Bacillus Cy15 with disease inhibiting and growth promoting functions and application thereof

Abstract

The invention belongs to the technical field of agricultural microorganism application, and particularly relates to a bacillus Cy15 with disease-inhibiting and growth-promoting functions and application of the bacillus Cy15. The bacillus sp. Cy15 disclosed by the invention is preserved in the China Center for Type Culture Collection on March 31, 2025, the preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 2025659. The bacillus Cy15 disclosed by the invention can be applied to the field of agricultural biological control, can generate a relatively strong inhibition effect on eight plant pathogenic fungi, and can generate substances such as IAA (Indoleacetic Acid) and siderophores to promote plant growth; meanwhile, the bacterial strain can also increase the content of vitamin C and soluble protein in vegetables and improve the nutritional value of plants, is a biocontrol bacterium with excellent performance, and has good development and application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of agricultural microbial applications, and specifically relates to a Bacillus Cy15 with disease inhibition and growth promotion functions and its applications. Background Art

[0002] The long-term and large-scale use of chemical pesticides can cause environmental pollution and ecological damage, leading to problems in food safety and endangering human health.

[0003] Biological control can, to a certain extent, supplement or even replace chemical control, achieving the effect of reducing chemical substances, and is a currently highly valuable green control method. The beneficial interaction between plants and microorganisms is an important factor determining crop health and soil fertility. Biocontrol bacteria, that is, biocontrol microorganisms, refer to probiotic groups that can inhibit the growth of plant pathogens and promote the health of hosts through mechanisms such as competition, antibiosis, and lysis. Currently, the beneficial effects of biocontrol bacteria have been proven in the production of various important crops.

[0004] Bacillus is a type of Gram-positive bacteria that can form spores. Most Bacillus mainly have functions such as producing indole acetic acid, dissolving phosphates, nitrogen fixation, and producing siderophores. Some currently used highly efficient Bacillus strains usually have multiple characteristics described above. Indole acetic acid (IAA) is a plant growth hormone that promotes the formation of the top bud ends of plant branches or buds, seedlings, etc. Siderophores are a class of low-molecular-weight chelates with high affinity for Fe 3+ with high affinity, and are low-molecular-weight substances that can bind ferric ions and supply them to microbial cells. Protease can promote proteolysis and decompose into amino acids to supply plant roots. Bacillus is widely distributed in nature and can be isolated from marine and river sediments, soil, rhizosphere of plants, and plant tissues. It is harmless to humans and animals, pollution-free to the environment, and can produce secondary metabolites with antagonistic effects against 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. There is currently no relevant report on Bacillus promoting plants to produce vitamins. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides a Bacillus Cy15 with disease inhibition and growth promotion functions and its applications.

[0006] The technical solution of the present invention is as follows: A Bacillus ( Bacillus sp.) Cy15, was deposited at the China Center for Type Culture Collection on March 31, 2025. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 2025659.

[0007] The Bacillus Cy15 has the abilities of producing IAA and siderophores, dissolving phosphorus, and decomposing potassium. At the same time, it 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.

[0008] The cultivation method of the above-mentioned Bacillus Cy15 specifically includes the following steps: Inoculate Bacillus Cy15 into a solid medium for activation culture to obtain single colonies; pick the single colonies into a liquid medium for fermentation culture to obtain a Bacillus Cy15 bacterial liquid.

[0009] Preferably according to the present invention, the solid medium is an LB solid medium, and the liquid medium is an LB liquid medium.

[0010] Preferably according to the present invention, the conditions for activation culture are: culturing at 28-30°C for 12-24 h.

[0011] Preferably according to the present invention, the conditions for fermentation culture are: culturing with shaking at 30-37°C and 160-180 rpm for 24-36 h.

[0012] A microbial inoculant includes the above-mentioned Bacillus Cy15.

[0013] The application of the above-mentioned Bacillus Cy15 or its inoculant in one or more of producing IAA and siderophores, dissolving phosphorus, decomposing potassium, and promoting plant growth.

[0014] The application of the above-mentioned Bacillus Cy15 or its inoculant in increasing the content of vitamin C or soluble protein in vegetables.

[0015] The application of the above-mentioned Bacillus Cy15 or its inoculant in preventing and controlling plant diseases caused by plant pathogenic fungi.

[0016] Preferably according to the present invention, the pathogenic fungi include: Trichothecium roseum ( Trichothecium roseum ), Fusarium solani ( Fusarium solani ), Botrytis cinerea ( Botrytis cinerea ), Fusarium oxysporum ( Fusarium oxysporum ), Alternaria solani ( Alternaria Nees ), Gibberella fujikuroi ( Gibberella fujikuroi ), Neocosmospora vasinfecta ( Neocosmospora vasinfecta ), Pythium catenulatum ( Pythium catenulatum ).

[0017] Beneficial effects: The Bacillus Cy15 of the present invention can be applied to the field of agricultural biological control, can strongly inhibit 8 kinds of plant pathogenic fungi, and can produce substances such as IAA and siderophores to promote plant growth. At the same time, this strain can also increase the contents of vitamin C and soluble protein in vegetables, improve the nutritional value of plants, is a biocontrol bacterium with excellent performance, and has good development and application prospects. Description of the Drawings

[0018] Figure 1 It is the colony morphology diagram of strain Cy15; Figure 2 It is the growth promotion effect diagram of strain Cy15 on lettuce. Among them, A is the Cy15 group and B is the CK group; Figure 3 It is the bar graph of the dry weight of lettuce; Figure 4 It is the bar graph of the soluble protein content of lettuce; Figure 5 It is the bar graph of the vitamin C content of lettuce. Detailed Embodiments

[0019] The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can all be obtained from commercial channels unless otherwise specified. The culture media in the following embodiments are all at natural pH unless otherwise specified. For the quantitative tests in the following embodiments, unless otherwise specified, three repeated experiments are set, and the results are averaged.

[0020] In the following embodiments, the used Trichothecium roseum ( Trichothecium roseum ), Fusarium solani ( Fusarium solani ), Botrytis cinerea ( Botrytis cinerea ), Fusarium oxysporum ( Fusarium oxysporum ), Alternaria solani ( Alternaria Nees ), Gibberella fujikuroi ( Gibberella fujikuroi ), Neocosmospora vasinfecta ( Neocosmospora vasinfecta ), Pythium catenulatum ( Pythium catenulatum ) are all isolated from the diseased parts of tomatoes. The above biological materials are only used for repeating the relevant experiments of the present invention and cannot be used for other purposes. Other technical personnel in the field can isolate the above strains from the diseased parts of tomatoes or obtain them from commercial channels for repeating the relevant experiments of the present invention.

[0021] Example 1 Isolation and Screening of Strains 1. Isolation: Soil samples were collected from the soil under pine trees on the mountainside in the southern suburbs of Jinan. 5 g of soil samples were placed into a triangular flask containing 45 mL of sterile water, shaken on a shaker at 180 rpm for 30 min, and then placed in a water bath at 80 °C and boiled for 20 min to kill other bacteria. A soil suspension sample with a concentration gradient of 10 -3 , 10 -4 , 10 -5 was prepared by the 10-fold dilution method. 0.1 mL of the soil suspension sample was spread on the LB solid medium plate and incubated at 37 °C in an inverted position for 48 h; The components of the above LB solid medium were: 10 g of peptone, 5 g of yeast extract, 10 g of NaCl, 15 g of agar, and distilled water was made up to 1000 mL; sterilized at 121.5 °C for 20 min.

[0022] 2. Screening: Single colonies obtained from isolation were inoculated into 100 mL of LB liquid medium and cultured on a shaker at 37 °C and 180 rpm for 24 h to obtain a bacterial suspension. A 5-mm-diameter Fusarium oxysporum mycelial disc was inoculated in the center of the PDA medium plate, and at the same time, 5 μL of the bacterial suspension was inoculated 3.5 cm away from the center of the plate and cultured at 28 °C for 5 d. According to the size of the inhibition zone, the strain with the best effect of inhibiting Fusarium oxysporum was selected, and a strain with grayish-white color, blurred edge, and irregular shape was obtained. The colony morphology of this strain is as shown in Figure 1 and was named Cy15.

[0023] The components of the above LB liquid medium were: 10 g of peptone, 5 g of yeast extract, 10 g of NaCl, and distilled water was made up to 1000 mL; sterilized at 121.5 °C for 20 min.

[0024] Example 2 Molecular biological identification of the strain After extracting the genomic DNA of strain Cy15 using a common bacterial genomic DNA extraction kit, PCR amplification was carried out using the universal primers for bacterial 16S rDNA. The sequences of the universal primers are as follows: 27F: 5’-AGAGTTTGATCCTGGCTCAG-3’, 1492R: 5’-ACGGCTACCTTGTTACGACTT-3’.

[0025] PCR reaction conditions: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 63°C for 30 s, extension at 72°C for 45 s, for 34 cycles; extension at 72°C for 10 min. The PCR amplification products were sent to Sangon Biotech for sequence determination, and the sequencing results are shown in SEQ ID NO.1. The obtained sequence was submitted to the NCBI database for similarity comparison with the 16S rDNA gene sequences in the nucleic acid database. The results showed that the similarity between strain Cy15 and Bacillus reached 99%. Combining the physiological characteristics of the strain, this strain was identified as Bacillus ( Bacillus sp.).

[0026] The above-mentioned strain Cy15 was taxonomically named Bacillus ( Bacillus sp.), which has been deposited in the China Center for Type Culture Collection. The deposit address is Wuhan University, Wuhan, China. The deposit date is March 31, 2025, and the deposit number is CCTCC NO: M 2025659.

[0027] Example 3 Fungal antibacterial spectrum test of strain Cy15 Two strains (Cy116, Cy128) isolated in Example 1, which also have the effect of inhibiting pathogenic bacteria, were selected to conduct a plate confrontation experiment with strain Cy15 at the same time to detect the inhibition rate of the isolated strains against pathogenic fungi.

[0028] The specific method is as follows: Single colonies of strains Cy15, Cy116, and Cy128 were respectively picked and inoculated into 100 mL of LB liquid medium, and cultured with shaking at 28°C and 180 rpm for 24 h to obtain bacterial suspensions; A pathogenic fungal cake with a diameter of 5 mm was inoculated in the center of the PDA medium plate. At the same time, 5 μL of the bacterial suspension was inoculated 3.5 cm away from the center of the plate (inoculating LB liquid medium as the control treatment group), and cultured at 28°C for 5 d, and then the diameter of the pathogenic fungal colony was measured. Each pathogenic fungus was set with 3 repeated treatments.

[0029] Inhibition rate (%) = 100% × (diameter of the pathogenic fungal colony in the control treatment - diameter of the pathogenic fungal colony treated with the bacterial suspension of the isolated strain) / diameter of the pathogenic fungal colony in the control treatment.

[0030] The antibacterial effects of strains Cy15, Cy116, and Cy128 against different pathogenic fungi are shown in Table 1.

[0031] Table 1. Inhibition rates of strains Cy15, Cy116, and Cy128 against different pathogenic fungi

[0032] The results showed that Cy15 had better antibacterial effects and a wider range of antibacterial species than other isolated strains (Cy116, Cy128).

[0033] Example 4 Functional Characteristic Detection of Strain Cy15 Pick a single colony of strain Cy15 and inoculate it into 100 mL of LB liquid medium. Incubate it at 28 °C and 180 rpm for 24 h to obtain a Cy15 bacterial suspension for the following tests.

[0034] 1. Detection of the Ability of the Strain to Produce IAA: Inoculate the Cy15 bacterial suspension into King's B liquid medium (B King) at a volume ratio of 1%. Incubate it on a constant temperature shaker at 30 °C and 180 rpm for 3 d. Centrifuge it at 12,000 rpm for 10 min. Take 1 mL of the supernatant and mix it evenly with 2 mL of Salkowski colorimetric solution. Separately, take 1 mL of standard plant growth hormone IAA with concentrations of 90, 70, 50, 30, and 10 mg / L, and add 2 mL of Salkowski colorimetric solution to each and mix evenly as gradient controls. Let it stand in the dark at room temperature for 30 min. Use a spectrophotometer to record the absorbance of the reaction solution at 530 nm, draw an IAA standard curve, and calculate the IAA concentration in the Cy15 bacterial suspension according to the curve. After detection, the IAA yield of the Cy15 bacterial suspension is 115.36 ± 2.35 mg / L.

[0035] The composition of the above King's B liquid medium is: 20 g of peptone, 1.5 g of dipotassium hydrogen phosphate, 1.5 g of magnesium sulfate heptahydrate, 1 g of tryptophan, 10 g of glycerol, add distilled water to a volume of 1000 mL, stir evenly, and adjust the pH to 7.2; The composition of the above Salkowski colorimetric solution is: 10 mL of 0.5 mol / L ferric chloride, 500 mL of 35% perchloric acid, mix evenly before use.

[0036] 2. Detection of the Function of Producing Siderophores: Inoculate the Cy15 bacterial suspension into MKB liquid medium at a volume ratio of 1%. After culturing at 30 °C and 180 rpm for 48 h, use an inoculation loop to spot inoculate it onto the CAS medium plate and culture it at 30 °C for 2 d. Observe the cultured CAS medium plate. An obvious orange-yellow halo appears around the Cy15 colony, indicating that strain Cy15 has the function of producing siderophores.

[0037] The composition of the above MKB liquid medium is: 5 g of casein amino acids, 15 mL of glycerol, 2.5 g of K2HPO4, 2.5 g of MgSO4·7H2O, add distilled water to a volume of 1 L; pH = 7.2.

[0038] The above-mentioned CAS culture medium components are as follows: Solution A: 60.5 mg of chrome azurol S is dissolved in 50 mL of deionized water, 10 mL of ferric ion solution (1 mmol / L FeCl3·6H2O, with 10 mmol / L hydrochloric acid as the solvent), 72.9 mg of CTAB (cetyltrimethylammonium bromide) is dissolved in 40 mL of deionized water. The above three solutions are mixed and made up to 100 mL, the pH is adjusted to neutral, and it is sterilized at 121°C for 20 min; Solution B: 30.24 g of PIPES (piperazine-1,4-diethanesulfonic acid), 4 g of NaOH, 11 g of agar, and made up to 900 mL with distilled water, pH = 6.8, and sterilized at 121°C for 20 min; Solutions A and B are mixed and then used.

[0039] 3. Detection of protease production function: The Cy15 bacterial suspension was inoculated into the casein medium at a volume ratio of 1% and cultured at 30°C for 2 d. Whether a clear zone was produced around the colonies was observed. The results showed that no clear zone was produced around the colonies, indicating that the strain Cy15 did not have the function of producing protease.

[0040] The above-mentioned casein medium components are as follows: Solution A: Weigh 1.07 g of Na2HPO4·7H2O and 4 g of casein, add an appropriate amount of distilled water, and heat to dissolve; Solution B: Weigh 0.36 g of KH2PO4 and dissolve it in water; after mixing Solutions A and B, add 0.3 mL of casein hydrolyzate, add 20 g of agar, and finally make up to 1000 mL with distilled water.

[0041] 4. Detection of phosphorus solubilization function: The Cy15 bacterial suspension was inoculated into the NBRIP liquid medium at a volume ratio of 1% and cultured at 30°C and 180 rpm for 7 d. After the culture was completed, it was centrifuged at 12000 rpm for 10 min. 1 mL of the supernatant was taken and diluted 10 times. The molybdenum antimony anti-colorimetric method was used to measure the phosphorus content in the supernatant. The NBRIP liquid medium without inoculation was used as a control, and it was repeated 3 times. After detection, the phosphorus solubilization amount of the strain Cy15 was 213.8 ± 7.3 mg / L.

[0042] The above-mentioned NBRIP liquid medium components are as follows: 10.0 g of glucose, 5.0 g of calcium phosphate, 0.1 g of ammonium chloride, 0.2 g of sodium chloride, 0.25 g of magnesium sulfate heptahydrate, 0.2 g of potassium chloride, 0.002 g of ferrous sulfate, 0.002 g of manganese sulfate, and made up to 1000 mL with distilled water.

[0043] 5. Detection of potassium solubilization function: The Cy15 bacterial suspension was inoculated into the potassium-solubilizing medium at a volume ratio of 1%, and cultured at 30 °C and 180 rpm for 7 days. After the culture, it was centrifuged at 500 rpm for 10 min to remove insoluble substances in the fermentation broth, and then centrifuged at 12,000 rpm for 10 min. The supernatant was collected, and the content of available potassium in the supernatant was determined by the flame spectrophotometer method. The potassium-solubilizing medium without inoculation was used as a control, and the experiment was repeated 3 times. The potassium-solubilizing amount of strain Cy15 was detected to be 19.4 ± 1.3 mg / L.

[0044] The components of the above potassium-solubilizing medium were as follows: 5 g of glucose, 0.5 g of ammonium sulfate, 0.5 g of yeast powder, 0.3 g of magnesium sulfate, 2 g of disodium hydrogen phosphate, 0.03 g of ferrous sulfate, 0.03 g of manganese sulfate, 2 g of potassium feldspar, and distilled water was added to make up 1000 mL.

[0045] The above results showed that the functional characteristics of strain Cy15 were as shown in Table 2.

[0046] Table 2. Functional characteristics of strain Cy15

[0047] Note: + indicates detected, ND indicates not detected Example 5 Activation of strain Cy15 and preparation of Cy15 bacterial agent Strain Cy15 was inoculated on LB solid medium and cultured at 30 °C for 24 h to obtain an activated strain. Then, plump activated strains were picked and inoculated into LB liquid medium, and cultured with shaking at 37 °C and 180 rpm for 24 h to obtain a fermentation broth; the fermentation broth was transferred to a sterile centrifuge bottle, centrifuged at 5000 rpm for 5 min to collect the bacteria, washed with sterile deionized water, and resuspended to make the bacterial liquid concentration reach 5×10 8 cfu / mL or more to obtain the Cy15 bacterial agent.

[0048] Example 6 Determination of the growth-promoting effect of strain Cy15 Strain Cy15 was applied to the lettuce pot experiment and carried out according to the following method: The pot for potting was 14 cm in side length × 11 cm in height and contained 1.5 kg of soil. The purchased lettuce seeds were surface-sterilized, 10 seeds were sown in each pot, and after germination, the seedlings were thinned to 5 plants per pot. The soil was regularly irrigated to keep it moist; the Cy15 bacterial agent prepared in Example 5 was diluted with sterile deionized water to a bacterial liquid concentration of 1×10 8cfu / mL; Two treatment groups were set up, one with deionized water added (CK group) and the other with bacteria added (Cy15 group), with 3 pots in parallel for each group. At the third leaf stage of lettuce, a ditch (1 - 2 cm deep) was dug around the roots, and the diluted bacterial agent or deionized water was added to the ditch, with the liquid addition amount being 20 mL / pot. The potted plants were cultivated in a greenhouse (temperature 10 - 22 °C, relative humidity 30 - 45%, normal light), and the entire cultivation period was 45 days. Figure 2 It is a morphological diagram of some lettuce after the cultivation ended.

[0049] After the cultivation ended, the edible tissues and roots of each pot of lettuce were collected for subsequent analysis; the lettuce roots and edible tissues were washed with distilled water and evenly divided into two parts. One part was inactivated at 105 °C for 30 min and dried to a constant weight at 65 °C, and the weight after drying was recorded; as Figure 3 shown, 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, proving that applying the Cy15 bacterial agent had a significant growth-promoting effect.

[0050] Example 7 Effect of strain Cy15 on increasing the vitamin C and soluble protein contents of lettuce For the other part of the edible tissues collected after the cultivation in Example 6, the vitamin C content was detected according to the method in GB 5009.86 - 2016.

[0051] Weighed 2.0 g of edible tissue, added 5 mL of distilled water and ground it into a homogenate, then centrifuged at 4 °C and 12 000×g for 20 min, and the supernatant collected was the soluble protein extract. The protein content in the above extract was determined according to the method in GB 5009.5 - 2016, which was the content of soluble protein.

[0052] The results of the soluble protein content were as Figure 4 shown. 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 Figure 5 shown. 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 applying the Cy15 bacterial agent could increase the vitamin C and soluble protein contents of 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 at Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 2025659.

2. The method for culturing Bacillus Cy15 according to claim 1, characterized in that: 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 up and placed in a liquid culture medium for fermentation culture to obtain a Bacillus Cy15 bacterial liquid.

3. The culture method according to claim 2, characterized in that 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, characterized in that The activation culture conditions are: culturing at 28-30° C. for 12-24 hours.

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

6. A microbial agent, characterized in that: Comprising the Bacillus Cy15 described in 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, dissolving phosphate, dissolving potassium, and promoting plant growth.

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

9. Use of the Bacillus Cy15 according to claim 1 or the microbial agent according to claim 6 in preventing and controlling plant diseases caused by plant pathogenic fungi.

10. The use according to claim 9, characterized in that The pathogenic fungi include: Pink Trichothecene ( Trichothecium roseum )、Fusarium solani( Fusarium solani )、Botrytis cinerea( Botrytis cinerea ), Fusarium oxysporum ( Fusarium oxysporum ), Alternaria solani ( Alternaria Nees )、Fujikura Gibberella Gibberella fujikuroi )、Invasion of new red shell ( Neocosmospora vasinfecta )、Pythium chain Pythium catenulatum ).

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