Composite microbial agent and application thereof

Abstract

The invention provides a compound microbial agent and application thereof, and belongs to the technical field of bioengineering. The compound microbial agent provided by the invention is prepared from the following bacterial strains: bacillus subtilis AF0907, bacillus amyloliquefaciens ZDS-1 and paenibacillus polymyxa AFHXD7. The microbial fertilizer can effectively control wheat stem rot and toxin pollution, promotes plant growth, and has important significance in improving the growth state of crops in the environment and the rhizosphere environment of the crops and improving the action effect of the microbial fertilizer.

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 grain crops with the largest planting area and the highest yield. Its planting and disease control issues are related to global food supply and security. Wheat crown rot (Fusarium crown rot), also known as dryland foot rot (Dryland Foot Rot), sickle root rot (Fusarium Root Rot), etc., is a global soil-borne disease caused by a variety of pathogenic fungi, which seriously limits wheat production. When wheat is infected with crown rot, the base of the stem turns brown and a pink mold layer grows between the diseased internodes. In severe cases, it can cause white ears and death during the filling period of wheat, resulting in shriveled or even seedless grains. In addition, it is contaminated with DON (deoxynivalenol) toxins and ZEN (zearalenone), which poses a serious threat to the quality and yield of wheat.

[0003] At present, wheat stem base rot is a new disease and lacks resistant resources. Agricultural prevention and control measures include crop rotation, fertilizer and water management, and straw treatment. Chemical control mainly prevents and controls wheat stem base rot by mixing seeds with pesticides and spraying during the greening and jointing period. Among them, seed mixing with pesticides is easily affected by soil conditions and climatic conditions and cannot achieve the expected prevention and control effect. Long-term application of chemical agents will also produce resistance and environmental impacts. The use of microbial agents to prevent and control wheat stem base rot has the characteristics of being green, environmentally friendly, and safe. It can better serve the sustainable development of agriculture and achieve the unity of social benefits, economic benefits, and ecological benefits. It meets the requirements of pollution-free, green environmental protection, and sustainable development in my country and has great development prospects.

[0004] Bacillus is a common microbial biocontrol resource with functions such as preventing and controlling pathogens, degrading biological toxins and promoting plant growth. Currently, most of the microbial agents on the market are single microbial agents, which have problems such as single function and poor yield-increasing effect. The composite agents prepared by combining different functional microbial strains have similar biological characteristics and action mechanisms. Their synergistic use can make up for the many shortcomings of single agents and effectively improve the crop yield-increasing effect, which is the development trend of microbial agents. Therefore, it is urgent to develop a microbial agent that can effectively inhibit wheat stem base rot, degrade Fusarium toxins and promote plant growth, thereby improving soil resource utilization and reducing the use of pesticides. 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 base 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 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 the 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 controlling 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 the wheat seedlings emerge 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 prepared by mixing Bacillus subtilis AF0907, Bacillus amyloliquefaciens ZDS-1 and Bacillus 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 state of crops and the rhizosphere environment of crops in the environment and enhancing the effect of microbial fertilizers, and provides the market with a composite microbial agent product with better growth promotion effect.

[0014] Biological Deposit Description

[0015] Bacillus subtilis AF0907, the strain is deposited in China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, the deposit date is: May 20, 2010, the deposit number is CCTCC NO: M2010124.

[0016] Bacillus sp. ZDS-1, the strain is deposited in China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, the deposit date is: March 13, 2010, the deposit number is CCTCCNO: M2010053.

[0017] Paenibacillus polymyxa AFHXD7, the strain is deposited in China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, the deposit date is: January 22, 2015, the deposit number is CCTCCNO: M2015059. DETAILED DESCRIPTION

[0018] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0019] Example 1

[0020] Bacillus subtilis AF0907 (viable count 3×10 8 cfu·mL -1 ), Bacillus amyloliquefaciens ZDS-1 (viable count 3×10 8 cfu·mL -1 ) and 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, 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 Pseudomonas graminearum strain KF20-15 were diluted to 10 5 pc·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 1% NaClO solution and placed at 25°C for germination for 2 days. The germinated wheat was soaked in the spore solution of the pseudograminearum Fusarium strain KF20-15 for 2 minutes and then taken out and sown in a pot filled with soil, with 10 seeds sown in each pot. The soil was covered to the mouth of the flower pot, and an appropriate amount of water was added to keep the soil humidity of each pot consistent, and the relative humidity of the soil was kept between 60% and 85%. The amount of watering for each pot was controlled to be the same every day. In the later stage, each flower pot was watered every 2 days.

[0026] One week after the seedlings emerged, the composite microbial agent of Example 2 was inoculated by irrigation. 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 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 inoculant were used as the blank control group ck, and other commercially available composite microbial inoculant products (Zhengzhou Bio Chemical Co., Ltd.; composite microbial inoculant EM bacteria) were used as the regular control group. The inoculation of single strain bacterial liquid A1, A2, and A3 were used 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. The white ear rate caused by stem base rot was investigated before the wheat matured, and the control effect 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 effects of wheat stem rot were 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 effects of composite microbial agents with different ratios, all of which were above 55%. Among them, 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, add farmland soil uniformly and press it to 1.5 cm from the pot mouth, evenly put 25 wheat seeds of basically the same size, and cover the soil to the pot mouth. In the later stage, water each flower pot every 2 days. One week after the emergence of the seedlings, inoculate the composite microbial agent in Example 1 by watering. When inoculating, take 1 mL of each composite agent M1, M2, M3, and M4, centrifuge at 12000r / min for 2 minutes to collect the bacteria, and wash with sterile water to adjust its concentration to 2×109 CFU / ml. Take 1ml and suspend the bacteria in 9mL 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 inoculant were used as the blank control group ck, and other commercially available composite microbial inoculant products (Zhengzhou Bio Chemical Co., Ltd.; composite microbial inoculant EM bacteria) were used as the regular control group. The inoculation of single strain bacterial liquid A1, A2, and A3 were used as the positive control group, and inoculated in the same way, and 3 replicates were set.

[0033] 30 days after sowing, the potted soil of each test group was collected, dried naturally, sieved and packaged into plastic bags. The content of nutrients such as pH, organic matter, total nitrogen, available potassium, and available phosphorus was measured, and the soil that had not been planted with wheat was used as the original soil group. The data were recorded in Table 3.

[0034] The soil pH value 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] Referring to NY / T87-1988 “Determination of total potassium in soil”, the total potassium content in soil was determined by NaOH alkali fusion-flame photometry.

[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 soil available nitrogen content.

[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 extraction with ammonium acetate solution and flame photometry.

[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 are shown in Table 3. The total nitrogen, total phosphorus, total potassium, available phosphorus, quick-acting potassium and organic matter content in the soil after inoculation with the composite microbial agent were significantly higher than those of the uninoculated control group (CK). The improvement effect of the experimental group inoculated with the composite microbial agent on the soil was higher than that of the blank control group, the conventional control group and the soil inoculated with a single strain, indicating that the different functional microbial strains in the composite microbial agent have a synergistic effect and can effectively make up for the shortcomings of a single microbial agent.

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

[0047] 30 days after wheat was sown in Example 3, all healthy wheat in the flowerpot was carefully pulled out, most of the soil adhering to the roots was shaken off, and the wheat was washed with tap water and dried with newspaper. The wheat was spread out on the table in turn, and the length from the cotyledon node to the growth 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 leaf length of the wheat.

[0048] Wash and dry the wheat plants, count the number of roots extending from the wheat seed embryo as its seed root number, and use a ruler to measure the length from the junction of the wheat rhizome to the longest root tip of the whole plant, that is, the length of the longest seed root in the seed embryo as its maximum root length.

[0049] After washing and drying the wheat plants, weigh them directly with a balance (accurate to 0.01 g) to obtain their fresh weight. Then dry the wheat plants in an oven at 100°C until constant weight is reached and weigh them to obtain their dry weight. Record the above data, see 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composite microbial agent, characterized in that: The following strains were included: Bacillus subtilis AF0907, Bacillus amyloliquefaciens ZDS-1 and Paenibacillus polymyxa AFHXD7.

2. The bacterial agent according to claim 1, characterized in that The live bacterial concentrations of Bacillus subtilis AF0907, Bacillus amyloliquefaciens ZDS-1 and Paenibacillus polymyxa AFHXD7 are all (1-5)×10 8 cfu·mL -1 .

3. The bacterial agent according to claim 2, characterized in that The volume ratio of the bacterial liquid of the Bacillus subtilis AF0907, the Bacillus amyloliquefaciens ZDS-1 and the Paenibacillus polymyxa AFHXD7 is 1-2:1-2:1-2.

4. Use of the composite microbial agent according to any one of claims 1 to 3 in preventing and controlling wheat stem rot, regulating soil physical and chemical properties and fertility, and promoting wheat growth.

5. The use according to claim 4, characterized in that The method for 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 the wheat seedlings emerge with a bacterial suspension containing the composite microorganisms.

6. The use according to claim 5, characterized in that The total concentration of microorganisms in the bacterial suspension containing the composite microorganisms is (1-3)×10 8 cfu·mL -1 .

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