A Bacillus Cy15 strain with disease-inhibiting and growth-promoting functions and its application

Abstract

The present invention belongs to the field of agricultural microbial application technology, and specifically relates to a strain of Bacillus Cy15 with the function of inhibiting disease and promoting growth and its application. Bacillus Bacillus sp.) Cy15 was deposited with the China Center for Type Culture Collection at Wuhan University, Wuhan, China, on March 31, 2025, under the accession number CCTCC NO: M 2025659. The Bacillus sp. Cy15 strain of the present invention can be used in agricultural biological control, exhibiting a strong inhibitory effect against eight plant pathogenic fungi and promoting plant growth by producing substances such as IAA and siderophores. Furthermore, the strain can increase the content of vitamin C and soluble protein in vegetables, improving the nutritional value of plants. This strain is an excellent biocontrol bacterium with promising development and application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of agricultural microbial application, and in particular relates to a Bacillus Cy15 strain with the function of inhibiting disease and promoting growth and an application thereof. Background Art

[0002] The long-term and large-scale use of chemical pesticides will cause environmental pollution and ecological damage, lead to food safety problems, and endanger human health.

[0003] Biological control can, to a certain extent, supplement or even replace chemical control, reducing the amount of chemical substances used, making it a highly valuable green pest control method. Beneficial plant-microbe interactions are crucial factors in determining crop health and soil fertility. Biocontrol bacteria, or biocontrol microorganisms, are probiotic bacteria that inhibit the growth of plant pathogens and promote host health through competitive, antibiotic, and bacteriolytic mechanisms. The beneficial effects of biocontrol bacteria have been demonstrated in the production of various important crops.

[0004] Bacillus is a type of Gram-positive bacteria that can form spores. Most Bacillus species mainly produce indoleacetic acid, dissolve phosphates, fix nitrogen, and produce siderophores. Some of the efficient Bacillus strains currently used usually have multiple characteristics described above. Indoleacetic acid (IAA) is a plant growth hormone that promotes the formation of top buds of plant branches or buds and seedlings. Siderophores are a type of low molecular weight, Fe 3+ A chelate with high affinity is a low molecular weight substance that can bind to ferric ion and supply to microbial cells. Protease can promote protein hydrolysis, breaking it down into amino acids to supply plant roots. Bacillus is widely distributed in nature and can be isolated from marine and river sediments, soil, plant rhizosphere, and plant tissues. It is harmless to humans and animals, does not pollute the environment, and can produce secondary metabolites that have antagonistic effects on a variety of pathogenic microorganisms. As a new type of biocontrol bacteria, Bacillus has attracted much attention in plant disease prevention and control, promoting plant growth, etc., but there are currently no reports on Bacillus promoting plant vitamin production. Summary of the Invention

[0005] In view of the deficiencies in the prior art, the present invention provides a Bacillus Cy15 strain having the function of inhibiting disease and promoting growth and its application.

[0006] The technical solutions of the present invention are as follows:

[0007] A strain of Bacillus ( Bacillussp.) Cy15, deposited on March 31, 2025, in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with the deposit number being CCTCC NO: M 2025659.

[0008] The Bacillus Cy15 has the ability to produce IAA and iron carriers, dissolve phosphorus and potassium, and also has the functions of promoting plant growth, increasing the vitamin C content of plants, increasing the soluble protein content of plants, and inhibiting pathogenic fungi.

[0009] The above-mentioned method for culturing Bacillus Cy15 specifically comprises the following steps:

[0010] The Bacillus Cy15 is inoculated into a solid culture medium for activation culture to obtain a single colony; the single colony is picked out and placed into a liquid culture medium for fermentation culture to obtain a Bacillus Cy15 bacterial liquid.

[0011] Preferably according to the present invention, the solid culture medium is LB solid culture medium, and the liquid culture medium is LB liquid culture medium.

[0012] Preferably, according to the present invention, the activation culture conditions are: culture at 28-30° C. for 12-24 hours.

[0013] According to the preferred embodiment of the present invention, the fermentation culture conditions are: 30-37° C., 160-180 rpm shaking culture for 24-36 hours.

[0014] A microbial agent comprises the above-mentioned Bacillus Cy15.

[0015] The above-mentioned Bacillus Cy15 or its bacterial agent is used in one or more of producing IAA and siderogen, dissolving phosphate, dissolving potassium, and promoting plant growth.

[0016] The application of the above-mentioned Bacillus Cy15 or its bacterial agent in increasing the content of vitamin C or soluble protein in vegetables.

[0017] Application of the above-mentioned Bacillus Cy15 or its bacterial agent in preventing and controlling plant diseases caused by plant pathogenic fungi.

[0018] According to the present invention, preferably, the pathogenic fungi include: Trichothecene roseum ( Trichothecium roseum )、Fusarium solani( Fusarium solani )、Botrytis cinerea( Botrytis cinerea ), Fusarium oxysporum ( Fusarium oxysporum ), Alternaria solani ( Alternaria Nees )、Fujikura Gibberellic acid( Gibberella fujikuroi )、Invasion of new red shell ( Neocosmospora vasinfecta )、Pythium chain ( Pythium catenulatum ).

[0019] Beneficial effects:

[0020] The Bacillus Cy15 of the present invention can be applied in the field of agricultural biological control, can produce a strong inhibitory effect on eight plant pathogenic fungi, and can produce substances such as IAA and siderophores to promote plant growth; at the same time, the strain can also increase the content of vitamin C and soluble protein in vegetables, improving the nutritional value of plants. It is a biocontrol bacterium with excellent performance and good development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the colony morphology of strain Cy15;

[0022] Figure 2 Figure 2 is the growth-promoting effect of strain Cy15 on lettuce, where A is the Cy15 group and B is the CK group;

[0023] Figure 3 This is a bar graph of lettuce dry weight;

[0024] Figure 4 This is a bar graph of soluble protein content in lettuce;

[0025] Figure 5 This is a bar chart of vitamin C content in lettuce. DETAILED DESCRIPTION

[0026] The experimental methods in the following examples, unless otherwise specified, are all conventional methods. Materials, reagents, etc. used in the following examples, unless otherwise specified, are commercially available. Culture media in the following examples, unless otherwise specified, were at natural pH. Quantitative experiments in the following examples, unless otherwise specified, were performed in triplicate, and the results were averaged.

[0027] In the following examples, the pink trichothecene spore ( Trichothecium roseum )、Fusarium solani( Fusarium solani )、Botrytis cinerea( Botrytis cinerea ), Fusarium oxysporum ( Fusarium oxysporum ) and Alternaria solani ( Alternaria Nees )、Fujikura Gibberellic acid( Gibberella fujikuroi )、Invasion of new red shell ( Neocosmospora vasinfecta )、Pythium chain ( Pythium catenulatum ) were isolated from diseased parts of tomatoes. These biological materials were used solely for repeating the experiments described herein and should not be used for other purposes. Other skilled artisans in this field can isolate these strains from diseased parts of tomatoes or purchase them commercially to replicate the experiments described herein.

[0028] Example 1

[0029] Isolation and screening of strains

[0030] 1. Isolation: Soil samples were collected from the soil under the pine trees on the hillside of Nanshan District, Jinan City. 5 g of soil sample was placed in a triangular flask filled with 45 mL of sterile water, shaken at 180 rpm for 30 minutes, and then placed in a water bath at 80°C for 20 minutes to kill other bacteria. A 10-fold dilution method was used to prepare a concentration gradient of 10 -3 , 10 -4 , 10 -5 Take 0.1 mL of soil suspension sample and spread it on LB solid culture medium plate, and incubate it upside down at 37℃ for 48 h;

[0031] The LB solid medium components are: peptone 10 g, yeast powder 5 g, NaCl 10 g, agar 15 g, distilled water to 1000 mL; sterilized at 121.5°C for 20 min.

[0032] 2. Screening: Pick the isolated single colony and inoculate it into 100mL LB liquid medium. Incubate it at 37℃ and 180rpm shaking for 24h to obtain a bacterial suspension. Inoculate a 5mm diameter Fusarium oxysporum cake in the center of the PDA medium plate. At the same time, inoculate 5μL of bacterial suspension 3.5cm away from the center of the plate and incubate it at 28℃ for 5d. According to the size of the inhibition zone, select the strain with the best inhibitory effect on Fusarium oxysporum. A grayish white strain with fuzzy edges and irregular shape is obtained. The colony morphology of this strain is as follows: Figure 1 As shown, it was named Cy15.

[0033] The LB liquid culture medium comprises the following components: 10 g of peptone, 5 g of yeast powder, 10 g of NaCl, and distilled water to a volume of 1000 mL; the mixture was sterilized at 121.5°C for 20 min.

[0034] Example 2

[0035] Molecular biological identification of strains

[0036] After extracting the genomic DNA of strain Cy15 using a common bacterial genome extraction kit, PCR amplification was performed using universal primers for bacterial 16S rDNA. The universal primer sequences are as follows:

[0037] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3',

[0038] 1492R: 5'-ACGGCTACCTTGTTACGACTT-3'.

[0039] PCR reaction conditions: 95°C pre-denaturation for 5 minutes; 34 cycles of 95°C denaturation for 30 seconds, 63°C annealing for 30 seconds, and 72°C extension for 45 seconds; and 72°C extension for 10 minutes. The PCR amplification product was sent to Sangon Biotech for sequencing, and the sequencing result is shown as SEQ ID NO. 1. The obtained sequence was submitted to the NCBI database and compared with the 16S rDNA gene sequence in the nucleic acid database. The results showed that strain Cy15 had a 99% similarity to Bacillus. Combined with the physiological characteristics of the strain, it was identified as Bacillus ( Bacillus sp.).

[0040] The above strain Cy15 is taxonomically named Bacillus ( Bacillus sp.), has been deposited in the China Center for Type Culture Collection, with the deposit address at Wuhan University, Wuhan, China, on March 31, 2025, with the deposit number CCTCCNO: M 2025659.

[0041] Example 3

[0042] Fungal inhibition spectrum test of strain Cy15

[0043] Two strains (Cy116 and Cy128) isolated in Example 1 that also have the effect of inhibiting pathogens were selected and used together with strain Cy15 to conduct a plate confrontation experiment to detect the inhibition rate of the isolated strains on pathogenic fungi.

[0044] The specific method is as follows: Single colonies of strains Cy15, Cy116, and Cy128 were inoculated into 100 mL of LB liquid medium and cultured at 28°C with shaking at 180 rpm for 24 hours to obtain a bacterial suspension. A 5-mm-diameter pathogenic fungal cake was inoculated in the center of a PDA medium plate. Simultaneously, 5 μL of the bacterial suspension was inoculated 3.5 cm from the center of the plate (LB liquid medium was used as a control treatment). The plates were incubated at 28°C for 5 days, and the diameters of the pathogenic fungal colonies were measured. Three replicates were set for each pathogenic fungus.

[0045] Inhibition rate (%) = 100% × (pathogenic fungal colony diameter of control treatment - pathogenic fungal colony diameter of isolated strain suspension treatment) / pathogenic fungal colony diameter of control treatment.

[0046] The antibacterial effects of strains Cy15, Cy116, and Cy128 against different pathogenic fungi are shown in Table 1.

[0047] Table 1. Inhibitory effects of strains Cy15, Cy116, and Cy128 on different pathogenic fungi

[0048]

[0049] The results showed that Cy15 had a better antibacterial effect than other isolates (Cy116, Cy128) and inhibited more types of bacteria.

[0050] Example 4

[0051] Functional characterization of strain Cy15

[0052] A single colony of strain Cy15 was picked and inoculated into 100 mL of LB liquid culture medium, and cultured at 28°C and 180 rpm for 24 h to obtain a Cy15 bacterial suspension for the following experiments.

[0053] 1. Detection of IAA production ability of strains:

[0054] A 1% volume ratio of the Cy15 suspension was inoculated into King's B liquid medium (B King) and incubated at 30°C, 180 rpm, and a constant temperature shaker for 3 days. The culture was centrifuged at 12,000 rpm for 10 minutes, and 1 mL of the supernatant was mixed with 2 mL of Salkowski colorimetric solution. Separately, 1 mL of a standard plant growth hormone (IAA) at concentrations of 90, 70, 50, 30, and 10 mg / L was added to 2 mL of Salkowski colorimetric solution and mixed as a gradient control. The mixture was incubated at room temperature in the dark for 30 minutes. The absorbance of the reaction solution at 530 nm was recorded using a spectrophotometer, and an IAA standard curve was constructed. The IAA concentration in the Cy15 suspension was calculated based on the curve. The IAA production of the Cy15 suspension was determined to be 115.36 ± 2.35 mg / L.

[0055] The above-mentioned King's B liquid medium is composed of: 20 g of peptone, 1.5 g of dipotassium hydrogen phosphate, 1.5 g of magnesium sulfate heptahydrate, 1 g of tryptophan, and 10 g of glycerol. Add distilled water to 1000 mL, stir well, and adjust the pH to 7.2.

[0056] The above-mentioned Salkowski colorimetric solution consists of: 10 mL of 0.5 mol / L ferric chloride and 500 mL of 35% perchloric acid, which are mixed evenly before use.

[0057] 2. Siderophore production function test:

[0058] A 1% volume suspension of Cy15 was inoculated into MKB liquid medium and cultured at 30°C and 180 rpm for 48 hours. The suspension was then spotted onto CAS medium plates using an inoculating loop and incubated at 30°C for 2 days. Upon observation of the incubated CAS medium plates, a distinct orange-yellow halo appeared around the Cy15 colonies, indicating that strain Cy15 produced siderophores.

[0059] The above-mentioned MKB liquid culture medium comprises: 5 g of casamino acids, 15 mL of glycerol, 2.5 g of K2HPO4, 2.5 g of MgSO4·7H2O, and distilled water to 1 L; pH = 7.2.

[0060] The CAS medium components are as follows: Solution A: 60.5 mg of chrome azurol S dissolved in 50 mL of deionized water, 10 mL of ferric iron solution (1 mmol / L FeCl3·6H2O, 10 mmol / L hydrochloric acid as solvent), and 72.9 mg of CTAB (cetyltrimethylammonium bromide) dissolved in 40 mL of deionized water. The three solutions were mixed and diluted to 100 mL, the pH was adjusted to neutral, and sterilized at 121°C for 20 min. Solution B: 30.24 g of PIPES (piperazine-1,4-diethanesulfonic acid), 4 g of NaOH, and 11 g of agar. The solution was diluted to 900 mL with distilled water, the pH was adjusted to 6.8, and sterilized at 121°C for 20 min. Solutions A and B were mixed before use.

[0061] 3. Protease function detection:

[0062] The Cy15 bacterial suspension was inoculated into casein culture medium at a volume ratio of 1%, cultured at 30℃ for 2 days, and observed to see whether a transparent zone was formed around the colonies. The results showed that no transparent zone was formed around the colonies, and the strain Cy15 did not have the ability to produce protease.

[0063] The casein culture medium comprises the following components: Solution A: 1.07 g of Na₂HPO₄·7H₂O and 4 g of casein are weighed, added with an appropriate amount of distilled water, and heated to dissolve; Solution B: 0.36 g of KH₂PO₄ is weighed, dissolved in water; after mixing Solutions A and B, 0.3 mL of casein hydrolyzate and 20 g of agar are added, and the volume is finally adjusted to 1000 mL with distilled water.

[0064] 4. Phosphate dissolving function test:

[0065] A 1% volume ratio of a Cy15 suspension was inoculated into NBRIP liquid medium and cultured at 30°C and 180 rpm for 7 days. After incubation, the suspension was centrifuged at 12,000 rpm for 10 minutes. One mL of the supernatant was diluted 10-fold and the phosphorus content in the supernatant was determined using the molybdenum antimony colorimetric method. Uninoculated NBRIP liquid medium was used as a control. This was repeated three times. The solubility of strain Cy15 was 213.8 ± 7.3 mg / L.

[0066] The NBRIP liquid culture medium consists of the following components: 10.0 g glucose, 5.0 g calcium phosphate, 0.1 g ammonium chloride, 0.2 g sodium chloride, 0.25 g magnesium sulfate heptahydrate, 0.2 g potassium chloride, 0.002 g ferrous sulfate, 0.002 g manganese sulfate, and the volume is adjusted to 1000 mL with distilled water.

[0067] 5. Potassium-dissolving function test:

[0068] A 1% volume ratio of a Cy15 suspension was inoculated into a potassium-solubilizing medium and cultured at 30°C and 180 rpm for 7 days. After incubation, the fermentation broth was centrifuged at 500 rpm for 10 minutes to remove insoluble matter. The supernatant was then centrifuged at 12,000 rpm for 10 minutes, and the available potassium content was determined using a flame spectrophotometer. This experiment was repeated three times, using uninoculated potassium-solubilizing medium as a control. The Cy15 strain was found to have a potassium-solubilizing capacity of 19.4 ± 1.3 mg / L.

[0069] The potassium-dissolving medium comprises the following components: 5 g glucose, 0.5 g ammonium sulfate, 0.5 g yeast powder, 0.3 g magnesium sulfate, 2 g disodium hydrogen phosphate, 0.03 g ferrous sulfate, 0.03 g manganese sulfate, 2 g potassium feldspar, and the volume is adjusted to 1000 mL with distilled water.

[0070] The above results showed that the functional characteristics of strain Cy15 are shown in Table 2.

[0071] Table 2. Functional characteristics of strain Cy15

[0072]

[0073] Note: + indicates detected, ND indicates not detected

[0074] Example 5

[0075] Activation of strain Cy15 and preparation of Cy15 bacterial agent

[0076] The strain Cy15 was inoculated on LB solid medium and cultured at 30°C for 24 hours to obtain an activated strain. Then, the full activated strain was picked and inoculated into LB liquid medium, and cultured at 37°C and 180 rpm for 24 hours to obtain a fermentation broth. The fermentation broth was transferred to a sterile centrifuge bottle and centrifuged at 5000 rpm for 5 minutes to collect the cells, which were washed with sterile deionized water and resuspended to a concentration of 5×10 8 cfu / mL or above, and the Cy15 bacterial agent was obtained.

[0077] Example 6

[0078] Growth-promoting effect assay of strain Cy15

[0079] The strain Cy15 was used in a lettuce pot experiment as follows: the pot was 14 cm long by 11 cm high and contained 1.5 kg of soil. Purchased lettuce seeds were surface sterilized and 10 seeds were sown per pot. After germination, the seedlings were thinned to 5 plants per pot. The soil was irrigated regularly to keep it moist. The Cy15 inoculum prepared in Example 5 was diluted with sterile deionized water to a bacterial solution concentration of 1×10 8cfu / mL; two treatment groups were set up: deionized water (CK group) and bacteria (Cy15 group), with three pots in each group. When the lettuce was in the third leaf stage, a trench (1-2 cm deep) was dug around the roots and diluted bacteria agent or deionized water was added to the trench at a rate of 20 mL / pot. The potted plants were cultivated in a greenhouse (temperature 10-22°C, relative humidity 30-45%, normal light) for a total of 45 days. Figure 2 This is a morphological picture of some lettuces after cultivation.

[0080] After the cultivation, the edible tissue and roots of each pot of lettuce were collected for subsequent analysis; the roots and edible tissue of the lettuce were washed with distilled water and divided into two equal parts. One part was inactivated at 105℃ for 30min and dried at 65℃ to constant weight, and the weight after drying was recorded; Figure 3 As shown in the figure, the average dry weight of the CK group was 1.68 g, and the average dry weight of the Cy15 group was 2.49 g, which proved that the application of Cy15 bacterial agent had a significant growth-promoting effect.

[0081] Example 7

[0082] Effect of strain Cy15 on increasing the content of vitamin C and soluble protein in lettuce

[0083] Another portion of edible tissue collected after the cultivation in Example 6 was tested for vitamin C content according to the method in national standard GB 5009.86-2016.

[0084] Weigh 2.0 g of edible tissue, add 5 mL of distilled water, grind into a homogenate, and centrifuge at 12,000 × g for 20 min at 4°C. Collect the supernatant as the soluble protein extract. Determine the protein content in the extract according to the method in GB 5009.5-2016, which is the soluble protein content.

[0085] The soluble protein content results are as follows Figure 4 As shown in Figure 2, the average content of the CK group was 1.22 mg / g, and the average content of the Cy15 group was 1.46 mg / g; the results of vitamin C were as follows: Figure 5 As shown in the figure, the average vitamin C content of the CK group was 0.15 mg / g, and the average content of the Cy15 group was 0.21 mg / g. The results showed that the application of Cy15 inoculant can increase the content of vitamin C and soluble protein in lettuce.

Claims

1. A strain of Bacillus ( Bacillus sp.) Cy15, characterized in that It was deposited in the China Center for Type Culture Collection on March 31, 2025, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M 2025659.

2. The method for culturing Bacillus Cy15 according to claim 1, wherein The specific steps include: The Bacillus Cy15 is inoculated into a solid culture medium for activation culture to obtain a single colony; the single colony is picked out and placed into a liquid culture medium for fermentation culture to obtain a Bacillus Cy15 bacterial liquid.

3. The culture method according to claim 2, wherein The solid culture medium is LB solid culture medium, and the liquid culture medium is LB liquid culture medium.

4. The culture method according to claim 2, wherein The activation culture conditions are: culture at 28-30° C. for 12-24 hours.

5. The culture method according to claim 2, wherein The fermentation culture conditions are: 30-37° C., 160-180 rpm shaking culture for 24-36 hours.

6. A microbial agent, characterized in that: The invention comprises the Bacillus Cy15 according to claim 1.

7. Use of the Bacillus Cy15 according to claim 1 or the microbial agent according to claim 6 in one or more of producing IAA and siderophore, solubilizing phosphate, solubilizing potassium, and promoting plant growth.

8. Use of the Bacillus Cy15 according to claim 1 or the microbial agent according to claim 6 for increasing the content of vitamin C or soluble protein in lettuce.

9. Use of the Bacillus Cy15 according to claim 1 or the microbial agent according to claim 6 in controlling plant pathogenic fungi, characterized in that: The pathogenic fungus is: Trichothecene pink ( Trichothecium roseum )、Fusarium solani( Fusarium solani )、Botrytis cinerea( Botrytis cinerea ), Fusarium oxysporum ( Fusarium oxysporum ), Alternaria solani ( Alternaria Nees )、Fujikura Gibberellic acid( Gibberella fujikuroi )、Invasion of new red shell ( Neocosmospora vasinfecta )、Pythium chain ( Pythium catenulatum ).

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