Bacillus subtilis B180 and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to bacillus subtilis B180, which is classified and named as bacillus subtilis, is preserved in the China Center for Type Culture Collection (CCTCC), and has the preservation number of CCTCC NO: M 20241919. The bacillus subtilis B180 disclosed by the invention has excellent seed germination promoting performance, seedling growth promoting performance and phosphate solubilizing performance; the bacillus subtilis B180 has the advantages of excellent growth promoting performance, phosphate solubilizing function and the like, and has a wide application prospect and a huge economic value in the aspect of agricultural production.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and specifically to a Bacillus subtilis B180 and its application. Background Art

[0002] With the increase in population, people's demand for agricultural product safety and yield has gradually increased. As the basis of agricultural production, chemical fertilizers play a crucial role in promoting grain yield increase. However, the excessive application of fertilizers in recent decades has led to a significant reduction in the input-output ratio of agriculture in China and even caused serious pollution of the agricultural ecological environment. To maintain the sustainable development of agriculture, the demand for new agricultural fertilizers is on the rise. The important role of microorganisms in aspects such as soil improvement and crop yield increase has been gradually recognized by people. Currently, emerging microbial fertilizers with beneficial microorganisms such as plant growth-promoting bacteria, rhizobia, and mycorrhizal fungi as the main body have been widely used in agricultural production and play an inestimable role in promoting green agriculture in China.

[0003] Among them, plant growth-promoting bacteria (PGPB) are a type of microorganisms that promote plant growth and development and prevent and control pests and diseases through complex direct or indirect mechanisms. PGPB can activate soil nutrients, help crops resist diseases, and promote the growth and reproduction of crops in agricultural production. Its action mechanisms mainly include regulating plant hormones (such as producing ACC deaminase, secreting indole acetic acid, gibberellin, etc.), activating soil nutrients (dissolving phosphorus, decomposing potassium, and nitrogen fixation), regulating the release of secondary metabolites, and preventing and controlling pathogens. Currently, it has been found that Bacillus subtilis, Penicillium, Pseudomonas, and Rhizobium genus, etc. all have the effect of promoting plant growth. Among them, Bacillus subtilis is a type of PGPB that is widely used in agricultural production. The solid bacterial agent invented in the patent CN118931807A can be stored for a long time, is convenient for field application, and can effectively improve the soil environment, thus effectively meeting the nutritional needs of crop growth. The patent invention of CN115960744A is a growth-promoting microbial agent that can significantly promote the growth of rice, can effectively dissolve the insoluble phosphorus in the soil, and improve the utilization of phosphorus by wheat, thereby promoting its growth and development. These studies indicate that certain Bacillus can significantly promote plant growth, and their growth-promoting mechanisms for plants are different. Therefore, analyzing the mechanism of Bacillus subtilis promoting plant growth has important guiding significance for making full use of Bacillus resources.

[0004] Bacillus is widely present in soil, plants, food, and the animal intestine. Its vegetative body divides and multiplies approximately every half hour. Changes in environmental factors such as nutritional status, temperature, pH value, oxygen content, and salt concentration can trigger the mutual transformation between spores and vegetative bodies. Bacillus subtilis has the characteristics of high temperature resistance, rapid resuscitation, and strong enzyme secretion, and can survive under both aerobic and anaerobic conditions. It forms spores under conditions such as nutrient deficiency and drought, and can germinate back into vegetative bodies when conditions are suitable.

[0005] Bacillus subtilis has many strains with special functions, and these strains show extensive application potential in many fields such as agriculture and medicine.

[0006] Phosphorus is one of the essential nutrients for plant growth and microbial activities. It can participate in the biogeochemical cycle coupled with carbon and nitrogen. Soil phosphorus is crucial for maintaining the services, structure, and functions of the ecosystem.

[0007] As an important sand-fixing plant in Inner Mongolia, how to better germinate the seeds of Caragana korshinskii and the growth of seedlings is crucial for improving the sand-fixing effect. In addition, phosphorus in the soil in Inner Mongolia mainly exists in the form of bound state, which seriously affects the absorption and utilization of plants, greatly restricts soil productivity, and phosphate-solubilizing bacteria can dissolve insoluble phosphorus in the soil, increase the content of soluble phosphorus, and promote plant growth. Summary of the Invention

[0008] The purpose of the present invention is to provide a Bacillus subtilis B180 and its application to solve the problems raised in the above background technology.

[0009] To achieve the above purpose, the present invention provides the following technical solutions:

[0010] A strain of Bacillus subtilis B180, whose taxonomic name is Bacillus subtilis. This strain was deposited at the China Center for Type Culture Collection on September 5, 2024. The deposit address is Wuhan University, Wuhan, and the deposit number is CCTCC NO: M 20241919.

[0011] The above-mentioned Bacillus subtilis B180 is used as an application of a growth-promoting bacterium agent for Caragana korshinskii plants.

[0012] Compared with the prior art, the beneficial effects of the present invention are:

[0013] The Bacillus subtilis B180 of the present invention has excellent performance in promoting seed germination, promoting seedling growth and phosphorus solubilization, and has some unique and significant effects on the germination of Caragana korshinskii seeds and the growth promotion of Caragana korshinskii plants; by utilizing the advantages of the excellent growth-promoting performance and phosphorus-solubilizing function of the Bacillus subtilis B180 of the present invention, it has broad application prospects and great economic value in agricultural production. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a phylogenetic tree of the 16S rDNA sequence of Bacillus subtilis B180;

[0015] Figure 2 It is a colony morphology diagram and a microscopic oil immersion diagram of Bacillus subtilis B180;

[0016] Figure 3 It is a phosphorus-solubilizing picture of Bacillus subtilis B180. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0017] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0018] Please refer to Figures 1 to 3 , the present invention provides:

[0019] Example 1: Molecular biological identification of Bacillus subtilis B180:

[0020] The pure bacteria of Bacillus subtilis B180 were inoculated into LB liquid medium and cultured with shaking at 37 °C until the logarithmic phase. The bacterial sludge was collected, and after repeated freezing and thawing, the universal bacterial 16S rDNA primers 27F: 5'-AGAGTTTGACCTGGCTAG-3' (SEQ ID NO: 2) and 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 3) were used for amplification; after the amplification products were detected by 0.1% agarose gel electrophoresis, they were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing; after comparing the obtained sequencing results with the sequences in the database, its homology with Bacillus subtilis was relatively high, reaching 99%, so B180 belongs to the genus Bacillus subtilis.

[0021] Its 16S rDNA sequence SEQ ID NO: 1 is described as follows:

[0022]

[0023] Example 2: Morphological characteristics of Bacillus subtilis B180

[0024] Macroscopic morphology: The colonies are off-white, opaque, wrinkled, and have rough edges;

[0025] Microscopic morphology: rod-shaped, some parts connected end to end to form strips, without flagella.

[0026] Example 3: Growth characteristics of Bacillus subtilis B180

[0027] (1) Effect of pH on the growth of Bacillus subtilis B180

[0028] Bacillus subtilis B180 was inoculated into LB liquid culture medium with pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 at an inoculation ratio of 1%, and cultured at 37°C for 24 hours. The absorbance values ​​(OD600) of the culture medium at 0 hours and 24 hours were measured respectively. The growth state of the bacteria was judged according to the absorbance values. The specific measurement results are shown in Table 1 below:

[0029] Table 1: Growth status of Bacillus subtilis B180 at different pH

[0030] pH 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Bacillus subtilis B180 - W + + + W -

[0031] Note: -, no growth; +, growth; W, weak growth;

[0032] It can be seen from Table 1 that Bacillus subtilis B180 grows well at pH 6.0, 7.0 and 8.0, grows weakly at pH 5.0 and pH 9.0, and does not grow at pH 4.0 and 10.0.

[0033] (2) Effect of temperature on the growth of Bacillus subtilis B180

[0034] Bacillus subtilis B180 was inoculated into LB liquid medium with a pH of 7.0 at an inoculation ratio of 1%, and the absorbance values ​​(OD600) of the culture solution at 0 h and 24 h were measured respectively at 22, 25, 28, 31, 34, 37, and 40 degrees Celsius for 24 hours. The growth state of the bacteria was determined based on the absorbance values. The results are shown in Table 2 below:

[0035] Table 2: Growth status of Bacillus subtilis B180 at different temperatures

[0036] Temperature (°C) 22 25 28 31 34 37 40 Bacillus subtilis B180 W + + + + + W

[0037] Note: -, no growth; +, growth; W, weak growth;

[0038] As can be seen from Table 2, Bacillus subtilis B180 grows well at temperatures of 25°C, 28°C, 31°C, 34°C, and 37°C, and grows weakly at temperatures of 22°C and 40°C.

[0039] Example 4: Physiological and Biochemical Characteristics of Bacillus subtilis B180

[0040] Inoculate Bacillus subtilis B180 into a physiological and biochemical identification tube, culture for 24 - 48 hours, observe the color change, and judge the biochemical characteristics of Bacillus subtilis B180 as shown in Table 3 below:

[0041] Table 3: Biochemical Detection Table of Bacillus subtilis B180

[0042]

[0043] Note: +, positive; -, negative;

[0044] As can be seen from Table 3 above, Bacillus subtilis B180 shows positive reactions with V-P, citrate, D-xylose, L-arabinose, growth in 7% NaCl, growth at pH 5.7, nitrate reduction, and starch hydrolysis reactions, and negative reactions with propionate, D-mannitol, gelatin liquefaction, and anaerobic growth reactions.

[0045] Example 5: Phosphorus-Solubilizing Characteristics of Bacillus subtilis B180

[0046] Use the phosphorus-solubilizing circle method: Take out the bacillus stored in 50% glycerol solution, activate it in LB solid medium, use a 5mm punch to pick up the whole single colony of bacillus on NBRIP solid medium for plate experiment, and culture it in a constant temperature incubator at 28°C for 5 - 7d, observe whether there is a transparent phosphorus-solubilizing circle around the colony; use a ruler to measure the diameter (D) of the phosphorus-solubilizing circle and the diameter (d) of the colony grown by the bacillus respectively, and calculate its phosphorus-solubilizing coefficient value (D / d) to preliminarily judge whether the bacillus has the ability to solubilize phosphorus; after measurement, the phosphorus-solubilizing coefficient of Bacillus subtilis B180 is 1.76.

[0047] Use the molybdenum antimony anti-colorimetric method. Transfer the seed liquid to NBRIP liquid medium at an inoculation amount of 1%, culture at 28°C and 180r / min for 7d, take the fermented bacterial liquid and centrifuge it at 8000r / min for 10min, take 100μL of the supernatant, dilute it to 10mL with distilled water, add 200μL of 0.1g / mL ascorbic acid solution and ammonium molybdate-sulfuric acid mixed solution in sequence, shake well and let it stand for 5min, measure the absorbance at a wavelength of 700nm using a spectrophotometer, and zero before measurement; substitute into the phosphorus standard curve y = 20.268X + 0.4095, and the phosphorus-solubilizing amount of B180 is 35.35 ± 5.41 μg / mL.

[0048] Example 6: Characteristics of Bacillus subtilis B180 in promoting the germination of Caragana korshinskii seeds:

[0049] Disinfect the Caragana korshinskii seeds with 75% alcohol solution and rinse them 2 - 3 times with distilled water; place the Caragana korshinskii seeds evenly in a petri dish lined with sterile filter paper at a density of about 100 seeds per dish, and add 7 mL of the bacterial solution; culture them in a plant room, supplement the bacterial solution once on the third day, and observe the number of germinated seeds every day; use the addition of a sterile medium as a control, and set three replicates.

[0050] Count the number of germinated seeds, measure the germination time of the seeds (record for a total of 7 days, represented by the germination time curve), and the germination rate. Use the criterion that the radicle length is greater than or equal to the seed length as the germination standard.

[0051] Germination rate = (number of germinated seeds / total number of seeds) * 100%.

[0052] The results showed that the germination rate of Caragana korshinskii seeds treated with the B180 bacterial solution increased by 8% compared with the control group.

[0053] Example 7: Growth - promoting characteristics of Bacillus subtilis B180 on Caragana korshinskii:

[0054] Plant the Caragana korshinskii seeds in plastic pots with a soil formula of V (nutrient soil): V (vermiculite) = 1:3, 4 plants per pot. Add 5 mL of Bacillus subtilis B180 bacterial solution every three days. Use Caragana korshinskii seedlings treated with a sterile medium as a control, and add 5 mL of sterile liquid medium every three days. Each treatment is repeated 3 times; observe and measure data such as the plant height, root length, stem diameter, and number of leaves of Caragana korshinskii every five days and analyze them; compare the growth status of Caragana korshinskii in the treatment group and the control group; as shown in Table 4 below;

[0055] Table 4: Table showing the growth - promoting effect of Bacillus subtilis B180 on Caragana korshinskii

[0056]

[0057] Note: Different lowercase letters represent significant differences.

[0058] As can be seen from Table 4, compared with the Caragana korshinskii seedlings treated with a sterile medium, the plant height of Caragana korshinskii seedlings treated with Bacillus subtilis B180 increased by 12.12%, the root length increased by 13.05%, the number of leaves increased by 23.59%, the fresh weight increased by 26.09%, and the dry weight increased by 20.34%.

[0059] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A strain of Bacillus subtilis B180, characterized in that: The deposit number of the Bacillus subtilis B180 is CCTCCNO:M 20241919, the deposit date is September 5, 2024, and it is deposited in the China Center for Type Culture Collection.

2. Use of the Bacillus subtilis B180 as claimed in claim 1 as a growth-promoting agent for Caragana korshinskii plants.

Images