Application of silicic acid degrading bacillus nissi L114 in promoting growth of eucalyptus

Abstract

The invention discloses a bacterial strain capable of degrading silicate, the bacterial strain is bacillus nissi L114 (Niallia nealsonii), the bacterial strain is preserved in the China General Microbiological Culture Collection Center, the preservation number is CGMCC No.32592, the preservation date is November 12, 2024, and the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No.1 yard, Beichen West Road, Chaoyang District, Beijing. The bacillus nissi L114 can normally grow in a silicate bacteria culture medium, has the effect of degrading silicic acid and can degrade silicate in soil, and experiments prove that the strain has the effect of promoting the growth of eucalyptus and meets the needs of forestry development.

Description

Technical Field

[0001] The invention discloses an application of Bacillus nissini L114 capable of degrading silicate in promoting the growth of eucalyptus, belonging to the technical fields of microbial application and environmental engineering. Background Art

[0002] Bacillus niger belongs to the genus Bacillus. The specific fertilizer effect of Bacillus not only effectively degrades potassium in the soil and releases soluble potassium and trace elements such as calcium, sulfur, magnesium, iron, zinc, molybdenum, and manganese, which not only improves soil fertility but also provides eucalyptus with a comprehensive range of nutrients that can be absorbed and utilized, promoting root growth and enhancing root absorption capacity, which is beneficial for increasing eucalyptus yields. It also enhances the stress resistance of crops. Bacillus has the ability to secrete antibiotic substances and various active enzymes, which can inhibit or kill pathogens, improve crop disease resistance, drought resistance, and drought tolerance, increase base soil nutrients, enhance soil microbial activity, inhibit the reproduction of pathogens inside and outside plants, kill harmful bacteria, and reduce pests and diseases. It improves soil nutrients. Effective Bacillus bacteria can promote the formation of soil aggregate structure, prevent soil compaction, disrupt soil capillary phenomena, and prevent soil moisture evaporation, thereby revitalizing the soil and improving soil fertility.

[0003] Potassium is a major nutrient essential for eucalyptus growth, playing a key role in the tree's physiological development and metabolism, and effectively enhancing the tree's resistance to biotic and abiotic stresses. The total amount of potassium in soil ranges from 0.04% to 3%, but most soil potassium (90-98%) exists in the form of insoluble or poorly soluble mineral potassium, which cannot be directly absorbed and utilized by plants. Finding new methods to convert mineral potassium into soluble potassium that is easily absorbed and utilized by crops, to meet plant growth needs and achieve the goal of "reducing fertilizer application and increasing efficiency" has become a current research hotspot.

[0004] Rhizosphere microorganisms are an extremely important component of the soil ecosystem, participating in the biochemical cycles and energy flows in the soil ecosystem, including the cycling of nutrients such as carbon, nitrogen, and phosphorus, and the decomposition of soil organic matter. Studies have shown that plant growth-promoting rhizobacteria can decompose insoluble minerals in the soil and convert them into compounds that are easily absorbed by trees, thereby helping to protect trees from nutritional stress and improving the soil microecological environment. Potassium-solubilizing bacteria, also known as silicate bacteria, are a type of rhizosphere microorganism that has the ability to decompose insoluble aluminosilicate minerals such as potassium feldspar and apatite, and can convert insoluble potassium in the soil into a soluble form. Therefore, potassium-solubilizing bacteria in the soil play a key role in the natural potassium cycle. Research on the isolation and screening process of potassium-solubilizing bacteria is of great practical significance for alleviating the current shortage of potassium resources, promoting the sustainable development of forestry, and reducing the use of chemical fertilizers. Summary of the Invention

[0005] The purpose of the present invention is to solve the deficiencies in the prior art and provide a Bacillus niger L114 that can degrade silicate and promote the growth of eucalyptus.

[0006] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0007] The first object of the present invention is to provide a strain having silicate degradation capability, which is Niallia nealsonii L114.

[0008] The second object of the present invention is to provide a Bacillus nissori L114 strain that degrades silicate.

[0009] Compared with the prior art, the Bacillus niger L114 of the present invention has a better effect on silicate degradation.

[0010] The third object of the present invention is to provide a Bacillus niger L114 strain that can promote the growth of eucalyptus.

[0011] Compared with the prior art, the Bacillus niger L114 of the present invention has a better effect on promoting the growth of eucalyptus BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments.

[0013] Figure 1 This is the reverse morphology of the colony of Bacillus niger L114 on LB medium;

[0014] Figure 2 This is a front view of the colony morphology of Bacillus niger L114 on LB medium;

[0015] Figure 3 This is the reverse morphology of the colony of Bacillus niger L114 in silicate bacterial culture medium;

[0016] Figure 4 This is a front view of the colony morphology of Bacillus niger L114 in silicate bacterial culture medium;

[0017] Figure 5 The reverse side of the colony of Bacillus niger L114 in CAS medium;

[0018] Figure 6 This is the front morphology of the colony of Bacillus niger L114 in CAS medium;

[0019] Figure 7 Schematic diagram of the growth-promoting effect of Bacillus niger L114 on eucalyptus. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below.

[0021] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0022] Example 1

[0023] A silicate-degrading strain, the strain being Bacillus nealsonii L114 (Niallia nealsonii), has a deposit number of CGMCC No. 32592 and a deposit date of November 12, 2024, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The specific screening method for Bacillus nealsonii L114 is as follows:

[0024] Isolation of endophytes

[0025] Isolation and purification of soil bacteria suspension by gradient dilution method

[0026] 1. Take soil

[0027] Take soil samples 5-10 cm below the surface and place them in a sterile bag for later use, or store them temporarily in a 4°C refrigerator.

[0028] 2. Preparation of dilution solution

[0029] (1) Preparation of soil suspension:

[0030] Weigh 0.5g of soil sample and quickly pour it into a 49.5mL sterile water bottle with glass beads. Oscillate for 5-10 minutes to fully disperse the soil sample. 2 of soil suspension.

[0031] (2) Dilution:

[0032] Use a sterile pipette to draw 10 2 0.5mL of soil suspension is placed in 4.5mL of sterile water to make 10 3 Repeat this process to make 10 dilutions. 3 ~10 8 Note: The tip of the pipette should not touch the liquid surface during operation. Use a new pipette for each dilution. After each aspiration of soil liquid, insert the pipette into the liquid surface and blow and aspirate 3 times. The liquid level aspirated each time should be higher than the previous one to reduce the error in dilution.

[0033] 3. Determination of colony count by mixed bacteria method

[0034] Take 10 7 , 10 6Add 1 mL of each dilution solution from two tubes to correspondingly labeled plates, using two plates for each dilution. Then, take beef extract agar culture medium cooled to 50°C and pour it into each of the above plates (the amount should be enough to cover the bottom of the plate by 1.5-2 mm). Quickly and gently shake the plates to thoroughly mix the bacterial solution and culture medium, but do not wet the edges of the plates. Once the agar solidifies, the bacterial plates are ready. Ensure sterile operation when pouring the plates.

[0035] 4. Cultivation

[0036] Invert the inoculated plate with the lid facing downward and incubate at 28-30°C for 1-2 days for bacteria, 5-7 days for actinomycetes, and 3-5 days for molds. Observe the growing colonies for further purification or direct transfer to a slant.

[0037] Flat plate production and separation

[0038] 1. Pour the flat plate

[0039] According to the requirements of aseptic operation, operate next to the flame

[0040] 2. Separation by lines

[0041] Use an inoculating loop to take a small amount of bacterial sample from the colony to be purified or the slant strain to be isolated, and streak it on the corresponding culture medium plate. There are various streaking methods, the purpose of which is to obtain a single colony.

[0042] Identification of strain L114

[0043] 1. Morphological identification

[0044] The strain L114 was inoculated on LB solid medium plates and the colony morphology was observed. The colony photos of the strain L114 are shown in Figure 1 and Figure 2 The colonies are white, opaque, moist, with neat edges and a sticky texture.

[0045] 2. Molecular Biological Identification

[0046] On the basis of the above morphological identification, the strain L114 was further identified by molecular biology.

[0047] DNA from strain L114 was extracted and used as a template. 16S rDNA universal primers were used to amplify the 16S rDNA nucleotide fragment of the strain. The amplified product was sent to Sangon for sequencing. The sequencing results are shown in the sequence listing. Homology analysis of the sequence with the 16S rDNA sequence in the NCBI website database showed that the sequence had a similarity of 99.85% with Niallia nealsonii. Based on these results, the strain of the present invention was preliminarily identified as Niallia nealsonii.

[0048] Example 2

[0049] The purpose of this example is to analyze the application potential of strain L114 in degrading silicate and to determine the silicate degradation performance of strain L114.

[0050] Silicate bacterial culture medium formula: sucrose 5g, Na2HPO4 2g, MgSO4·7H2O 0.5g, FeCl3 0.005g, CaCO3 0.1g, bauxite 0.5g, agar 10-20g, water 1000ml, pH 7.0-7.5

[0051] L114 was cultured in shake flasks for activation and expansion. Silicate bacterial culture medium was prepared according to the above formula. Sterilized plates were prepared. After the culture medium was sterilized, it was poured onto the plates. After solidification, 1 μL of bacterial solution was aspirated and spotted onto various plates. The plates were sealed and placed upside down at 28°C for culture. When a transparent hydrolysis zone or colony appeared, the experimental results were observed. Figure 3 Figure 4 shown.

[0052] Example 3

[0053] The purpose of this example is to analyze the siderophore detection of strain L114 using CAS medium, and to determine whether the microorganism can produce siderophore by observing the color change (such as orange halo) around the colony. Figure 5 Figure 6 shown.

[0054] CAS medium formula:

[0055] Solution A: Dissolve 60.5 mg of Chrome azurol S (CAS) in 50 mL of deionized water.

[0056] Solution B: Dissolve 10 mL of ferric iron solution (containing 1 mmol / L FeCl3.6H2O and 10 mmol / L hydrochloric acid as solvent) and 72.9 mg of CTAB (Cetyltrimethylammonium Bromide) in 40 mL of deionized water.

[0057] Mixing and dilution: Mix solution A and solution B and dilute to 100 mL. Adjust the pH to neutral. Sterilization: Sterilize at 121°C for 20 minutes. Observe the color change around the colony (such as an orange halo) to determine whether the L114 strain can produce siderophores. The results are as follows: Figure 5 Figure 6 shown.

[0058] Example 4

[0059] The purpose of this example is to analyze the application of the inoculant prepared by strain L114 in promoting the growth of eucalyptus and to determine the growth-promoting function of strain L114.

[0060] Soil sterilization: Soil was collected from the natural environment and sterilized at 121°C and high pressure for 30 min.

[0061] Seed sowing: Soak eucalyptus seeds in 40°C water for 3 hours, spread evenly on the soil surface, and place in a 25°C climate chamber for germination. Water every 3 days and observe the germination of the seeds.

[0062] Preparation of inoculants: Culture strain L114 overnight in a shaker at 37°C, centrifuge at 4500 rpm for 10 minutes, pour off the supernatant, rinse with sterile deionized water, resuspend, and mix equal volumes according to the inoculant composition. The resuspension was evenly applied to the roots of eucalyptus seedlings in the treatment group, while sterile deionized water was used as a control. Inoculants were then applied every two weeks, and the growth of the eucalyptus seedlings was observed.

[0063] After four weeks of growth, the height of the eucalyptus seedlings was measured. The strain L114 significantly promoted the growth of eucalyptus, as shown in the figure.

Claims

1. A bacterial strain capable of degrading silicate, the strain being Bacillus niger L114, characterized in that: The strain is Niallia nealsonii, the preservation number is: CGMCC No. 32592, the preservation date is: November 12, 2024, and the preservation address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. Use of the silicate-degrading strain Bacillus niger L114 according to claim 1 in silicate degradation.

3. Use of the silicate-degrading strain Bacillus niger L114 according to claim 1 in promoting the growth of eucalyptus.

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