Disease-preventing and growth-promoting microbial agent as well as preparation method and application thereof

Abstract

The invention discloses a microbial agent for preventing diseases and promoting growth as well as a preparation method and application thereof, and belongs to the technical field of microorganisms. The microbial agent disclosed by the invention comprises bacillus aryabhattai, pseudomonas oryzae and actinoplanes dark orange. Bacillus aryabhattai, pseudomonas oryzae and actinoplanes darkorange are screened to form functional microorganisms, and after the three bacterial liquids are mixed in equal proportion, various antibacterial substances are generated to synergistically inhibit the growth of pathogenic bacteria, and the bacillus aryabhattai, pseudomonas oryzae and actinoplanes darkorange have good antagonistic effects on soybean root rot, soybean anthracnose and soybean gray speck disease. The microbial agent provided by the invention can promote soybean growth, increase soybean yield, reduce the occurrence probability of soybean root rot, soybean anthracnose and soybean gray speck disease, solve the problems of low yield and high incidence of diseases of part of soybeans, is safe and environment-friendly in components, is non-toxic to soybeans, and has a positive effect on reducing environmental pollution and pesticide residues.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a disease-preventing and growth-promoting microbial agent and a preparation method and application thereof. Background Art

[0002] Soybeans are high in protein and one of the highest in protein content among plant foods. The amino acid composition of its protein is close to what the human body needs and is easy to digest and absorb. In addition, soybeans are rich in unsaturated fatty acids, calcium, potassium and vitamin E, and have many health benefits such as lowering cholesterol, promoting muscle growth, and enhancing immunity. Soybeans can not only be eaten directly, but also processed into soy products such as tofu, soy milk, and soy sauce, and can also be used to make meat substitutes. In addition, soybeans have important economic value in agriculture. It can coexist with nitrogen-fixing bacteria in its roots to convert nitrogen in the atmosphere into a form that can be used by plants, thereby reducing the need for chemical fertilizers and improving soil health and structure. In addition, soybeans are also widely used in animal feed, especially soybean meal, a byproduct of oil extraction, which is an important ingredient in poultry and pig feed.

[0003] Heilongjiang Province is my country's high-quality soybean production and supply base. 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 adult 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 and affect 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, a microbial mixed agent for promoting soybean growth and its application, the microbial mixed agent includes the fermentation liquid of Azospirillum brasiliensis and Hydrogenophilus parviphilus. The microbial mixed agent of the present invention can stimulate plant cells to secrete various growth hormones, strongly promote root growth, rooting, root strengthening, and hairy roots, and its length or width is increased, which enhances the ability of crops to absorb water and fertilizer, so that crops have effects such as long and thick stems and resistance to lodging, thereby increasing crop yield. Chinese patent CN102925387A, a simple Bacillus with inducing soybean resistance to soybean cyst nematodes and its application, after the strain is cultured by conventional liquid fermentation, the metabolites of the bacteria can be used to develop a seed treatment agent for treating soybean seeds, and the treated soybean seeds can induce soybeans to produce resistance to soybean cyst nematodes. The simple Bacillus (Bacillussimplex) Sneb545 metabolites of the present invention can induce soybeans to produce obvious resistance to the first generation of cyst nematodes in the seedling stage after treating soybean seeds.

[0005] However, the microbial agents currently studied for soybeans have single functions, are used in large quantities, and have limited effects on promoting soybean growth, making them difficult to effectively apply and promote in agricultural production. Summary of the invention

[0006] The present invention aims at the problems existing in the prior art and provides a disease-preventing and growth-promoting microbial agent, which can effectively prevent and control soybean root rot, anthracnose and gray spot disease, and effectively increase soybean yield.

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

[0008] A disease prevention 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, which was purchased from 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, which was purchased from China General Microorganism Collection Center, and the original deposit date is February 25, 2015; the Actinoplanes atraurantiacus has a deposit number of CGMCC No.4.6857, which was purchased from 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 carry out repeated biological preservation.

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

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

[0011] S1 Preparation of Bacillus arginifera and Pseudomonas gushiguanis bacterial liquid: Bacillus arginifera and Pseudomonas gushiguanis were thawed and activated in LB solid medium, and then single colonies were picked and inoculated into LB liquid medium, and cultured 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 number was detected by the dilution plate method, and the number 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, and then a single colony was picked and inoculated into ISP-2 liquid medium, and cultured at 28°C and 200 r / min 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 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, the Pseudomonas guguanyi suspension in step S1 and the dark orange actinomycetes fermentation liquid in step S2 are mixed in a volume ratio of 1:1:1 to obtain a 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 40 L / mu to wet the roots or spray the leaves.

[0018] Bacillus arginii can induce systemic resistance in plants and improve their resistance to a variety of pathogens. At the same time, Bacillus arginii can synthesize plant hormones such as indoleacetic acid (IAA) and cytokinin (CTK), which can directly promote the development of plant roots, improve photosynthesis efficiency, and enhance plant resistance to environmental stress. In addition, through rapid reproduction and secretion of lysozyme, it can not only improve the bacterial environment in the soil, but also effectively eliminate harmful pathogens such as fungi and bacteria.

[0019] Pseudomonas guguanis can produce a variety of plant hormones, such as indoleacetic acid (IAA), gibberellins (GA) and cytokinins, which can stimulate the development of plant roots and increase soybean biomass and yield. At the same time, Pseudomonas guguanis can produce antibiotics and other antibacterial substances, which can inhibit or kill pathogenic fungi and bacteria in the soil and reduce the occurrence of soybean diseases. In addition, Pseudomonas guguanis can activate the systemic resistance response of plants. When one part is attacked by pathogens, other parts will also produce a defense response, thereby improving the disease resistance of plants as a whole.

[0020] Dark orange actinomycetes and their metabolites have significant antibacterial activity and can effectively inhibit the growth of a variety of plant pathogens. They can also produce plant hormones such as indoleacetic acid (IAA) and gibberellins, which directly stimulate plant cell division and tissue formation. In addition, actinomycetes can improve soil nutrient supply by dissolving phosphorus and fixing nitrogen, thereby improving the plant's ability to absorb nutrients.

[0021] Beneficial Effects

[0022] The present invention selects Bacillus agglomerans, Pseudomonas glutinosus and Actinomycetes 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 pathogenic bacteria, 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 a small amount, can promote soybean growth, increase soybean yield, and reduce the occurrence probability of soybean root rot, soybean anthracnose, and soybean gray spot disease, thereby 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 anthracnose 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 is further described below in conjunction with specific embodiments, but is not limited thereto.

[0026] Example 1

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

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

[0029] S1 Preparation of Bacillus arginifera and Pseudomonas gushiguanis bacterial liquid: Bacillus arginifera and Pseudomonas gushiguanis were thawed and activated in LB solid medium, and then single colonies were picked and inoculated into LB liquid medium, and cultured 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 number was detected by the dilution plate method, and the number 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, and then a single colony was picked and inoculated into ISP-2 liquid medium, and cultured at 28°C and 200 r / min 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 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, the Pseudomonas guguanyi suspension in step S1 and the dark orange actinomycetes fermentation liquid in step S2 are mixed in a volume ratio of 1:1:1 to obtain a final product microbial agent.

[0032] Comparative Example 1

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

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

[0035] S1 Preparation of Bacillus arginifera and Pseudomonas gushiguanis bacterial liquid: Bacillus arginifera and Pseudomonas gushiguanis were thawed and activated in LB solid medium, and then single colonies were picked and inoculated into LB liquid medium, and cultured 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 number was detected by the dilution plate method, and the number 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, and then a single colony was picked and inoculated into ISP-2 liquid medium, and cultured at 28°C and 200 r / min 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 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, the Pseudomonas guguanyi suspension in step S1 and the dark orange actinomycetes fermentation liquid in step S2 are mixed in a volume ratio of 1:1:2 to obtain a 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 guguanyi suspension and dark orange actinomycetes fermentation broth is changed to 1:1:2, and 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 agglutinosa, Pseudomonas guineensis and Actinomyces aurantii.

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

[0042] S1 Preparation of Bacillus arginifera and Pseudomonas gushiguanis bacterial liquid: Bacillus arginifera and Pseudomonas gushiguanis were thawed and activated in LB solid medium, and then single colonies were picked and inoculated into LB liquid medium, and cultured 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 number was detected by the dilution plate method, and the number 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, and then a single colony was picked and inoculated into ISP-2 liquid medium, and cultured at 28°C and 200 r / min 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 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, the Pseudomonas guguanyi suspension in step S1 and the dark orange actinomycetes fermentation liquid in step S2 are mixed in a volume ratio of 1:2:1 to obtain a 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 guguanyi 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 agglutinosa, Pseudomonas guineensis and Actinomyces aurantii.

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

[0049] S1 Preparation of Bacillus arginifera and Pseudomonas gushiguanis bacterial liquid: Bacillus arginifera and Pseudomonas gushiguanis were thawed and activated in LB solid medium, and then single colonies were picked and inoculated into LB liquid medium, and cultured 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 number was detected by the dilution plate method, and the number 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, and then a single colony was picked and inoculated into ISP-2 liquid medium, and cultured at 28°C and 200 r / min 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 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, the Pseudomonas guguanyi suspension in step S1 and the dark orange actinomycetes fermentation liquid in step S2 are mixed in a volume ratio of 2:1:1 to obtain a 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 guguanyi suspension and dark orange actinomycetes fermentation broth is changed to 2:1:1, and 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 uses Bacillus aeruginosa suspension and Pseudomonas guguanyi suspension, and the volume ratio of the two is 1:1. The rest is the same as Example 1.

[0055] Comparative Example 5

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

[0057] Comparative Example 6

[0058] Compared with Example 1, this comparative example only used the Pseudomonas guguanis bacterial suspension and the Pseudomonas guguanis bacterial suspension, and the volume ratio of the two was 1:1. The rest was the same as Example 1.

[0059] Comparative Example 7

[0060] Compared with Example 1, this comparative example only uses the Bacillus agni suspension, and the rest is the same as Example 1.

[0061] Comparative Example 8

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

[0063] Comparative Example 9

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

[0065] Performance Testing

[0066] Determination of the ability to inhibit pathogens:

[0067] The flat plate confrontation culture method was used to determine the antagonistic effect of microbial agents on soybean root rot, soybean anthracnose, and soybean gray spot. Soybean root rot, anthracnose, and gray spot bacterial cakes with a diameter of 5 mm were inoculated in the center of the culture dish, respectively. 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 the pathogenic bacteria cake was inoculated but not the microbial agent of the present invention was used as the control. 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 the pathogens in each treatment was determined, and the mycelium growth inhibition rate was calculated by the following formula (see Table 1). The graphs of each inhibition of pathogens are shown in Table 1. Figure 1 .

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

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

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

[0071] Root rot pathogen control test:

[0072] A blank control (without adding microbial agents), 11 groups of microbial agents of Example 1 and Comparative Examples 1-9 were tested, each of which treated 5 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 agents and Fusarium oxysporum were sown together. The spore concentration of Fusarium oxysporum was 1×10 5 Each plant was injected with 5 ml of the microbial agent 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 extent of the roots 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 taproot and stem base, and the area of ​​the diseased spots accounts for less than 20% of the root and stem base, and the plant grows normally;

[0076] Level 2: The main root becomes diseased and black, but the plant can continue to grow. The root 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 taproot turns black and cannot continue to grow. The number of fibrous roots is small or stops growing. The lesions occupy 40%-60% of the total area of ​​the soybean root and stem base. The plant grows poorly.

[0078] Level 4: The lesions on the taproot and stem base are connected, forming a phenomenon of surrounding the soybean stem. The lesions account for 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 parts wilt or die. Some of the 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), Example 1 and Comparative Examples 1-9 microbial agent 11 groups 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 outside each treatment group. 6 Each milliliter of the suspension of flat-headed anthracnose spores was evenly sprayed on the front and back sides of soybean leaves at the two-leaf one-heart stage (the first pair of true leaves were fully expanded), and the plastic film was covered for 2 days after inoculation, and then the plastic film was uncovered. Five days after inoculation, the microbial agents of Example 1 and Comparative Examples 1-9 diluted 500 times were sprayed, and the spraying amount was 40L / mu. After 15 days of inoculation, 15 plants were continuously taken from the middle of each row to investigate the disease situation.

[0084] Soybean Anthracnose Disease Grading Standards:

[0085] Level 0: no lesions on leaves;

[0086] Level 1: There are a few scattered spots on the leaves;

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

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

[0089] Level 4: The lesions on the leaves expand over a large area, accompanied by a large area of ​​yellow halos, 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), Example 1 and Comparative Examples 1-9 microbial agent 11 groups 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 outside each treatment group. 6 The suspension of gray leaf spot fungus spores was sprayed evenly on the front and back of soybean leaves at the two-leaf one-heart stage (the first pair of true leaves were fully expanded), and the inoculation was selected in the evening or on a cloudy day. Five days after the inoculation, the microbial agents of Example 1 and Comparative Examples 1-9 diluted 500 times were sprayed, and the spraying amount was 40L / mu. After 15 days of inoculation, 15 plants were continuously taken from the middle of each row to investigate the disease situation.

[0095] Gray spot disease grading standards:

[0096] Level 1: Soybean leaf lesions account for 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 spots occupy 6%-20% of the leaf area;

[0099] Level 4: Soybean leaf lesions occupy 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 Effect of microbial agents on the control of various pathogens

[0104]

[0105]

[0106] From the data in the table, it can be seen that the microbial agent of the present invention can effectively prevent and control soybean root rot, anthracnose and gray spot, and the control effect on the three pathogens is more than 84%, among which the microbial agent of the present invention has the best control effect on root rot. At the same time, the comparative examples 1-9 with changed agent composition were measured, and it was found that the synergistic balance between the three strains was broken, and the disease resistance of soybean was significantly reduced.

[0107] Field trials:

[0108] The experiment was conducted in the southeast of Heilongjiang Province. The previous crop was corn. The basic nutrient indicators were organic matter 24.2g / kg, pH 6.35, alkaline nitrogen 125.60mg / kg, available phosphorus 76.43mg / kg, and available potassium 182.46mg / kg. The soybean variety tested 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 adopted for planting, single seed per hole, and plant spacing of 5 cm. Artificial sowing was carried out in early May, harvesting was carried out in October, and field management was consistent with local management. The bacterial agents obtained in Example 1 and Comparative Examples 1-9 were diluted 500 times and irrigated roots or sprayed on leaves at 40L / mu.

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

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

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

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

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

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

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

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

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

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

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

[0122] Agronomic traits determination:

[0123] At the soybean seedling stage, 20 plant samples were taken from each plot, and the aboveground dry weight and root dry weight were measured by drying method; 20 plant samples were taken from each plot at the flowering stage to measure plant height. The number of pods per plant, the number of grains per plant and the yield were measured at the harvest stage. The measured data are shown in Table 3.

[0124] Table 3 Agronomic traits of soybean

[0125]

[0126] It can be seen from the data in Table 3 that, compared with conventional fertilization, the microbial agent of the present invention significantly increased the aboveground dry weight and root dry weight of soybeans in the seedling stage, with the aboveground dry matter increasing by 13.1% and the root dry weight increasing by 20%. Compared with conventional fertilization, the microbial agent of the present invention can increase the height of soybean plants during the flowering period of soybeans. 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, the number of grains per plant and the soybean yield, wherein the number of soybean pods increased by 14.7%, the number of grains per plant increased by 10.4%, and the soybean yield increased by 12.9%. In addition, it can be seen from the data in the table that, 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 embodiments of the preferred methods of implementing the present invention, rather than all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field 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 composite microorganism consists of Bacillus aryabhattai, Pseudomonas guguanensis and Actinoplanes atraurantiacus; the Bacillus aryabhattai has a collection number of CGMCC No. 1.15821, which is purchased from China General Microorganism Collection Center; the Pseudomonas guguanensis has a collection number of CGMCC No. 1.15627, which is purchased from China General Microorganism Collection Center; and the Actinoplanes atraurantiacus has a collection number of CGMCC No. 4.6857, which is purchased from China General Microorganism Collection Center.

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

3. The method for preparing the disease-preventing and growth-promoting microbial agent according to any one of claims 1 to 2, characterized in that: The following steps are involved: S1 Preparation of Bacillus arginifera and Pseudomonas gushiguanis bacterial liquid: Bacillus arginifera and Pseudomonas gushiguanis were thawed and activated in LB solid medium, and then single colonies were picked and inoculated into LB liquid medium, and cultured 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 number was detected by the dilution plate method, and the number 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, and then a single colony was picked and inoculated into ISP-2 liquid medium, and cultured at 28°C and 200 r / min 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 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 inoculants: Mix the Bacillus aeruginosa suspension and the Pseudomonas guguanyi suspension prepared in step S1 and the The dark orange actinomycete fermentation broth in S2 was mixed in a volume ratio of 1:1:1 to obtain the final product microbial inoculant.

4. Use of the microbial agent according to any one of claims 1 to 3 in soybean disease prevention and growth promotion.

5. The use of the microbial agent in soybean disease prevention and growth promotion according to claim 4, characterized in that: The diseases are soybean root rot, soybean anthracnose and soybean gray spot.

6. The use of the microbial agent in soybean disease prevention and growth promotion according to claim 4, characterized in that: The microbial agent is used for promoting the growth of soybean.

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