Compound microbial agent for improving yield of red soil peanuts as well as preparation method and application of compound microbial agent

Abstract

The invention discloses a compound microbial agent for improving the yield of red soil peanuts and a preparation method and application thereof, and relates to the technical field of microorganisms, the compound microbial agent is composed of photosynthetic bacteria ISP-1 and burkholderia cepacia ISOP 5, and the mass part ratio of the photosynthetic bacteria ISP-1 to the burkholderia cepacia ISOP 5 is 1: 1. The Burkholderia cepacia ISOP 5 is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC NO: M 2020482, and the Burkholderia cepacia ISOP 5 has high acid resistance and can produce alkali and solubilize phosphorus. The invention not only provides an efficient microbial inoculum product, but also provides a complete solution from soil improvement to plant health care to scientific application, and has great application value for promoting green, high-yield and efficient planting in a red lozenia production area. Wide market development prospects and application and popularization values are realized in the field of peanut planting.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a compound microbial agent for improving the yield of red soil peanuts, and a preparation method and application thereof. BACKGROUND

[0002] Red soil is a typical soil type in southern China, which is characterized by high acidity, low organic matter content, and soil compaction. These problems have become one of the main degradation problems in southern red soil, leading to low and unstable crop yields. With the extensive use of chemical fertilizers, the soil structure and pH have further deteriorated, resulting in a further decline in crop yields and posing a great risk to the ecological system. Peanut is a major economic crop in China, especially in red soil regions such as Jiangxi and Hunan. There are various special fertilizers for peanuts on the market, but the utilization rate of these fertilizers is generally low. In order to increase production, a large amount of chemical fertilizers are used, which increases the risk of environmental pollution. In recent years, microbial agents have shown great potential in improving soil, promoting crop growth, and increasing fertilizer utilization as an environmentally friendly and sustainable agricultural technology. Previous studies have reported the application of single or compound microbial agents such as rhizobium, phosphorus solubilizing bacteria, potassium solubilizing bacteria, and biocontrol bacteria in agricultural production. However, most existing microbial agents have the problems of single function, weak specificity of strain compatibility, low survival rate of colonization in specific acidic stress of red soil, and low symbiotic efficiency with peanuts. Therefore, it is of great significance to develop a special microbial agent that can efficiently adapt to red soil environment, has multiple functions, and significantly improves peanut yield, for breaking through the yield bottleneck of peanuts in red soil regions and promoting the green and efficient planting of peanuts. Therefore, the present application provides a compound microbial agent for improving the pH of red soil, increasing the utilization rate of fertilizers, and improving the yield of peanuts, and a method for applying the same. The use of the compound microbial agent can not only increase the utilization rate of fertilizers, but also reduce the acidification of soil to some extent, increase the yield of peanuts, and reduce the use of chemical fertilizers, thereby avoiding waste and environmental pollution caused by excessive fertilization, improving the economic and environmental benefits of peanut planting, and promoting the sustainable development of agriculture in red soil regions and increasing farmers' income. SUMMARY

[0003] The present application aims to provide a compound microbial agent for improving the yield of red soil peanuts, and a preparation method and application thereof, which can be applied to peanut planting in red soil regions, improve the pH of red soil, and increase the yield of peanuts.

[0004] Technical solution: A compound microbial agent for increasing the yield of red soil flowers, which is composed of photosynthetic bacteria ISP-1 and Burkholderia cepacian ISOP 5, and the mass fraction ratio of the photosynthetic bacteria ISP-1 to the Burkholderia cepacian ISOP 5 is 1:1. The Burkholderia cepacian ISOP 5 is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC NO: M 2020482, the preservation date is September 11, 2020, and the preservation address is Wuhan University, Wuhan, China. The strain has high acid tolerance and can produce alkali and decompose phosphorus.

[0005] The photosynthetic bacteria ISP-1 is a laboratory preserved strain, which can produce EPS and has the ability to promote the growth of plants (sweet leaf), and has been recorded in the following non-patent literature: Wu J. Mechanism of Purple Photosynthetic Bacteria on Sweet Leaf Yield Increase [D]. Chinese Academy of Sciences, 2013.

[0006] Preferably, the effective viable bacterial count of the compound microbial agent is not less than 1×10 9 CFU / g.

[0007] A method for preparing the compound microbial agent, comprising the following steps: S1, preparing photosynthetic bacteria ISP-1 liquid and Burkholderia cepacian ISOP 5 liquid respectively; S2, mixing the photosynthetic bacteria ISP-1 liquid and the Burkholderia cepacian ISOP 5 liquid prepared in step S1 uniformly according to a volume ratio of 1:1 to obtain the compound microbial agent.

[0008] Preferably, the preparation method of the photosynthetic bacteria ISP-1 liquid in step S1 is as follows: inoculating photosynthetic bacteria ISP-1 strain into photosynthetic bacteria liquid medium, and culturing under light conditions at 25-35°C for 10-16 days until the culture solution is dark red to obtain the photosynthetic bacteria ISP-1 liquid.

[0009] Preferably, the photosynthetic bacteria liquid medium comprises sodium dihydrogen phosphate 5.0-6.0 g / L, sodium acetate 1.5-2.5 g / L, ammonium chloride 0.8-1.2 g / L, magnesium chloride 0.2-0.3 g / L, DL-malic acid 1.2-1.8 g / L, and vitamin B1, nicotinic acid and biotin.

[0010] Preferably, the preparation method of the Burkholderia cepacian ISOP 5 liquid in step S1 is as follows: inoculating Burkholderia cepacian ISOP 5 strain into LB liquid medium, and oscillating and culturing at 25-35°C and 150-200 r / min for 2-4 days to obtain the Burkholderia cepacian ISOP 5 liquid.

[0011] Application of the above-mentioned complex microbial agent in red soil peanut planting.

[0012] A method for improving the yield of red soil peanuts, comprising applying the above-mentioned complex microbial agent to the red soil peanut planting system, and the application amount of the complex microbial agent is 20-30 L / acre.

[0013] Preferably, the application method of the above-mentioned complex microbial agent is: applying at the time of peanut seeding, and the application amount is 20-30 L / acre; first applying fermented cow dung organic fertilizer 100 kg / acre in the planting ditch, the fermented cow dung organic fertilizer has 30% organic matter and pH<8, and then uniformly spraying the complex microbial agent on the fermented cow dung, covering a thin layer of soil and then seeding.

[0014] Beneficial effects: The Burkholderia cepacia ISOP 5 in the microbial agent of the application is screened from red soil, can better adapt to the red soil environment, rapidly colonize in the red soil and efficiently play a role. When the microbial agent is applied, it is sprayed on the fermented cow dung in the planting ditch, so that the fermented cow dung is used as the nutrient carrier of the microbial agent, greatly improving the survival and propagation of the microbial agent in the soil. Meanwhile, the peanut is seeded on the microbial agent after covering the soil, which is beneficial to the microbial agent adhering to the root system when the peanut root system grows through the soil layer, so that the microbial agent fully plays a role in promoting growth. Meanwhile, the functional bacteria in the microbial agent can convert the fixed ineffective phosphorus in the soil into effective phosphorus that can be absorbed by peanuts, improving the utilization rate of phosphorus fertilizer. The microorganisms in the microbial agent can secrete plant hormones such as auxin, stimulate root development, enhance root activity, and expand the nutrient absorption range. This not only promotes the healthy growth of the plant, but also provides sufficient nutrients for the swelling and fullness of the pods, effectively increases the number of results per plant and the weight of 100 fruits, and significantly improves the yield. The application of the microbial agent is not only inoculation, but also repair and reconstruction of the red soil micro-ecosystem. The beneficial microorganisms become the dominant population in the soil, improve the soil microbial flora, increase the soil biological activity and diversity, and thus create a soil environment suitable for the healthy growth of peanuts, promoting the yield improvement of peanuts. The application method provided by the application closely combines the peanut farming operation, ensures that the functional microorganisms are accurately delivered to the peanut rhizosphere. The method is simple to operate, easy to be accepted and popularized by farmers, and can maximize the colonization of the root system of peanuts and play its beneficial effects. The application not only provides an efficient microbial agent product, but also provides a complete solution from soil improvement to plant health care to scientific application, which has great application value for promoting green, high-yield and efficient planting of red soil peanuts. In the field of peanut planting, it has broad market development prospects and application popularization value. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Growth of the strain ISOP 5 under different pH conditions.

[0016] Figure 2 pH changes of culture solution after cultivation of strain ISOP 5 under different initial pH conditions.

[0017] Figure 3 Microbial community structure (genus-level species relative abundance map) of different treatment soils in Ji'an, Jiangxi. DETAILED DESCRIPTION

[0018] The application will be further described in detail below in combination with the following specific embodiments:

[0019] Example 1: Cultivation method of photosynthetic bacteria ISP-1 and Burkholderia cenocepacia ISOP 5

[0020] Burkholderia cenocepacia ISOP 5 was isolated from red soil, and the specific isolation and cultivation method is as follows:

[0021] Isolation method of Burkholderia cenocepacia ISOP 5: take 5 g of fresh red soil sample and add 45 mL of sterile water, shake well for 20 min, and gradient dilute to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 6 concentrations, spread the dilution on the phosphorus-solubilizing bacteria screening plate, and incubate at 30°C for 3-5 days until colonies grow, then pick the colonies with obvious hydrolysis ring (maximum diameter of hydrolysis ring) to fresh LB medium for purification 3 times, obtain pure bacteria, and inoculate the obtained bacteria into LB liquid medium, cultivate at 25-30°C, 180 r / min on a shaker for 24 hours, to obtain phosphorus-solubilizing bacteria ISOP 5. The formula of the phosphorus-solubilizing bacteria culture medium is: glucose 10 g, ammonium sulfate 0.5 g, sodium chloride 0.3 g, potassium chloride 0.3 g, magnesium sulfate 0.3 g, ferrous sulfate 0.03 g, manganese sulfate 0.03 g, calcium phosphate 10 g, agar powder 20 g, distilled water 1000 mL, adjust pH to 7.0-7.5, and sterilize at 115°C for 30 min. The formula of the LB culture medium is: sodium chloride 10 g, peptone 10 g, yeast extract 5 g, distilled water 1000 mL, adjust pH to 7.0-7.2, and sterilize at 121°C for 20 min.

[0022] The specific cultivation method of photosynthetic bacteria ISP-1 is as follows:

[0023] Photosynthetic bacteria ISP-1 culture method: 1 mL of photosynthetic bacteria ISP-1 was taken from the preserved culture solution, added to 15 mL of photosynthetic bacteria culture medium, placed in an anaerobic state under a 60 W daylight lamp, and cultured at 30 degrees Celsius for 7-14 days until the culture solution turned dark red. Then 10 mL was taken and transferred into 100 mL of fresh photosynthetic bacteria culture medium, and the photosynthetic bacteria ISP-1 seed solution was obtained after further culture for subsequent tests. The photosynthetic bacteria culture medium formula is: sodium dihydrogen phosphate 5.5 g, sodium acetate 2.0 g, calcium chloride 0.05 g, ammonium chloride 1.0 g, magnesium chloride 0.25 g, beef extract 0.2 g, DL-malic acid 1.5 g, the above medicines were sequentially dissolved in 1000 mL of distilled water, and the pH was adjusted to 6.5-7.0 with sodium hydroxide solution, then 1 mL of VB1 solution (1000 mg / L), 1 mL of nicotine solution (100 mg / L), 1 mL of biotin solution (10-50 mg / L), 0.3 mL of 1% boric acid solution, 0.05 mL of 1.6% sodium molybdate solution, 0.2 mL of 0.8% manganese sulfate solution, 0.1 mL of 0.24% zinc sulfate solution, and 0.2 mL of 0.02% copper sulfate solution were sequentially added. The above solutions were prepared in a transparent glass container and sterilized at 115°C for 30 min.

[0024] Example 2 Verification of acid tolerance and alkali production ability of Burkholderia cepacia ISOP 5

[0025] Burkholderia cepacia ISOP 5 was cultured under different pH conditions, and the OD600 of the bacterial solution was determined at regular time intervals to determine the acid tolerance of the strain. The pH of the culture solution was measured after 60 hours of culture, and the results are shown in Table 1. Figure 1 、 Figure 2 As shown in Table 1, strain ISOP 5 can grow vigorously under pH 4.0, 5.0, 6.0, and 7.0 conditions, and the pH of the culture solution after completion of the culture can reach about 8.0, indicating that strain ISOP 5 has good acid tolerance and alkali production ability.

[0026] Example 3 Effect of photosynthetic bacteria ISP-1 and Burkholderia cepacia ISOP 5 respectively and in combination on peanut planting

[0027] A long-term positioning peanut planting test was conducted, and 4 treatments were set, each with 4 replicates: (1) basic fertilization: application of peanut special fertilizer (N: 14.2%, P2O5: 11.7%, K2O: 19.2%) 40 kg / acre, fermented cow dung (organic matter: 30%) 100 kg / acre; (2) basic fertilization + ISOP 5: application of 20 L / acre ISOP 5 bacterial liquid after application of basic fertilizer; (3) basic fertilization + ISP-1: application of 20 L / acre ISP-1 bacterial liquid after application of basic fertilizer; (4) basic fertilization + ISOP 5 + ISP1: application of 20 L / acre ISOP 5 bacterial liquid and 20 L / acre ISP-1 bacterial liquid after application of basic fertilizer. 70% of the peanut special fertilizer and all the fermented cow dung were applied at the time of sowing, and the remaining 30% of the peanut special fertilizer was applied 45 days after sowing. The method of applying microbial agents: evenly spread the fermented cow dung in the planting ditch, and evenly spray the bacterial liquid on it, then cover a thin layer of soil, and then sow the peanut seeds, and finally cover the soil to complete the peanut planting.

[0028] The yield of peanuts in different treatments was determined after harvesting, and the results are shown in Table 1. The mixed bacterial agent treatment showed a stable yield-increasing effect, with an increase of 18.5%, 20.3%, 24.7% and 18.0% in 2014, 2016, 2024 and 2025, respectively, compared with the equal-nutrient control (basic fertilization) treatment; it was also higher than the single-bacterial treatment. In 2024, the yield of the compound bacterial agent treatment (basic fertilization + ISOP 5 + ISP-1) was 8.6% and 7.1% higher than that of the ISOP 5 and ISP-1 single-application treatments, respectively; in 2025, the yield of the compound bacterial agent treatment (basic fertilization + ISOP 5 + ISP-1) was 3.5% and 7.2% higher than that of the ISOP 5 and ISP-1 single-application treatments, respectively.

[0029] Soil samples were collected to determine the contents of hydrolytic nitrogen, available phosphorus and available potassium, and the protein content in peanut seeds was determined, and the results are shown in Table 2. Inoculation of ISOP 5 and ISP-1 significantly promoted the absorption of nitrogen by peanuts and increased the protein content in peanut seeds. Regardless of inoculation of ISOP 5, ISP-1 or compound bacterial agent, the soil pH increased by 0.1-0.2 units compared with the control (basic fertilization) treatment. Compared with the basic fertilization treatment, the bacterial agent treatments increased the contents of available phosphorus and potassium, with the available potassium (AK) in the ISOP 5, ISP-1 and ISOP 5+ISP-1 treatments increasing by 12%, 10% and 14%, respectively, and reaching a significant level; the content of available phosphorus in the compound bacterial agent treatment also increased by 14.02 mg / kg compared with the control (basic fertilization) treatment. The ISOP 5+ISP-1 compound bacterial agent treatment significantly improved the contents of available phosphorus and potassium in the soil and improved the utilization rate of fertilizers.

[0030] Example 4 verification of the application effect of the complex microbial inoculant

[0031] The application effect of the microbial inoculant was verified by the test, which included 2 treatments with 4 parallel samples for each treatment: (1) basic fertilization: applying peanut special fertilizer (N: 14.2%, P2O5: 11.7%, K2O: 19.2%) 40 kg / acre, and fermented cow dung (organic matter: 30%) 100 kg / acre; (2) basic fertilization + microbial inoculant: applying basic fertilizer (peanut special fertilizer (N: 14.2%, P2O5: 11.7%, K2O: 19.2%) 40 kg / acre, and fermented cow dung (organic matter: 30%) 100 kg / acre, and applying 20 L / acre of the microbial inoculant after applying the basic fertilizer. 70% of the peanut special fertilizer and all the fermented cow dung were applied at the time of sowing, and the remaining 30% of the peanut special fertilizer was applied 45 days after sowing. The application method of the microbial inoculant: the fermented cow dung was applied to the planting ditch, and then the microbial inoculant was uniformly sprayed on it, followed by covering a thin layer of soil, and then sowing the peanut seeds, and finally covering the soil to complete the planting of peanuts.

[0032] The yield of peanuts in different treatments was determined after harvesting, and plant samples were collected to determine the fruit weight, pulp weight, and hundred-grain weight, and soil samples were collected to determine the contents of hydrolyzable nitrogen, available phosphorus, and available potassium, and the pH value. The results are shown in Tables 3 and 4: compared with the basic fertilization treatment, the fruit weight, pulp weight, and plot yield of the treatment applying the complex microbial inoculant increased by 11%, 21%, and 13%, respectively. In terms of soil physical and chemical properties, compared with the basic fertilization treatment, the soil pH increased by 0.47 units after applying the complex microbial inoculant, while the basic fertilization only increased by 0.15 units. At the same time, the contents of soil hydrolyzable nitrogen, available phosphorus, and available potassium were also improved, and compared with the basic fertilization, the contents of hydrolyzable nitrogen, available phosphorus, and available potassium in the treatment applying the microbial inoculant increased by 32%, 58%, and 16%, respectively. In summary, the application of the complex microbial inoculant can significantly improve the fertilizer utilization rate, increase the pH of red soil, and increase the yield of peanuts.

[0033] The peanut rhizosphere soil was collected, and the soil microbial community structure was analyzed by high-throughput sequencing. The results of the relative abundance of the top 10 species at the genus level are as follows Figure 3As shown: compared with the base fertilization treatment, after applying the compound microbial inoculant, the relative abundance of Burkholderia-Caballeronia-Paraburkholderia, Candidatus_Nitrosotalea, Dyella, Candidatus_Solibacter in the soil was significantly increased, and these genera were reported to have the functions of phosphorus solubilization, potassium solubilization, IAA production, etc., which can promote plant growth and inhibit the growth of pathogenic bacteria. The above experimental results show that the addition of the compound microbial inoculant improves the soil microbial community and increases the abundance of beneficial microorganisms in the soil, thereby indirectly promoting the growth of peanut plants.

[0034] Table 1: Yield and growth of peanuts under different treatments

[0035]

[0036] Table 2: Quality of peanuts and analysis of soil chemical properties under different treatments

[0037]

[0038] Table 3: Determination results of peanut biomass and yield under different treatments

[0039]

[0040] Table 4: Determination results of soil available nutrients and pH under different treatments

[0041]

[0042] Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein can be applied to other embodiments without departing from the spirit or scope of the application. Accordingly, the application is not to be restricted based on the specific embodiments illustrated herein, but is to be accorded the broadest scope consistent with the principles and novel features disclosed herein.

Claims

1. A compound microbial inoculant for increasing peanut yield in red soil, characterized in that, It consists of photosynthetic bacteria ISP-1 and Burkholderia cepacia ISOP 5 with accession number CCTCC NO: M 2020482, wherein the mass ratio of photosynthetic bacteria ISP-1 to Burkholderia cepacia ISOP 5 is 1:

1.

2. The compound microbial agent according to claim 1, characterized in that, The effective viable bacteria count of the compound microbial agent is not less than 1×10⁻⁶. 9 CFU / g.

3. A method for preparing the composite microbial agent as described in claim 1 or 2, characterized in that, Includes the following steps: S1. Prepare photosynthetic bacteria ISP-1 bacterial solution and Burkholderia cepacia ISOP 5 bacterial solution respectively; S2. Mix the photosynthetic bacteria ISP-1 bacterial solution and Burkholderia cepacia ISOP 5 bacterial solution prepared in step S1 at a volume ratio of 1:1 to obtain the composite microbial agent.

4. The method according to claim 3, characterized in that, The preparation method of the photosynthetic bacteria ISP-1 bacterial solution in step S1 is as follows: the photosynthetic bacteria ISP-1 strain is inoculated into photosynthetic bacteria liquid culture medium, and cultured statically at 25-35℃ for 10-16 days under light conditions until the culture medium turns dark red, thereby obtaining the photosynthetic bacteria ISP-1 bacterial solution.

5. The method according to claim 4, characterized in that, The photosynthetic bacteria liquid culture medium contains: sodium dihydrogen phosphate 5.0-6.0 g / L, sodium acetate 1.5-2.5 g / L, ammonium chloride 0.8-1.2 g / L, magnesium chloride 0.2-0.3 g / L, DL-malic acid 1.2-1.8 g / L, as well as vitamin B1, niacin and biotin.

6. The method according to claim 3, characterized in that, The method for preparing the Burkholderia cepacia ISOP 5 bacterial suspension in step S1 is as follows: Burkholderia cepacia ISOP 5 bacterial strain is inoculated into LB liquid medium and cultured with shaking at 25-35℃ and 150-200 r / min for 2-4 days to obtain the Burkholderia cepacia ISOP 5 bacterial suspension.

7. The application of the compound microbial agent according to claim 1 or 2 in peanut cultivation in red soil.

8. A method for increasing peanut yield in red soil, characterized in that, This includes applying the compound microbial agent as described in claim 1 or 2 to a red soil peanut planting system, wherein the amount of the compound microbial agent is 20-30 L / mu.

9. The method according to claim 8, characterized in that, The application method is as follows: 100 kg / mu of fermented cow manure organic fertilizer is evenly spread in the planting furrow. The fermented cow manure organic fertilizer has 30% organic matter and pH < 8. Then, the compound microbial agent is evenly sprayed on the organic fertilizer, covered with a thin layer of soil, and then sowing is carried out.

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