Baijiang soil straw degrading bacterium and application thereof

Abstract

The invention relates to a high-efficiency corn straw degrading bacterium, which is an actinomycete Streptomyces sp.NC5, an actinomycete Nomuraea sp.CP82, an actinomycete Pseudomonas miguae MD68, an actinomycete Streptomyces broussonetiae JD30 and a compound bacterium group, and is characterized in that the compound bacterium group comprises an actinomycete Streptomyces sp.NC5, an actinomycete Nomuraea sp.CP82, an actinomycete Pseudomonas miguae MD68 and an actinomycete Streptomyces broussonetiae JD30. The degrading bacteria or the complex microbial inoculant obtained through screening can tolerate low-temperature, high-temperature and complex acidic and alkaline environments, is suitable for the complex environment of straw returning to the field, remarkably improves the straw degradation rate, has the degradation rate of corn straw directly returned to the field in white slurry soil as high as 55%, and improves the organic matter content by about 20%.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to albicans straw-degrading bacteria and applications thereof. Background Art

[0002] White pulp soil is a soil with a dark humus surface layer (black soil layer), a grayish-white subsurface layer (white pulp layer) and a dark brown clay sediment layer formed by soil-forming processes such as white pulping under forest and meadow vegetation in temperate semi-humid and humid areas, on slightly sloping hills, on light upper and lower clay parent materials. It is mainly distributed in the northeast of Heilongjiang and Jilin provinces. White pulp soil is the main cultivated land soil in the Sanjiang Plain area and the largest obstacle soil in Heilongjiang Province, with a cultivated land area of ​​nearly 30 million mu, accounting for 12% of the province's area. The white pulp layer soil is located 20-30cm below the surface, with low organic matter content, only 1 / 3-1 / 2 of the organic matter in the black soil layer; the total nitrogen and phosphorus content is low, only about 1 / 2 of the black soil layer; the abundance of microorganisms is low and the uniformity is poor. The above characteristics lead to a shallow arable layer and low nutrient content of white pulp soil, and the crop yield is 30% lower than that of normal soil. It is not drought-resistant or waterlogged, and the yield is low and unstable. Therefore, how to improve the fertility of the white pulp layer of albic soil is an urgent problem to be solved.

[0003] Corn straw return to the field is a common method to improve soil fertility. However, due to the cold climate in the albic soil area, the degradation rate of the straw directly returned to the field is slow, and the degradation rate in one year is only about 40%. Therefore, it is necessary to study efficient albic soil straw degradation bacteria. Summary of the invention

[0004] The invention aims to provide an efficient corn stalk degrading bacterium, which is actinomycete Streptomyces p. NC5 (preservation number GDMCC No: 63431).

[0005] The invention aims to provide an efficient corn stalk degrading bacterium, which is actinomycete Nonomuraea sp. CP82 (preservation number GDMCC No: 65858).

[0006] The invention aims to provide an efficient corn stalk degrading bacterium, which is actinomycete Pseudomonas migulae MD68 (preservation number GDMCC No: 65846).

[0007] The invention aims to provide an efficient corn straw degrading bacterium, which is actinomycete Streptomyces broussonetiae JD30 (preservation number GDMCC No: 65841).

[0008] The invention aims to provide a high-efficiency corn stalk degradation composite bacterial agent, comprising actinomycete Streptomyces sp. NC5 (preservation number GDMCC No: 63431), actinomycete Nonomurae asp. CP82 (preservation number GDMCC No: 65858), actinomycete Pseudomonas migulae MD68 (preservation number GDMCC No: 65846) and actinomycete Streptomyces broussonetiae JD30 (preservation number GDMCC No: 65841).

[0009] In the preferred technical solution of the present invention, the preparation method of the composite bacterial agent is to prepare each actinomycete in the composite bacterial agent so that the effective viable count is not less than 10 9 The bacterial liquids are mixed in equal proportions to obtain a composite bacterial agent. Preferably, the effective viable bacterial count of the composite bacterial agent is not less than 10 9 Pieces / ml.

[0010] In the preferred technical solution of the present invention, the method for preparing the actinomycete liquid is as follows: streak the actinomycete strain on ISP 2 solid culture medium, culture at 28-30°C for 5-7 days, wash the spores with sterile water after spore production, and then inoculate them into ISP 2 liquid culture medium at an inoculum rate of 3-5%, culture at 30-40°C for 5-10 days under a shaking table at 180-210rpm, and obtain an effective viable count of not less than 10 9 Preferably, the effective viable count is not less than 10 9 The actinomycete liquid with a concentration of 100 μg / mL is added to the fermentation medium at an inoculation rate of 3-5%, and cultured at 180-210 rpm and 30-40° C. for 7-10 days.

[0011] In the preferred technical solution of the present invention, the I SP 2 solid culture medium formula consists of: 0.4% glucose, 1% malt extract, 0.4% yeast extract, 2% agar, pH 7.0-7.4.

[0012] In the preferred technical solution of the present invention, the I SP 2 liquid culture medium has a formula of: 0.4% glucose, 1% malt extract, 0.4% yeast extract, pH 7.0-7.4.

[0013] In the preferred technical solution of the present invention, the fermentation medium formula consists of: 0.3% yeast extract, 0.3% dipotassium hydrogen phosphate, 0.03% magnesium sulfate, 0.01% ferrous sulfate, 0.04% zinc sulfate, 0.01% calcium chloride and 0.0015% Tween 20.

[0014] Another object of the present invention is to provide the use of the efficient corn straw degrading bacteria or composite bacterial agent of the present invention in returning corn straw to the field.

[0015] In the preferred technical solution of the present invention, the specific steps of the application are: burying the corn stalks inoculated with corn stalk degrading bacteria or composite bacterial agent in the soil at a depth of 20-30 cm 10-15 days before corn sowing.

[0016] In the preferred technical solution of the present invention, the effective viable bacteria count of the corn straw degrading bacteria or composite bacterial agent is not less than 10 9 Pieces / ml.

[0017] In a preferred technical solution of the present invention, the inoculation amount of the corn straw degrading bacteria or the composite bacterial agent on the corn straw is 3-5%.

[0018] In a preferred technical solution of the present invention, the corn stalks are flattened and cut into small segments of 1-5 cm before being inoculated with the composite bacterial agent, and dried at 70-80°C.

[0019] In a preferred technical solution of the present invention, the soil is albic soil.

[0020] In a preferred technical solution of the present invention, the soil is albic soil, and the albic soil layer at a depth of 20-30 cm is albic soil.

[0021] In a preferred technical solution of the present invention, the organic matter content of the white pulp layer of the white pulp soil is less than 12 g / kg.

[0022] In a preferred technical solution of the present invention, the total nitrogen in the white pulp layer of the white pulp soil is less than 1g / kg.

[0023] In a preferred technical solution of the present invention, the total phosphorus in the white pulp layer of the white pulp soil is less than 0.5 g / kg.

[0024] In a preferred technical solution of the present invention, the total potassium of the white pulp layer of the white pulp soil is less than 7g / kg.

[0025] In the preferred technical solution of the present invention, the temperature of the white pulp layer of the white pulp soil is 1-25°C.

[0026] In a preferred technical solution of the present invention, the pH value of the white pulp layer of the white pulp soil is 5-11.

[0027] Unless otherwise specified, when the present invention relates to the percentage between liquids, the percentage is volume / volume percentage; when the present invention relates to the percentage between liquids and solids, the percentage is volume / weight percentage; when the present invention relates to the percentage between solids and liquids, the percentage is weight / volume percentage; the rest are weight / weight percentages.

[0028] Unless otherwise stated, the present invention is tested using the following methods:

[0029] (1) The determination of main components such as cellulose, lignin, hemicellulose and ash shall refer to the Fan washing method.

[0030] (2) Cellulose degradation rate (%) = (cellulose amount before treatment - cellulose amount after treatment) / cellulose amount before treatment × 100%;

[0031] Hemicellulose degradation rate (%) = (hemicellulose amount before treatment - hemicellulose amount after treatment) / hemicellulose amount before treatment × 100%;

[0032] Lignin degradation rate (%) = (lignin amount before treatment - lignin amount after treatment) / lignin amount before treatment × 100%.

[0033] Straw degradation rate (%) = (weight of straw before returning to the field - weight of residual straw after returning to the field) / weight of straw before returning to the field × 100%.

[0034] (3) Soil composition detection method

[0035] Organic matter (SOM): the method in "Soil Agrochemical Analysis (3rd Edition)" (Edited by Bao Shidan, published by China Agriculture Press in 2000, pages 34-35); total nitrogen (TN) was determined using the LY / T 1228-2015 standard ("Determination of Nitrogen in Forest Soils"); total phosphorus (TP) and total potassium (TK) were determined using the HJ 832-2017 standard ("Digestion of the Total Metal Elements in Soil and Sediment by Microwave Digestion / Inductively Coupled Plasma Mass Spectrometry"); soil moisture content: determined using a water meter.

[0036] Compared with the prior art, the present invention has the following beneficial technical effects:

[0037] 1. The four strains of corn straw degrading bacteria selected by the present invention have strong degradation capabilities of cellulose, hemicellulose and lignin. The composite bacterial agent obtained by compounding the four strains has a higher straw degradation efficiency than a single strain. The degrading bacteria or the compound bacterial agent can tolerate low temperature, high temperature (10-40°C) and complex acidic and alkaline environments (pH tolerance range 5-11), are suitable for the complex environment of returning straw to the field in albic soil, and significantly improve the straw degradation rate. In the cold albic soil area of ​​Heilongjiang Province, the direct return of corn straw degradation rate can reach a maximum of 55% from corn sowing to harvesting in one growing season, which is about 35% higher than the degradation rate of ordinary corn straw; the soil organic matter is increased by about 20%.

[0038] 2. The present invention realizes the resource utilization of waste, is easy to operate, has low cost, and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The degradation effect of the high-efficiency corn stalk degrading bacteria of the present invention on corn stalks;

[0040] Figure 2 Field test results of the high-efficiency corn stalk degradation composite microbial agent of the present invention;

[0041] Figure 3 The high-efficiency corn stalk degradation composite bacterial agent of the present invention has an improvement effect on soil fertility. DETAILED DESCRIPTION

[0042] The present invention is further described below with reference to the embodiments.

[0043] The formula of I SP 2 solid culture medium is composed of: 0.4% glucose, 1% malt extract, 0.4% yeast extract, 2% agar, and pH 7.0-7.4.

[0044] The formula of I SP No. 2 liquid culture medium is composed of: 0.4% glucose, 1% malt extract, 0.4% yeast extract, pH 7.0-7.4.

[0045] The fermentation medium formula consists of: 0.3% yeast extract, 0.3% potassium hydrogen phosphate, 0.03% magnesium sulfate, 0.01% ferrous sulfate, 0.04% zinc sulfate, 0.01% calcium chloride and 0.0015% Tween 20.

[0046] Example 1 Screening of efficient corn stalk degrading bacteria

[0047] 1. Screening of actinomycetes with straw lignin degradation ability

[0048] The soil samples were collected from the soil under the straw pile in Mohe, Heilongjiang Province. The soil samples were naturally air-dried in a cool place, ground into fine powder in a mortar, and placed in a conical flask. An appropriate amount of sterilized glass beads were added, and then oscillated in an air oscillator at a speed of 180 rpm for 0.5 hours. Then, they were diluted and spread on the surface of I SP2 solid culture medium for separation. The pH range was 7.0-7.4. The culture was inverted and cultured in an incubator at 28°C. The above strains were pure cultured, subcultured for 3-5 generations, stained with Congo red, and the strain NC5 that produced a larger transparent circle was selected.

[0049] Five sampling points were selected from the white soil layer in the white soil area of ​​the 852 Farm in Baoqing County, Shuangyashan City, Heilongjiang Province, the soil under the straw pile in the white soil area, and the soil under the straw pile in Beiji Town, Mohe City, Heilongjiang Province. Five points were randomly selected from each sampling point, and the samples were combined and processed. The soil samples were naturally air-dried in a cool place, ground into fine powder in a mortar, and placed in a conical flask. An appropriate amount of sterilized glass beads were added, and then oscillated in an air oscillator at a speed of 180 rpm for 0.5 hours, and then diluted and spread on the surface of I SP2 solid culture medium for separation. The pH range was 7.0-7.4. The culture was inverted and cultured in an incubator at 28°C. The above strains were pure cultured, subcultured for 3-5 generations, stained with Congo red, and strains CP82, MD68, and JD30 that produced larger transparent circles were selected.

[0050] The filter paper decomposition results showed that strain numbers CP82, MD68, JD30, and NC5 showed excellent performance (+++), and the filter paper lost its original shape and almost decomposed into a paste.

[0051] Molecular biological identification was performed on these four active strains, and the specific results are as follows: NC5 strain belongs to the genus Streptomyces sp., CP82 strain belongs to the genus Nonomuraea sp., MD68 strain belongs to the genus Pseudomonas migulae, and JD30 strain belongs to the genus Streptomyces broussonetiae.

[0052] Strain 1 was named as actinomycete Streptomyces sp.NC5, with the deposit number GDMCCNo:63431. The depository unit is Guangdong Provincial Microbiological Culture Collection Center. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The deposit date is July 19, 2023.

[0053] Strain 2 was named as actinomycete Nonomuraea sp. CP82. The deposit number is GDMCC No: 65858), the depository is Guangdong Microbiological Culture Collection Center, the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit date is January 24, 2025.

[0054] The strain 3 was named as actinomycete Pseudomonas migulae MD68. The deposit number is GDMCCNo:65846, the deposit unit is: Guangdong Microbiological Culture Collection Center, the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit date is January 22, 2025.

[0055] Strain 4 was named as Streptomyces broussonetiae JD30. The deposit number is GDMCC No: 65841, the deposit unit is Guangdong Microbiological Culture Collection Center, the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit date is January 22, 2025.

[0056] 2. Study on antagonism between strains

[0057] In order to determine the antagonistic effect between the strains, strains CP82, MD68, and JD30 were streaked onto I SP2 solid culture medium and then cultured at 30°C for 5 days. The growth of the colonies was then observed. The results showed that the four strains did not have antagonistic effects and could be used to construct a mixed bacterial community.

[0058] The corn stalks were collected from the Science and Technology Park of Heilongjiang Academy of Agricultural Sciences. The main components of corn stalks are shown in Table 1. The corn stalks were flattened and cut into 1 cm segments and dried at 80 °C for later use.

[0059] Table 1

[0060]

[0061] 1. Preparation of bacterial solution

[0062] Preparation of CP82 bacterial solution: The strain CP82 stored in a -80°C refrigerator was streaked and inoculated on I SP 2 solid medium, cultured at 28°C for 5 days, and after spore production, the spores were washed with sterile water, and then inoculated into I SP 2 liquid medium at a 3% inoculum amount, and cultured at 30°C for 10 days under a shaking table at 180 rpm to obtain CP82 bacterial solution, the number of effective live bacteria in the bacterial solution was not less than 10 9 Pieces / mL.

[0063] Preparation of MD68 bacterial solution: The strain MD68 stored in a -80℃ refrigerator was streaked and inoculated on I SP 2 solid medium, cultured at 28℃ for 5 days, and after spore production, the spores were washed with sterile water, and then inoculated into I SP 2 liquid medium at a 3% inoculum, and cultured at 30℃ for 10 days under a shaking table at 180rpm to obtain MD68 bacterial solution, the number of effective live bacteria in the bacterial solution was not less than 10 9 Pieces / mL.

[0064] Preparation of JD30 bacterial solution: The strain JD30 stored in a -80℃ refrigerator was streaked and inoculated on I SP 2 solid medium, cultured at 28℃ for 5 days, and after spore production, the spores were washed with sterile water, and then inoculated into I SP 2 liquid medium at a 3% inoculum amount, and cultured at 30℃ for 10 days under a shaking table at 180rpm to obtain JD30 bacterial solution, the number of effective live bacteria in the bacterial solution was not less than 10 9 Pieces / mL.

[0065] Preparation of NC5 bacterial solution: The strain NC5 stored in a -80℃ refrigerator was streaked and inoculated on I SP 2 solid medium, cultured at 28℃ for 5 days, and after spore production, the spores were washed with sterile water, and then inoculated into I SP 2 liquid medium at a 3% inoculum, and cultured at 30℃ for 10 days under a shaking table at 180rpm to obtain NC5 bacterial solution, the number of effective live bacteria in the bacterial solution was not less than 10 9 Pieces / mL.

[0066] Preparation of composite bacterial agent: CP82 bacterial solution, MD68 bacterial solution, JD30 bacterial solution and NC5 bacterial solution were mixed in equal volume ratio to obtain composite bacterial agent, and the number of effective live bacteria in the bacterial solution was not less than 10 9 Pieces / mL.

[0067] 3. Degradation test study

[0068] CP82 bacterial solution, MD68 bacterial solution, JD30 bacterial solution, NC5 bacterial solution and composite bacterial agent were added to 150 ml fermentation medium at an inoculation rate of 3%, and 3 g of crushed 40-mesh corn stalks were added. The pH value was adjusted to 7.0. After culturing at 30°C at a speed of 210 rpm for 7 days, the fermented corn stalks were taken and dried. The contents of cellulose, hemicellulose and lignin in the dried products were determined, and the degradation rate was calculated. Figure 1 .

[0069] Experimental Example 2 Field test study on high-efficiency corn straw degradation composite microbial agent

[0070] 1. Preparation of compound bacterial agents for field trials

[0071] The CP82 bacterial solution, MD68 bacterial solution, JD30 bacterial solution, and NC5 bacterial solution prepared in Test Example 1 were added to 150 ml of fermentation medium at an inoculum rate of 3%, respectively, the pH value was adjusted to 7.0, and the culture was carried out at 30° C. at a rotation speed of 210 rpm for 7 days to obtain expanded cultured CP82 bacterial solution, MD68 bacterial solution, JD30 bacterial solution, and NC5 bacterial solution;

[0072] The expanded culture CP82 bacterial solution, the expanded culture MD68 bacterial solution, the expanded culture JD30 bacterial solution, and the expanded culture NC5 bacterial solution were mixed in equal volumes to obtain a composite bacterial agent for field trials.

[0073] 2. Field return experiment

[0074] The field experiment was conducted in the Science and Technology Park of Heilongjiang Academy of Agricultural Sciences, which has a cold temperate continental climate, with an annual precipitation of 500-550 mm, a frost-free period of about 140 days, and an accumulated temperature of ≥10°C of about 2700°C. During the experiment, the temperature of the 20 cm soil plow layer was between 1.55 and 24.22°C. The experimental field was divided into four experimental areas, each with a soil area of ​​7.14 m 2 The soil was collected from the Science and Technology Park of Heilongjiang Academy of Agricultural Sciences, and the soil pH value was 6.66.

[0075] The corn stalks were collected from the Science and Technology Park of Heilongjiang Academy of Agricultural Sciences. The composition of corn stalks is shown in Table 1. The corn stalks were flattened and cut into 1 cm segments and dried at 80 °C for later use.

[0076] Set up 4 groups:

[0077] Group A: Take 600g of soil, add 24ml of water and stir to mix;

[0078] Group B: 40 g corn stalks were added to 24 ml water and stirred, then added to 600 g soil and stirred;

[0079] Group C: Take 40g corn stalks and 0.18g Wobao stalk compost (purchased from Henan Wobao Biotechnology Co., Ltd., with an effective viable count of not less than 100 million / g), add 24ml water and stir to mix, then add to 600g soil and mix;

[0080] Group D: 1.2 ml of the compound bacterial agent for field test prepared in step 1 was diluted with 22.8 ml of water, mixed with 40 g of straw, and then added to 600 g of soil and mixed evenly;

[0081] Water was added to each group to adjust the soil moisture content to 60%. The nylon mesh bag method was used. The size of the nylon mesh bag was 34cm×36cm and the aperture was 80 mesh. The samples of groups A, B, C, and D were placed in nylon mesh bags, sealed with nylon thread, and buried in a 20cm white pulp soil layer. Straw samples and soil samples were taken every 28 days for analysis.

[0082] Under the condition of direct return of corn straw to the field, the degradation rate of corn straw buried in albic soil for 28d, 56d, 84d, 112d and 140d is as follows: Figure 2 After one quarter of direct corn straw return to the field, the straw degradation rate of group D was as high as 56%, which was approximately 10% and 30% higher than that of group C and group B, respectively. The difference in corn straw degradation rate among different groups reached a significant level.

[0083] The results of soil organic matter (SOM), total nitrogen (TN), total phosphorus (TP) and total potassium (TK) in each group are shown in Figure 3 Compared with the soil blank group, the addition of the composite bacterial agent of the present invention significantly increased the levels of soil organic matter, total nitrogen, total phosphorus and total potassium.

[0084] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art may make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should all fall within the scope of protection of the claims of the present invention.

Claims

1. An efficient corn straw degrading bacterium, which is the actinomycete Streptomyces sp. NC5 (preservation number GDMCC No: 63431).

2. A highly efficient corn straw degrading bacterium, which is the actinomycete Nonomuraea sp. CP82 (deposit number GDMCC No: 65858).

3. An efficient corn straw degrading bacterium, which is the actinomycete Pseudomonas migulae MD68 (deposit number GDMCC No: 65846).

4. An efficient corn straw degrading bacterium is the actinomycete Streptomyces broussonetiae JD30 (deposit number GDMCC No: 65841).

5. A highly efficient corn straw degradation composite bacterial agent, comprising actinomycetes Streptomyces sp. NC5 (preservation number GDMCC No: 63431), actinomycetes Nonomuraea sp. CP82 (preservation number GDMCC No: 65858), actinomycetes Pseudomonas migulae MD68 (preservation number GDMCC No: 65846) and actinomycetes Streptomyces broussonetiae JD30 (preservation number GDMCC No: 65841).

6. The composite bacterial agent according to claim 5, wherein the preparation method of the composite bacterial agent is to prepare the four actinomycetes in the composite bacterial agent so that the effective viable count is not less than 10 9 / mL of actinomycete liquid, and then mix the various liquids in equal proportions to obtain a composite bacterial agent, preferably the effective viable bacterial count of the composite bacterial agent is not less than 10 9 Pieces / ml.

7. The composite bacterial agent according to claim 6, wherein the preparation method of the actinomycete liquid is as follows: streak the actinomycete strain on ISP2 solid culture medium, culture at 28-30°C for 5-7 days, wash the spores with sterile water after spore production, and then inoculate them into ISP2 liquid culture medium at an inoculum rate of 3-5%, culture at 30-40°C for 5-10 days under a shaking table at 180-200 rpm to obtain a bacterial liquid, wherein the number of effective viable bacteria in the bacterial liquid is not less than 10 9 Pieces / mL.

8. Use of the efficient corn straw degrading bacteria according to any one of claims 1 to 4 or the composite bacterial agent according to claim 5 in returning corn straw to the field.

9. The use according to claim 8, wherein the specific steps of the use are: burying the corn stalks inoculated with degradation bacteria or composite bacterial agent in the soil at a depth of 20-30 cm 10-15 days before corn sowing.

10. The use according to claim 9, wherein the soil is albic soil, and the albic soil layer at a depth of 20-30 cm is albic soil.

Images