Acid-resistant growth-promoting bacillus altitudinis and application thereof in crop seed coating

Abstract

The invention provides acid-resistant growth-promoting bacillus altitudinis and application thereof in crop seed coating, and belongs to the technical field of microorganisms. The acid-resistant growth-promoting bacterium bacillus altitudinis FAFU1 and the microbial coating preparation thereof provided by the invention can effectively improve the physiological adaptability of rice in an acid stress environment. Under the condition of acid red soil, key growth indexes such as plant height, overground part dry weight, root biomass and total root length of rice plants subjected to strain inoculation or microorganism coating treatment are all remarkably improved. The strain and the prepared coating product provide innovative technical support for microbial remediation of an acid soil farming system, and have important application value for improving the crop productivity of marginal land.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly to an acid-tolerant growth-promoting bacterium, Bacillus altitudinis, and its application in crop seed coating. Background Art

[0002] Acidic soil, as an important limiting factor in global agricultural production, is prone to trigger the toxic effects of aluminum, manganese and other ions when its pH value is lower than 5.5, and at the same time, it causes a decrease in the availability of essential elements such as phosphorus and potassium. In such soils, the increase in hydrogen ion concentration will accelerate the leaching of base nutrients, forming a vicious cycle of nutrient poverty and metal toxicity. Especially in long-term intensive farming systems, the unreasonable application of chemical fertilizers further exacerbates the process of soil acidification. Research shows that when the soil pH drops below 4.5, the microbial activity is significantly inhibited, the soil aggregate structure is damaged, and a hardening phenomenon occurs.

[0003] Rice, as a crop that prefers a weakly acidic environment, shows the best growth state during its seedling stage within the pH range of 4.5 - 5.5. Under this condition, elements such as phosphorus and iron can be effectively activated, root development can be promoted, and the proliferation of pathogenic bacteria can be inhibited. However, when the soil pH continuously drops below 5.0, the absorption of calcium and magnesium by the plants is blocked, and the capillary roots show brown rot, manifested as typical nutrient deficiency symptoms such as reduced tillering and decreased effective panicle number. It is worth noting that although rice has a certain acid tolerance, when the degree of soil acidification exceeds its physiological tolerance threshold, the yield loss will still reach more than 20%.

[0004] At present, the improvement of acidic soil mainly relies on two types of technologies: chemical neutralization and microbial remediation. Although the application of lime can quickly increase the soil pH, its improvement effect only lasts for 3 - 5 planting cycles, and excessive use will exacerbate soil hardening and reduce the cation exchange capacity. Although microbial remediation technology has the characteristics of environmental friendliness, existing acid-tolerant strains generally have the defect of single function: some strains can only survive in an acidic environment but lack growth-promoting metabolic functions, or their activity decays rapidly when the pH < 5.0. More importantly, the colonization success rate of commercial microbial inoculants in acidic red soil is less than 30%, and the dual problems of alleviating aluminum toxicity and activating nutrients cannot be solved simultaneously. Recent research has tried to improve the acid tolerance of strains through genome shuffling and genetic engineering transformation, but the transformed engineered bacteria have new problems such as increased metabolic burden and poor field stability. Summary of the Invention

[0005] The purpose of the present invention is to provide an acid-tolerant growth-promoting bacterium, Bacillus altitudinis, and its application in crop seed coating, which has better effects.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a strain of Bacillus altitudinis, characterized in that the Bacillus altitudinis is Bacillus altitudinis FAFU1, and the deposit number is CCTCC NO: M 20242716.

[0008] The present invention also provides a fermentation medium for culturing the above-mentioned Bacillus altitudinis, and the fermentation medium is LB liquid medium or Bacillus selective liquid medium.

[0009] The present invention also provides a method for culturing the above-mentioned Bacillus altitudinis, comprising the following steps:

[0010] Inoculate Bacillus altitudinis FAFU1 into the above-mentioned fermentation medium, culture it to obtain a Bacillus altitudinis fermentation broth.

[0011] The present invention also provides a Bacillus altitudinis fermentation broth obtained by the above-mentioned culture method.

[0012] The present invention also provides a dry bacterial powder containing the above-mentioned Bacillus altitudinis with biological activity.

[0013] The present invention also provides the application of the above-mentioned Bacillus altitudinis, fermentation medium, Bacillus altitudinis fermentation broth or dry bacterial powder in the preparation of a crop growth-promoting bactericide.

[0014] Preferably, the dosage form of the crop growth-promoting bactericide is crop seed coating.

[0015] The present invention also provides a seed coating, and the seed coating contains the above-mentioned Bacillus altitudinis.

[0016] Preferably, the seed coating further contains auxiliary materials, and the auxiliary materials include insecticides, film-forming agents, thickeners and dyes.

[0017] The present invention also provides the application of the above-mentioned Bacillus altitudinis or Bacillus altitudinis fermentation broth in the preparation of a rice seed germination promoter under acidic conditions or a product for improving the acid tolerance characteristics of rice.

[0018] Advantages of the present invention:

[0019] The acid-tolerant growth-promoting bacterium Bacillus altitudinis FAFU1 and its microbial coating preparation provided by the present invention can effectively improve the physiological adaptability of rice in an acidic stress environment. Under acidic red soil conditions, the key growth indexes of rice plants treated by strain inoculation or microbial coating, such as plant height, above-ground dry weight, root biomass and total root length, are all significantly improved. The strain and the prepared coating product provide innovative technical support for the microbial remediation of acidic soil crop systems, and have important application value for improving the productivity of marginal land crops. Description of the drawings

[0020] Figure 1 This is a diagram showing the colonization of the strain with RFP tag in rice roots observed by a laser confocal microscope in the present invention;

[0021] Figure 2 This is a Gram staining result diagram of Bacillus altitudinis in the present invention. The staining result is blue-violet, indicating that it is a positive bacterium;

[0022] Figure 3 This is the rice growth phenotype of seeds treated differently in acidic red soil for 21 days in the present invention;

[0023] Figure 4 This is the plant height of rice with seeds treated differently in acidic red soil for 21 days in the present invention;

[0024] Figure 5 This is the above-ground dry weight of rice with seeds treated differently in acidic red soil for 21 days in the present invention;

[0025] Figure 6 This is the root dry weight of rice with seeds treated differently in acidic red soil for 21 days in the present invention;

[0026] Figure 7 This is the root length of rice with seeds treated differently in acidic red soil for 21 days in the present invention;

[0027] Figure 8 This is the total root length of rice with seeds treated differently in acidic red soil for 21 days in the present invention;

[0028] Figure 9 This is a comparison diagram of the rice root phenotypes of different coated varieties in acidic red soil (a is the root diagram of the control rice seeds cultured for 21 days at the seedling stage, b is the root diagram of the control coated rice seeds cultured for 21 days at the seedling stage, c is the root diagram of the microbially coated rice seeds cultured for 21 days at the seedling stage, with three replicates set).

[0029] Biological deposit description

[0030] The Bacillus altitudinis provided by the present invention is named Bacillus altitudinis FAFU1, and was deposited at the China Center for Type Culture Collection (CCTCC) on December 4, 2024. The address is: Wuhan University, Wuhan, China, Zip Code: 430072, and the deposit number is CCTCC NO: M 20242716. Detailed implementation manners

[0031] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0032] Embodiment

[0033] Colonization assay of the strain in rice and Gram staining

[0034] The specific experimental steps for the colonization assay of the strain in rice are as follows: The acid-tolerant plant growth-promoting bacterium Bacillus altitudinis (preservation number CCTCC NO: M 20242716) screened was used for the experiment. This strain was colonized in rice, and confocal microscopy was used for microscopic examination. The results are shown in Figure 1 , confirming that Bacillus altitudinis was successfully colonized in rice.

[0035] Steps for Gram staining identification:

[0036] Smear fixation: When smearing the bacterial solution, it should not be too thick, and then dry and fix. When fixing, pass it through the flame 1 - 2 times, not overheating, and it is appropriate when the glass slide is not hot to the touch.

[0037] Staining generally includes four steps: primary staining, mordanting, decolorization, and counterstaining. The specific operation methods are as follows:

[0038] (1) After adding crystal violet, stain for 1 minute, then wash with water.

[0039] (2) After adding iodine solution, stain for 1 minute, then wash with water.

[0040] (3) Add 95% alcohol, shake the glass slide, and decolorize for about 20 - 60 seconds according to the thickness of the smear, then wash with water and blot the water. This step requires strict control of the alcohol decolorization degree.

[0041] (4) After adding safranin, stain for 1 minute, then wash with water.

[0042] (5) After blotting dry or air-drying, examine under an oil immersion microscope.

[0043] Result observation: The microscopic examination result shows purple, evenly distributed linearly, indicating that this Bacillus altitudinis is a Gram-positive bacterium. The results of multiple repetitions are all Gram-positive bacteria, and the results are shown in Figure 3 .

[0044] Development of seed coating formulation:

[0045] The specific coating operation is as follows: Inoculate the Bacillus altitudinis FAFU1 into an LB liquid medium with a pH value of 7.0 and culture overnight at 37 °C with a shaker oscillation rate of 180 - 200 rpm, and then transfer and ferment to OD 600It is 1.0. The cultured bacterial liquid was centrifuged at 7000 rpm for 10 min, and the supernatant was discarded. Centrifugation and removal of the supernatant were repeated to obtain a precipitate, i.e., the bacterial cells. It was sealed with sterile gauze and placed in a constant temperature incubator at 37 °C for 3 days until the bacterial cells were completely dry. Then it was poured into a mortar and ground into a powder, which was the growth-promoting bacterial powder. 5 parts of Lorsban insecticide, 3 parts of polyvinyl alcohol film-forming agent, 0.3 part of bentonite thickener, 0.3 part of permanent red dye, and 10 parts of Bacillus altitudinis powder were put into a container and mixed and stirred evenly according to the ratio. The control coating did not contain strains, and the other components were the same as those of the microbial coating.

[0046] Artificial simulated acid-resistant pot experiment:

[0047] 1 Materials and methods

[0048] 1.1 Reagents and culture media: NaClO, sterile water, LB liquid medium, Lorsban insecticide, polyvinyl alcohol film-forming agent, bentonite thickener, permanent red dye, etc.

[0049] 1.2 Methods

[0050] 1.2.1 Seed materials

[0051] a. Select plump and uniform rice seeds (Nipponbare), and disinfect them with 1% NaClO for 3 - 5 min;

[0052] b. Wash with sterile water 3 - 5 times and dry for later use.

[0053] 1.2.2 Treatment of rice seeds with microbial coating

[0054] The microbial-coated seeds include the above microbial coating and rice seeds. Preparation method of microbial-coated seeds: Mix the control coating, microbial coating, the rice seeds, and distilled water in a ratio of 1:200:19 parts, stir evenly until the surface of each seed is evenly wrapped with the microbial coating, and then leave it to dry at room temperature for 6 - 8 h.

[0055] 1.2.3 Conduct a rice pot experiment under acid stress of red soil

[0056] Set 3 treatments, CK (control, normal rice seeds), control coating (coating without added strains), microbial coating (coating with added strains);

[0057] Inoculate wild-type Nipponbare rice, control-coated, and microbial-coated rice seeds of the present invention into a moist seedling tray. After the rice takes root, select rice seedlings with consistent growth and transfer them to the pre-prepared red soil. Water regularly every day, with 6 biological replicates for each setting, and culture for 21 days.

[0058] 1.2.4 Rice biomass

[0059] Sampling, using a long ruler to measure the distance from the base of the plant to the highest leaf of the plant to obtain the plant height, and the distance from the base of the plant to the root tip to obtain the total root length, and then conducting analysis and processing;

[0060] After harvesting the plants, separate the above-ground parts from the roots, use a root scanner to obtain the total root length of the roots, dry them to a constant weight at 45 °C in an oven to obtain the dry weights of the above-ground parts and the roots, and then conduct analysis and processing.

[0061] Results: The effects of microbial coating on rice growth under acidic red soil

[0062] As Figure 2 and Figures 4 to 8 shown, the growth agronomic trait indexes of rice seedlings were measured, including plant height, main root length, total root length, above-ground dry weight, and underground dry weight. The potting results showed that the plant heights of both the conventional coating and microbial coating seeds were significantly higher than that of the control (p < 0.01), and the plant height of the microbial coating seeds was significantly higher than that of the conventional coating seeds (p < 0.01). Compared with the control seeds and the conventional coating seeds, the plant height of the microbial coating seeds was significantly increased, by 46% and 14% respectively, indicating that both coating seed treatments could promote rice growth, and the effect of microbial coating was more significant. The above-ground dry weight and underground dry weight of the microbial coating seeds were significantly increased by 27% and 68% compared with the conventional coating seeds, and the total root amount was significantly increased; at the same time, the total root length of the microbial coating seeds was also increased by 68%. The conventional coating treatment could significantly increase the total root amount, and the effect of microbial coating was more prominent. In summary, the above experimental results show that microbial coating can significantly improve the growth of rice in acidic soil, and can further improve the agricultural production efficiency and promote the sustainable development of the agricultural economy.

[0063] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A strain of Bacillus altitudinis, characterized in that, The Bacillus altitudinis is Bacillus altitudinis FAFU1, and the preservation number is CCTCC NO: M 20242716.

2. The fermentation medium for culturing the Bacillus altitudinis as claimed in claim 1, characterized in that, The fermentation medium is LB liquid medium or Bacillus selective liquid medium.

3. A cultivation method of Bacillus altitudinis according to claim 1, characterized in that, It includes the following steps: Inoculate Bacillus altitudinis FAFU1 into the fermentation medium described in claim 2, and culture it to obtain a Bacillus altitudinis fermentation broth.

4. The Bacillus altitudinis fermentation broth obtained by the culture method described in claim 3.

5. A dry bacterial powder, characterized in that, It contains the Bacillus altitudinis described in claim 1 with biological activity.

6. Use of the Bacillus altitudinis described in claim 1, the fermentation medium described in claim 2, the Bacillus altitudinis fermentation broth described in claim 4, or the dried bacterial powder described in claim 5 in the preparation of a crop growth-promoting bactericide.

7. The application according to claim 6, wherein The dosage form of the crop growth-promoting bactericide is crop seed coating.

8. A seed coating, characterized in that, The seed coating contains the Bacillus altitudinis described in claim 1.

9. The seed coating according to claim 8, characterized in that, The seed coating also contains excipients, and the excipients include insecticides, film-forming agents, thickeners, and colorants.

10. Use of the Bacillus altitudinis described in claim 1 or the Bacillus altitudinis fermentation broth described in claim 4 in the preparation of a rice seed germination promoter under acidic conditions or a product for enhancing the acid tolerance of rice.

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