Disease-preventing and growth-promoting compound microbial agent special for traditional Chinese medicinal materials as well as preparation method and application of compound microbial agent

Abstract

The invention provides a disease-preventing and growth-promoting compound microbial agent special for traditional Chinese medicinal materials as well as a preparation method and application of the compound microbial agent, and belongs to the technical field of biocontrol microbial agents. The disease-preventing and growth-promoting compound microbial agent special for the traditional Chinese medicinal materials, disclosed by the invention, is prepared from cellulomicrobium fennellii MHP3, brevibacterium brevicola MHP56, bacillus tequilensis MHP82 and bacillus cereus MHP107. The disease-preventing and growth-promoting compound microbial agent disclosed by the invention has a relatively good prevention and treatment effect on root rot of traditional Chinese medicinal materials, and also can promote the growth of the traditional Chinese medicinal materials and increase the yield. The disease-preventing and growth-promoting compound microbial agent disclosed by the invention can also improve rhizosphere soil of traditional Chinese medicinal materials and reduce the salt and alkali content in the soil.

Description

Technical Field

[0001] The present invention relates to the technical field of biocontrol agents, and in particular to a disease prevention and growth-promoting composite microbial agent special for traditional Chinese medicines, as well as a preparation method and application thereof. Background Art

[0002] As an integral part of traditional medicine, Chinese medicinal materials (TCMs) not only occupy a central position in the Chinese medical system but also attract widespread attention worldwide. TCMs such as Astragalus, Codonopsis, and Angelica are highly sought after for their unique medicinal properties and extensive health benefits. With the growing demand for TCMs, their cultivation has expanded annually. However, due to limited land resources, continuous cropping and alternating cropping patterns are common, leading to the frequent occurrence of soil-borne diseases.

[0003] Root rot is a common soil-borne disease that occurs in crop-growing areas. It is primarily caused by a combination of pathogens, including Fusarium and Alternaria, and severely hinders the sustainable development of modern agriculture, earning it the nickname "plant cancer." Root rot has been found in medicinal plants worldwide. Lycium barbarum root rot causes 5% of wolfberry trees to die annually, with a disease rate of 15%. In severely affected areas, the disease rate reaches 37.6% and the death rate reaches 26.5%. In low-lying, poorly drained fields, the incidence of Astragalus root rot ranges from 32% to 41%, with severely affected fields reaching 55%. Isatis root rot causes yield reductions of 11.2% to 21.6% in medicinal areas with mild disease, over 35% in more severe areas, and even 50% in severe years. The field incidence of Bupleurum chinense root rot ranges from 15% to 30%, with the incidence exceeding 60% in severely affected areas. Planting disease-resistant varieties and using chemical pesticides are the primary measures for controlling crop diseases and insect pests. However, due to the long breeding cycle and difficulty in selecting disease-resistant varieties, major planting areas still rely primarily on agronomic and chemical control. While the long-term use of chemical pesticides and fertilizers can control diseases in the short term, their long-term application not only damages soil structure and leads to soil ecological deterioration, but may also cause environmental pollution and drug safety issues. Therefore, exploring environmentally friendly and efficient biological control technologies for root rot of traditional Chinese medicines is of great scientific value for protecting the ecological environment and promoting the high-quality development of the traditional Chinese medicine industry. Summary of the Invention

[0004] In view of this, the present invention provides a disease prevention and growth-promoting composite microbial agent specifically for Chinese medicinal materials, as well as its preparation method and application, to address the problems of soil-borne diseases and soil degradation faced in the cultivation of Chinese medicinal materials and promote the sustainable development of the Chinese medicinal materials industry.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a disease prevention and growth-promoting composite microbial agent for Chinese medicinal materials, comprising Cellulobacterium finschmidtii MHP3, Brevibacterium frost-resistant MHP56, Bacillus tequilae MHP82 and Bacillus cereus MHP107;

[0007] The Cellulosomyces finneyii MHP3 is deposited in the China Center for Type Culture with the deposit number CCTCC M2025737;

[0008] The cold-resistant Brevibacterium MHP56 was deposited in the China Center for Type Culture with the deposit number CCTCC M2025738;

[0009] The Bacillus tequila MHP82 is deposited in the China Center for Type Culture with the deposit number CCTCC M2025739;

[0010] The Bacillus cereus MHP107 is deposited in the China Center for Type Culture, with the deposit number being CCTCC M2025740.

[0011] Preferably, the number of effective viable bacteria in the disease prevention and growth promotion composite microbial agent is ≥ 1×10 12 CFU / mL.

[0012] The present invention also provides a method for preparing the disease prevention and growth promotion composite microbial agent, comprising the following steps:

[0013] S1. After activation culture of Cellulobacterium finnii MHP3, Brevibacterium psychrotrophicum MHP56, Bacillus tequilae MHP82, and Bacillus cereus MHP107, select colonies from each strain and transfer them to LB broth. Incubate at 30-34°C and 180-200 rpm for 36-48 hours to obtain seed solution for each strain.

[0014] S2. Inoculate the seed culture medium with a mixture of Cellulosomyces finchii MHP3 seed solution, Brevibacterium frost-resistant MHP5 seed solution, Bacillus tequila MHP82 seed solution, and Bacillus cereus MHP107 seed solution at inoculum levels of 2.8-3.2%, 2.8-3.2%, 0.8-1.2%, and 1.8-2.2%, respectively. Cultivate the mixture at 30-34°C and 180-200 rpm for 36-48 hours to obtain a mixed bacterial seed solution.

[0015] S3. Inoculate the mixed bacterial seed solution into the fermentation culture medium at an inoculum rate of 1.5-2.5%, and ferment and culture at 30-34°C, 180-200 r / min, for 58-62 hours.

[0016] Preferably, the effective viable count of the seed solution of Cellulobacterium finschmidt MHP3, the seed solution of Brevibacterium frost-resistant MHP5, the seed solution of Bacillus tequila MHP82 and the seed solution of Bacillus cereus MHP107 is 10 8 CFU / mL.

[0017] Preferably, the seed culture medium and fermentation culture medium are LB culture medium.

[0018] The present invention also provides the use of the disease prevention and growth promotion composite microbial agent in at least one of the following:

[0019] (1) Application in inhibiting pathogens;

[0020] (2) Application in the prevention and control of plant diseases;

[0021] (3) Application in promoting the growth and / or increasing the yield of Chinese medicinal materials;

[0022] (4) Application in improving crop rhizosphere soil.

[0023] Preferably, the pathogenic bacteria include at least one of Fusarium solani, Fusarium avenae, Fusarium solani, Fusarium odoriferum, Fusarium oxysporum, Fusarium equisetum and Fusarium trilineata.

[0024] Preferably, the plant diseases include astragalus root rot, codonopsis root rot and / or angelica root rot.

[0025] Preferably, the Chinese medicinal materials include Astragalus, Codonopsis and / or Angelica.

[0026] Preferably, the disease-preventing and growth-promoting composite microbial agent is used by root irrigation, seed mixing or spraying.

[0027] By adopting the above technical solution, the present invention has the following beneficial effects: The composite microbial agent for disease prevention and growth promotion specifically for Chinese medicinal materials comprises Cellulobacterium finschmidtii MHP3, Brevibacterium frostii MHP56, Bacillus tequila MHP82, and Bacillus cereus MHP107. The composite microbial agent for disease prevention and growth promotion has a good preventive and therapeutic effect on root rot of Chinese medicinal materials, and can also promote the growth of Chinese medicinal materials and increase their yield. The composite microbial agent for disease prevention and growth promotion can also improve the rhizosphere soil of Chinese medicinal materials and reduce the salinity and alkali content in the soil. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 OD values ​​of mixed fermentation broths of different treatments with different inoculation amounts of each strain.

[0029] Figure 2 OD values ​​of fermentation broths under different fermentation conditions.

[0030] Biological Deposit Description

[0031] Cellulosimicrobium funkei MHP3 was deposited in the China Center for Type Culture, Wuhan University, on April 10, 2025, with the deposit number CCTCC M2025737;

[0032] Brevibacterium frigoritolerans MHP56 was deposited in the China Center for Type Culture, Wuhan University, on April 10, 2025, with the accession number CCTCC M2025738;

[0033] Bacillus tequilensis MHP82 was deposited in the China Center for Type Culture, Wuhan University, on April 10, 2025, with the accession number CCTCC M2025739.

[0034] Bacillus cereus MHP107 was deposited in the China Center for Type Culture, Wuhan University on April 10, 2025, with the deposit number CCTCC M 2025740. DETAILED DESCRIPTION

[0035] The present invention provides a disease prevention and growth-promoting composite microbial agent for Chinese medicinal materials, comprising Cellulobacterium finschmidtii MHP3, Brevibacterium frost-resistant MHP56, Bacillus tequilae MHP82 and Bacillus cereus MHP107;

[0036] The Cellulosomyces finneyii MHP3 is deposited in the China Center for Type Culture with the deposit number CCTCC M2025737;

[0037] The cold-resistant Brevibacterium MHP56 was deposited in the China Center for Type Culture with the deposit number CCTCC M2025738;

[0038] The Bacillus tequila MHP82 is deposited in the China Center for Type Culture with the deposit number CCTCC M2025739;

[0039] The Bacillus cereus MHP107 is deposited in the China Center for Type Culture, with the deposit number being CCTCC M2025740.

[0040] The number of effective living bacteria in the disease prevention and growth promotion composite microbial agent of the present invention is ≥1×10 12 CFU / mL.

[0041] The present invention also provides a method for preparing the disease prevention and growth promotion composite microbial agent, comprising the following steps:

[0042] S1. After activation culture of Cellulobacterium finnii MHP3, Brevibacterium psychrotrophicum MHP56, Bacillus tequilae MHP82, and Bacillus cereus MHP107, select colonies from each strain and transfer them to LB broth. Incubate at 30-34°C and 180-200 rpm for 36-48 hours to obtain seed solution for each strain.

[0043] S2. Inoculate the seed culture medium with a mixture of Cellulosomyces finchii MHP3 seed solution, Brevibacterium frost-resistant MHP5 seed solution, Bacillus tequila MHP82 seed solution, and Bacillus cereus MHP107 seed solution at inoculum levels of 2.8-3.2%, 2.8-3.2%, 0.8-1.2%, and 1.8-2.2%, respectively. Cultivate the mixture at 30-34°C and 180-200 rpm for 36-48 hours to obtain a mixed bacterial seed solution.

[0044] S3. Inoculate the mixed bacterial seed solution into the fermentation culture medium at an inoculum rate of 1.5-2.5%, and ferment and culture at 30-34°C, 180-200 r / min, for 58-62 hours.

[0045] The present invention first activates and cultures Bacillus cereus MHP3, Brevibacterium frost-resistant MHP56, Bacillus tequila MHP82, and Bacillus cereus MHP107, and then picks each colony and cultures it in LB culture medium to obtain a seed solution of each strain; the culture temperature is 30-34°C, preferably 31-33°C, and more preferably 32°C; the culture speed is 180-200 r / min, preferably 185-195 r / min, and more preferably 190 r / min; the culture time is 36-48 hours, and the effective viable bacteria count of each strain is 10 8 CFU / mL, seed solution was obtained;

[0046] The present invention inoculates a seed solution of Bacillus finnsii MHP3, a seed solution of Brevibacterium frostii MHP5, a seed solution of Bacillus tequila MHP82, and a seed solution of Bacillus cereus MHP107 into a seed culture solution to obtain a mixed bacterial seed solution; the inoculation amount of the seed solution of Bacillus finnsii MHP3 is 2.8-3.2%, preferably 2.9-3.1%, and more preferably 3%; the inoculation amount of the seed solution of Brevibacterium frostii MHP5 is 2.8-3.2%, preferably 2.9-3.1%, and more preferably 3%; the inoculation amount of the seed solution of Bacillus tequila MHP82 is 0 .8-1.2%, preferably 0.9-1.1%, more preferably 1%; the inoculation amount of the Bacillus cereus MHP107 seed liquid is 1.8-2.2%, preferably 1.9-2.1%, more preferably 2%; the culture temperature is 30-34°C, preferably 31-33°C, more preferably 32°C; the culture speed is 180-200r / min, preferably 185-195r / min, more preferably 190r / min; the culture time is 36-48h, preferably 40-44h, more preferably 42h, to obtain a mixed bacteria seed liquid;

[0047] The present invention inoculates the mixed seed liquid into the fermentation culture medium and cultivates the culture medium until the effective viable bacteria count is ≥1×10 12 CFU / mL, that is, the inoculation amount of the mixed seed liquid is 1.5-2.5%, preferably 1.8-2.2%, more preferably 2%; the culture temperature is 30-34°C, preferably 31-33°C, more preferably 32°C; the culture speed is 180-200r / min, preferably 185-195r / min, more preferably 190r / min; the culture time is 58-32h, and the culture is cultured until the effective viable count is ≥1×10 12 CFU / mL, the disease prevention and growth promotion composite microbial agent is obtained. The seed culture medium and fermentation culture medium are LB culture medium.

[0048] The present invention also provides the use of the disease prevention and growth promotion composite microbial agent in at least one of the following:

[0049] (1) Application in inhibiting pathogens;

[0050] (2) Application in the prevention and control of plant diseases;

[0051] (3) Application in promoting the growth and / or increasing the yield of Chinese medicinal materials;

[0052] (4) Application in improving crop rhizosphere soil.

[0053] The pathogens include at least one of Fusarium spp., Fusarium avenae, Fusarium solani, Fusarium odoriferum, Fusarium oxysporum, Fusarium equisetum and Fusarium trilineatum; the plant diseases include astragalus root rot, Codonopsis pilosula root rot and / or Angelica sinensis root rot; the Chinese medicinal materials include astragalus, Codonopsis pilosula and / or Angelica sinensis; the composite microbial agent is used by root irrigation, seed mixing or spraying.

[0054] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0055] Example 1. Performance determination of each strain

[0056] 1. Determination of the inhibitory rate of each strain against different Fusarium species

[0057] The inhibition rate of each functional strain was determined by referring to the method in "Screening of disease-preventing and growth-promoting bacteria for barley root rot and their control efficacy" (Xu Shiyang et al. 2022. Acta Microbiologica Sinica). The specific method is as follows:

[0058] Each functional strain was activated on LB plates for 24 hours and then inoculated into LB broth (50 mL / 150 mL Erlenmeyer flask) and incubated in a shaker at 30°C and 180 rpm for 48 hours. The fermentation broth was then centrifuged at 10,000 × g for 10 minutes. The supernatant was filtered through a 0.22 μm microporous filter and evenly plated onto a polydimethylsiloxane (PDA) plate. A PDA plate coated with an equal amount of sterile LB broth served as a control. Each test pathogen was then inoculated onto the center of the plate. Four replicates were used for each treatment. After incubation at 25°C in the dark for 5 days, the diameter of the pathogen was measured to calculate the inhibition rate. Inhibition rate (%) = (pathogen colony diameter on the control plate – pathogen colony diameter on the infected plate) / (control plate colony diameter – inoculated cake diameter) × 100%. The inhibition rates of each strain against different Fusarium species are shown in Table 1.

[0059] Table 1 Inhibition rate of each strain against different Fusarium species

[0060]

[0061] The results in Table 1 show that the inhibition rates of Cellulosomyces finneyii MHP3, Brevibacterium psychrotrophicum MHP56, Bacillus tequila MHP82, and Bacillus cereus MHP107 against the pathogens were 44.61% to 91.32%, indicating that each strain had a good inhibitory effect on different Fusarium species.

[0062] 2. Determination of the ability of each strain to solubilize phosphate, fix nitrogen, solubilize potassium, secrete IAA and produce siderophores

[0063] The molybdenum antimony colorimetric method was used to determine the amount of soluble organic phosphorus and soluble inorganic phosphorus in the strains. The specific method was as follows: after activating each strain on an LB plate, inoculate it into LB culture medium (liquid volume: 50mL / 150mL Erlenmeyer flask), culture it on a shaker at 30℃ and 180r / min for 36h~48h, and take 1mL of the solution with a concentration of 10 8 The fermentation broth of each strain with a CFU / mL was inoculated into the culture medium of Montina organophosphate and PKO inorganic phosphorus. After incubation at 30℃ and 180r / min in a shaking incubator for 10 days, the solubilized phosphorus content was determined by ultraviolet spectrophotometer. The culture medium of Montina organophosphate and PKO inorganic phosphorus inoculated with an equal amount of LB culture medium was used as the control. Each strain was replicated three times.

[0064] The Kjeldahl method was used to determine the nitrogen fixation capacity of the strains. The specific method was as follows: after activating each strain on an LB plate, inoculate it into LB culture medium (liquid volume: 50 mL / 150 mL Erlenmeyer flask), culture it on a shaker at 30°C and 180 rpm for 36 to 48 hours, and take 1 mL of the solution with a concentration of 10 8 The fermentation broth of each strain with a CFU / mL was inoculated into NFM culture medium, with three replicates for each strain. After culturing at 30°C and 180 rpm for 7 days, the nitrogen fixation capacity was determined using a Kjeldahl nitrogen analyzer. The NFM culture medium inoculated with an equal amount of LB culture medium was used as the control, with three replicates for each strain.

[0065] The potassium-dissolving capacity of the strains was determined by atomic absorption flame spectrophotometry. The specific method was as follows: after activation of each strain on LB plate, inoculate into LB culture medium (liquid volume: 50mL / 150mL Erlenmeyer flask), culture on a shaking table at 28°C and 180r / min for 36h-48h, take 1mL of the solution with a concentration of 10 8 The fermentation broth of each strain with a CFU / mL was inoculated into potassium feldspar culture medium, with three replicates for each strain. The potassium feldspar culture medium inoculated with an equal amount of LB culture medium was used as a control. After culturing in a shaking incubator at 28°C and 180 rpm for 7 days, the potassium solubilization capacity was determined by flame atomic absorption spectrometry.

[0066] The Salkowski colorimetric method was used to determine the IAA secretion ability of the strains. Specifically, after activating each strain on an LB plate, it was inoculated into 4 mL of LB culture medium containing 100 mg / L tryptophan and cultured on a shaker at 30°C and 180 rpm for 24 h. The bacterial liquid was then centrifuged at 16,500 rpm for 5 min. This was repeated three times for each strain. An equal amount of LB culture medium was inoculated as a control. The supernatant was aspirated and an equal amount of Salkowski colorimetric solution was added. After mixing, the culture was allowed to stand in the dark for 30 min. The content of IAA secreted was determined by a spectrophotometer.

[0067] The ability of the strains to produce siderophores was quantitatively determined using CAS siderophore detection solution. The specific method was as follows: each strain was activated on an LB plate and then transferred to 1 mL of CAS culture medium. The culture was shaken at 30°C and 180 rpm for 48 h. An equal amount of LB culture medium was inoculated into CAS culture medium as a control. After centrifugation at 10,000 × g for 10 min, the siderophore production content was determined using a microplate reader. This was repeated three times for each strain.

[0068] The phosphate solubilization, nitrogen fixation, potassium solubilization, IAA secretion and siderophore production abilities of each strain are shown in Table 2 .

[0069] Table 2 Phosphate solubilization, nitrogen fixation, potassium solubilization, IAA secretion and siderophore production abilities of each strain

[0070]

[0071] As shown in Table 2, Brevibacterium frostii MHP56 had the highest amounts of soluble organic and inorganic phosphorus, which were 322.06 g / L and 969.32 g / L, respectively; Bacillus cereus MHP107 had the highest nitrogen fixation capacity, which was 0.209 g / L; Cellulosus finnsii MHP3 had the highest potassium solubility, which was 86.22 mg / L; Bacillus cereus MHP107 had the highest ability to secrete IAA, which was 9.65 mg / L; Brevibacterium frostii MHP56 had the highest ability to produce siderophores, with an su value of 0.38.

[0072] From Table 1 and Table 2, we can see that MHP3 has the best antibacterial activity against Fusarium solani, Fusarium solani, Fusarium aromaticum, and Fusarium trilineatum, with an inhibition rate of over 90%. At the same time, its nitrogen fixation ability, potassium solubilization ability, and IAA secretion ability are all good, with a nitrogen fixation capacity of 0.602 g / L, a potassium solubilization capacity of 86.22 mg / L, and an IAA secretion capacity of 9.27 mg / L.

[0073] The cold-resistant Brevibacterium MHP56 has the best phosphorus solubility, dissolving 969.32 g / L of inorganic phosphorus and 322.06 g / L of organic phosphorus. It also has good siderophore production ability, with a su value of 0.38.

[0074] Bacillus tequilae MHP82 had the strongest antibacterial activity against Fusarium acuminate, with an inhibition rate of 85.36%. It also had good siderophore production ability with an su value of 0.30.

[0075] Bacillus cereus MHP107 has strong antibacterial activity against Fusarium oxysporum, Fusarium equisetum and Fusarium trilineatum, with an inhibition rate of over 90%. It also has the strongest ability to secrete IAA, which is 9.65 mg / L.

[0076] Example 2. Optimization of fermentation conditions and preparation of composite microbial agents

[0077] 1. Inoculation ratio of each strain

[0078] By L 16 (4 4 ) Orthogonal experiment, set up 1% , 2% , 3% , 4% of the total inoculum size gradient, a total of 16 treatments (A1 to A16). After each strain was activated on LB plate, it was inoculated into LB culture medium (liquid volume: 50mL / 150mL Erlenmeyer flask), and cultured at 30℃, 180r / min on a shaking table for 36h. The concentration of each fermentation culture medium was measured to reach 10 8 After the cells were grown in LB culture medium according to the treatment ratios in Table 3, the mixed fermentation culture was carried out at 30 ° C and 180 r / min for 48 h, and the OD was measured by spectrophotometry. 660 Value, the result is Figure 1 shown.

[0079] Table 3 Inoculation ratio of each strain L 16 (4 4 ) Orthogonal experimental design

[0080]

[0081]

[0082] Figure 1 The results show that A11 handles OD 660 The value was significantly the highest (P<0.05), which was 1.52, corresponding to the inoculation ratio of strains MHP3:MHP56:MHP82:MHP107 of 3:3:1:2.

[0083] 2. Optimization of fermentation conditions

[0084] Further adoption of L 16 (4 5 ) Orthogonal design was used to optimize the process by setting different inoculum sizes (2%, 4%, 6%, 8%), different temperatures (28℃, 30℃, 32℃, 34℃), different fermentation times (36h, 48h, 60h, 72h), different rotation speeds (160r / min, 180r / min, 200r / min, 220r / min) and different pH values ​​(6.0, 6.5, 7.0, 7.5). The specific steps were as follows: after activating each strain of the composite bacterial system on an LB culture plate, they were inoculated into LB culture medium (liquid volume: 50mL / 150mL Erlenmeyer flask), cultured in a shaking incubator at 30℃ and 180r / min for 36h, and the concentration of the fermentation culture medium was measured to determine whether it reached 10 8After the CFU / mL was measured, the mixed bacteria seed solution was inoculated into LB culture medium (liquid volume: 50mL / 150mL triangular flask) according to the optimal ratio of MHP3:MHP56:MHP82:MHP107 = 3:3:1:2, and cultured in a shaking incubator at 30°C and 180r / min for 48h to obtain the mixed bacteria seed solution. The mixed bacteria seed solution was inoculated into LB culture medium (liquid volume: 600mL / 1000mL triangular flask) according to the inoculum amount of each treatment in Table 4, and repeated 3 times for each treatment, and the pH was adjusted. The culture was carried out according to the corresponding speed, culture temperature and time. After the culture was completed, the OD was measured. 660 Value, the result is Figure 2 shown.

[0085] Table 4 Optimization of fermentation conditions of composite bacteria system 16 (4 5 )Orthogonal design

[0086]

[0087]

[0088] Figure 2 The results showed that B3 treatment OD 660 The value was significantly the highest (P<0.05), which was 1.82, and the corresponding fermentation conditions were inoculation amount 2%, pH value 7.0, fermentation temperature 32℃, rotation speed 200r / min, and fermentation time 60h.

[0089] 3. Preparation of disease prevention and growth promotion composite microbial agents

[0090] After activating strains MHP3, MHP56, MHP82, and MHP107 on LB plates for 24 h, they were inoculated into LB culture medium (liquid volume: 50 mL / 150 mL Erlenmeyer flask) and cultured in a shaking incubator at 32°C and 200 rpm for 36 to 48 h. The concentration of the fermentation medium was determined to be 10 8 CFU / mL, MHP3, MHP56, MHP82, and MHP107 were inoculated into LB culture medium (liquid volume: 50mL / 150mL Erlenmeyer flask) at an inoculum size of 3%, 3%, 1%, and 2%, respectively. After incubation at 32°C and 200r / min on a shaking table for 48h, a mixed bacterial seed solution was formed. The mixed bacterial seed solution was then inoculated into LB culture medium at an inoculum size of 2%, and fermented for 60h under the conditions of initial pH 7.0, fermentation temperature 32°C, and rotation speed 200r / min. The culture was stopped after culturing. The effective viable bacteria count in the culture medium was determined to be 10 12 CFU / mL, which is named "Tuweiwei No. 8". After storage at room temperature for 6 months, the number of viable bacteria was 8.92×10 9CFU / mL, in line with the requirements of the Agricultural Industry Standard of the People's Republic of China (NY / T 1113-2006).

[0091] Example 3. Effect of the disease prevention and growth promotion composite microbial agent on the prevention and growth promotion of Astragalus root rot

[0092] Field experiments were conducted to determine the disease prevention and growth promotion effects of the composite microbial agent for preventing and promoting growth of Astragalus root rot. The test site was a continuous cropland in Weiyuan County, Dingxi City, Gansu Province, where root rot was severely occurring. The composite microbial agent for preventing and promoting growth was prepared using the method in Example 2.

[0093] There are 2 groups of processing:

[0094] For the treatment group (DT), the root strips of Astragalus membranaceus were immersed in the disease prevention and growth promotion composite microbial agent of the present invention for 30 minutes using the root dipping method. After naturally drying in a cool place, the plants were planted using conventional field planting methods. During the planting process, an electric sprayer was used to evenly spray the disease prevention and growth promotion composite microbial agent onto the planting furrows and the surface of the root strips, and then covered with soil. The spraying rate was 10 L / mu.

[0095] Control group (CK1): The Astragalus root strips were soaked in clean water for 30 minutes using the root dipping method. After drying naturally in a cool place, they were planted using conventional field planting methods. During this period, an electric sprayer was used to evenly spray clean water to the planting furrow and the root strip surface, and then covered with soil. The spraying rate was 10L / mu. The row spacing was 10cm×30cm, and the area of ​​each group was 20m 2 , 3 repeats.

[0096] Planting began on April 10th and continued until harvest. Management procedures before and during planting were the same as those for other non-experimental plots. Seedling emergence and settling rates were calculated after seedling emergence, following the method described in "Effects of Different Basal Fertilizers and Their Application Rates on Astragalus Yield and Quality" (Zhao Yalan et al., 2018).

[0097] During the harvest period, the incidence and disease index of Astragalus root rot in each treatment were observed and recorded, as well as the efficacy of the application of the disease prevention and growth promotion composite microbial bacteria on Astragalus root rot. The survey method was based on the "Guidelines for Field Efficacy Tests of Pesticides". The root rot survey grading standard is:

[0098] Level 0: no disease;

[0099] Level 1: The lesion area accounts for 1% to 5% of the root surface area;

[0100] Level 2: The lesion area accounts for 6% to 10% of the root surface area;

[0101] Level 3: The lesion area accounts for 11% to 20% of the root surface area;

[0102] Level 4: The lesion area accounts for 21% to 40% of the root surface area;

[0103] Level 5: The lesion area accounts for 41% to 60% of the root surface area;

[0104] Level 6: The lesion area accounts for 61% to 80% of the root surface area;

[0105] Level 7: The lesion area accounts for more than 80% of the root surface area.

[0106] Incidence rate (%) = (incidence rate of control group - incidence rate of treatment group) / incidence rate of control group × 100%

[0107] Disease index = 100 × ∑ (number of diseased plants at each level × representative value of each level) / (total number of plants × representative value of the highest disease level)

[0108] Preventive effect (%) = (disease index of control group - disease index of treatment group) / disease index of control group × 100%

[0109] Urease activity in the soil of each treatment was determined using the phenol-sodium hypochlorite colorimetric method, catalase activity in the soil of each treatment was determined using the modified KMnO4 titration method, and phosphatase activity in the soil of each treatment was determined using the 0.5% disodium phenyl phosphate colorimetric method. Soil organic matter content in each treatment was determined using the potassium dichromate volumetric method-dilution heat method, ammonium nitrogen content in each treatment was determined using the HCl extraction-indophenol blue colorimetric method, soil pH was determined using a pH meter, and soil salinity was determined using a soil nutrient rapid tester. The results are shown in Tables 5 and 6.

[0110] Table 5 The preventive effect and growth-promoting effect of the composite microbial bacteria on Astragalus root rot

[0111]

[0112]

[0113] As shown in Table 5, the field incidence of Astragalus root rot was significantly reduced after application of the disease prevention and growth promotion composite microorganisms, which was 71.47% lower than the control (CK1). The root rot disease index was also significantly reduced, with a field prevention efficacy of up to 80.51% for Astragalus root rot. Simultaneously, Astragalus plant height, aboveground dry weight, underground dry weight, taproot length, and root diameter all increased significantly, with increases of 2.46%, 1.07 times, 39.57%, 15.89%, and 49.26% compared to the control, reaching 30.38 cm, 2.54 g, 1.94 g, 31.58 cm, and 7.09 cm, respectively. This indicates that the disease prevention and growth promotion composite microorganisms of the present invention have excellent prevention efficacy against Astragalus root rot and a significant growth-promoting effect.

[0114] Table 6 Effects of disease prevention and growth promotion composite microorganisms on enzyme activities and physicochemical properties of rhizosphere soil of Astragalus membranaceus

[0115] deal with Urease (mg / g) Catalase (mg / g) Phosphatase (mg / g) Organic matter (g / kg) Ammonium nitrogen (g / kg) pH Salt content (mg / kg) CK1 6.35±0.07b 1.07±1.05b 0.47±0.0051b 29.65±0.05b 1.23±0.39b 7.99±0.09a 0.25±0.0048a DT 12.88±0.03a 11.33±1.17a 0.61±0.0302a 40.28±0.11a 2.54±0.24a 7.04±0.11b 0.11±0.0029b

[0116] As shown in Table 6, after the application of the disease prevention and growth promotion composite microorganisms, the activities of enzymes such as urease, catalase, and phosphatase in the rhizosphere soil of Astragalus membranaceus in the treatment group (DT), as well as the nutrient contents such as organic matter and ammonium nitrogen, were significantly improved. Compared with the control group (CK1), the increases in the activities of urease, catalase, and phosphatase in the soil of the DT group were 1.02 times, 9.59 times, and 0.3 times, respectively, and the nutrient contents such as organic matter and ammonium nitrogen were increased by 35.85% and 1.07 times, respectively, indicating that the disease prevention and growth promotion composite microorganisms of the present invention have the effect of activating the rhizosphere soil of Astragalus membranaceus and improving soil nutrients. At the same time, the soil pH value and salt content in the DT group were significantly lower than those in CK1, respectively, by 0.95 and 0.14 mg / kg, indicating that the disease prevention and growth promotion composite microorganisms have an excellent effect on improving saline-alkali soil.

[0117] Example 4. Effect of the disease prevention and growth promotion composite microbial agent on the prevention and growth promotion of Codonopsis root rot

[0118] Selected Codonopsis root strips with healthy taproots, no lateral roots, and uniform size were washed and dried, then disinfected with 75% alcohol for 1 minute, 15% sodium hypochlorite solution for 20 minutes, and the surface rinsed three times with sterile water. Five even wounds were then made on the root epidermis using a sterile insect needle and set aside. Pot and field trials were conducted to comprehensively evaluate the preventive and growth-promoting effects of the disease-preventive and growth-promoting composite microbial strain on Codonopsis root rot. The disease-preventive and growth-promoting composite microbial strain was prepared using the method described in Example 2.

[0119] 1. Potted plant experiment:

[0120] There are two treatment groups in the pot experiment.

[0121] CK-A1, control group, 2×10 6 The roots were irrigated with a mixed spore suspension of Fusarium oxysporum, F. solani, F. tricinctum, and F. equiseti at a concentration of CFU / mL (20 mL / pot);

[0122] DS1, 2×10 6 The roots were irrigated with a mixed spore suspension of Fusarium oxysporum, Fusarium solani, Fusarium tricinctum, and Fusarium equiseti at a concentration of CFU / mL (20 mL / pot) and a disease prevention and growth promotion composite microbial fungus (20 mL / pot); each treatment was repeated in 3 pots, and 4 plants were planted in each pot.

[0123] The above treatments were all carried out 2 days after the root strips were planted. After 10 days of cultivation, the root rot disease index of the Codonopsis pilosula plants under each treatment was counted, and the control effect of the composite microbial bacteria for disease prevention and growth promotion was calculated.

[0124] The grading standard for Codonopsis root rot is: Grade 0, healthy roots, no lesions;

[0125] Level 1: There are a few light brown spots on the roots, and the diseased area accounts for 1% to 15%;

[0126] Level 3: Part of the roots turn brown, and the diseased area accounts for 16% to 30%; Level 5: Most of the roots turn brown, and the diseased area accounts for 31% to 60%;

[0127] Level 7: The roots become soft or even rotten, and the root area affected accounts for more than 60%.

[0128] Example 3 of the calculation method of disease index and prevention effect.

[0129] 2. Field experiment:

[0130] The field experiment was conducted in a continuous cropping plot with severe root rot in Weiyuan County, Dingxi City, Gansu Province. Two treatments were set up:

[0131] CK-A2: soak the root strips of Codonopsis pilosula in clean water for 30 minutes using the root dipping method. After drying naturally in a cool place, the conventional field planting method is used for planting. During this period, an electric sprayer is used to evenly spray clean water to the planting ditch and the surface of the root strips, and then cover with soil. The spraying rate is 10L / mu.

[0132] DS2: The root strips of Codonopsis pilosula were immersed in the disease prevention and growth promotion composite microorganisms of the present invention for 30 minutes by the root dipping method. After natural drying in a cool place, the conventional field planting method was adopted. During the planting process, the disease prevention and growth promotion composite microorganisms were evenly sprayed on the planting ditch and the surface of the root strips by an electric sprayer, and then covered with soil. The spraying rate was 10L / mu; the row spacing was 10cm×30cm, and the area of ​​each group was 30m 2 , 3 repeats.

[0133] Root cuttings of roughly uniform growth were selected and planted on April 2nd, maintaining conventional management until harvest. At harvest, the root rot disease index and the efficacy of the microbial control and growth-promoting composite were calculated for each treatment, along with plant survival rate and underground dry weight. Disease grading criteria were the same as those used in the potted Codonopsis pilosula experiment. The results are shown in Table 7.

[0134] Table 7 The preventive effect and growth-promoting effect of the composite microbial bacteria on Codonopsis root rot

[0135]

[0136] As shown in Table 7, after application of the disease prevention and growth promotion composite microbial bacteria (DS1 / DS2), the disease index of Codonopsis root rot was significantly reduced in the pot test and field test, and the pot and field control efficacy against Codonopsis root rot was 86.47% and 86.61%, respectively. The field survival rate and underground dry weight of Codonopsis plants were significantly increased, increasing by 97.97% and 26.17%, respectively, compared with the control group CK-A2. This shows that the disease prevention and growth promotion composite microbial bacteria of the present invention have excellent control efficacy against Codonopsis root rot and have an outstanding growth-promoting effect on Codonopsis.

[0137] Example 5. Effect of the composite microbial agent on the prevention and growth promotion of Angelica root rot

[0138] Field experiments were conducted to determine the disease prevention and growth promotion effects of the composite microorganisms for astragalus root rot. The test site was a continuous cropping plot where root rot was severely occurring in Weiyuan County, Dingxi City, Gansu Province. The composite microorganisms for disease prevention and growth promotion were prepared using the method in Example 2.

[0139] The experiment set up 2 groups of treatments:

[0140] For the treatment group (DG), the root strips of angelica sinensis were immersed in the disease prevention and growth promotion composite microorganisms of the present invention for 30 minutes using the root dipping method. After naturally drying in a cool place, they were planted using conventional field planting methods. During this period, an electric sprayer was used to evenly spray the disease prevention and growth promotion composite microorganisms onto the planting furrows and the surface of the root strips, and then covered with soil. The spraying rate was 10 L / mu.

[0141] Control group (CKG): Astragalus root strips were soaked in clean water for 30 minutes using the root dipping method. After drying naturally in a cool place, they were planted using conventional field planting methods. During this period, an electric sprayer was used to evenly spray clean water to the planting furrow and the surface of the root strips, and then covered with soil. The spraying rate was 10L / mu. The row spacing was 10cm×30cm, and the area of ​​each group was 18m. 2 , 3 replicates, routine management before sowing and during planting.

[0142] Refer to the disease grading standard of "Effects of Bacillus subtilis B1 and B2 strains on the prevention and growth promotion of angelica and astragalus" (Xin Zhongyao et al. 2008):

[0143] Level 1 - healthy, no illness;

[0144] Level 2 - the lesion area accounts for 1% to 5% of the root surface area;

[0145] Level 3 - lesion area accounts for 6% to 10% of the root surface area;

[0146] Level 4 - lesion area accounts for 11% to 20% of the root surface area;

[0147] Level 5 - lesion area accounts for 21% to 40% of the root surface area;

[0148] Level 6: The lesion area accounts for 41% to 60% of the root surface area;

[0149] Level 7 - lesion area accounts for 61% to 80% of the root surface area;

[0150] Level 8 - The lesion area accounts for more than 80% of the root surface area.

[0151] During the maturity period of Angelica sinensis, the root rot disease index of Angelica sinensis in different treatments was statistically recorded, and the disease prevention and growth promotion composite microbial bacteria control efficacy was calculated. The calculation method was the same as in Example 3. A five-point sampling method was used to randomly collect plant and rhizosphere soil samples in each treatment to measure the diameter of the Angelica sinensis rhizome, root length, underground dry weight and yield in different treatments. Referring to the method of Lu Rukun (1999), the potassium dichromate oxidation-external heating method was used to determine the soil organic matter content, the alkaline hydrolysis diffusion method was used to determine the soil alkaline hydrolyzable nitrogen content, the sodium bicarbonate extraction-molybdenum antimony colorimetric method was used to determine the soil available phosphorus content, the ammonium acetate extraction-flame photometry method was used to determine the soil available potassium content, and the acidity meter method was used to determine the soil pH value. The results are shown in Tables 8 and 9.

[0152] Table 8 Disease prevention and growth promotion effect of composite microorganisms on Angelica root rot and its growth promotion effect

[0153] deal with Disease index Prevention effect (%) Reed head diameter (mm) Root length (cm) Underground dry weight (g) <![CDATA[Yield (kg / hm 2 ) <!-- 10 -->]]> CKG 48.58±1.03a — 16.65±0.05b 21.24±0.39b 7.12±0.28b 1127.25±5.48b DG 12.32±0.67b 74.64±0.85 22.38±0.07a 25.44±0.24a 10.24±0.25a 1331.75±5.16a

[0154] As shown in Table 8, the disease index of angelica root rot was significantly reduced after application of the disease prevention and growth promotion composite microbial bacteria, achieving a 74.64% control efficacy against angelica root rot. Furthermore, the rhizome diameter, root length, underground dry weight, and yield of angelica were significantly increased, with increases of 40.42%, 19.77%, 43.82%, and 18.14%, respectively. This demonstrates that the disease prevention and growth promotion composite microbial bacteria of the present invention have excellent control efficacy against angelica root rot and a significant growth-promoting and yield-increasing effect on angelica.

[0155] Table 9 Effects of disease prevention and growth promotion composite microorganisms on the physical and chemical properties of Angelica rhizosphere soil

[0156] deal with Organic matter (g / kg) Alkaline nitrogen (mg / kg) Available phosphorus (mg / kg) Fast-acting potassium (mg / kg) pH CKG 18.72±0.32a 74.64±0.78b 84.02±1.31b 184.24±1.22b 7.86±0.24a DG 25.82±0.35b 128.76±0.58a 185.42±0.97a 267.16±1.64a 7.11±0.20b

[0157] As shown in Table 5, after the application of the disease prevention and growth promotion composite microbial bacteria in the treatment group (DG), the organic matter, alkaline nitrogen, available phosphorus, and available potassium contents in the rhizosphere soil of Angelica sinensis were significantly increased compared with the control group (CKG), with increases of 37.93%, 72.51%, 1.20 times, and 45.01%, respectively, reaching 25.82 g / kg, 128.76 mg / kg, 185.42 mg / kg, and 267.16 mg / kg, respectively. This indicates that the disease prevention and growth promotion composite microbial bacteria of the present invention have the effect of activating the nutrients in the rhizosphere soil of Angelica sinensis, and the effect is outstanding. At the same time, the pH value of the rhizosphere soil of Angelica sinensis in the treatment group (DG) was significantly lower than that in the control group (CKG), with a decrease of 0.75 to 7.11, indicating that the disease prevention and growth promotion composite microbial bacteria are also outstanding in alkali resistance and improving alkaline soil.

[0158] It can be seen from the above embodiments that the present invention provides a disease-preventing and growth-promoting composite microbial agent specifically for Chinese medicinal materials, and its preparation method and application. The disease-preventing and growth-promoting composite microbial agent has a good preventive and therapeutic effect on root rot of Chinese medicinal materials.

[0159] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A disease prevention and growth-promoting composite microbial agent for Chinese medicinal materials, characterized in that: These include Cellulobacterium finschmidtii MHP3, Brevibacterium psychrotrophicum MHP56, Bacillus tequilae MHP82, and Bacillus cereus MHP107; The Cellulosomyces finneyii MHP3 is deposited in the China Center for Type Culture with the deposit number CCTCC M 2025737; The Brevibacterium psychrophilum MHP56 was deposited in the China Center for Type Culture with the deposit number CCTCC M 2025738; The Bacillus tequila MHP82 is deposited in the China Center for Type Culture with the deposit number CCTCC M2025739; The Bacillus cereus MHP107 is deposited in the China Center for Type Culture, with the deposit number being CCTCC M 2025740.

2. The disease prevention and growth promotion composite microbial agent according to claim 1, characterized in that The number of effective living bacteria in the disease prevention and growth promotion composite microbial agent is ≥1×10 12 CFU / mL.

3. The method for preparing the disease prevention and growth promotion composite microbial agent according to claim 1 or 2, characterized in that: The following steps are involved: S1. After activation culture of Cellulobacterium finnii MHP3, Brevibacterium psychrotrophicum MHP56, Bacillus tequilae MHP82, and Bacillus cereus MHP107, select colonies from each strain and transfer them to LB broth. Incubate at 30-34°C and 180-200 rpm for 36-48 hours to obtain seed solution for each strain. S2. Inoculate the seed culture medium with a mixture of Cellulosomyces finchii MHP3 seed solution, Brevibacterium frost-resistant MHP5 seed solution, Bacillus tequila MHP82 seed solution, and Bacillus cereus MHP107 seed solution at inoculum levels of 2.8-3.2%, 2.8-3.2%, 0.8-1.2%, and 1.8-2.2%, respectively. Cultivate the mixture at 30-34°C and 180-200 rpm for 36-48 hours to obtain a mixed bacterial seed solution. S3. Inoculate the mixed bacterial seed solution into the fermentation culture medium at an inoculum rate of 1.5-2.5%, and ferment and culture at 30-34°C, 180-200 r / min, for 58-62 hours.

4. The preparation method according to claim 3, characterized in that The effective viable count of the seed liquid of Cellulobacterium finschmidt MHP3, the seed liquid of Brevibacterium frost-resistant MHP5, the seed liquid of Bacillus tequila MHP82 and the seed liquid of Bacillus cereus MHP107 is 10 8 CFU / mL.

5. The preparation method according to claim 3, characterized in that The seed culture medium and fermentation culture medium are LB culture medium.

6. Use of the disease prevention and growth promotion composite microbial agent according to claim 1 or 2 in at least one of the following: (1) Application in inhibiting pathogens; (2) Application in the prevention and control of plant diseases; (3) Application in promoting the growth and / or increasing the yield of Chinese medicinal materials; (4) Application in improving crop rhizosphere soil.

7. The use according to claim 6, characterized in that The pathogenic bacteria include at least one of Fusarium acuminate, Fusarium avenae, Fusarium solani, Fusarium odoratum, Fusarium oxysporum, Fusarium equisetum and Fusarium trilineatum.

8. The use according to claim 6, characterized in that The plant diseases include astragalus root rot, codonopsis root rot and / or angelica root rot.

9. The use according to claim 6, characterized in that The Chinese medicinal materials include astragalus, codonopsis and / or angelica.

10. The use according to any one of claims 6 to 9, characterized in that: The disease prevention and growth promotion composite microbial agent is used in the form of root irrigation, seed mixing or spraying.

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