A microbial agent for preventing disease and promoting growth, and its preparation method and application

Abstract

The present invention discloses a microbial agent for preventing diseases and promoting growth, as well as a preparation method and application thereof, and belongs to the field of microbial technology. The microbial agent of the present invention includes Bacillus argyi, Pseudomonas guguanyi, and Actinomycetes aurantii. Bacillus argyi, Pseudomonas guguanyi, and Actinomycetes aurantii are screened to form functional microorganisms. After the three bacterial liquids are mixed in equal proportions, a variety of antibacterial substances are produced, which synergistically inhibit the growth of pathogens and have good antagonistic effects on soybean root rot, soybean anthracnose, and soybean gray spot. The microbial agent of the present invention can promote soybean growth and increase soybean yield, while reducing the probability of soybean root rot, soybean anthracnose, and soybean gray spot, solving the problems of low soybean yield and high incidence of diseases in some soybeans, and the ingredients are safe and environmentally friendly, non-toxic to soybeans, and have a positive effect on reducing environmental pollution and pesticide residues.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and in particular relates to a disease-preventing and growth-promoting microbial agent, a preparation method and an application thereof. Background Art

[0002] Soybeans are high in protein, one of the highest protein sources among plant foods. The amino acid composition of their protein closely matches human needs, making them easy to digest and absorb. They are also rich in unsaturated fatty acids, calcium, potassium, and vitamin E, offering numerous health benefits, including lowering cholesterol, promoting muscle growth, and boosting immunity. Soybeans can be eaten directly or processed into soy products like tofu, soy milk, and soy sauce, and can also be used to make meat substitutes. Soybeans also hold significant economic value in agriculture. They can coexist with nitrogen-fixing bacteria in their roots, converting atmospheric nitrogen into a form that plants can use, reducing the need for chemical fertilizers and improving soil health and structure. Soybeans are also widely used in animal feed, particularly soybean meal, a byproduct of oil extraction, which is a key ingredient in poultry and pig feed.

[0003] Heilongjiang Province is my country's base for the production and supply of high-quality soybeans. However, soybeans are often threatened by diseases during their growth, which seriously affects their yield and quality. Root rot is one of the main diseases in soybean production. It is a disease that can occur throughout the entire growth period of soybeans and is mainly caused by Fusarium fungi. Root rot can cause seed rot, seedling death, and browning and rot at the base of the stems of mature plants, seriously affecting the growth and yield of soybeans. In addition, soybean anthracnose and gray spot are also common, which can also cause the death of soybean plants, affecting soybean growth and yield. In addition, soybean diseases not only affect the crops themselves, but may also cause certain damage to the ecological environment.

[0004] For example, Chinese patent CN113046260A discloses a microbial mixture for promoting soybean growth and its application, wherein the microbial mixture comprises a fermentation broth of Azospirillum brasiliensis and Hydrogenophilus parviflora. The microbial mixture of the present invention can stimulate plant cells to secrete various growth hormones, strongly promoting root growth, rooting, root strengthening, and capillary roots, which increase in length or width, thereby enhancing the crop's ability to absorb water and fertilizer, and making the crop have effects such as long and thick stems and resistance to lodging, thereby increasing crop yield. Chinese patent CN102925387A discloses a simple Bacillus strain capable of inducing soybean resistance to soybean cyst nematodes and its application. After the strain is cultured by conventional liquid fermentation, the metabolites of the strain can be used to develop a seed treatment agent for treating soybean seeds. The treated soybean seeds can induce soybeans to produce resistance to soybean cyst nematodes. The simple Bacillus (Bacillus simplex) Sneb545 metabolites of the present invention can induce soybeans to produce significant resistance to the first generation of cyst nematodes in the seedling stage after treating soybean seeds.

[0005] However, the microbial agents currently studied in soybean research have single functions, large usage amounts, and limited effects on soybean growth promotion, making them difficult to effectively apply and promote in agricultural production. Summary of the Invention

[0006] The present invention aims to solve the problems existing in the prior art and provides a disease-preventing and growth-promoting microbial agent, which effectively prevents and controls soybean root rot, anthracnose, and gray spot, while effectively increasing soybean yield.

[0007] In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

[0008] A disease-preventing and growth-promoting microbial agent comprises Bacillus aryabhattai, Pseudomonas guguanensis, and Actinoplanes atraurantiacus; the Bacillus aryabhattai has a deposit number of CGMCC No. 1.15821, was purchased from the China General Microorganism Collection Center, and the original deposit date is September 10, 2016; the Pseudomonas guguanensis has a deposit number of CGMCC No. 1.15627, was purchased from the China General Microorganism Collection Center, and the original deposit date is February 25, 2015; and the Actinoplanes atraurantiacus has a deposit number of CGMCC No. 4.6857, was purchased from the China General Microorganism Collection Center, and the original deposit date is January 15, 2011. The three strains used in the present invention can be purchased publicly by searching the strain catalog of the China General Microorganism Collection Center, so there is no need to conduct repeated biological preservation.

[0009] Furthermore, the microbial agent is a liquid agent.

[0010] A method for preparing a microbial agent for preventing disease and promoting growth comprises the following steps:

[0011] Preparation of Bacillus arguta and Pseudomonas guguanis bacterial suspension: Thaw Bacillus arguta and Pseudomonas guguanis and activate them in LB solid medium. Then pick a single colony and inoculate it into LB liquid medium. Cultivate at 30℃ and 180r / min until OD 600 =0.6 to obtain seed solution, and the seed solution was evenly inoculated into 200 mL LB liquid medium at 1% and cultured at 30°C and 180 r / min. The bacterial colony activity was detected by the dilution plate method, and the number of colonies reached 1×10 8 cfu·mL -1 After the culture was terminated, the cells were washed three times with sterile PBS buffer solution and then resuspended to obtain two bacterial suspensions;

[0012] Preparation of S2 dark orange swimming actinomycete fermentation broth: The dark orange swimming actinomycetes were thawed and activated in ISP-2 solid medium. Then, a single colony was picked and inoculated into ISP-2 liquid medium and cultured at 28°C and 200 rpm until OD 600 =0.6 to obtain seed solution, and the seed solution was evenly inoculated into 200 mL ISP-2 liquid culture medium at 1% and cultured at 28°C and 200 r / min. The bacterial colony activity was detected by the dilution spread plate method, and the number of colonies reached 1×10 8 cfu·mL -1 Finally, the culture was terminated to obtain a dark orange actinomycete fermentation liquid;

[0013] S3 Preparation of microbial agent: The Bacillus aeruginosa suspension and the Pseudomonas guguanyi suspension in step S1 and the dark orange actinomycetes fermentation broth in step S2 are mixed in a volume ratio of 1:1:1 to obtain the final product microbial agent.

[0014] Application of a microbial agent in soybean disease prevention and growth promotion.

[0015] Furthermore, the diseases are soybean root rot, soybean anthracnose, and soybean gray spot.

[0016] Furthermore, the microbial agent is used to promote the growth of soybeans.

[0017] The method for using the disease-preventing and growth-promoting microbial agent of the present invention is as follows: starting from the soybean seedling stage, the microbial agent is diluted 500 times and applied at a rate of 40 L / mu, and then the roots are wetted or the leaves are sprayed.

[0018] Bacillus argonieffii can induce systemic resistance in plants, improving their resistance to a variety of pathogens. It also synthesizes plant hormones such as indoleacetic acid (IAA) and cytokinin (CTK), which directly promote root development, increase photosynthesis efficiency, and enhance plant resistance to environmental stress. Furthermore, through its rapid reproduction and secretion of lysozyme, it not only improves the soil microbial environment but also effectively eliminates harmful pathogens such as fungi and bacteria.

[0019] Pseudomonas gujianensis produces a variety of plant hormones, such as indoleacetic acid (IAA), gibberellins (GA), and cytokinins. These hormones can stimulate root development, increasing soybean biomass and yield. Furthermore, Pseudomonas gujianensis produces antibiotics and other antimicrobial substances that can inhibit or kill pathogenic fungi and bacteria in the soil, reducing the incidence of soybean diseases. Furthermore, Pseudomonas gujianensis can activate a plant's systemic resistance response. When one part of the plant is attacked by a pathogen, other parts also respond with a defensive response, thereby improving the plant's overall disease resistance.

[0020] Dark orange motile actinomycetes and their metabolites possess significant antimicrobial activity, effectively inhibiting the growth of a variety of plant pathogens. They also produce plant hormones such as indoleacetic acid (IAA) and gibberellins, which directly stimulate cell division and tissue formation in plants. Furthermore, actinomycetes can improve soil nutrient availability by dissolving phosphorus and fixing nitrogen, thereby enhancing plant nutrient absorption.

[0021] Beneficial effects

[0022] The present invention screens Bacillus agglomerans, Pseudomonas guineensis, and Actinomyces aurantii to form functional microorganisms. After the three bacterial liquids are mixed in equal proportions, multiple antibacterial substances are produced to synergistically inhibit the growth of pathogens, and have good antagonistic effects on soybean root rot, soybean anthracnose, and soybean gray spot.

[0023] The microbial agent of the present invention, when used in small amounts, can promote soybean growth and increase soybean yield, while reducing the incidence of soybean root rot, soybean anthracnose, and soybean gray spot, solving the problems of low soybean yield and high incidence of diseases. The ingredients are safe and environmentally friendly, non-toxic to soybeans, and have a positive effect on reducing environmental pollution and pesticide residues. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 These are the inhibition graphs of Example 1 of the present invention on three pathogens; Note: A is the inhibition graph of the microbial agent on root rot pathogens, B is the inhibition graph of the microbial agent on anthrax pathogens, and C is the inhibition graph of the microbial agent on gray spot pathogens. DETAILED DESCRIPTION

[0025] The technical solution of the present invention will be further described below with reference to specific embodiments, but is not limited thereto.

[0026] Example 1

[0027] A microbial agent for preventing diseases and promoting growth, comprising Bacillus agglomerans, Pseudomonas guguanyi, and Actinomyces aurantii.

[0028] A method for preparing a microbial agent for preventing disease and promoting growth comprises the following steps:

[0029] Preparation of Bacillus arguta and Pseudomonas guguanis bacterial suspension: Thaw Bacillus arguta and Pseudomonas guguanis and activate them in LB solid medium. Then pick a single colony and inoculate it into LB liquid medium. Cultivate at 30℃ and 180r / min until OD 600 =0.6 to obtain seed solution, and the seed solution was evenly inoculated into 200 mL LB liquid medium at 1% and cultured at 30°C and 180 r / min. The bacterial colony activity was detected by the dilution plate method, and the number of colonies reached 1×10 8 cfu·mL-1 After the culture was terminated, the cells were washed three times with sterile PBS buffer solution and then resuspended to obtain two bacterial suspensions;

[0030] Preparation of S2 dark orange swimming actinomycete fermentation broth: The dark orange swimming actinomycetes were thawed and activated in ISP-2 solid medium. Then, a single colony was picked and inoculated into ISP-2 liquid medium and cultured at 28°C and 200 rpm until OD 600 =0.6 to obtain seed solution, and the seed solution was evenly inoculated into 200 mL ISP-2 liquid culture medium at 1% and cultured at 28°C and 200 r / min. The bacterial colony activity was detected by the dilution spread plate method, and the number of colonies reached 1×10 8 cfu·mL -1 Finally, the culture was terminated to obtain a dark orange actinomycete fermentation liquid;

[0031] S3 Preparation of microbial agent: The Bacillus aeruginosa suspension and the Pseudomonas guguanyi suspension in step S1 and the dark orange actinomycetes fermentation broth in step S2 are mixed in a volume ratio of 1:1:1 to obtain the final product microbial agent.

[0032] Comparative Example 1

[0033] A microbial agent for preventing diseases and promoting growth, comprising Bacillus agglomerans, Pseudomonas guguanyi, and Actinomyces aurantii.

[0034] A method for preparing a microbial agent for preventing disease and promoting growth comprises the following steps:

[0035] Preparation of Bacillus arguta and Pseudomonas guguanis bacterial suspension: Thaw Bacillus arguta and Pseudomonas guguanis and activate them in LB solid medium. Then pick a single colony and inoculate it into LB liquid medium. Cultivate at 30℃ and 180r / min until OD 600 =0.6 to obtain seed solution, and the seed solution was evenly inoculated into 200 mL LB liquid medium at 1% and cultured at 30°C and 180 r / min. The bacterial colony activity was detected by the dilution plate method, and the number of colonies reached 1×10 8 cfu·mL -1 After the culture was terminated, the cells were washed three times with sterile PBS buffer solution and then resuspended to obtain two bacterial suspensions;

[0036] Preparation of S2 dark orange swimming actinomycete fermentation broth: The dark orange swimming actinomycetes were thawed and activated in ISP-2 solid medium. Then, a single colony was picked and inoculated into ISP-2 liquid medium and cultured at 28°C and 200 rpm until OD 600=0.6 to obtain seed solution, and the seed solution was evenly inoculated into 200 mL ISP-2 liquid culture medium at 1% and cultured at 28°C and 200 r / min. The bacterial colony activity was detected by the dilution spread plate method, and the number of colonies reached 1×10 8 cfu·mL -1 Finally, the culture was terminated to obtain a dark orange actinomycete fermentation liquid;

[0037] S3 Preparation of microbial agent: The Bacillus aeruginosa suspension and the Pseudomonas guguanyi suspension in step S1 and the dark orange actinomycetes fermentation broth in step S2 are mixed in a volume ratio of 1:1:2 to obtain the final product microbial agent.

[0038] Compared with Example 1, this comparative example is the same as Example 1, except that the volume ratio of Bacillus aeruginosa suspension, Pseudomonas guguanycin suspension and dark orange actinomycetes fermentation broth is changed to 1:1:2. The other raw materials and steps are the same as Example 1.

[0039] Comparative Example 2

[0040] A microbial agent for preventing diseases and promoting growth, comprising Bacillus agglomerans, Pseudomonas guguanyi, and Actinomyces aurantii.

[0041] A method for preparing a microbial agent for preventing disease and promoting growth comprises the following steps:

[0042] Preparation of Bacillus arguta and Pseudomonas guguanis bacterial suspension: Thaw Bacillus arguta and Pseudomonas guguanis and activate them in LB solid medium. Then pick a single colony and inoculate it into LB liquid medium. Cultivate at 30℃ and 180r / min until OD 600 =0.6 to obtain seed solution, and the seed solution was evenly inoculated into 200 mL LB liquid medium at 1% and cultured at 30°C and 180 r / min. The bacterial colony activity was detected by the dilution plate method, and the number of colonies reached 1×10 8 cfu·mL -1 After the culture was terminated, the cells were washed three times with sterile PBS buffer solution and then resuspended to obtain two bacterial suspensions;

[0043] Preparation of S2 dark orange swimming actinomycete fermentation broth: The dark orange swimming actinomycetes were thawed and activated in ISP-2 solid medium. Then, a single colony was picked and inoculated into ISP-2 liquid medium and cultured at 28°C and 200 rpm until OD 600 =0.6 to obtain seed solution, and the seed solution was evenly inoculated into 200 mL ISP-2 liquid culture medium at 1% and cultured at 28°C and 200 r / min. The bacterial colony activity was detected by the dilution spread plate method, and the number of colonies reached 1×10 8 cfu·mL -1Finally, the culture was terminated to obtain a dark orange actinomycete fermentation liquid;

[0044] S3 Preparation of microbial agent: The Bacillus aeruginosa suspension and the Pseudomonas guguanyi suspension in step S1 and the dark orange actinomycetes fermentation broth in step S2 are mixed in a volume ratio of 1:2:1 to obtain the final product microbial agent.

[0045] Compared with Example 1, this comparative example is the same as Example 1, except that the volume ratio of Bacillus aeruginosa suspension, Pseudomonas guguanycin suspension and dark orange actinomycetes fermentation broth is changed to 1:2:1, and the other raw materials and steps are the same as Example 1.

[0046] Comparative Example 3

[0047] A microbial agent for preventing diseases and promoting growth, comprising Bacillus agglomerans, Pseudomonas guguanyi, and Actinomyces aurantii.

[0048] A method for preparing a microbial agent for preventing disease and promoting growth comprises the following steps:

[0049] Preparation of Bacillus arguta and Pseudomonas guguanis bacterial suspension: Thaw Bacillus arguta and Pseudomonas guguanis and activate them in LB solid medium. Then pick a single colony and inoculate it into LB liquid medium. Cultivate at 30℃ and 180r / min until OD 600 =0.6 to obtain seed solution, and the seed solution was evenly inoculated into 200 mL LB liquid medium at 1% and cultured at 30°C and 180 r / min. The bacterial colony activity was detected by the dilution plate method, and the number of colonies reached 1×10 8 cfu·mL -1 After the culture was terminated, the cells were washed three times with sterile PBS buffer solution and then resuspended to obtain two bacterial suspensions;

[0050] Preparation of S2 dark orange swimming actinomycete fermentation broth: The dark orange swimming actinomycetes were thawed and activated in ISP-2 solid medium. Then, a single colony was picked and inoculated into ISP-2 liquid medium and cultured at 28°C and 200 rpm until OD 600 =0.6 to obtain seed solution, and the seed solution was evenly inoculated into 200 mL ISP-2 liquid culture medium at 1% and cultured at 28°C and 200 r / min. The bacterial colony activity was detected by the dilution spread plate method, and the number of colonies reached 1×10 8 cfu·mL -1 Finally, the culture was terminated to obtain a dark orange actinomycete fermentation liquid;

[0051] S3 Preparation of microbial agent: The Bacillus aeruginosa suspension and the Pseudomonas guguanyi suspension in step S1 and the dark orange actinomycetes fermentation broth in step S2 are mixed in a volume ratio of 2:1:1 to obtain the final product microbial agent.

[0052] Compared with Example 1, this comparative example is the same as Example 1, except that the volume ratio of Bacillus aeruginosa suspension, Pseudomonas guguanycin suspension and dark orange actinomycetes fermentation broth is changed to 2:1:1. The other raw materials and steps are the same as Example 1.

[0053] Comparative Example 4

[0054] Compared with Example 1, this comparative example only used the Bacillus aeruginosa suspension and the Pseudomonas guguanis suspension, with the volume ratio of the two being 1:1, and the rest were the same as Example 1.

[0055] Comparative Example 5

[0056] Compared with Example 1, this comparative example used only the Bacillus aeruginosa suspension and the dark orange actinomycetes fermentation broth, with the volume ratio of the two being 1:1, and the rest were the same as Example 1.

[0057] Comparative Example 6

[0058] Compared with Example 1, this comparative example only used the Pseudomonas glycyrrhizic acid suspension and the Pseudomonas glycyrrhizic acid suspension, with a volume ratio of 1:1, and the rest were the same as Example 1.

[0059] Comparative Example 7

[0060] Compared with Example 1, this comparative example only used the Bacillus aeruginosa suspension, and the rest were the same as Example 1.

[0061] Comparative Example 8

[0062] Compared with Example 1, this comparative example only used the Pseudomonas guguanylicus suspension, and the rest were the same as Example 1.

[0063] Comparative Example 9

[0064] Compared with Example 1, this comparative example used only the dark orange actinomycete fermentation broth, and the rest were the same as Example 1.

[0065] Performance Testing

[0066] Determination of the ability to inhibit pathogens:

[0067] The antagonistic effect of microbial agents on soybean root rot, soybean anthracnose and soybean gray spot was determined by the plate confrontation culture method. Soybean root rot, anthracnose and gray spot cakes with a diameter of 5 mm were inoculated in the center of the culture dish respectively, and a 5 mm sterilized filter paper was attached about 2 cm away from the center of the pathogen. 2.5 uL of the microbial agent of Example 1 of the present invention was dripped on the filter paper. The treatment in which only pathogen cakes were planted but not the microbial agent of the present invention was used as the control, and each treatment was repeated 3 times. The inoculated culture dish was placed in an incubator at 28°C for culture. After culturing for 4-5 days, the colony diameter (mm) of each pathogen was determined, and the mycelium growth inhibition rate was calculated by the following formula (see Table 1). The graph of each inhibited pathogen is shown in Table 1. Figure 1 .

[0068] Mycelial growth inhibition rate (%) = (control colony diameter - treated colony diameter) / (control colony diameter - cake diameter) × 100 Table 1 Inhibitory effect of microbial agents on various pathogens

[0069] germs Colony diameter (mm) Mycelial growth inhibition rate (%) Soybean root rot pathogen 22.5 76.7 Soybean Anthracnose 28.2 69.2 Gray leaf spot pathogen 27.3 70.3

[0070] Tests on the control effect of various pathogens:

[0071] Root rot pathogen control test:

[0072] Eleven groups of experiments were conducted using blank control (no microbial agent), Example 1, and Comparative Examples 1-9. Each treatment had five rows, with a total row length of 5 m and a row spacing of 0.5 m. 100 seeds were sown in each row. Each group of microbial agent and Fusarium oxysporum were sown together. The spore concentration of Fusarium oxysporum was 1×10 5 Each plant was injected with 5 ml, and the microbial agent was diluted 500 times and injected with 30 ml per plant. About 35 days after soybean sowing, 5 soybean seedlings were dug out from each group. When sampling, the whole root was dug out to ensure the integrity of the soybean root system, and then rinsed with clean water. Finally, the disease degree of the root was observed.

[0073] Soybean root rot disease grade grading standards:

[0074] Level 0: The whole plant has no disease spots and can grow normally;

[0075] Level 1: There is no disease or slight brown spots on the main root and stem base, and the area of ​​the diseased spots accounts for less than 20% of the root and stem base. The plant grows normally.

[0076] Level 2: The main root is diseased and blackened, but the plant can continue to grow. The tips of the fibrous roots turn black, and the lesions occupy 20%-40% of the total area of ​​the soybean root and stem base. The plant grows normally.

[0077] Level 3: The main root turns black and cannot continue to grow. The number of fibrous roots is small or has stopped growing. The lesions occupy 40%-60% of the total area of ​​the soybean root and stem base, and the plant grows poorly.

[0078] Level 4: The lesions on the main root and stem base are connected, forming a phenomenon around the soybean stem. The lesions occupy 60%-80% of the total area of ​​the soybean root and stem base, but the root system is not completely necrotic;

[0079] Level 5: The entire soybean root system is necrotic, and the above-ground part wilts or dies. Some seeds are already infected and rotten.

[0080] Disease index = Σ(disease level × number of plants at that disease level) / (highest disease level × total number of plants) × 100%

[0081] Preventive effect = (disease index of control group - disease index of treatment group) / disease index of control group × 100%.

[0082] Anthracnose pathogen control test:

[0083] A blank control (no microbial agent) and 11 groups of microbial agents of Example 1 and Comparative Examples 1-9 were set up for testing. Soybeans were planted in groups of 20 rows, with a row length of 5 m, a plant spacing of 5 cm, and a row spacing of 60 cm. A protective row was set around each treatment group. 6 A suspension of Colletotrichum flatulum spores at a concentration of 100 ml was evenly sprayed onto the front and back surfaces of soybean leaves at the two-leaf, one-heart stage (when the first pair of true leaves were fully expanded). The leaves were covered with plastic film for 2 days after inoculation and then the film was removed. Five days after inoculation, the microbial agents of Example 1 and Comparative Examples 1-9, diluted 500 times, were sprayed at a rate of 40 L / mu. Fifteen days after inoculation, 15 plants were sampled from the center of each row to investigate the incidence of disease.

[0084] Soybean anthracnose disease grade grading standards:

[0085] Level 0: no lesions on leaves;

[0086] Level 1: There are a few scattered spot-like lesions on the leaves;

[0087] Level 2: There are dotted lesions on the leaves, which are brown and nearly circular, and the lesions on the leaves account for less than 30% of the total leaf area;

[0088] Level 3: The lesions on the leaves expand over a small area, accompanied by a small yellow halo, and the lesions on the leaves occupy 30%-50% of the total leaf area;

[0089] Level 4: The lesions on the leaves are widely spread, accompanied by a large area of ​​yellow halo, and the lesions on the leaves account for 50%-70% of the total leaf area;

[0090] Level 5: The lesions on the leaves expand over a large area, accompanied by large yellow halos, and the lesions on the leaves account for more than 70% of the total leaf area.

[0091] Disease index = Σ(disease level × number of plants at that disease level) / (highest disease level × total number of plants) × 100%

[0092] Preventive effect = (disease index of control group - disease index of treatment group) / disease index of control group × 100%.

[0093] Gray spot disease control test:

[0094] A blank control (no microbial agent) and 11 groups of microbial agents of Example 1 and Comparative Examples 1-9 were set up for testing. Soybeans were planted in groups of 20 rows, with a row length of 5 m, a plant spacing of 5 cm, and a row spacing of 60 cm. A protective row was set around each treatment group. 6 A suspension of gray leaf spot fungi containing 100 ml of spores was evenly sprayed onto the front and back surfaces of soybean leaves at the two-leaf, one-heart stage (when the first pair of true leaves were fully expanded). Inoculation was performed in the evening or on a cloudy day. Five days after inoculation, a 500-fold diluted microbial agent from Example 1 and Comparative Examples 1-9 was sprayed at a rate of 40 L / mu. Fifteen days after inoculation, 15 plants were sampled from the middle of each row to investigate the incidence of disease.

[0095] Gray spot disease grading standards:

[0096] Level 1: Soybean leaf lesions occupy less than 1% of the leaf area;

[0097] Level 2: Soybean leaf lesions occupy 1%-5% of the leaf area;

[0098] Level 3: Soybean leaf lesions cover 6%-20% of the leaf area;

[0099] Level 4: Soybean leaf lesions cover 21%-50% of the leaf area;

[0100] Level 5: Soybean leaf spots occupy more than 51% of the leaf area.

[0101] Disease index = Σ(disease level × number of plants at that disease level) / (highest disease level × total number of plants) × 100%

[0102] Preventive effect = (disease index of control group - disease index of treatment group) / disease index of control group × 100%.

[0103] Table 2 The control effect of microbial agents on various pathogens

[0104]

[0105]

[0106] The data in the table demonstrates that the microbial agent of the present invention effectively controls soybean root rot, anthracnose, and gray leaf spot, achieving a control efficiency of over 84% against all three pathogens. The microbial agent of the present invention exhibits the greatest control effect against root rot. Simultaneous testing of comparative examples 1-9, which varied the composition of the agent, revealed that the synergistic balance between the three strains was disrupted, significantly reducing soybean disease resistance.

[0107] Field trials:

[0108] The experiment was conducted in southeastern Heilongjiang Province. The previous crop was corn. Basic nutrient indicators were organic matter 24.2 g / kg, pH 6.35, alkaline-hydrolyzable nitrogen 125.60 mg / kg, available phosphorus 76.43 mg / kg, and available potassium 182.46 mg / kg. The soybean variety used was Kenfeng 17.

[0109] Treatment group:

[0110] The experiment adopted a randomized block design with 11 treatment groups and 3 replicates. Each plot had 5 rows and a row width of 0.65m. 2 , row length 6m, area 19.5m 2 , artificial spot sowing was used for planting, with a single seed per hole and a plant spacing of 5 cm. Artificial sowing was carried out in early May and harvested in October. Field management was consistent with local management. The bacterial agents obtained in Example 1 and Comparative Examples 1-9 were diluted 500 times and applied to the roots or leaves at a rate of 40 L / mu.

[0111] CK: Conventional fertilization (diammonium phosphate 150kg / hm 2 , urea 60kg / hm 2 , potassium chloride 50kg / hm 2 );

[0112] T0: 50% conventional fertilizer + microbial agent prepared in Example 1;

[0113] T1: 50% conventional fertilizer + microbial agent prepared in Comparative Example 1;

[0114] T2: 50% conventional fertilizer + microbial agent prepared in Comparative Example 2;

[0115] T3: 50% conventional fertilizer + microbial agent prepared in Comparative Example 3;

[0116] T4: 50% conventional fertilizer + microbial agent prepared in Comparative Example 4;

[0117] T5: 50% conventional fertilizer + microbial agent prepared in Comparative Example 5;

[0118] T6: 50% conventional fertilizer + microbial agent prepared in Comparative Example 6;

[0119] T7: 50% conventional fertilizer + microbial agent prepared in Comparative Example 7;

[0120] T8: 50% conventional fertilizer + microbial agent prepared in Comparative Example 8;

[0121] T9: 50% conventional fertilizer + microbial agent prepared in Comparative Example 9.

[0122] Agronomic traits determination:

[0123] Twenty soybean plants were sampled from each plot at the seedling stage and their aboveground dry weight and root dry weight were measured using the oven-drying method. Twenty plants were sampled from each plot at the peak flowering stage and their plant height was measured. At harvest, the number of pods per plant, the number of grains per plant, and yield were measured. These data are shown in Table 3.

[0124] Table 3 Agronomic traits of soybean

[0125]

[0126] As can be seen from the data in Table 3, compared with conventional fertilization, the microbial agent of the present invention significantly increased the aboveground dry weight and root dry weight of soybeans at the seedling stage, with the aboveground dry matter increasing by 13.1% and the root dry weight increasing by 20%. During the flowering period of soybeans, the microbial agent of the present invention was able to increase soybean plant height compared with conventional fertilization. During the soybean harvest period, compared with conventional fertilization, the application of the microbial agent of the present invention significantly increased the number of soybean pods per plant, the number of grains per plant, and soybean yield, with the number of soybean pods per plant increasing by 14.7%, the number of grains per plant increasing by 10.4%, and the soybean yield increasing by 12.9%. In addition, as can be seen from the data in the table, compared with the microbial agent of the embodiment of the present invention, the synergistic balance between the three strains was broken in Comparative Examples 1-9, in which the composition of the microbial agent was changed, resulting in a decrease in yield.

[0127] It should be noted that the above embodiments are only some of the preferred embodiments of the present invention, and not all of them. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work should fall within the scope of protection of the present invention.

Claims

1. A microbial agent for preventing disease and promoting growth, characterized in that: The microbial agent is composed of Bacillus arguta ( Bacillus aryabhattai ), Pseudomonas guguanyi ( Pseudomonas guguanensis ), dark orange motile actinomycetes ( Actinoplanes atraurantiacus ); the Bacillus aeruginosa has a deposit number of CGMCC No. 1.15821, purchased from the China General Microorganism Collection Center; the Pseudomonas guguanyi has a deposit number of CGMCC No. 1.15627, purchased from the China General Microorganism Collection Center; the dark orange actinomycetes has a deposit number of CGMCC No. 4.6857, purchased from the China General Microorganism Collection Center.

2. The microbial agent for preventing disease and promoting growth according to claim 1, characterized in that: The microbial agent is a liquid agent.

3. The method for preparing the disease prevention and growth promotion microbial agent according to any one of claims 1 to 2, characterized in that: The following steps are involved: Preparation of Bacillus arguta and Pseudomonas guguanis bacterial suspension: Bacillus arguta and Pseudomonas guguanis were thawed and activated in LB solid medium. Then, single colonies were picked and inoculated into LB liquid medium and cultured at 30°C and 180 rpm until OD 600 =0.6 to obtain seed solution, and the seed solution was evenly inoculated into 200 mL LB liquid medium at 1% of the amount and cultured at 30 ° C and 180 r / min. The bacterial colony activity was detected by the dilution spread plate method, and the number of bacterial colonies reached 1×10 8 cfu·mL -1 After the culture was terminated, the cells were washed three times with sterile PBS buffer solution and then resuspended to obtain two bacterial suspensions; Preparation of S2 dark orange swimming actinomycete fermentation broth: The dark orange swimming actinomycetes were thawed and activated in ISP-2 solid medium. Then, a single colony was picked and inoculated into ISP-2 liquid medium and cultured at 28°C and 200 rpm until OD 600 =0.6 to obtain seed solution, and the seed solution was evenly inoculated into 200 mL ISP-2 liquid medium at 1% and cultured at 28°C and 200 r / min. The bacterial colony activity was detected by the dilution plate method, and the number of colonies reached 1×10 8 cfu·mL -1 Finally, the culture was terminated to obtain a dark orange actinomycete fermentation liquid; S3 Preparation of microbial agent: The Bacillus aeruginosa suspension and the Pseudomonas guguanyi suspension in step S1 and the dark orange actinomycetes fermentation broth in step S2 are mixed in a volume ratio of 1:1:1 to obtain the final product microbial agent.

4. A use of the disease-preventing and growth-promoting microbial agent according to any one of claims 1 to 2, characterized in that: The microbial agent prevents and controls soybean root rot, soybean anthracnose and soybean gray spot.

5. A use of the disease-preventing and growth-promoting microbial agent according to any one of claims 1 to 2, characterized in that: The microbial agent is used for promoting soybean growth.

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