CN114907152B

CN114907152B - Application of bacillus megatherium RL-126 strain in promoting crop straw decomposition

Info

Publication number: CN114907152B
Application number: CN202210717760.9A
Authority: CN
Inventors: 张冬冬; 肖嘉文; 杨静; 刘兆厦; 樊晨曦; 孙尚仪
Original assignee: Heibei Agricultural University; Hebei Jijun Microbial Technology Co ltd
Current assignee: Heibei Agricultural University; Hebei Jijun Microbial Technology Co ltd
Priority date: 2022-06-23
Filing date: 2022-06-23
Publication date: 2023-07-18
Anticipated expiration: 2042-06-23
Also published as: CN114907152A

Abstract

The invention relates to the technical field of microorganisms and environmental protection, in particular to application of a bacillus megatherium RL-126 strain in promoting crop straw decomposition, wherein the strain is preserved in China general microbiological culture Collection center (CGMCC) No.24619 in the 3 rd month 31 th 2022; the preservation address is North Chen Silu No. 1 and No. 3 in the Chaoyang area of Beijing city. The bacillus megatherium RL-126 strain has the capability of degrading lignocellulose, can improve the degradation rate of straw, promote straw decomposition and the release of nutrient components, and increase the enzymatic activity of soil. In addition, the bacillus megaterium RL-126 strain can also antagonize crop pathogenic bacteria. After straw decomposed by bacillus megaterium RL-126 strain is returned to the field, the crop yield can be improved.

Description

Application of bacillus megatherium RL-126 strain in promoting crop straw decomposition

Technical Field

The invention relates to the technical field of microorganisms and environmental protection, in particular to application of a bacillus megaterium RL-126 strain in promoting crop straw decomposition.

Background

The Chinese is agricultural large country, the theoretical resource amount of the straw reaches 8.86 hundred million tons, the collectable resource amount is about 7.38 hundred million tons, and the corn straw accounts for about 32.5 percent. The straw is used as an agricultural solid waste, the recycling comprehensive utilization mode is various, but the transportation and storage cost as an industrial raw material is too high, so that the direct returning of the straw to the field and the conversion of the straw into an organic fertilizer are still one of the best modes of straw utilization in China at present. Crop straw is rich in organic matters and inorganic nutrients, and is an excellent organic fertilizer raw material. According to the measurement, the contents of nitrogen, phosphorus and potassium in the corn straw are respectively 0.61%, 0.21% and 2.28%, and after the straw is returned to the field and decomposed by microorganisms in the nature, N, P, K and other mineral elements and the like in the straw are released into soil, so that element circulation is realized, and the corn straw has very important significance in relieving contradiction of imbalance of nitrogen, phosphorus and potassium fertilizer proportion in China and making up for shortage of phosphorus and potassium fertilizer. Meanwhile, the straw decomposition can effectively increase the organic matter content of the soil, improve the aggregate structure and physical and chemical properties of the soil, improve the soil, fertilize the soil and increase the crop yield.

The straw is mainly composed of cellulose, hemicellulose, lignin and other straw 'three elements', and in addition, the straw also contains a certain amount of pectin, nitride and a small amount of inorganic salt nutrient. Cellulose is a polysaccharide formed by condensing a plurality of glucoses, and has relatively complex structure and large molecular weight. Hemicellulose is a heteromultimer composed of a plurality of monosaccharides. Lignin is a phenolic polymer and is mainly composed of alcohol monomers. In the straw, three elements of the straw are mutually coated and wound to form a complex and intermittent layered structure, and the complex structure makes the degradation of the straw difficult under natural conditions, so that a method for developing and utilizing the straw by utilizing the degradation has a plurality of problems. For example, if straw is directly returned to the field, the degradation speed is slow, so that soil is too loose, gaps are unbalanced, seeds cannot be fully contacted with the soil after sowing, germination growth is affected, seedlings are uneven, especially in corn-wheat rotation areas, phenomena of non-germination, weak seedlings, lodging and withering often occur after sowing wheat, freezing injury is easy to occur in winter wheat, and high yield of the wheat is seriously affected. Meanwhile, if the previous crop is ill, the straw is directly returned to the field, so that pathogenic microorganisms are spread in the field, and the disease is aggravated. The method is also one of the important reasons that a large amount of straws are not utilized properly every year in China and the phenomena of discarding and burning the straws are serious.

However, the random discarding and burning of the straw can bring a series of environmental problems. The environmental protection has long-term influence on ecological environment, economic development, human health and the like. Therefore, the method effectively degrades the straws, accelerates the comprehensive utilization of the propelled straws, is beneficial to stabilizing the ecological balance of agriculture, relieving the resource constraint, and has very important positive significance for reducing the environmental protection problems caused by discarding and burning the straws and relieving the environmental protection pressure.

Disclosure of Invention

Aiming at the technical problem that the straw is not easy to degrade, the invention provides application of bacillus megatherium RL-126 strain in promoting crop straw decomposition. The bacillus megaterium RL-126 strain can improve the degradation rate of straw and promote the decomposition of the straw, and can promote the growth of crops and improve the yield after being applied to straw returning.

In order to solve the technical problems, the embodiment of the invention adopts the following technical scheme:

in one aspect, the embodiment of the invention provides application of bacillus megaterium RL-126 strain in promoting crop straw decomposition. The bacillus megaterium RL-126 strain is separated from the rhizosphere soil of the healthy wheat seedlings in the wheat producing area, and the classification name is bacillus megateriumBacillus megaterium) The microbial strain is preserved in China general microbiological culture collection center (CGMCC) at 3/31/2022, and the preservation number is CGMCC No.24619; the preservation address is North Chen Silu No. 1 and No. 3 in the Chaoyang area of Beijing city.

Proved by experimental study, the bacillus megatherium RL-126 strain has higher cellulase activity and stronger lignin degradation capability, can improve the degradation rate of straw, promote straw decomposition and the release of nutrient components, and increase the enzymatic activity of soil. In addition, the bacillus megaterium RL-126 strain can also antagonize crop pathogenic bacteria, such as wheat take-all, wheat sheath blight and wheat root rot. After straw decomposed by bacillus megaterium RL-126 strain is returned to the field, the crop yield can be improved, and the spread of pathogenic microorganisms in the field can be reduced.

In a second aspect, the embodiment of the invention also provides a method for promoting crop straw to be thoroughly decomposed, which comprises the steps of crushing the crop straw, spraying an aqueous solution containing a nitrogen source, spraying a bacterial suspension of the bacillus megaterium RL-126 strain, and fermenting for more than 30 days.

The bacteria grow by using a carbon source and a nitrogen source, and the straw can provide the carbon source, and the injected nitrogen source-containing aqueous solution is combined, so that the growth and propagation of the bacillus megaterium RL-126 strain are facilitated, the bacterial load is increased, and the straw degradation is promoted. The nitrogen source can be urea, and the urea can be supplemented to provide nitrogen source required by bacterial growth and fertilizer required by wheat growth.

Preferably, the viable bacteria concentration of the bacterial suspension of the bacillus megaterium RL-126 strain is 1X 10 ⁷ ~2×10 ⁷ cfu/mL。

In a third aspect, the embodiment of the invention also provides a microbial inoculum comprising the live strain of the bacillus megaterium strain RL-126.

The microbial inoculum can be used for turning soil together with the straw and the fertilizer when the straw is returned to the field, promoting the degradation and decomposition of the straw in the soil, releasing nutrient components, improving the enzymatic activity of the soil, antagonizing pathogenic bacteria in the soil and promoting the healthy growth of crops.

Preferably, the microbial inoculum is a liquid microbial inoculum.

In a fourth aspect, the embodiment of the invention also provides a method for returning straw to the field, wherein the straw is crushed, the liquid microbial inoculum is applied, then the straw and the fertilizer are turned and pressed underground together, and crops are planted after 3-7 days.

After the straw and the fertilizer which are applied with the microbial inoculum containing the bacillus megaterium RL-126 live strain are jointly turned and pressed underground, the fertilizer can provide nutritional ingredients required by the growth of the bacillus megaterium RL-126 live strain, promote the growth and propagation of the bacillus megaterium RL-126 strain, further promote the degradation, fermentation and decomposition of the straw underground, release the nutrients, and can improve the soil enzyme activity, thereby improving the crop yield.

Preferably, the fertilizer is a compound fertilizer, and the application amount is 50-70 kg/mu.

Preferably, the compound fertilizer comprises the following elements in proportion: n: P ₂ O ₅ :K ₂ O=15:15:15; the total nutrient is more than or equal to 45 percent.

Preferably, the straw dosage is 3000-5000 jin/mu, and the viable bacteria concentration of the bacillus megaterium RL-126 bacterial suspension in the liquid microbial inoculum is 1 multiplied by 10 ⁹ ~2×10 ⁹ cfu/mL。

The invention has the beneficial effects that: the invention improves the degradation rate of the straw by utilizing the bacillus megaterium RL-126 strain, promotes the straw to be decomposed, reduces the spread of pathogenic microorganisms in the field caused by returning the straw to the field, is beneficial to promoting the proper utilization of the agricultural solid waste straw, and has very important positive significance for reducing the environmental damage phenomena such as straw discarding, burning and the like and reducing the environmental protection pressure.

Drawings

FIG. 1 shows the results of the detection of cellulase activity of Bacillus megaterium strain RL-126 in example 1;

FIG. 2 is a glucose content calibration curve in example 1;

FIG. 3 shows the result of cellulase activity detection of Bacillus megaterium RL-126 strain in example 1;

FIG. 4 shows the result of the detection of the filter paper enzyme activity of the Bacillus megaterium RL-126 strain in example 1;

FIG. 5 shows the results of the detection of the activity of Bacillus megaterium RL-126 strain in degrading aniline blue in example 1;

FIG. 6 shows the results of laccase viability assay of Bacillus megaterium RL-126 strain in example 1;

FIG. 7 shows the manganese peroxidase activity test result of the Bacillus megaterium RL-126 strain of example 1;

FIG. 8 shows the lignin peroxidase activity test results of Bacillus megaterium strain RL-126 of example 1;

FIG. 9 shows antagonism of the Bacillus megaterium RL-126 strain of example 1 against Rhizoctonia cerealis;

FIG. 10 shows antagonism of the Bacillus megaterium RL-126 strain of example 1 against wheat take-all;

FIG. 11 shows antagonism of Bacillus megaterium RL-126 strain in example 1 against wheat root rot.

Detailed Description

The present invention will be described in further detail with reference to specific embodiments in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

The experimental methods used in the following examples are conventional in the art unless otherwise specified.

The raw materials, reagents and the like used in the following examples were obtained from commercial sources unless otherwise specified.

In the following examples, excel 2003 software was used to process data and draw tables, SPSS13.0 software was used for statistical analysis, and Least Significant Difference (LSD) was used for differential significance checkingP＜0.05）。

Example 1

The embodiment of the invention provides application of bacillus megatherium RL-126 strain in promoting crop straw decomposition.

1. Materials and methods

1.1 corn stalk powder

Cutting corn straw into small sections of 2-3 cm, putting the small sections into a baking oven for baking, crushing the small sections by a plant crusher, and sieving the crushed small sections with a 40-mesh sieve.

1.2 Medium

All media were autoclaved for 30min at 121 ℃.

Nutrient Agar medium (NA): beef extract 5.0 g, peptone 10.0 g,NaCl 5.0 g, agar 15.0 g, water 1000 mL,pH 7.0~7.2.

NB medium: beef extract 5.0 g, peptone 10.0 g,NaCl 5.0 g, water 1000 mL,pH 7.0~7.2.

Carboxymethylcellulose (CMC) identification medium: CMC-Na 7.5 g, KH ₂ PO ₄ 1.0 g, peptone 1.0. 1.0 g, yeast extract 0.5 g, mgSO ₄ 0.5 g, naCl 1.25, g, agar 20.0, g, water 1000 mL,pH 7.0~7.2.

Aniline blue (Azure-B) medium: 5 g yeast extract, 10g tryptone, 10g NaCl, aniline blue 0.1 g, agar 20 g and distilled water 1L.

Liquid fermentation medium: adding 40 mesh corn stalk powder (10.0 g/L) into inorganic salt culture medium. Inorganic salt culture medium: KH (KH) ₂ PO ₄ 1.0 g，NaCl 0.5 g，MgSO ₄ ·7H ₂ O 0.3 g，NaNO ₃ 2.5 g，CaCl ₂ 0.1 g，(NH ₄ ) ₂ SO ₄ 1.2 g，FeSO ₄ 0.01 0g, water 100 mL,pH 7.0~7.2.

Seed culture medium: naH (NaH) ₂ PO ₄ ·2H ₂ O 2.0 g，Na ₂ HPO ₄ ·2H ₂ O4.0. 4.0 g, glucose 10.0 g, mgSO ₄ ·7H ₂ O0.5 g, peptone 10.0 g, water 1000 mL,pH 7.0~7.2.

1.3 reagents and methods of formulation

3, 5-dinitrosalicylic acid (DNS) reagent: weighing 10.0. 10.0 g of 3, 5-dinitrosalicylic acid, placing in 600 mL water, gradually adding 0.0 g of NaOH, magnetically stirring in a water bath at 50 ℃ to dissolve, sequentially adding 200.0 g of potassium sodium tartrate, 2.0 g of phenol (re-steaming) and 5.0 g of anhydrous sodium sulfite, cooling to room temperature after all the materials are dissolved and clarified, fixing the volume to 1000 mL by water, and filtering. Stored in brown reagent bottles and used after leaving 7 d in the dark.

0.05 mol/L pH 4.8 citrate buffer: weighing citric acid monohydrate 4.83 and g, dissolving in water of about 750 and mL, adding trisodium citrate 7.94 and g under stirring, fixing the volume to 1000 and mL with water, and adjusting pH to 4.8 for standby.

1.0% CMC-Na solution: weighing 2.0 g CMC-Na to 1.0 mg, slowly adding citric acid buffer solution to about 150 mL, heating to 80-90 ℃, magnetically stirring while heating until CMC-Na is completely dissolved, cooling, diluting to 200 mL with corresponding buffer solution, adjusting pH to 4.8 with 2.0 mol/L HCl or NaOH, fixing volume to 200 mL, mixing uniformly, and storing in a refrigerator at 4 ℃ for standby.

1.4 qualitative detection of cellulase Activity of Bacillus megaterium RL-126 Strain

The bacillus megaterium RL-126 strain is inoculated on a CMC differential medium plate, cultured at the constant temperature of 37 ℃ until a colony grows, poured into a Congo red solution of 1.0 g/L, and after dyeing for 1 h, congo red is discarded, 1.0 mol/L NaCl is used for soaking for 1 h decoloration, and whether a hydrolysis zone exists around the colony of the plate is observed.

As shown in FIG. 1, the hydrolysis band shows that the bacillus megaterium RL-126 strain has higher cellulase activity.

The size of the bacterial strain cellulose hydrolysis zone represents the strength of the cellulase production capability of the bacterial strain to a certain extent, and in order to further verify the reliability of test results, the fermentation broth cellulase and filter paper enzyme activities are detected in a continuous sampling mode in the research.

1.5 quantitative detection of cellulase Activity synthesized by Bacillus megaterium RL-126 Strain

Inoculating Bacillus megaterium RL-126 strain into NB medium, shake culturing at 37deg.C overnight, inoculating into liquid fermentation medium at 10deg.C (V/V), culturing at 25deg.C, collecting 5.0 mL fermentation liquid when culturing 3,5, 7, 9, 11 and d respectively, centrifuging 8000 r/min for 10 min, collecting supernatant as crude enzyme solution, and measuring cellulase activity and filter paper enzyme activity with non-inoculated blank fermentation medium as reference. The cellulase and the filter paper enzyme activity are measured by adopting a 3, 5-dinitrosalicylic acid (DNS) method.

1.5.1 glucose content Standard Curve

Accurately weighing glucose 2.5 g which is dried to constant weight at 105 ℃ and is accurate to 0.1 mg, and fixing the volume to 50mL by distilled water to prepare a glucose standard stock solution of 50 mg/mL. The standard stock solutions of glucose are respectively sucked into 50mL volumetric flasks of 0.0, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mL, distilled water is used for constant volume to 50mL, and the standard solutions with glucose concentration of 0.00, 0.50, 1.00, 1.50, 2.00, 2.50 and 3.00 mg/mL are obtained by shaking. Glucose standard solution, buffer and DNS reagent were separately aspirated into each tube (3 replicates per tube number) and mixed well as per table 1. The test tube was placed in a boiling water bath for reaction for 10 min, cooled rapidly to room temperature, and mixed well with distilled water to a volume of 25 mL, and absorbance was measured at 540 nm. And drawing a standard curve by taking the glucose amount as an abscissa and the absorbance as an ordinate to obtain a linear regression equation, wherein the linear regression coefficient can be used (or needs to be reworked) when the linear regression coefficient is more than 0.9990.

TABLE 1 glucose standard curve

The glucose content standard curve obtained is shown in FIG. 2. The regression equation of the standard curve is y=0.166 x-0.163, variance R ² =0.999, indicating that this curve has a good linear relationship.

1.5.2 Measurement of cellulase Activity

To a 25 mL stoppered tube was added 2.0 mL of a 1.0% CMC-Na solution in 0.05 mol/L pH 4.8 in citric acid buffer, and the mixture was preheated in a water bath at 50℃for 5 min. Adding 0.5 mL bacillus megaterium RL-126 strain fermentation supernatant, shaking, reacting at 50 ℃ for 30min, taking out, rapidly adding a DNS reagent 3.0 mL, placing into a boiling water bath for color development for 10 min, immediately placing into ice water for cooling to room temperature, fixing the volume to 25.0 mL with water, and shaking. The fermentation supernatant inactivated by boiling water bath under the same condition for 5min is used as a blank, the absorbance value is measured by 540 and nm, and the content of reducing sugar is obtained by looking up a glucose content standard curve in 1.5.1.

Definition of enzyme activity unit: under the above conditions, the amount of enzyme required to catalyze the substrate to 1. Mu. Mol of glucose per mL of enzyme solution in 1 min, i.e., 1U/mL. Cellulase activity calculation formula:

X=（5.56×M×n）/V×t；

wherein X, MC enzyme activity (U/mL);

m, the amount of glucose (mg) as found (or calculated) on the standard curve for absorbance;

n, dilution times of enzyme solution;

v, enzyme liquid volume (mL);

t, time (min);

5.56 is the μmol number of 1 mg glucose (1000/180=5.56).

1.5.3 Determination of Filter paper enzyme Activity

Adding 50.0 mg non-starch Xinhua filter paper and 2.0 mL 0.05 mol/L citric acid buffer solution with pH of 4.8 into a 25 mL plug test tube, preheating in a water bath at 50 ℃ for 5min, adding 0.5 mL of fermentation supernatant of bacillus megaterium RL-126 strain, inactivating the fermentation supernatant in a boiling water bath under the same condition for 5min as a blank control, preserving heat in the water bath at 50 ℃ for 1 h, immediately adding 3.0 mL of DNS solution into the test tube, standing in the boiling water bath for 10 min after shaking fully, immediately cooling, fixing the volume to 25.0 mL by using distilled water, and fully and uniformly mixing. 540 The OD value of the sample is measured at nm, the corresponding glucose amount is found on a glucose standard curve in 1.5.1, and the filter paper enzyme activity is calculated according to the glucose amount. The definition and calculation formula of the enzyme activity unit are the same as the calculation formula of the cellulase activity of 1.5.2.

1.5.4 Results

The result shows that the cellulase activity of the bacillus megaterium RL-126 strain reaches 18.97U/mL (shown in figure 3) when the strain is fermented 11 and d, and the filter paper enzyme activity is 19.34U/mL (shown in figure 4).

1.6 qualitative detection of lignin degrading Capacity of Bacillus megaterium RL-126 Strain

The lignin degrading capacity of the bacillus megaterium RL-126 strain is carried out by adopting an Azure-B plate decoloring method. Inoculating bacillus megaterium RL-126 strain on an aniline blue culture medium by a cross streaking method, culturing in a dark place at 37 ℃ for 5 d, and qualitatively detecting lignin degradation condition according to decolorization circles of bacterial colonies in the culture medium. As shown in FIG. 5, the Bacillus megaterium RL-126 strain has high aniline blue degradation activity.

To further verify the reliability of the test results, the present study examined fermentation broth laccase, manganese peroxidase and lignin peroxidase activities using a continuous sampling format.

1.7 Quantitative detection of lignin degradation capability of bacillus megaterium RL-126 strain

Inoculating Bacillus megaterium RL-126 strain into NB medium, shake culturing at 37deg.C overnight, inoculating into liquid fermentation medium at 10deg.C (V/V), culturing at 25deg.C, collecting 5.0 mL fermentation liquid when culturing 3,5, 7, 9, 11 and d respectively, centrifuging 4000 r/min for 10 min, collecting supernatant as crude enzyme liquid, and measuring laccase, manganese peroxidase and lignin peroxidase activities with non-inoculated blank fermentation medium as reference.

Laccase activity determination: bacillus megaterium RL-126 strain laccase activity was determined by monitoring the oxidation of ABTS (2, 2-azino-di-3-ethylbenzothiazole-6-sulfonic acid). The reaction system was 3 mL, and the reaction mixture contained 2 mL of 0.5 mmol/L ABTS (dissolved in 0.1 mmol/L acetic acid-sodium acetate buffer solution having pH 5.0), and the reaction was started by adding 1mL to the fermentation supernatant of the RL-126 strain at 37℃and the change in absorbance was measured at 420 nm. One enzyme activity unit (U) is defined as the amount of enzyme required to oxidize 1. Mu. Mol of ABTS per minute.

Manganese peroxidase activity determination: 50 mmol/L sodium lactate buffer (pH 4.5) 3.4 mL,1.6 mmol/L MnSO ₄ 0.1mL of solution, 0.4 mL of fermentation supernatant of Bacillus megaterium RL-126 strain, and adding 1.6 mmol/L of H by preheating to 37 DEG C ₂ O ₂ The solution 0.1mL starts the reaction and the change in absorbance at λ=240 nm is measured over the first 3 minutes of the reaction, one enzyme activity unit being expressed as the increase in absorbance per minute by 0.1.

Measurement of lignin peroxidase activity: the assay was performed by monitoring the oxidation of veratrole to veratraldehyde at 310 nm. The reaction system was 3 mL, the reaction mixture contained 1.85 mL 0.24 mmol/L resveratrol and 1.0 mL fermentation supernatant, and 0.1mL of 6 mmol/L H was added after preheating to 37 ℃ ₂ O ₂ The reaction was started and the increase in absorbance at 310nm after 3 min was measured. One viability unit (U) is defined as the amount of enzyme required to oxidize 1. Mu. Mol of veratrole per minute.

The detection result shows that the laccase activity reaches the maximum value of 11.68U/mL (shown in FIG. 6) when the bacillus megaterium RL-126 strain is cultured to 9 d; manganese peroxidase activity reached a maximum of 54.66U/mL at 7 in d culture (as shown in FIG. 7); at 9 d culture, lignin peroxidase activity reached a maximum of 6.84U/mL (as shown in FIG. 8).

1.8 Straw decomposition rate detection method by bacillus megatherium RL-126 strain

Selecting complete corn straw with approximate thickness and length, cutting into about 2cm small sections, weighing 40 g straw (oven-dried weight), spraying deionized water 10 mL dissolved with 0.4 g urea, treating 1 group (T1), and uniformly spraying living bacteria content 1×10 on the straw ⁷ cfu/mL bacillus megatherium suspension 10 mL, 2 groups (T2) treated, and the content of viable bacteria evenly sprayed on straw is 2 multiplied by 10 ⁷ cfu/mL of Bacillus megaterium suspension 10 mL sprayed with deionized water as Control (CK). And (3) filling the straws into a 40-mesh nylon mesh bag, and horizontally burying the straws into a soil layer with about 5-10 cm (based on the soil covering thickness of 5 cm). Sampling time is 30, 90 and 150 d after burying, 3 repetitions are taken each time, the sample is washed to be colorless by water, and the sample is dried and weighed, so that the decomposition rate of the straw is calculated. Straw decomposition rate% = straw post-decomposition dry mass/pre-decomposition dry mass.

The results are shown in Table 2:

TABLE 2 influence of Bacillus megaterium RL-126 Strain on straw degradation Rate

Note that: data in the table are mean ± standard deviation. The data of different processing groups under the same detection time and represented by different letters after the data of the same column are checked by LSD methodP<The 0.05 level difference was significant.

The results show that the difference between 30 d and 3 treatments of straw decomposition is remarkable, the degradation rate of the T2 treated straw is highest and reaches 46.17 percent, the degradation rate of the T1 treated straw is remarkably lower than that of the T2 treated straw but is remarkably higher than that of a control group, and the degradation rate of the corn straw is remarkably improved after 30 d of the bacillus megaterium RL-126 strain is utilized, so that the inoculation amount of the bacillus megaterium is improved, and the degradation rate of the corn is remarkably improved. After the bacillus megaterium RL-126 strain is utilized to digest the corn straw 90 d, compared with a control, the straw degradation rate is obviously improved, and the T2 treatment is higher than the T1 treatment, but the difference between the two is not obvious. After the bacillus megaterium treatment of 150 d, the differences between the three treatments were not significant. Test results show that the bacillus megaterium RL-126 strain remarkably improves the degradation rate of corn straws, improves the inoculation dosage of the microbial inoculum and remarkably improves the degradation rate of the straws. However, after long-time treatment, the promoting effect of the microbial inoculum on straw decomposition is weakened. The addition of the bacillus megatherium RL-126 has remarkable action effect in short-time treatment (30 d), which shows that the application of the microbial inoculum can rapidly promote the straw to be decomposed and degraded, thereby avoiding the adverse effect of straw returning on wheat seedling emergence and promoting the wheat seedling emergence and growth.

1.9 Field effect test for promoting corn stalk decomposition by bacillus megaterium RL-126 strain

The test site is Zhang Zhuangcun in Qingyuan area of baoding city in Hebei province, the topography is flat, and the water pouring condition is good. Corn stalk is returned to field after mechanical crushing, the test is totally provided with 3 treatments, each treatment 666.7 m ² . Treatment 1, cleaning up straws after corn is harvested manually, and turning over and pressing the fertilizer into the ground when the fertilizer is turned over, wherein the fertilizer is 60 kg/mu mixed with Shi Fu; treatment 2: mechanically harvesting corns, crushing the straws to a grain size smaller than 2cm, mixing the crushed straws with a fertilizer by 60 kg/mu based on Shi Fu, and turning the crushed straws and the fertilizer into the ground when turning over the land; treatment 3, mechanically harvesting corns, crushing the straws to a grain size smaller than 2cm, and spraying viable bacteria content of 1X 10 per mu of land ⁹ cfu/mL of bacillus megaterium RL-126 liquid microbial inoculum 20L and a base Shi Fu mixed fertilizer 60 kg/mu, and the microbial inoculum, crushed straws and fertilizer are turned and pressed into the ground together during turning. And 5, planting wheat on the 5 th day after the soil turning, and then uniformly managing. Wherein, the compound fertilizer applied by each treatment group is the same, and N is P ₂ O ₅ :K ₂ O=15:15:15, total nutrient not less than 45%; the amount of the straws in the treatment 2 and the treatment 3 groups is 4000 jin/mu.

During the period of wheat milk ripening, the number of ears and the thousand grain weight were measured by sampling, the mu yield was reduced according to the total yield of the obtained plants, and the effect of straw returning and bacillus megaterium strain RL-126 on the yield of wheat was analyzed, and the results are shown in Table 3.

TABLE 3 influence of different treatments on wheat yield

Treatment of	Spike number/m 2	Spike and grain number	Thousand grain weight/g	Yield/(g/m 2)
					Process 1	792.17±44.46a	32.69±3.18a	31.76±1.89a	822.46±35.37b
Process 2	794.65±37.21a	33.46±2.73a	32.19±2.68a	855.90±23.51ab
					Process 3	824.89±40.17a	32.19±2.96a	34.02±2.24a	903.34±37.92a

Note that: data in the table are mean ± standard deviation. After the data in the same column, the data with different letters representing the same detection index and different treatment groups are tested by LSD methodP<The 0.05 level difference was significant.

The results show that the spike number, thousand kernel weight and yield of the wheat in treatment 2 and 3 are all increased compared with treatment 1, wherein the increase in spike number, spike number and thousand kernel weight is not significant, and the yield of the wheat in treatment 3 is significantly increased compared with treatment 1. Compared with treatment 1, the wheat yield of treatment 2 reaches 855.90 g/m ² Increase the volumeYield 4.07%; the yield of the treated 3 wheat is 903.34 g/m ² Yield is increased by 9.83%. Compared with the treatment 2, the yield of the wheat is increased by 5.54 percent, and the results show that the yield can be improved by improving the effective tillering number and the grain weight by applying the bacillus megaterium RL-126 in the straw returning field, and the effect is remarkable.

1.10 Soil physicochemical property and enzyme activity detection

And (3) collecting soil with a depth of 5-10 cm in each treatment by adopting a 5-point sampling method when the wheat in the step (1.9) is harvested. The pH value of soil is measured by a water-soil ratio leaching method of 2.5:1 (mass ratio), the content of potassium dichromate is measured by a soil organic matter measuring method, the total nitrogen measuring method is a Kjeldahl nitrogen measuring method, the alkaline hydrolysis nitrogen measuring method is an alkaline diffusion method, the quick-acting phosphorus measuring method is a sodium bicarbonate dissolution molybdenum antimony colorimetry, and the effective potassium measuring method is an ammonium acetate dissolution flame photometer method. Urease activity was detected using sodium phenolate-sodium hypochlorite colorimetric method: NH in soil at 1 g after 24 h ₃ -milligrams of N; the alkaline phosphatase activity is detected by adopting a disodium phosphate colorimetric method: expressed as mass (mg) of phenol released in the soil at 1 g after 24 h; the cellulase activity detection adopts a 3, 5-nitro salicylic acid colorimetric method: expressed in milligrams of glucose produced by 72 hours, 1 g of dry soil; the catalase activity is detected by adopting a potassium permanganate titration method: 0.1 mol/L KMnO consumed in 1/g dry soil 1 h ₄ Volume (in mL). The results of the physical and chemical properties of the soil are shown in Table 4, and the results of the biological enzyme activity of the soil are shown in Table 5.

TABLE 4 influence of different treatments on the physicochemical properties of the soil

Treatment of

Total nitrogen (%)

Alkaline hydrolysis nitrogen (mg/kg)

Quick-acting phosphorus (mg/kg)

Quick-acting potassium (mg/kg)

Organic matter (g/kg)

pH

Process 1

2.32±0.06b

147.72±11.92b

33.72±3.46b

210.48±5.24c

11.68±2.87b

8.68±0.08a

Process 2

2.54±0.05a

182.47±7.57a

41.28±3.75a

240.62±4.73b

16.77±1.28a

8.39±0.02b

Process 3

2.48±0.04a

179.90±13.40a

43.69±2.89a

261.93±5.18a

15.93±1.13a

8.22±0.03c

Note that: watch (watch)Data are mean ± standard deviation. After the data in the same column, the data with different letters representing the same detection index and different treatment groups are tested by LSD methodP<The 0.05 level difference was significant.

TABLE 5 Effect of different treatments on soil enzyme Activity

Treatment of	Urease Activity [ mg/(g. D)]	Cellulase activity [ mg/(10 g. 72 h)]	Alkaline phosphatase Activity [ mg/(g. D)]	Catalase Activity [ mL (KMnO 4)/(h.g)]
					Process 1	1.52±0.17c	3.38±0.39c	5.13±0.47c	1.21±0.09b
Process 2	2.14±0.33b	4.12±0.24b	6.34±0.56b	1.55±0.18a
					Process 3	2.95±0.42a	4.91±0.31a	7.05±0.35a	1.51±0.24a

The detection result of the physical and chemical properties of the soil shows that the straw returning to the field increases the content of total nitrogen, alkaline hydrolysis nitrogen, quick-acting phosphorus, quick-acting potassium and organic matters in the soil, promotes the pH of the soil to be reduced, and has remarkable effect. The application of bacillus megaterium RL-126 further increases the content of quick-acting phosphorus and quick-acting potassium in the soil, causing the pH of the soil to decrease, wherein the quick-acting potassium and the pH change are significant. Total nitrogen, alkaline nitrogen and organic matter were reduced, but the difference was not significant. The application of the bacillus megaterium RL-126 promotes the degradation of straws and the release of nutrient components, and has remarkable effect.

The detection result of the soil biological enzyme activity shows that the straw returning increases the activities of soil urease, cellulase, alkaline phosphatase and catalase, and the effect is remarkable. The application of bacillus megaterium RL-126 further increased the soil urease, cellulase and alkaline phosphatase activities, with significant differences in urease and cellulase activities and insignificant differences in alkaline phosphatase and catalase activities compared to treatment 2 without the addition of the microbial inoculum. Treatment 3 increased urease activity by 94.08%, cellulase activity by 45.27%, and alkaline phosphatase activity by 37.43% compared to treatment 1 without straw returning, indicating that the application of bacillus megaterium RL-126 significantly increased soil enzyme activity.

1.11 Antagonism detection of bacillus megaterium RL-126 strain on pathogenic bacteria of wheat root diseases

Wheat take-all and sheath blight pathogenic bacteria cultured at constant temperature of 26 ℃ for 5 d are inoculated in the center of a PDA plate by selecting small blocks with the diameter of 0.7 cm by a puncher. Wheat root rot pathogen is inoculated to PDA inclined plane and cultured at constant temperature of 26 deg.c for 7 d. Adding sterile water into the pathogen bevel, and scraping the pathogen with sterile glass rod to suspend the sporesAnd (5) floating. Adjusting the number of spores in the suspension to a concentration of 1X 10 with sterile water ⁶ Per mL, the number of spores was counted by means of a hemocytometer. 10 mL pathogen spore suspension and 100 mL PDA medium were mixed and poured into plates. 4 wells were punched uniformly in a crisscross manner on each plate containing 3 pathogenic bacteria, wherein 3 wells were placed with 50. Mu.L of sterile supernatant of Bacillus megaterium and the other well was added with sterile medium as a negative control. The strain was inoculated into NB medium, shake-cultured overnight at 37℃and inoculated into seed medium at 10% of the inoculum size, shake-cultured at 37℃for 48 hours. And (3) sucking 1mL of culture solution 10000r/min, centrifuging for 5min, and filtering the supernatant by a hydrophilic microporous filter membrane with the pore diameter of 0.22 mu m to obtain the sterile supernatant. And placing the flat plate in a 26 ℃ incubator for constant temperature culture overnight, and then culturing the flat plate upside down for 5-7 d. Observing the formation condition of the inhibition zone or zone.

The detection result shows that the bacillus megatherium RL-126 strain has remarkable inhibition effect on wheat take-all germ, the antibacterial band width is 1.21 cm (shown in figure 9), the antibacterial band width on Rhizoctonia cerealis is 1.12 cm (shown in figure 10), the antibacterial band has remarkable inhibition activity on wheat root rot germ, and the diameter of the antibacterial band is 2.41 cm (shown in figure 11).

Example 2

The embodiment provides a method for promoting crop straw decomposition, which comprises the following steps:

after the straw is crushed to the grain size smaller than 2cm, 250mL of 40mg/mL urea aqueous solution is sprayed per 1kg of straw, and then 250mL of viable bacteria with the content of 1 multiplied by 10 is sprayed ⁷ ~2×10 ⁷ cfu/mL of bacillus megaterium RL-126 strain suspension is fermented for more than 30 days.

Example 3

The embodiment provides a liquid microbial inoculum, which is a liquid preparation containing viable strain of bacillus megaterium RL-126, the viable bacteria content is 1 multiplied by 10 ⁹ ~2×10 ⁹ cfu/mL。

Example 4

The embodiment provides a method for returning straw to the field, which comprises the steps of crushing straw to a grain size smaller than 2cm, and spraying 20L of the liquid microbial inoculum in the embodiment 3 per mu (the straw dosage is 3000-5000 jin/mu)Then, the mixed fertilizer is mixed with a base Shi Fu fertilizer (N: P ₂ O ₅ :K ₂ O=15:15:15; total nutrient is more than or equal to 45%, the dosage is 60 kg/mu), the crops are planted after 3-7 days.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, or alternatives falling within the spirit and principles of the invention.

SEQUENCE LISTING

<110> Hebei Ji microbiological technology Co., ltd., hebei university of agriculture

<120> Bacillus megaterium RL-126 and application thereof in crop growth promotion

<130> 20220511

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1408

<212> DNA

<213> 16SrDNA

<400> 1

gcaagtcgag cgaactgatt agaagcttgc ttctatgacg ttagcggcgg acgggtgagt 60

aacacgtggg caacctgcct gtaagactgg gataacttcg ggaaaccgaa gctaataccg 120

gataggatct tctccttcat gggagatgat tgaaagatgg tttcggctat cacttacaga 180

tgggcccgcg gtgcattagc tagttggtga ggtaacggct caccaaggca acgatgcata 240

gccgacctga gagggtgatc ggccacactg ggactgagac acggcccaga ctcctacggg 300

aggcagcagt agggaatctt ccgcaatgga cgaaagtctg acggagcaac gccgcgtgag 360

tgatgaaggc tttcgggtcg taaaactctg ttgttaggga agaacaagta caagagtaac 420

tgcttgtacc ttgacggtac ctaaccagaa agccacggct aactacgtgc cagcagccgc 480

ggtaatacgt aggtggcaag cgttatccgg aattattggg cgtaaagcgc gcgcaggcgg 540

tttcttaagt ctgatgtgaa agcccacggc tcaaccgtgg agggtcattg gaaactgggg 600

aacttgagtg cagaagagaa aagcggaatt ccacgtgtag cggtgaaatg cgtagagatg 660

tggaggaaca ccagtggcga aggcggcttt ttggtctgta actgacgctg aggcgcgaaa 720

gcgtggggag caaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta 780

agtgttagag ggtttccgcc ctttagtgct gcagctaacg cattaagcac tccgcctggg 840

gagtacggtc gcaagactga aactcaaagg aattgacggg ggcccgcaca agcggtggag 900

catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat cctctgacaa 960

ctctagagat agagcgttcc ccttcggggg acagagtgac aggtggtgca tggttgtcgt 1020

cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct tgatcttagt 1080

tgccagcatt tagttgggca ctctaaggtg actgccggtg acaaaccgga ggaaggtggg 1140

gatgacgtca aatcatcatg ccccttatga cctgggctac acacgtgcta caatggatgg 1200

tacaaagggc tgcaagaccg cgaggtcaag ccaatcccat aaaaccattc tcagttcgga 1260

ttgtaggctg caactcgcct acatgaagct ggaatcgcta gtaatcgcgg atcagcatgc 1320

cgcggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccacga gagtttgtaa 1380

cacccgaagt cggtggagta actagccg 1408

Claims

1. The application of the bacillus megatherium RL-126 strain in promoting crop straw decomposition is characterized in that the preservation number of the bacillus megatherium RL-126 strain is CGMCC No.24619; the method for promoting the crop straw to be decomposed by using the bacillus megaterium RL-126 comprises the following steps: crushing straw, spraying an aqueous solution containing a nitrogen source, spraying a bacterial suspension of the bacillus megaterium RL-126 strain, and fermenting for more than 30 days.

2. The use according to claim 1, characterized in that said giant budThe viable bacteria concentration of the bacterial suspension of the bacillus RL-126 strain is 1 multiplied by 10 ⁷ ～2×10 ⁷ cfu/mL。

3. The use according to claim 1, wherein the nitrogen source is urea.

4. A microbial agent comprising a viable strain of bacillus megaterium RL-126 strain according to claim 1.

5. The microbial inoculant of claim 4, wherein the microbial inoculant is a liquid microbial inoculant.

6. A method for returning straw to field is characterized in that the straw is crushed, the liquid microbial inoculum of claim 5 is applied, then the straw and the fertilizer are turned and pressed underground together, and crops are planted after 3-7 days.

7. The method according to claim 6, wherein the fertilizer is a compound fertilizer applied in an amount of 50-70 kg/mu.

8. The method according to claim 7, wherein the compound fertilizer comprises the following elements in proportion: n: P ₂ O ₅ :K ₂ O=15:15:15; the total nutrient is more than or equal to 45 percent.

9. The method according to claim 6, wherein the straw is used in an amount of 3000-5000 jin/mu, and the viable bacteria concentration of the bacillus megaterium RL-126 strain suspension in the liquid microbial inoculum is 1X 10 ⁹ ～2×10 ⁹ cfu/mL。