Bacillus sp. sga510 and application thereof

Abstract

The present application relates to the technical field of microorganism, in particular to a bacillus SGA510 and application thereof, which is classified and named as Bacillus sp., has been preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO:M20241840.The bacillus SGA510 has good plant growth promoting function and phosphorus solubilizing performance, and can secrete auxin, promote seed germination, promote seedling growth and degrade insoluble phosphorus in soil when used as a single strain in preparation of a compound microbial agent, so that the bacillus SGA510 has wide application prospect and great economic value in the field of biological fertilizer and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a bacillus SGA510 and its application. BACKGROUND

[0002] With the increase of population, people's demand for agricultural safety and yield is gradually increasing. As the basis of agricultural production, chemical fertilizer plays a crucial role in promoting food production. However, in recent decades, the excessive use of fertilizer has led to a significant decrease in the input-output ratio of agriculture in China, and even caused serious pollution of the agricultural ecological environment. In order to maintain the sustainable development of agriculture, the demand for new types of agricultural fertilizers is on the rise. The important role of microorganisms in soil improvement and crop yield increase has been gradually recognized by people. At present, the emerging microbial fertilizer using plant growth-promoting bacteria (PGPB), rhizobium and mycorrhizal fungi as the main body has been widely used in agricultural production and has played an invaluable role in promoting green agriculture in China.

[0003] Among them, plant growth-promoting bacteria (PGPB) is a kind of microorganism that promotes plant growth and development through complex direct or indirect mechanisms, and can activate soil nutrients, help crops resist diseases and promote the growth and reproduction of crops. Its mechanism mainly includes regulation of plant hormones (such as production of ACC deaminase, secretion of indole acetic acid, gibberellin, etc.), activation of soil nutrients (phosphorus dissolution, potassium dissolution and nitrogen fixation), regulation of secondary metabolite release and pathogen control, etc. It has been found that bacillus, penicillium, pseudomonas and rhizobium have the function of promoting plant growth. Among them, bacillus is a kind of growth-promoting bacteria that is more commonly used in agricultural production. The solid bacterial agent invented in CN118931807A can be stored for a long time, is convenient for field application, and can also effectively improve the soil environment, thereby effectively meeting the nutritional needs of crop growth. The growth-promoting microbial preparation invented in CN115960744A can significantly promote the growth of rice, and can effectively dissolve the insoluble phosphorus in the soil, improve the utilization of phosphorus by wheat, and thereby promote its growth and development. These studies show that certain bacillus can significantly promote plant growth, and the mechanism of its growth-promoting effect on plants is different.

[0004] Bacillus is widely present in soil, plants, food and animal intestines, and its vegetative cell divides and proliferates about every half hour. Changes in environmental factors such as nutrition, temperature, pH, oxygen content and salt concentration can all trigger the mutual transformation between spores and vegetative cells. Bacillus has the characteristics of high temperature resistance, rapid revival and strong enzyme secretion, and can survive under aerobic and anaerobic conditions. In the conditions of nutrient deficiency, drought and the like, spores are formed, and when the conditions are suitable, they can germinate into vegetative cells again.

[0005] Bacillus has many strains with special functions, which have shown wide application potential in many fields such as agriculture and medicine.

[0006] Phosphorus is one of the nutrients necessary for plant growth and microbial activity, and can be coupled with carbon and nitrogen to participate in the biogeochemical cycle. Soil phosphorus is crucial to maintaining the service, structure and function of the ecosystem. In addition, the phosphorus in the soil of Inner Mongolia mainly exists in the form of combined state, which seriously affects the absorption and utilization of plants and greatly limits the productivity of the soil. Phosphorus-solubilizing bacteria can dissolve the insoluble phosphorus in the soil, increase the content of soluble phosphorus, and promote plant growth. The caragana is distributed in Inner Mongolia, Shaanxi, Gansu, Ningxia and other places. As an important sand-fixing plant in Inner Mongolia, how to better make its seeds germinate and seedlings grow is crucial to improve the sand-fixing effect. The existing Bacillus genus has poor growth-promoting effect on caragana, and there is no special growth-promoting agent for caragana plants at present. SUMMARY

[0007] The purpose of the present application is to provide a Bacillus SGA510 and its application, in order to solve the problems raised in the above background art.

[0008] In order to achieve the above purpose, the present application provides the following technical scheme:

[0009] A Bacillus SGA510, which is classified and named as Bacillus sp., has been preserved in the China Center for Type Culture Collection on August 26, 2024, and the preservation address is Wuhan University in Wuhan City, with the preservation number CCTCC NO: M20241840.

[0010] The application of the Bacillus SGA510 as a caragana plant growth-promoting agent.

[0011] Compared with the prior art, the present application has the beneficial effects that:

[0012] The bacillus SGA510 has excellent growth promotion performance, growth performance and phosphorus solubilization performance for caragana plants, and has some unique and significant effects on caragana seed germination and growth promotion of caragana plants; the composite microbial agent prepared by using the bacillus SGA510 has excellent growth promotion performance and phosphorus solubilization function, and has wide application prospect and great economic value in agricultural production. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 A 16S rDNA sequence phylogenetic tree of the bacillus SGA510;

[0014] Figure 2 A colony morphology and microscope oil lens picture of the bacillus SGA510;

[0015] Figure 3 A culture picture of the bacillus SGA510 in a phosphorus-containing medium;

[0016] Figure 4 An IAA color development picture of the bacillus SGA510. DETAILED DESCRIPTION

[0017] The technical solutions in the embodiments of the present application will be described clearly and completely in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0018] Please refer to Figures 1 to 4 The present application provides:

[0019] Embodiment 1: Molecular biological identification of bacillus SGA510

[0020] The pure bacillus SGA510 was inoculated into LB liquid medium, and cultured at 37℃ with shaking until the logarithmic phase, then the bacterial slurry was collected and repeatedly frozen and thawed, and then the bacterial universal 16S rDNA primer

[0021] 27F: 5'-AGAGTTTGACCTGGCTAG-3' (SEQ ID NO: 2) and

[0022] 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 3) for amplification. After the amplification product was detected by 0.1% agarose gel electrophoresis, it was sent to Shenguo Bioengineering (Shanghai) Co., Ltd. for sequencing. After the obtained sequencing results were compared with the sequences in the database, they had high homology (99%) with Bacillus filamentosus and Bacillus endophyticus, so SGA510 could not determine its species, and it was a kind of Bacillus, and its phylogenetic tree diagram is shown in Figure 1

[0023] The sequence of SEQ ID NO: 1 thereof is described as follows:

[0024] ​

[0025] Example 2: Morphological characteristics of Bacillus SGA510

[0026] Macroscopic morphology: The colony is beige, opaque, with wrinkles on the surface, and the edge is slightly rough;

[0027] Microscopic morphology: Rod-shaped, partially connected end to end to form a strip, no flagellum;

[0028] The colony morphology and oil microscope chart of Bacillus SGA510 are shown in Figure 2 .

[0029] Example 3: Growth characteristics of Bacillus SGA510

[0030] (1) Effect of pH on the growth of Bacillus SGA510

[0031] According to the inoculation ratio of 1%, Bacillus SGA510 was inoculated into LB liquid medium with pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0, respectively, and the absorbance value (OD600) of the culture solution at 0h and 24h was measured when it was cultured at 37°C for 24h. The growth state of the bacteria was determined according to the absorbance value, and the specific determination results are shown in Table 1:

[0032] Table 1: Growth state table of Bacillus SGA510 at different pH

[0033] pH 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Bacillus sp. SGA510 - W + + + W W

[0034] Note: -, no growth; +, growth; W, weak growth;

[0035] From Table 1, it can be seen that Bacillus SGA510 grows well at pH 6.0, 7.0 and 8.0, grows weakly at pH 5.0, 9.0 and 10.0, and does not grow at pH 4.0;

[0036] (2) Effect of temperature on the growth of Bacillus SGA510

[0037] According to the inoculation ratio of 1%, Bacillus SGA510 was inoculated into LB liquid medium with pH 7.0, and the absorbance value (OD600) of the culture solution at 0h and 24h was measured when it was cultured at 22, 25, 28, 31, 34, 37 and 40 for 24h. The growth state of the bacteria was determined according to the absorbance value, and the determination results are shown in Table 2:

[0038] Table 2: Growth state table of Bacillus SGA510 at different temperatures

[0039] Temperature (°C) 22 25 28 31 34 37 40 Bacillus sp. SGA510 W + + + + + +

[0040] Note: -, no growth; +, growth; W, weak growth;

[0041] As can be seen from Table 2, Bacillus SGA510 grows well at temperatures of 25, 28, 31, 34, 37, and 40°C, and grows weakly at a temperature of 22°C.

[0042] Example 4: Physiological and biochemical characteristics of Bacillus SGA510

[0043] Bacillus SGA510 was inoculated into physiological and biochemical identification tubes and cultured for 24-48 hours, and color changes were observed to determine the biochemical characteristics of Bacillus SGA510, as shown in Table 3 below:

[0044] Table 3: Biochemical detection table of Bacillus SGA510

[0045]

[0046] Note: +, positive; -, negative;

[0047] As can be seen from Table 3, Bacillus SGA510 grows with V-P, L-arabinose, D-mannitol, 7% NaCl, at pH 5.7, and has a positive starch hydrolysis reaction, and is negative for reactions with citrate, propionate, D-xylose, gelatin, nitrate, and anaerobic growth.

[0048] Example 5: Phosphorus solubilization characteristics of Bacillus SGA510

[0049] The phosphorus solubilization ring method was used: Bacillus SGA510 stored in a 50% glycerol solution was activated in LB solid medium, a 5mm puncher was used to pick up a single colony of Bacillus SGA510 on NBRIP solid medium for plate experiments, and the plate was cultured in a 28°C constant temperature incubator for 5-7 days. Whether a transparent phosphorus solubilization ring was produced around the colony was observed. A ruler was used to measure the phosphorus solubilization ring diameter (D) and the colony diameter (d) of Bacillus SGA510, and the phosphorus solubilization coefficient value (D / d) was calculated to preliminarily determine whether Bacillus SGA510 had phosphorus solubilization ability. After measurement, the phosphorus solubilization coefficient of Bacillus SGA510 was 2.13, and the culture picture of Bacillus SGA510 on the phosphorus-containing medium is shown in Figure 3 .

[0050] Example 6: Characteristics of Bacillus SGA510 in promoting seed germination of Caragana

[0051] The seed of Caragana seed is sterilized with 75% alcohol solution and then washed with distilled water for 2-3 times. The Caragana seed is evenly placed in a culture dish with sterile filter paper at a density of about 100 seeds per dish, and 7% (mL / seed) of Bacillus SGA510 bacterial solution is added. The culture dish is placed in a plant room and cultured. On the third day, the bacterial solution is supplemented once a day. The number of seeds germination is observed every day. The sterile medium is added as a control, and three groups are repeated.

[0052] The number of seeds germination is counted, the germination time of seeds (a total of 7 days is recorded, and the germination time curve is represented), the germination rate and the relative germination rate are measured. The radicle length is greater than or equal to the seed length as the germination standard. The three strains with fast germination speed and high germination rate are selected.

[0053] The germination rate = the number of germinated seeds / the total number of seeds * 100%

[0054] The results show that the germination rate of Caragana seed treated by SGA510 bacterial solution is increased by 4% compared with the control group.

[0055] Example 7: IAA production function of Bacillus SGA510

[0056] Salkowski colorimetric method is used. Bacillus SGA510 is streak inoculated on LB solid medium and cultured in dark for 24h for activation. A single colony is inoculated into a 150mL triangular flask containing 50mL of NB liquid medium. The inoculation amount is 1%, the final concentration of tryptophan is 500mg / L, and the control group without inoculation is set. The culture is carried out at 28℃ with 180r / min shaking for 72h. The fermented bacterial solution is centrifuged at 10000r / min for 10min. The supernatant is taken for colorimetric analysis. 1000μL of IAA color developing agent is added, mixed thoroughly, and developed at room temperature for 30min in dark. Compared with the blank control, red color appears, indicating that IAA is produced. The IAA color developing picture of Bacillus SGA510 is shown in Figure 4 .

[0057] A standard sample is prepared using IAA standard with a purity of 99.5%. The OD450 nm of each concentration standard sample is measured. According to the IAA concentration gradient change and the absorbance of each concentration gradient standard sample, the IAA standard curve is drawn with the concentration of IAA standard as the X axis and the OD450 nm of standard sample as the Y axis. The formula of the obtained standard curve is Y=0.0499x+0.2171, R 2 =0.9841. The absorbance of Bacillus SGA510 at OD450 nm is substituted to obtain the IAA production capacity of Bacillus SGA510, which is 16.01±2.39μg / mL.

[0058] Example 8: The growth-promoting characteristics of Bacillus SGA510 on Caragana

[0059] Caragana seeds were planted into plastic pots with soil formula of V (nutrient soil) : V (vermiculite) = 1 : 3, 4 plants per pot, 5 mL of Bacillus SGA510 bacterial solution was added every three days, and Caragana seedlings with added sterile medium were used as controls, 5 mL of sterile liquid medium was added every three days, and each treatment was repeated 3 times.

[0060] The height, root length, stem diameter, and leaf number of Caragana were observed and measured every five days, and the growth conditions of Caragana in the treatment and control groups were compared and analyzed, as shown in Table 4 below:

[0061] Table 4: Growth-promoting display of Bacillus SGA510 on Caragana

[0062]

[0063] Note: Lowercase letters are significantly different.

[0064] As can be seen from Table 4 above, the height of Caragana seedlings treated with Bacillus SGA510 was increased by 14.23%, the root length was increased by 20.17%, the leaf number was increased by 8.97%, the fresh weight was increased by 29.1%, and the dry weight was increased by 28.98%, and the growth-promoting effect of Bacillus SGA510 on Caragana was obvious.

[0065] Although embodiments of the present application have been shown and described, it is to be understood that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the present application, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A strain of Bacillus SGA510, characterized in that: The Bacillus SGA510 has the accession number CCTCC NO:M20241840, the accession date is August 26, 2024, and it is deposited at the China Center for Type Culture Collection.

2. The application of Bacillus SGA510 as described in claim 1 in the preparation of a growth-promoting bacterial agent for Caragana korshinskii plants.

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