complex bacterial liquid was inoculated into the medium containing different inorganic salts, and placed in a constant temperature shaking incubator at a culture temperature of 28°C and a shaking speed of 180 rpm for continuous culture for 24h. The OD 600 value was measured to evaluate the growth of the bacteria, and the optimal inorganic salt type was selected. The inoculation method was the same as step (1). The results are shown in Figure 10 , and the OD 600 values of the fermentation broth from left to right are 1.22, 0.19, 1.31, 1.24, 0.11, 1.33, 0.95, and K2HPO4 is determined to be the best inorganic salt source.

[0106] The obtained medium is: 10g peptone, 5g yeast powder, 5g K2HPO4 and 1L distilled water, pH 7.0, which is recorded as medium 3.

[0107] Optimization of K2HPO4 concentration: On the basis of medium 3, only the concentration of K2HPO4 was adjusted, and the concentration of K2HPO4 was set to 0.25%, 0.5%, 1%, 1.5%, 2% and 3% respectively. After inoculation with H1 complex bacterial liquid, it was cultured at 28°C and 180 rpm for 24h. The growth of the complex bacteria was evaluated by measuring the OD 600The OD value of the complex bacteria was evaluated to determine the optimal addition concentration and inoculation mode of step (1). Each concentration was set in triplicate, and the results are shown in Table 1. Figure 11 As shown in Table 1, the OD value of the complex bacteria was 1.21, 1.26, 1.42, 1.15, 1.08, and 0.92 from left to right. Figure 11 It can be seen that the OD value of the fermentation broth was 1.21, 1.26, 1.42, 1.15, 1.08, and 0.92 from left to right. 600 The OD value of the complex bacteria was 1.21, 1.26, 1.42, 1.15, 1.08, and 0.92 from left to right. 600 The OD value of the complex bacteria was 1.21, 1.26, 1.42, 1.15, 1.08, and 0.92 from left to right.

[0108] 2. Orthogonal test of medium components

[0109] The medium composition was as follows: 20 g sucrose, 10 g yeast powder, 10 g K2HPO4, and 1 L distilled water, pH 7.0, which was designated as medium 4.

[0110] Based on medium 4, the carbon source content (A1-A3), organic nitrogen source content (B1-B3), and inorganic salt content (C1-C3) were set. The complex bacteria H1 was inoculated into a conical flask (100 mL / 250 mL conical flask) at a 1% (v / v) inoculation amount and cultured at 28°C and 180 rpm for 24 h. The OD value was measured to evaluate the growth status of the complex bacteria, thereby determining the optimal medium formula. The medium composition was as follows: sucrose, yeast powder, K2HPO4, and water, and the test design levels are shown in Table 2. 600 The OD value was measured to evaluate the growth status of the complex bacteria, thereby determining the optimal medium formula. The medium composition was as follows: sucrose, yeast powder, K2HPO4, and water, and the test design levels are shown in Table 2.

[0111] Table 2 Orthogonal test of complex bacteria L9 (3 3 ) orthogonal test

[0112]

[0113] The orthogonal test results showed that the influence of the three factors on the growth of the antagonistic bacteria H1 was C>A>B, and the optimal level combination was A2B2C2 (Table 3). The variance analysis results showed that factor B (yeast powder) had a significant effect on the growth and development of the complex bacteria, and therefore the optimal formula of the medium was determined as follows: sucrose 1%, yeast powder 1%, and 1% K2HPO4.

[0114] Table 3 Orthogonal test results of salt-tolerant Bacillus

[0115]

[0116] 3. Effect of culture time on the growth of complex strains

[0117] The optimal medium formula obtained based on the orthogonal test was as follows: medium composition: 10 g sucrose, 10 g yeast powder, 10 g K2HPO4, and 1 L distilled water, pH 7.0, which was designated as medium 5.

[0118] Experimental setup 3 groups of parallel repeats, inoculated with H1 complex bacteria liquid, in 28℃ constant temperature conditions, with 180 rpm rotation speed for 96h batch culture, inoculation method step 2. In the rapid growth period (0~12h) every 2h sampling, stable period and decline period (12~96h) every 12h sampling. Using UV spectrophotometer to determine the absorbance of culture at 600nm wavelength, through three repeated measurements to take the average value. With the culture time (h) as the abscissa, OD 600 value as the ordinate, draw high time resolution growth curve, see Figure 12 .

[0119] According to Figure 12 , H1 complex bacteria growth lag phase is about 8h, into the logarithmic growth phase at 12h, the fastest growth. When the cell increases, the nutrients are consumed, and the toxic substances increase, the reproduction and death of bacteria reach a balance, and the highest OD 600 value is 1.21 at 34h. After 34h, it decreased and entered the decline phase.

[0120] 4, the effect of liquid volume on the growth of complex strains

[0121] Based on medium 5, set the initial liquid volume 50 / 250 mL bottle, 75 / 250 mL bottle, 100 / 250 mL bottle, 125 mL / 250 mL bottle, 50 / 250 mL bottle contains 50 mL medium 5, the rest is the same. Each treatment group was inoculated with 400 μL H1 complex bacteria liquid and cultured at 28℃, 180 rpm for 24h. The growth of complex bacteria was evaluated by measuring OD 600 value, and the inoculation method was the same as step 2. Three replicates were set, see Figure 13 .

[0122] According to Figure 13 , the OD 600 value of the fermentation broth from left to right is 1.46, 1.36, 1.27, 1.15, and the complex bacteria grow best in 50 mL liquid volume, with OD 600 nm value of 1.36. Increasing the liquid volume will affect the cell volume. With the increase of liquid volume, the growth of antagonistic bacteria gradually decreases.

[0123] 5, the effect of initial pH on the growth of complex strains

[0124] Based on medium 5, the initial pH value of medium 5 was set to 4, 5, 6, 7, 8 and 9. 400 μL of H1 complex bacteria liquid was inoculated into 100 mL of medium with different pH values, and then cultured at 28℃, 180 rpm for 24h. The growth of complex bacteria was evaluated by measuring OD 600To assess the growth status of the compound bacteria, the inoculation method is the same as in step 2. The growth of the culture medium at 600 nm is measured using a UV spectrophotometer. nm Calculate the absorbance at each wavelength and plot it on a graph. See the results below. Figure 14 It can be seen that the OD of the fermentation broth... 600 The values ​​from left to right are 0.15, 0.79, 1.36, 1.17, 1.09, and 0.83. The H1 composite bacterial solution grows best at pH 6. OD 600 The maximum value is 1.36, indicating that the strain is suitable for growth in a weakly acidic environment.

[0125] 6. Box-Behnken experimental design for compound bacteria

[0126] Based on the results of single-factor experiments, this study selected three key factors as independent variables: culture time (A, h), liquid volume (B, mL), and initial pH (C). The OD of the fermentation broth was used as the variability. 600 The response index was used, and a three-factor, three-level Box-Behnken central composite design was employed for response surface methodology. A quadratic polynomial regression model was established to analyze the effects of each factor and its interactions on bacterial growth, ultimately determining the optimal combination of culture conditions for the antagonistic strains.

[0127] The experimental factor levels for the compound strain Box-Behnken are shown in Table 4.

[0128] Table 4. Levels of experimental factors for the Box-Behnken composite strain.

[0129]

[0130] The experimental results of the Box-Behnken compound strain are shown in Table 5. Analysis of the experimental results using Design-Expert 10 software yielded the following quadratic polynomial regression equation model for the OD600 value (Y) of the biocontrol bacteria H1 fermentation broth against time (A), volume (B), and initial pH (C): Y = 1.53 + 0.0187A + 0.045B + 0.0438C + 0.055AB + 0.0125AC - 0.06BC - 0.16A 2 -0.1058B 2 -0.0733C 2 As shown in Table 6 of the regression model analysis, the Box-Behnken model for the H1 complex bacteria showed extremely significant regression (P<0.01), indicating that this model can be used for the study of H1 complex bacteria OD. 600 The F-value is a prediction of the OD value for the complex bacteria H1. 600 The influence strength of the values ​​can be seen from the data in the table below, which shows the effect of the three factors on the OD of the compound bacteria H1. 600The order of the effect size of the three factors was liquid volume > initial pH > time. The regression equation coefficients significance test showed that A2, B2 and C2 were extremely significant (P < 0.01). The equation misfit term was 0.0123, indicating that the Box-Behnken model of the composite strain H1 was very stable and could be well predicted. The determination coefficient R 2 was 0.9115, indicating that the model fitting degree was good and could better reflect the prediction.

[0131] Table 5 Box-Behnken test scheme and results of the composite strain

[0132]

[0133] Table 6 Regression model analysis results of the composite strain

[0134]

[0135] Note: * indicates significant, ** indicates extremely significant.

[0136] The Design-Expert 10 software was used to draw the 3D response surface analysis graph and the corresponding 2D contour graph to explore the mutual relationship of time (A), liquid volume (B) and initial pH (C). Among them, the contour lines of time (A), liquid volume (B) and time (A), initial pH (C) showed a near circular shape, indicating that the mutual relationship of time (A) and liquid volume (B), time (A) and initial pH (C) was not significant. The contour lines of liquid volume (B) and initial pH (C) showed a near elliptical shape, indicating that the mutual relationship of liquid volume (B) and initial pH (C) was significant. Similarly, through the change trend of the 3D surface graph of the three factors of time, liquid volume and pH, the Design-Expert 10 software was used for further analysis, and it was found that there was a maximum point of time, liquid volume and initial pH within the test range. The predicted value of OD 600 of the composite strain was 1.51 under the conditions of time of 34.854 h, liquid volume of 44.509 mL and initial pH of 6.415. In order to simplify the operation feasibility, the optimal fermentation conditions of the strain were as follows: fermentation time of 35 h, liquid volume of 45 mL and initial pH of 6.4.

[0137] Example 3 Effect of the composite strain on the control of root rot of potted P. saxicola

[0138] 1. Test site and materials

[0139] The potting test was completed in the greenhouse of the Science and Technology Park of the College of Horticulture and Plant Protection of Inner Mongolia Agricultural University from August 2024 to January 2025, and P. saxicola seedlings were used as experimental materials.

[0140] 2. Preparation of bacterial liquid. (1) Preparation of complex bacterial liquid. The microbial inoculant used in the test was H1 complex bacterial liquid, and the preparation method was as follows:

[0141] The composition of the liquid medium was 10 g sucrose, 10 g yeast powder, 10 g K2HPO4 and 1 L distilled water, pH 6.0.

[0142] M4 was inoculated into the liquid medium for culture, 50 mL of liquid medium was placed in a 250 mL conical flask, the initial pH of the liquid medium was 6.0, and after inoculation, it was cultured at 28°C, 180 rpm for 24 h, and M4 bacterial liquid was obtained.

[0143] L21 was inoculated into the liquid medium for culture, 50 mL of liquid medium was placed in a 250 mL conical flask, the initial pH of the liquid medium was 6.0, and after inoculation, it was cultured at 28°C, 180 rpm for 24 h, and L21 bacterial liquid was obtained.

[0144] W1 was inoculated into the liquid medium for culture, 50 mL of liquid medium was placed in a 250 mL conical flask, the initial pH of the liquid medium was 6.0, and after inoculation, it was cultured at 28°C, 180 rpm for 24 h, and W1 bacterial liquid was obtained.

[0145] The preparation method of H1 complex bacterial liquid was as follows: W1 bacterial liquid, M4 bacterial liquid and L21 bacterial liquid were mixed according to the volume ratio of 1:1:1 to obtain H1 complex bacterial liquid. All strains were activated by LB plate and inoculated with single colony to ensure the consistency of fermentation.

[0146] (2) The pathogenic fungus was treated with spore suspension of Fusarium oxysporum, and the concentration of the spore suspension was adjusted to 1×10 8 CFU / mL.

[0147] The Fusarium oxysporum used in the test was provided by the xerophytic vegetable research group of Inner Mongolia Agricultural University. The standard process was used for activation of the pathogenic fungus: in a clean bench, sterile punchers (6 mm) were used to obtain bacterial cakes from the edge of the pre-cultured PDA plate, and 5 bacterial cakes were randomly selected to inoculate 200 mL PDA liquid medium in a 500 mL conical flask, which was placed in a constant temperature shaker (28°C, 180 rpm) for dark culture for 7 days. After the culture was completed, the spore counting was carried out by using a hemocytometer (0.1 mm depth), and the final preparation was made into a standardized spore suspension with a concentration of 1×10 8 spores / mL by centrifugal washing (4000 rpm, 10 min) and resuspension with sterile water, which was stored at 4°C for standby use.

[0148] 3. Test design

[0149] Single factor randomized block design was adopted, and the uniform and robust potted Eutrema sibiricum seedlings were selected as the test materials. The high temperature and high pressure sterilized nutrient soil was loaded into standard culture pots (diameter 15 cm x height 12 cm), and 2 seedlings were planted in each pot, and the soil was compacted to ensure that the root system was evenly contacted. All treatments were started from the four-leaf one-heart stage of Eutrema sibiricum, and the test was divided into 4 treatment groups, and the specific design was as follows:

[0150] The control group (CK): first irrigate with equal amount of sterile water 50 mL, and then irrigate with sterile water 50 mL after 7 days;

[0151] Biological prevention group (T4): first inoculate with antagonistic bacteria fermentation broth (T4: H1 complex bacteria liquid) 50 mL, and then inoculate with pathogenic bacteria fermentation broth 50 mL after 7 days, namely antagonistic bacteria + pathogenic bacteria;

[0152] Biological treatment group (T8): first inoculate with pathogenic bacteria fermentation broth 50 mL, and then inoculate with H1 complex bacteria liquid 50 mL after 7 days, namely pathogenic bacteria + antagonistic bacteria:

[0153] Pathogenic stress group (T9): single inoculation of pathogenic bacteria fermentation broth; 20 pots per treatment, 3 biological repeats (60 pots per treatment).

[0154] The root injury irrigation method was used for inoculation: 3 1 cm deep wounds were made in the rhizosphere of the plants, and 50 mL of antagonistic bacteria fermentation broth or 50 mL of pathogenic bacteria fermentation broth was quantitatively injected, and the control group was treated with equal amount of sterile water at the same time.

[0155] After 30 days of the second inoculation, the following statistics were performed:

[0156] (1) Disease incidence statistics

[0157] After the plant root rot symptoms appeared, the pathogenic bacteria were again isolated from the diseased roots, and verified according to the Koch's rule. The disease grading standard of Eutrema sibiricum was counted according to the following standard:

[0158] 0 level: no disease in root system;

[0159] 1 level: root disease rate ≤20%, leaf normal, root has small disease spot, and plant is healthy without wilting;

[0160] 2 level: 20%≤root disease rate≤40%, root has disease spot, but plant is healthy without wilting;

[0161] 3 level: 40%≤root disease rate≤60%, leaf normal, root and leaf slightly wilted, and lower leaves have few shedding;

[0162] 4 level: 60%≤root disease rate≤80%, root disease spot reaches 1.0-2.0 cm, seedling leaf is obviously wilted or shed or whole plant is wilting;

[0163] Grade 5: root disease incidence ≥ 80%, leaf wilting, root blackening, and even whole plant death.

[0164] Disease incidence (DI) and disease severity index (DSI) and control effect were calculated using the following formula:

[0165] ;

[0166] ;

[0167] .

[0168] The results are shown in Table 7:

[0169] Table 7 Control effect of different treatments on potted P. saxicola

[0170]

[0171] (2) Determination of biomass index

[0172] After 30 days of treatment, P. saxicola samples were collected for biomass determination: (1) the plants were separated into aboveground and root parts using sterilized blades; (2) the fresh weight of each part was immediately weighed; (3) after being placed in a 105℃ oven for 30 min, the temperature was adjusted to 80℃ and dried to constant weight; (4) the dry weight was weighed using an analytical balance.

[0173] The results are shown in Table 8 and Figure 15 In terms of aboveground fresh weight, treatment T4 had a slight increase compared to CK, with an aboveground fresh weight of 1.74g; T9 had the largest decrease, with an average fresh weight of 0.58g. Treatment T9 decreased by 66.5% compared to CK. In terms of aboveground dry weight, T9 had the smallest aboveground dry weight, with an average of 0.23g, a decrease of 72.3% compared to CK; the other treatments had a slight decrease compared to CK. In terms of underground fresh weight, treatment T4 had a slight increase, with an average of 0.37g, an increase of 15.6% compared to CK, with no obvious effect; treatment T9 had a decrease in underground fresh weight, with an average of 0.15g, a decrease of 53.1% compared to CK; the other treatments decreased the underground fresh weight of P. saxicola. In terms of underground dry weight, treatment T9 had the smallest underground dry weight, a decrease of 66.7% compared to CK.

[0174] After 30 days, it can be seen that T4 (application of antagonistic bacteria + pathogenic bacteria) treatment can reduce the decrease rate of P. saximontana biomass caused by root rot, and T9 (application of pathogenic bacteria + antagonistic bacteria) treatment can also reduce the decrease rate of P. saximontana biomass caused by root rot, but the effect is not as good as that of antagonistic bacteria + pathogenic bacteria. The performance of single pathogenic bacteria showed a significant decrease in biomass. It shows that antagonistic bacteria can inhibit the growth of P. saximontana root rot pathogen and reduce the impact of disease on P. saximontana.

[0175] Table 8 Effect of different treatments on the biomass of P. saximontana in pots

[0176]

[0177] (3) Antagonistic bacteria regulate the disease defense enzyme system of P. saximontana seedlings

[0178] Select P. saximontana plants grown in pots, use systematic sampling method, randomly select 3 representative plants in each treatment group, take 3 functional leaves from each plant as test samples, set 3 biological replicates, and use for subsequent determination and analysis of physiological and biochemical indicators.

[0179] SOD activity determination uses nitro blue tetrazolium method.

[0180] POD activity determination uses guaiacol method.

[0181] CAT activity determination uses ultraviolet absorption method.

[0182] MDA (malondialdehyde) content determination uses spectrophotometric method.

[0183] Different treatments have different effects on the SOD, POD, CAT enzyme activity and MDA content of P. saximontana plants, and the results are shown in Table 8. Figure 16~Figure 19

[0184] According to the results, the SOD enzyme activity of T4 treatment is 227.70 U / g. T8 treatment has little difference with CK, and T9 treatment is reduced by 45.76 U / g compared with CK. Figure 16 T8 treatment has little effect on POD activity, and T9 treatment is reduced by 60.02 U / g compared with CK. Figure 17 T4 treatment has a higher increase in CAT activity, which can increase the CAT activity of P. saximontana plants at a certain level, and there is a significant difference. T9 treatment will reduce the CAT activity of P. saximontana plants at a certain level, which is 158 U / g, and the rest of the treatments are close to CK. Figure 18 ​It can be seen that the application of the antagonistic bacteria fermentation liquor first and then the pathogenic bacteria fermentation liquor can enhance the defense system of the plant before it encounters the pathogenic bacteria. The antagonistic bacteria fermentation liquor can activate the systemic resistance (ISR) of the plant, and prepare the antioxidant enzyme in advance, so that the plant can respond more quickly and remove more ROS when the pathogenic bacteria invade, so that the SOD activity is improved more obviously. On the contrary, if the pathogenic bacteria fermentation liquor is applied first and then the antagonistic bacteria fermentation liquor is applied, the plant may have been invaded by the pathogenic bacteria, and the cells are damaged, so that the repair effect of the antagonistic bacteria fermentation liquor is limited, or the accumulation of ROS is too much, which exceeds the regulation capacity of the antagonistic bacteria fermentation liquor, so that the activity is not improved as obviously as the former. Each treatment has a certain influence on the content of malondialdehyde (MDA), compared with the treatment T9 of only inoculating the pathogenic bacteria, the content of MDA of the rest of the treatments is improved to a certain extent Figure 19 If the antagonistic bacteria fermentation liquor is applied first, the defense mechanism of the plant can be activated in advance, so that the plant has stronger antioxidant capacity when it encounters the pathogenic bacteria, so as to more effectively reduce the accumulation of ROS and reduce MDA. On the contrary, if the pathogenic bacteria are inoculated first, a large amount of ROS can be generated, so that the application of the antagonistic bacteria fermentation liquor can not effectively activate the defense system in time, so that the MDA is not reduced effectively.

[0185] In summary, the application of Bacillus mojavensis W1, Bacillus halodurans L21 and Bacillus subtilis M4 further improves the prevention and treatment effect on the root rot of Pterocypsela indica.

[0186] Although the above embodiment describes the present application in detail, it is only a part of the embodiments of the present application, but not all the embodiments, and other embodiments can be obtained according to the present embodiment without creativity, and these embodiments all belong to the protection scope of the present application.

Claims

1. A complex microbial agent, characterized in that, The compound microbial agent is composed of Bacillus mojavei ( Bacillus mojavensis W1, Salt-resistant Bacillus ( Bacillus halotolerans L21 and Bacillus subtilis ( Bacillus subtilis The composition is M4; the *Bacillus mojavemi* W1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35479; the *Salmonella salina* L21 is deposited at the CGMCC with accession number CGMCC No. 35478; and the *Bacillus subtilis* M4 is deposited at the CGMCC with accession number CGMCC No. 35477.

2. The complex bacterial agent according to claim 1, characterized by, The viable cell number of Bacillus mojavensis W1 in the complex microbial agent is ≥1×10 8 CFU / g or ≥1×10 8 CFU / mL; the viable cell number of Bacillus halodurans L21 in the complex microbial agent is ≥1×10 8 CFU / g or ≥1×10 8 CFU / mL; the viable cell number of Bacillus subtilis M4 in the complex microbial agent is ≥1×10 8 CFU / g or ≥1×10 8 CFU / mL.

3. A complex liquid inoculant, characterized in that, The complex liquid bacterial agent is a fermentation broth obtained by fermentation culture of the complex bacterial agent of claim 1 or 2.

4. The complex liquid inoculant according to claim 3, characterized in that, The preparation method of the fermentation broth comprises: mixing Mojave Bacillus W1 fermentation broth, salt-tolerant Bacillus L21 fermentation broth and Bacillus subtilis M4 fermentation broth to obtain the complex liquid bacterial agent.

5. The complex liquid inoculant according to claim 4, characterized in that, The volume ratio of the Mojave Bacillus W1 fermentation broth, the salt-tolerant Bacillus L21 fermentation broth and the Bacillus subtilis M4 fermentation broth is (1-2):(1-2):(1-2) during the mixing.

6. The complex liquid inoculant of claim 4, wherein, The preparation method of the Mojave Bacillus W1 fermentation broth comprises: inoculating the Mojave Bacillus W1 into a fermentation medium for fermentation culture; The preparation method of the salt-tolerant Bacillus L21 fermentation broth comprises: inoculating the salt-tolerant Bacillus L21 into a fermentation medium for fermentation culture; The preparation method of the Bacillus subtilis M4 fermentation broth comprises: inoculating the Bacillus subtilis M4 into a fermentation medium for fermentation culture.

7. The complex liquid inoculant according to claim 6, characterized in that, The fermentation culture is performed for 32-38 hours at a rotation speed of 160-190 rpm and a temperature of 25-32℃.

8. The complex liquid inoculant according to claim 6, characterized in that, The medium composition used in the fermentation culture comprises: 9-11 g / L sucrose, 9-11 g / L yeast powder and 9-11 g / L K2HPO4; the initial pH of the medium is 6-7; and the liquid loading amount of the medium is 16%-22% of the culture container.

9. The complex bacterial agent of claim 1 or 2 or the complex liquid bacterial agent of any one of claims 3-8 for use in preventing and treating root rot of Pugionium cornutum caused by Fusarium oxysporum.

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