Application of bacillus aerophilus strain in promoting growth of alfalfa

Abstract

The invention belongs to the technical field of microorganisms, and relates to application of an aerobacillus strain in promoting alfalfa growth, in particular to application of the aerobacillus strain in promoting plant growth by accelerating soil matrix mineralization and soil matrix humification. According to the method disclosed by the invention, the growth environment of alfalfa is improved by utilizing an aerobacillus strain, the aerobacillus still keeps relatively high activity in a drought and high-temperature environment, the survival rate and the emergence rate of seeds can be improved by accelerating mineralization and humification of a soil matrix, the dosage is small, and acidification of the soil matrix is avoided.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology and relates to the application of an Aerobacillus strain in promoting the growth of alfalfa. Background Art

[0002] Alfalfa is a perennial herbaceous plant with a fibrous root system. Alfalfa contains a large amount of protein, dietary fiber, various minerals, trace elements, active polysaccharides and other nutrients, and is a nutritious food with high edible value; alfalfa also has high medicinal value and can be used to prevent and treat arthritis, gout, hepatitis, cholecystitis, kidney stones, diabetes, cardiovascular and cerebrovascular diseases, cancer and other symptoms. In the process of alfalfa cultivation, how to improve soil fertility and promote the healthy growth of plants has always been the focus of attention.

[0003] Since plant growth generally requires strict management of light, watering frequency and irrigation method, nutrient supply, pruning, temperature and humidity control, seedling management, pest and disease control, etc. Nutrient supply, as an important link in plant growth, can provide appropriate nutrients for plants, so nutrient supply is crucial for growth. The way of nutrient supply is: using suitable fertilizers to provide the necessary nitrogen, phosphorus, potassium and other nutrient elements for plants to promote their growth.

[0004] At present, the fertilizers for promoting the growth of alfalfa include chemical fertilizers, organic fertilizers and trace element fertilizers; although chemical fertilizers have significant effects and are convenient to use, they will cause environmental pollution, soil acidification and other problems; although organic fertilizers have good effects on improving alfalfa planting soil, their nutrient release is slow, the application amount is large, and they may bring pests and diseases; trace element fertilizers are often suitable for soils lacking certain elements and need to be used with caution to avoid overdose.

[0005] At present, microbial agents, as a new type of agent for promoting plant growth, can promote plant growth; since alfalfa is a crop with high environmental requirements, especially alfalfa needs to grow in a dry and relatively high-temperature arid environment, but the arid environment with relatively high temperature has high requirements for the performance of microbial agents, because the activity of microbial agents is easily inactivated in the arid environment, affecting the growth rate of plants.

[0006] In summary, due to the technical defects such as pollution and acidification, large application amount and inactivation at high temperature in the existing methods for promoting the growth of alfalfa, there is an urgent need to find a new method for promoting the growth of alfalfa. Summary of the Invention

[0007] Based on the technical problems such as pollution and acidification, large application amount and inactivation of the fertilizers for promoting the growth of alfalfa in the above background art, the present invention provides the application of an Aerobacillus strain in promoting the growth of alfalfa.

[0008] The present invention applies an Aerobacillus strain to the soil substrate for alfalfa cultivation. The Aerobacillus remains highly active in arid and high-temperature environments, can improve the survival rate and emergence rate of seeds by accelerating the mineralization and humification of the soil substrate, uses less amount, and avoids soil substrate acidification.

[0009] To achieve the above object, the technical solution adopted by the present invention is:

[0010] Application of an Aerobacillus strain in promoting alfalfa growth.

[0011] Application of an Aerobacillus strain in promoting alfalfa growth by accelerating the mineralization of the soil substrate.

[0012] Application of an Aerobacillus strain in promoting alfalfa growth by accelerating the humification of the soil substrate.

[0013] Further defined, the application of the Aerobacillus strain in promoting alfalfa growth by improving the germination rate, above-ground height, root length, root number, and fresh weight of alfalfa.

[0014] Further defined, the preservation number of the Aerobacillus strain is CCTCC M 20232075.

[0015] Further defined, the application process is:

[0016] S1. Form a soil substrate from coal gangue and sand; in the soil substrate, the mass ratio of coal gangue is 0% - 90%;

[0017] S2. Sow the pre-soaked alfalfa seeds in the soil substrate;

[0018] S3. Continue to add the bacterial liquid of Aerobacillus to the soil substrate. The addition amount of the bacterial liquid for every (950g ± 10g) soil substrate is 3ml - 12ml;

[0019] S4. Plant the alfalfa seeds according to conventional operations.

[0020] Further defined, in the soil substrate, the mass ratio of coal gangue is 30%.

[0021] Further defined, the addition amount of the bacterial liquid for every (950g ± 10g) soil substrate is 6ml - 9ml.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] 1. In the present invention, due to the fact that Bacillus aerius can maintain a high activity even in a relatively high-temperature environment and has extremely strong heat resistance, Bacillus aerius can accelerate the mineralization of the soil matrix in the soil, converting nutrients from the invalid and slow-acting states into the effective and rapid-acting states. At the same time, it accelerates the humification of the soil matrix, secretes phytase, degrades most of the phytates in the soil matrix, thereby generating auxin, stimulating the growth of alfalfa, improving the survival rate and emergence rate of alfalfa seeds, and enabling alfalfa to grow better in an arid environment. Thus, it provides a new idea for the use of Bacillus aerius.

[0024] 2. In the present invention, when spraying the bacterial solution of Bacillus aerius into the soil matrix for alfalfa planting, it can promote the formation of aggregate structure, improve the soil's fertilizer and water retention capacity, increase soil looseness, and promote root growth.

[0025] 3. In the present invention, the dosage of Bacillus aerius is optimized. When the addition amount of Bacillus aerius in the soil matrix is 6 ml to 9 ml, the germination rate of alfalfa can be maximally improved.

[0026] 4. In the present invention, through the optimization of the composition of the soil matrix, it is found that when the soil matrix contains coal gangue, the combination of coal gangue and Bacillus aerius can have a synergistic promoting effect on the improvement of the soil matrix, maximally improving the germination rate of plants, and thus accelerating the growth rate of alfalfa. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the absorbance change curve of Bacillus aerius Y-33 at 450 nm;

[0028] Figure 2 is the three-dimensional fluorescence result of the soil matrix (original sample);

[0029] Figure 3 is the three-dimensional fluorescence result of the degradation of the soil matrix by Bacillus aerius Y-33;

[0030] Figure 4 is the scanning electron microscope image of the soil matrix (original sample);

[0031] Figure 5 is the scanning electron microscope image of the degradation of the soil matrix by Bacillus aerius Y-33;

[0032] Figure 6 is the infrared spectrum of the degradation of the soil matrix by Bacillus aerius Y-33;

[0033] Figure 7 is the germination rate of alfalfa by Bacillus aerius Y-33 at 5 days;

[0034] Figure 8 is the germination rate of alfalfa by Bacillus aerius Y-33 at 25 days;

[0035] Figure 9 Comparison of the rhizomes per plant of alfalfa at 15 d and 30 d

[0036] Figure 10 Comparison of the germination rate of alfalfa by Bacillus aerius Y-33 at 7 d

[0037] Figure 11 Comparison of the root lengths of alfalfa by Bacillus aerius Y-33 at different degradation times

[0038] Figure 12 Comparison of the fresh weights of alfalfa by Bacillus aerius Y-33 at different degradation times

[0039] Figure 13 Comparison of the number of roots of alfalfa by Bacillus aerius Y-33 at different degradation times

[0040] Figure 14 Comparison of the above-ground heights of alfalfa by Bacillus aerius Y-33 at different degradation times

[0041] Figure 15 Growth conditions of alfalfa under different amounts of coal gangue Detailed implementation mode

[0042] The technical solutions of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0043] Unless otherwise defined, the technical terms or scientific terms used in the present invention shall have the ordinary meanings understood by those of ordinary skill in the art to which the present invention belongs.

[0044] For technologies, methods, and equipment known to those of ordinary skill in the relevant fields, they may not be discussed in detail, but where appropriate, the said technologies, methods, and equipment should be regarded as part of the specification.

[0045] In addition, it should be understood that although this specification is described in terms of embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art. These other embodiments are also covered by the protection scope of the present invention.

[0046] The present invention uses Bacillus aerius to improve the soil matrix for alfalfa planting and promote the growth of alfalfa.

[0047] Preferably, the preservation number of Bacillus aerius is CCTCC M 20232075, which is preserved in the China Center for Type Culture Collection and denoted as strain Y-33. This Bacillus aerius can maintain high activity in a relatively high-temperature environment and has extremely strong high-temperature resistance, enabling alfalfa to grow better in arid environments.

[0048] In the present invention, the application of Bacillus aerius strain in promoting the growth of alfalfa.

[0049] In the present invention, the application of Bacillus aerius strain in promoting the growth of alfalfa by accelerating the mineralization of soil matrix.

[0050] In the present invention, the application of Bacillus aerius strain in promoting the growth of alfalfa by accelerating the humification of soil matrix.

[0051] In the present invention, the application of Bacillus aerius strain in promoting the growth of alfalfa by increasing the germination rate, above-ground height, root length, root number, and fresh weight of alfalfa.

[0052] The application of Bacillus aerius strain in promoting the growth of alfalfa in the present invention has the following application process:

[0053] S1. Form a soil matrix with coal gangue and sandy soil; in the soil matrix, the mass ratio of coal gangue is 0% - 90%.

[0054] S2. Sow the pre-soaked alfalfa seeds into the soil matrix.

[0055] S3. Continuously add the bacterial liquid of Bacillus aerius to the soil matrix. The addition amount of the bacterial liquid is 3 ml - 12 ml per (950 g ± 10 g) of soil matrix.

[0056] S4. Plant the alfalfa seeds according to conventional operations.

[0057] Preferably, in the soil matrix, the mass ratio of coal gangue is 30%.

[0058] Preferably, the addition amount of the bacterial liquid is 6 ml - 9 ml per (950 g ± 10 g) of soil matrix.

[0059] The following explains the promotion mechanism and promotion effect of Bacillus aerius on the growth of alfalfa through aspects such as the biodegradation activity and humification effect of Bacillus aerius on the soil matrix, the promotion of plants by Bacillus aerius and the soil matrix, and the optimization of promotion conditions.

[0060] I. Biodegradation activity and humification effect

[0061] The humus-like substances in the soil matrix treated with bacteria and the control samples were characterized using a fluorescence spectrophotometer.

[0062] Mix the bacterial culture (the bacterial solution of Bacillus aerius) with the soil matrix (the mass ratio of coal gangue to sandy soil is 1:1), with 3 ml of the bacterial solution of Bacillus aerius in every 1000 g of the soil matrix, and culture it on a shaker at 30 °C and 160 rpm. Use the ultraviolet-visible absorption spectrometry at 450 nm to measure the absorbance of the supernatant and evaluate the biodegradation activity of the soil matrix.

[0063] The humification of the soil matrix was measured using a Duetta fluorescence spectrophotometer with a 75 W xenon lamp as the light source. Briefly, the excitation light wavelength was 225 - 600 nm, the emission light wavelength was 270 - 600 nm, the increment was 5 nm, and the response time was set to automatic. Data analysis was performed using Origin software to generate a contour map. To correct for the Raman scattering of water, ultrapure water was used as the blank.

[0064] See Figure 1 , the biodegradation activity increases with the increase of the culture time. When the Y-33 bacteria and the soil matrix are cultured for 6 days, the biodegradation activity is significantly enhanced. And after 15 days of culture, the A450 value reaches the maximum, indicating that the Y-33 bacteria can promote the degradation of organic matter in the soil matrix.

[0065] See Figure 2 and Figure 3 , the three-dimensional (3D) fluorescence data show that the humic acid-like fluorescence peak (Ex / EM: 340 nm / 470 nm) in the bacterial-treated soil matrix containing coal gangue is much stronger than that of the control sample. These data indicate that the Y-33 bacteria can promote the humification and biodegradation of the soil matrix.

[0066] II. Analysis of coal gangue before and after bacterial treatment

[0067] The surface morphology and composition of the original coal gangue and the bacterial-treated sample were characterized using a scanning electron microscope (SEM) (Zeiss Sigma 500, Germany) combined with a Bruker XFlash 6130 detector. The surface functional groups of the coal gangue samples were measured using a Fourier transform infrared spectrometer (NicoletiS20, Thermo Scientific, USA) (FTIR). The scanning range was 4000 - 400 cm -1 , and the resolution was 0.06 cm -1 . The FTIR spectra and data were analyzed using Origin 2018 software.

[0068] See Figure 4 and Figure 5, The SEM analysis of the original and bacteria-treated coal gangue is shown in the figure. The microstructure of the raw coal gangue shows an obvious planar flaky structure with a rough surface. In addition, the internal mineral particles in the coal gangue seem to be closely combined. After bacteria treatment, it can be clearly seen that Y-33 bacteria adhere to the surface of the coal gangue, and the surface of the coal gangue is slightly smoother than that of the original coal gangue. In summary, Y-33 bacteria can promote the biodegradation of coal gangue by adhering to its surface.

[0069] See Figure 6 As shown in the infrared result graph of -1 , the absorption peak at the wavenumber of 3422 cm -1 increases. While the absorption peaks at the wavenumbers of 1617 cm -1 and 1443 cm -1 , representing the stretching vibration of aromatic ring C=C and aliphatic - methylene respectively, decrease, indicating that C=C and -CH2 may be degraded by Y-33 bacteria. In addition, at 1029 cm

[0070] III. Verification of the effect of promoting alfalfa growth

[0071] The test soil and coal gangue were collected from Yulin, Shaanxi. Plastic pots with a diameter of 15.5 cm and a height of 10.9 cm were used, and each pot contained 500 cm 3 (950 g ± 10 g) of soil matrix. Clean alfalfa seeds were sown and the experiment was carried out at room temperature after pre-soaking the seeds. A blank group, treatment group 1 and treatment group 2 were established, and each group had 3 parallel samples.

[0072] The blank group was not added with 3 ml of the bacterial solution of Bacillus aerius, and the soil matrix was a 1:1 ratio of coal gangue to sandy soil.

[0073] Treatment group 1 was added with 3 ml of the bacterial solution of Bacillus aerius, and the soil matrix was sandy soil.

[0074] Treatment group 2 was added with 3 ml of the bacterial solution of Bacillus aerius, and the soil matrix was a 1:1 ratio of coal gangue to sandy soil.

[0075] The bacterial solution of Bacillus aerius was obtained by culturing Bacillus aerius in LB liquid medium for 24 hours and measuring its absorbance at 600 nm to be 1.357 with a spectrophotometer.

[0076] Further analyze the performance of each group after treatment.

[0077] (1) Remove weeds according to the principles of early, small-scale, and clean removal. Before watering, level the soil to ensure consistent seedling growth. After watering, use a measuring cylinder for quantitative irrigation, with the same amount of water for each treatment. After 15 days of planting, collect the soil from three groups for testing respectively. The results are shown in Table 1.

[0078] Table 1 Comparison of soil indicators for each group

[0079]

[0080] The main function of urease is to decompose urea, release ammonia, and promote nitrogen cycling and plant nitrogen uptake. It is widely used in agriculture and helps with the nitrogen release of urea fertilizers. The main function of phosphatase is to decompose organic phosphorus compounds, release inorganic phosphate, and promote phosphorus cycling and plant phosphorus uptake. It plays a crucial role in soil fertility and plant growth. Humic acid, humic acid, and fulvic acid are three main organic substances, widely present in natural resources such as soil, peat, compost, coal, and humus. They are all part of humus and have important environmental and agricultural functions, especially playing a key role in improving soil quality, promoting plant growth, and the health of the ecosystem. IAA is an important plant hormone in the plant life cycle, belonging to the auxin class of substances, widely present in plants, and playing a key role in plant growth and development, especially playing an important regulatory role in aspects such as growth, development, and morphogenesis.

[0081] It can be seen from Table 1 that compared with the blank group, in treatment group 1, Bacillus aerius is applied to the soil matrix containing sandy soil, which can increase the contents of urease, phosphatase, humic acid, humic acid, fulvic acid, and IAA. Bacillus aerius can also improve the organic matter in the soil. This shows that adding Bacillus aerius to the soil matrix containing sandy soil improves the soil for alfalfa growth.

[0082] Furthermore, compared with the blank group and treatment group 1, in treatment group 2, Bacillus aerius is applied to the soil matrix containing coal gangue and sandy soil. Bacillus aerius can degrade coal gangue, enabling the synergistic effect of the microbial action of Bacillus aerius and the degradation of coal gangue, accelerating the humification of the soil matrix. Furthermore, the mineral components in coal gangue become available elements for plants, and the production of auxin has a promoting effect on plant growth.

[0083] (2) Remove weeds according to the principles of early, small-scale, and clean removal. Before watering, level the soil to ensure consistent seedling growth. After watering, use a measuring cylinder for quantitative irrigation, with the same amount of water for each treatment. After 15 days of planting, uproot the alfalfa seedlings of the blank group and treatment group 2, and wash the rhizosphere soil.

[0084] Measure various parameters such as the germination rate, above-ground height, root length, number of roots, fresh weight, etc. of individual seedlings. The results are as Figures 7 - 14 . In the figure: *, **, *** indicate significant differences at the P<0.05, P<0.01, and P<0.001 levels, respectively.

[0085] See Figures 7 - 9 , Bacillus aerius has an obvious promoting effect on the growth of alfalfa. This is because when the bacterial solution of Bacillus aerius is sprayed into the soil matrix for alfalfa planting, it can promote the formation of aggregate structure, improve the soil's fertilizer and water retention capacity, increase soil looseness, and promote root growth.

[0086] See Figures 10 - 14 , it can be found from the results of the germination rate, above-ground height, root length, number of roots, and fresh weight that after treatment with Bacillus aerius, the germination rate, above-ground height, root length, number of roots, and fresh weight of alfalfa can be improved.

[0087] IV. Influence of Bacterial Addition Amount on Plant Promotion

[0088] The test soil and coal gangue were collected from Yulin, Shaanxi. Plastic pots with a diameter of 15.5 cm and a height of 10.9 cm were used, and each pot contained 500 cm 3 (950 g ± 10 g) of soil matrix. Clean alfalfa seeds were sown, and the experiment was carried out at room temperature after soaking the seeds in advance. A control group and an experimental group were established, and the bacterial solution of Bacillus aerius was added, with 3 parallel samples set in each group.

[0089] Experimental group 1: The soil matrix is sandy soil; Experimental group 2: The soil matrix is a mixture of sandy soil and coal gangue with a mass ratio of 1:1.

[0090] Both experimental group 1 and experimental group 2 were set with 5 soil matrices, and the addition amounts of the bacterial solution of Bacillus aerius were 0, 3 ml, 6 ml, 9 ml, and 12 ml in sequence.

[0091] The bacterial solution of Bacillus aerius was obtained by culturing Bacillus aerius in LB liquid medium for 24 hours and measuring its absorbance at 600 nm with a spectrophotometer to be 1.357.

[0092] For the seeds sown in each soil matrix of the above two groups, weeds were removed according to the principles of early, small, and clean removal. Before watering, the soil was leveled to ensure the consistent growth of seedlings. After watering, a measuring cylinder was used for quantitative irrigation, and the amount of water for each treatment was the same. After 15 days of planting, the growth of alfalfa was observed and the germination rate was calculated. The results are shown in Table 2.

[0093] Table 2 Germination Rate Results of Different Groups (%)

[0094]

[0095]

[0096] As can be seen from Table 2: In the soil matrix composed of sandy soil, after using the bacterial solution, the germination rate of alfalfa can be improved. However, when the dosage of the bacterial solution is too much, it will inhibit the growth of alfalfa. At the same time, under the same dosage, when adding coal gangue to the soil matrix, it can produce a synergistic promotion effect on the improvement of the soil matrix, maximize the germination rate of plants, and then accelerate the growth rate of alfalfa.

[0097] V. Influence of Coal Gangue Content on Plant Promotion

[0098] The test soil and coal gangue were collected from Yulin, Shaanxi. Plastic pots with a diameter of 15.5 cm and a height of 10.9 cm were used, and each pot contained 500 cm 3 (950 g ± 10 g) of soil matrix. Clean alfalfa seeds were sown, and the experiment was carried out at room temperature after soaking the seeds in advance.

[0099] The soil matrix was mixed with sandy soil and coal gangue. When mixing, the mass ratio of coal gangue in the soil matrix was 10%, 30%, 60%, and 90% respectively. 3 ml of the bacterial solution of Bacillus aerius was added.

[0100] The bacterial solution of Bacillus aerius was obtained by culturing Bacillus aerius in LB liquid medium for 24 hours and measuring its absorbance at 600 nm with a spectrophotometer to be 1.357.

[0101] For the seeds sown in the above four groups of soil matrices, weeds were removed according to the principles of early, small-scale, and clean removal. Before watering, the soil was leveled to ensure the consistent growth of seedlings. After watering, quantitative irrigation was carried out using a measuring cylinder, and the amount of water for each treatment was the same. After 15 days of planting, the growth of alfalfa was observed and the germination rate was calculated. The results are as Figure 15 shown in Table 3.

[0102] Table 3 Germination Rate Results under Different Coal Gangue Contents

[0103] Proportion of coal gangue 10％ 30％ 60％ 90％ Germination rate 67.77 79.98 70.81 43.33

[0104] From Figure 15 and Table 3, it can be seen that: as the dosage of coal gangue increases, the germination rate first increases and then decreases. When the content of coal gangue is preferably 30%, that is, when coal gangue and sandy soil are mixed at a mass ratio of 3:7, the germination rate is the best.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions of the present invention or make equivalent replacements, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Application of an air bacillus strain in promoting the growth of alfalfa.

2. Application of an aerobic Bacillus strain in promoting alfalfa growth by accelerating soil matrix mineralization.

3. Application of an airborne Bacillus strain in promoting alfalfa growth by accelerating soil matrix humification.

4. According to claim 1, the use of the aerobic bacillus strain in promoting the growth of alfalfa is characterized in that: The application of aerobic Bacillus strains in promoting the growth of alfalfa by increasing the germination rate, above-ground height, root length, root number and fresh weight of alfalfa.

5. The use according to claim 1, 2 or 3, characterized in that: The deposit number of the aerobic Bacillus strain is CCTCC M 20232075.

6. The use of the aerobic Bacillus strain in promoting the growth of alfalfa according to claim 1, characterized in that: The application process is: S1. Forming a soil matrix with coal gangue and sand; in the soil matrix, the mass proportion of coal gangue is 0% to 90%; S2, sowing the pre-soaked alfalfa seeds in the soil matrix; S3. Continue to add the bacterial solution of Aerobacillus to the soil matrix, and the amount of bacterial solution added to each (950g±10g) of soil matrix is ​​3ml-12ml; S4. Alfalfa seeds are planted according to conventional operations.

7. The use of the aerobic Bacillus strain in promoting the growth of alfalfa according to claim 6, characterized in that: In the soil matrix, the mass proportion of coal gangue is 30%.

8. The use of the aerobic Bacillus strain in promoting the growth of alfalfa according to claim 6, characterized in that: The amount of bacterial solution added to each (950g±10g) of soil matrix is ​​6ml-9ml.

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