A composite microbial agent and its application

Abstract

The present invention provides a composite microbial agent and its application, belonging to the field of bioengineering technology. The composite microbial agent provided by the present invention comprises the following strains: Bacillus subtilis AF0907, Bacillus amyloliquefaciens ZDS-1, and Paenibacillus polymyxa AFHXD7. It can effectively control wheat stem rot and toxin contamination while promoting plant growth. This is of great significance for improving crop growth and the rhizosphere environment, and enhancing the effectiveness of microbial fertilizers.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a composite microbial agent and application thereof. Background Art

[0002] Wheat is one of the world's largest and most productive food crops, and its cultivation and disease control are crucial to global food supply and security. Wheat crown rot (Fusarium crown rot), also known as dryland foot rot and Fusarium root rot, is a global soil-borne disease caused by a variety of pathogenic fungi that severely limits wheat yields. When wheat is infected with crown rot, the base of the stem turns brown, and a pink mold layer grows between the affected internodes. In severe cases, this can cause white ears and death during the grain filling stage, resulting in shriveled or even seedless grains. Furthermore, wheat is contaminated with toxins such as DON (deoxynivalenol) and ZEN (zearalenone), posing a serious threat to wheat quality and yield.

[0003] Currently, wheat stem rot is a newly identified disease, and resistance resources are scarce. Agricultural control measures include crop rotation, fertilizer and water management, and straw treatment. Chemical control primarily involves seed dressing with pesticides and spraying during the greening and jointing stages. However, seed dressing with pesticides is easily affected by soil and climatic conditions, and may not achieve the desired control effect. Long-term application of chemical agents can also lead to resistance and environmental impacts. Using microbial agents to control wheat stem rot is environmentally friendly, safe, and environmentally friendly. It can better serve the sustainable development of agriculture, achieving the unity of social, economic, and ecological benefits. It meets my country's requirements for pollution-free, green, and sustainable development, and has great development prospects.

[0004] Bacillus is a relatively common microbial biocontrol resource with functions such as preventing and controlling pathogens, degrading biotoxins, and promoting plant growth. Currently, most commercially available microbial agents are single microbial agents, which have problems such as single function and poor yield-increasing effects. However, composite agents prepared by combining different functional microbial strains have similar biological characteristics and mechanisms of action. Their synergistic use can make up for the many shortcomings of single agents and effectively improve crop yields, which is the development trend of microbial agents. Therefore, there is an urgent need to develop a microbial agent that can effectively inhibit wheat stem rot, degrade Fusarium toxins, and promote plant growth, thereby improving soil resource utilization and reducing pesticide use. Summary of the Invention

[0005] The object of the present invention is to provide a composite microbial agent and its application, which can effectively inhibit wheat stem rot, degrade Fusarium toxin, and promote plant seed germination and seedling growth.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a composite microbial agent, comprising the following strains: Bacillus subtilis AF0907, Bacillus amyloliquefaciens ZDS-1 and Paenibacillus polymyxa AFHXD7.

[0008] Preferably, the viable bacterial concentrations of Bacillus subtilis AF0907, Bacillus amyloliquefaciens ZDS-1 and Paenibacillus polymyxa AFHXD7 are all (1-5)×10 8 cfu·mL -1 .

[0009] Preferably, the volume ratio of the bacterial liquid of Bacillus subtilis AF0907, Bacillus amyloliquefaciens ZDS-1 and Paenibacillus polymyxa AFHXD7 is 1-2:1-2:1-2.

[0010] The present invention also provides an application of the composite microbial agent in preventing and treating wheat stem rot, regulating soil physical and chemical properties and fertility, and promoting wheat growth.

[0011] Preferably, the method for using the composite microbial agent to prevent and control wheat stem rot, regulate soil physical and chemical properties and fertility, and promote wheat growth is: watering the wheat one week after emergence with a bacterial suspension containing the composite microorganisms.

[0012] Preferably, the total concentration of microorganisms in the bacterial suspension containing the composite microorganisms is (1-3)×10 8 cfu·mL -1 .

[0013] The present invention obtains a composite microbial agent made by mixing Bacillus subtilis AF0907, Bacillus amyloliquefaciens ZDS-1 and Paenibacillus polymyxa AFHXD7 through strain screening, disease control and attenuation, and plant growth promotion experiments. The composite microbial agent can effectively control wheat stem base rot and toxin pollution, and promote plant growth. It is of great significance for improving the growth status of crops and the rhizosphere environment of crops in the environment, and enhancing the effect of microbial fertilizers. It provides the market with a composite microbial agent product with better growth promotion effect.

[0014] Biological Deposit Description

[0015] Bacillus subtilis AF0907 was deposited in China Center for Type Culture Collection, Wuhan University, Wuhan, China, on May 20, 2010, with a deposit number of CCTCC NO: M2010124.

[0016] Bacillus sp. ZDS-1 was deposited in China Center for Type Culture Collection, Wuhan University, Wuhan, China, on March 13, 2010, with a deposit number of CCTCCNO: M2010053.

[0017] Paenibacillus polymyxa AFHXD7, the strain was deposited in China Center for Type Culture Collection, Wuhan University, Wuhan, China, on January 22, 2015, with a deposit number of CCTCCNO: M2015059. DETAILED DESCRIPTION

[0018] 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.

[0019] Example 1

[0020] Bacillus subtilis AF0907 (viable cell count 3×10 8 cfu·mL -1 ), Bacillus amyloliquefaciens ZDS-1 (viable count 3×10 8 cfu·mL -1 ), Paenibacillus polymyxa AFHXD7 (viable count 3×10 8 cfu·mL -1 ) The bacterial liquids of the three strains were mixed in the volume ratios of 1:1:1, 1:2:1, 1:1:2, and 2:1:1 to produce liquid composite microbial agents, and were named CM1, CM2, CM3, and CM4 respectively. At the same time, the bacterial liquids of a single strain were retained and recorded as A1, A2, and A3, and used as single strain agents, as shown in Table 1.

[0021] Table 1 Composite microbial agents and single microbial agents

[0022]

[0023] Example 2 Effect of composite microbial agent on preventing and controlling wheat stem rot

[0024] Conidia of the pseudograminearum Fusarium strain KF20-15 were diluted to 10 5 mL -1 Before inoculation, add an appropriate amount of Tween-20 to a concentration of 0.1% (v / v); fill a pot with a diameter of 12 cm, a height of 15 cm, and a hole at the bottom with sterilized soil;

[0025] Wheat seeds (Jimai 22) were disinfected with a 1% NaClO solution and then germinated at 25°C for two days. The germinated wheat was then soaked in a spore solution of Fusarium graminearum strain KF20-15 for two minutes, removed, and sown in soil-filled pots, with 10 seeds per pot. The pots were covered with soil up to the top, and an appropriate amount of water was added to maintain a consistent soil humidity in each pot, maintaining a relative humidity between 60% and 85%. The amount of water applied to each pot was maintained at the same rate daily. Later, each pot was watered every two days.

[0026] One week after the seedlings emerged, the composite microbial agent of Example 2 was inoculated by watering. When inoculating, 1 mL of each of the composite microbial agents M1, M2, M3, and M4 was taken, centrifuged at 12000 r / min for 2 min to collect the bacteria, and washed with sterile water to adjust the concentration to 2×10 9 CFU / ml. Take 1mL and suspend the bacteria in 9mL of sterile distilled water, then inoculate 5mL into the flower pot. Inoculate each flower pot once, and set 3 replicates. The potted plants without inoculation of the inoculum were used as the blank control group ck, and other commercially available composite microbial inoculum products (Zhengzhou Bio Chemical Co., Ltd.; composite microbial inoculum EM bacteria) were used as the conventional control group. The single strain bacterial liquid A1, A2, and A3 were inoculated as the positive control group, and inoculated in the same way, and 3 replicates were set.

[0027] Potted wheat was placed in an open-air nursery for cultivation. Before the wheat matured, the white ear rate caused by stem rot was investigated and the control efficacy was calculated. The results are shown in Table 2.

[0028] Table 2 Control effect of composite microbial agent on wheat stem rot (artificial inoculation test)

[0029]

[0030] The results showed that the prevention effect of wheat stem rot was different after treatment with different bacterial suspensions. Compared with inoculation of single bacterial agents and other commercially available bacterial agents, inoculation of composite bacterial agents had a significant inhibitory effect on wheat stem rot. There was no significant difference in the prevention effect of composite microbial agents with different ratios, all above 55%, among which inoculation of composite bacterial agent CM2 had the best prevention and control effect.

[0031] Example 3 Effects of composite microbial agents on soil physical and chemical properties

[0032] In a plastic flower pot with a length and width of 10 cm, farmland soil was uniformly added and pressed to 1.5 cm from the pot mouth, 25 wheat seeds of basically the same size were evenly placed, and the soil was covered to the pot mouth. In the later stage, each flower pot was watered every 2 days. One week after the seedlings emerged, the composite microbial agent in Example 1 was inoculated by irrigation. When inoculating, 1 mL of each composite agent M1, M2, M3, and M4 was taken, centrifuged at 12000 r / min for 2 minutes to collect the bacteria, and washed with sterile water to adjust its concentration to 2×109 CFU / ml. Take 1ml and suspend the bacteria in 9mL of sterile distilled water, then inoculate 5mL into the flower pot. Inoculate each flower pot once, and set 3 replicates. The potted plants without inoculation of the microbial agent were used as the blank control group ck, and other commercially available composite microbial agent products (Zhengzhou Bio Chemical Co., Ltd.; composite microbial agent EM bacteria) were used as the conventional control group. The single strain bacterial liquid A1, A2, and A3 were inoculated as the positive control group, and inoculated in the same way, and 3 replicates were set.

[0033] Thirty days after sowing, potted soil from each experimental group was collected, air-dried, sieved, and packaged in plastic bags. The pH, organic matter, total nitrogen, available potassium, available phosphorus, and other nutrient contents were measured. Soil that had not been previously planted with wheat was used as the original soil group. The data are recorded in Table 3.

[0034] Soil pH was determined according to NY / T 1377-2007 “Determination of soil pH”.

[0035] With reference to NY / T 1121.24-2012 “Soil Testing Part 24”, the total nitrogen content of the soil was determined using the automatic nitrogen analyzer method.

[0036] With reference to NY / T 88-1988 “Determination of total phosphorus in soil”, the total phosphorus content in soil was determined by the NaOH alkali fusion-molybdenum antimony spectrophotometric method.

[0037] With reference to NY / T87-1988 “Determination of total potassium in soil”, the total potassium content in soil was determined by the NaOH alkali fusion-flame photometry method.

[0038] Referring to Bao Shidan's "Soil Agrochemical Analysis", the NH4F-HCl method was used to determine the content of available phosphorus in acidic soil.

[0039] With reference to Bao Shidan's "Soil Agrochemical Analysis", the alkaline diffusion method was used to determine the available nitrogen content in the soil.

[0040] With reference to NY / T 889-2004 “Determination of soil available potassium and slow-release potassium content”, the available potassium content in soil was determined by flame photometry using ammonium acetate solution extraction.

[0041] With reference to NY / T 1121.6-2006 “Soil Testing Part 6”, the potassium dichromate oxidation-external heating method was used to determine the soil organic matter content.

[0042] Table 3 Effects of inoculation of composite microbial agents on soil fertility

[0043]

[0044]

[0045] The experimental results, shown in Table 3, show that the total nitrogen, total phosphorus, total potassium, available phosphorus, available potassium, and organic matter content in the soil inoculated with the composite inoculum were significantly higher than those in the uninoculated control group (CK). The soil improvement effect of the composite inoculum experimental group was greater than that of the blank control group, the conventional control group, and the soil inoculated with a single bacterial strain, indicating that the different functional microbial strains in the composite inoculum work synergistically and can effectively compensate for the shortcomings of a single inoculum.

[0046] Example 4 Effect of composite microbial agent on wheat growth

[0047] 30 days after wheat sowing in Example 3, all healthy wheat in the flowerpot was carefully pulled out, most of the soil stuck to the roots was shaken off, and the wheat was washed with tap water and dried with newspaper. The wheat was then spread flat on the table in turn, and the length from the cotyledon node to the growing point was measured with a ruler as the wheat plant height; the length of the third true cotyledon was measured with a ruler as the wheat leaf length.

[0048] The wheat plants were washed and dried, and the number of roots extending from the wheat seed embryo was counted as its seed root number. The length from the wheat rhizome junction to the longest root tip of the whole plant was measured with a ruler, that is, the length of the longest seed root in the seed embryo was taken as its maximum root length.

[0049] After washing and drying the wheat plants, weigh them directly on a scale (accurate to 0.01 g) to determine their fresh weight. Then, dry the wheat plants in an oven at 100°C until constant weight is reached, and weigh them to determine their dry weight. Record these data in Table 4.

[0050] Table 4 Effects of inoculation with composite microbial agents on wheat growth

[0051]

[0052]

[0053] Taking into account the prevention and control effects on wheat stem rot, the improvement of wheat growth shape and the improvement of soil properties, the composite bacterial agent CM2 with a composition ratio of AF0907:ZDS-1:AFHXD7=1:2:1 has the best effect.

[0054] 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. Application of a composite microbial agent in preventing and controlling wheat stem rot, regulating soil physical and chemical properties and fertility, and promoting wheat growth; The composite microbial agent is composed of the following strains: Bacillus subtilis AF0907 with a deposit number of CCTCC NO: M2010124, Bacillus amyloliquefaciens ZDS-1 with a deposit number of CCTCC NO: M2010053, and Paenibacillus polymyxa AFHXD7 with a deposit number of CCTCC NO: M2015059; The viable bacterial concentrations of Bacillus subtilis AF0907, Bacillus amyloliquefaciens ZDS-1 and Paenibacillus polymyxa AFHXD7 are all (1-5)×10 8 cfu·mL -1 ; The volume ratio of the bacterial liquid of Bacillus subtilis AF0907, Bacillus amyloliquefaciens ZDS-1 and Paenibacillus polymyxa AFHXD7 is 1-2:1-2:1-2; The method of regulating the physical and chemical properties and fertility of the soil is to increase the content of total nitrogen, total phosphorus, total potassium, available phosphorus, available potassium and organic matter in the soil.

2. The use according to claim 1, characterized in that The method for preventing and controlling wheat stem rot, regulating soil physical and chemical properties and fertility, and promoting wheat growth by the composite microbial agent comprises watering wheat seedlings one week after emergence with a bacterial suspension containing the composite microbial agent.

3. The use according to claim 2, characterized in that The total concentration of microorganisms in the bacterial suspension containing the composite microbial agent is (1-3)×10 8 cfu·mL -1 .

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