Bacillus subtilis zhaochanglin000439 and application thereof

Abstract

The invention provides bacillus subtilis zhaochanglin000439 and application thereof, and relates to the technical field of microbial prevention and control. The preservation number of the bacillus subtilis zhaochanglin000439 is CGMCC (China General Microbiological Culture Collection Center) No. 34828, and the bacillus subtilis zhaochanglin000439 is applied to the prevention and treatment of the fruit rot of the roxburgh rose caused by fusarium circulans. According to the bacillus subtilis zhaochinglin000439, the defects in the prior art are overcome, the bacillus subtilis zhaochinglin000439 can have a good inhibition effect on pathogenic bacteria fusarium circulans of the rosa roxburghii fruit rot, and a good enlightenment effect is achieved for microbial prevention and control of the rosa roxburghii fruit rot.

Description

Technical Field

[0001] This invention relates to the field of microbial control technology, specifically to a strain of Bacillus subtilis zhaochanglin000439 and its applications. Background Technology

[0002] The prickly pear, a perennial deciduous shrub belonging to the genus Rosa in the family Rosaceae, is also known as the silk-reeling flower, the spring-sending fruit, or the Wenxian fruit. This species possesses both medicinal and edible value. Its fruit is rich in various bioactive components, including amino acids, vitamins, and minerals. In terms of vitamin C content, the prickly pear significantly surpasses other fruits and vegetables, ranking first in the plant kingdom. Simultaneously, its rutin and superoxide dismutase (SOD) content are among the highest in fruits, earning it the reputation of being a "three-king fruit" in the scientific community. The medicinal value of the prickly pear is not limited to its fruit; its flowers, leaves, and seeds also possess significant medicinal effects. Studies have shown that the prickly pear exhibits good effects in anti-tumor activity, free radical scavenging, and reducing radiation damage.

[0003] However, as one of the major diseases restricting the development of the prickly pear industry, the prevention and control of prickly pear fruit rot is particularly important. This disease is caused by various pathogenic fungi, leading to water-soaked lesions on the fruit surface, which rapidly expand into brown to dark brown rot, accompanied by a foul odor, and in severe cases, even causing fruit drop. Surveys show that the incidence of prickly pear fruit rot in some areas reaches 30% to 50%, resulting in a 20% to 40% reduction in fruit yield and direct economic losses exceeding tens of millions of yuan annually. Furthermore, this disease reduces the commercial value of the fruit, further affecting the quality and market competitiveness of processed prickly pear products.

[0004] In the cultivation of prickly pear, there are numerous pathogenic fungi causing fruit rot, and the pathogenic mechanisms and control methods of different pathogens vary significantly. Prickly pear fruit rot may be caused by pathogens such as *Colletotrichum oryzae*, *Fusarium* spp., *Penicillium* spp., and *Alternaria* spp. These pathogens invade the fruit through wounds and stomata under suitable temperature and humidity conditions, leading to disease. However, the pathogens causing prickly pear fruit rot may differ in different regions, and the specific pathogen species need to be accurately identified based on local conditions. Therefore, it is necessary to further explore the diversity of pathogens causing prickly pear fruit rot and comprehensively utilize ecological, chemical, and biological control methods to formulate a scientific and reasonable integrated control strategy to ensure the sustainable development of the prickly pear industry. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a strain of Bacillus subtilis zhaochanglin000439 and its applications. This strain has a good antagonistic effect on the pathogen of prickly pear fruit rot, effectively inhibiting the growth of the pathogen and achieving the effect of microbial control.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A strain of Bacillus subtilis zhaochanglin000439, characterized in that the preservation number of Bacillus subtilis zhaochanglin000439 is CGMCC No. 34828.

[0007] The Bacillus subtilis strain zhaochanglin000439 was used to control prickly pear fruit rot.

[0008] Preferably, the pathogen causing the prickly pear fruit rot is Fusarium rings (Fusarium moniliforme). Fusarium annulatum ).

[0009] This invention provides a strain of Bacillus subtilis zhaochanglin000439 and its applications, which have the following advantages compared with the prior art: The newly preserved Bacillus subtilis zhaochanglin000439 of this invention has a good antibacterial effect on Fusarium moniliforme, the pathogen of prickly pear fruit rot, and effectively inhibits the growth of Fusarium moniliforme, thereby achieving the effect of preventing and controlling prickly pear fruit rot. This provides a good inspiration for the microbial control of prickly pear fruit rot. Attached Figure Description

[0010] Figure 1 This is a schematic diagram illustrating the morphological characteristics of Bacillus subtilis zhaochanglin000439 in an embodiment of the present invention; Figure 2 This is a phylogenetic tree of Bacillus subtilis zhaochanglin000439 in an embodiment of the present invention; Figure 3 This is a schematic diagram of the plate confrontation experiment results between Bacillus subtilis zhaochanglin000439 and the pathogen Fusarium circophyllum in an embodiment of the present invention; Figure 4 This is a schematic diagram of the surface lesions on the prickly pear in the control group and experimental group 7 days after inoculation with pathogens in Example 3 of the present invention. Detailed Implementation

[0011] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0012] The Bacillus subtilis strain zhaochanglin000439 provided below is deposited at the China General Microbiological Culture Collection Center on June 10, 2025. The suggested classification name is Bacillus subtilis. Bacillus subtilis The accession number is: CGMCC No. 34828. Example 1:

[0013] Identification of Bacillus subtilis zhaochanglin000439: Identification of Bacillus subtilis zhaochanglin000439 isolated from soil: 1. Morphological identification: Liquid culture medium: 22.2 g / L glucose, 6.55 g / L yeast extract, 7.15 g / L peptone and 1.95 g / L potassium chloride, diluted to 1000 mL with distilled water, sterilized at 121℃ for 20 min, and set aside for later use (control the initial pH of the culture medium to 6.0). LB solid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar 15 g / L, bring the volume to 1000 mL with distilled water, sterilize at 121 °C for 20 min, and set aside. Bacillus subtilis zhaochanglin000439 was inoculated into liquid culture medium, and the culture temperature was controlled at 31℃, the shaking speed was 180 r / min, and the culture was carried out for 3 days to obtain a suspension of Bacillus subtilis zhaochanglin000439. Streaking a suspension of Bacillus subtilis (zhaochanglin000439) onto a solid culture medium for activation, and then incubating at 28°C for 24 hours. When single colonies form, observe and record their morphological characteristics, such as color, transparency, texture, and size. Figure 1 As shown on the left side of the middle; 2. Gram staining: Smear preparation: First, a suspension of Bacillus subtilis zhaochanglin000439 was smeared onto a microscope slide, and then the bacteria were fixed by flame burning. Crystal violet staining: The smear was stained with crystal violet dye for 1 minute to stain the bacteria. Iodine treatment: Iodine solution was added to combine with the crystal violet dye to form a crystal violet-iodine complex.

[0014] Decolorization: Gently rinse the smear with alcohol to remove excess dye. Gram-positive bacteria retain the violet-iodine complex, while Gram-negative bacteria, due to their thinner peptidoglycan layer and outer membrane, cause the complex to be washed away.

[0015] Safran staining: Finally, a red dye is used for contrast staining. Gram-positive bacteria, having already been stained with the violet-iodine complex, remain purple in this step; while Gram-negative bacteria will be stained red or pink.

[0016] Specific staining results are as follows: Figure 1 As shown on the right, Bacillus subtilis zhaochanglin000439 is Gram-positive.

[0017] 3. Phylogenetic tree: A phylogenetic tree was constructed using 16S rDNA gene fragment analysis, as shown in the following figure. Figure 2 As shown. Example 2:

[0018] Verification of Bacillus subtilis zhaochanglin000439 against Fusarium rings, the pathogen of prickly pear fruit rot ( Fusarium annulatum The antibacterial rate of ) Fusarium rings, the pathogen, was extracted and isolated from prickly pear fruits suffering from fruit rot. Using this pathogen as a test organism, the inhibitory effect of Bacillus subtilis (zhaochanglin000439) on Fusarium rings was verified using the plate confrontation culture method. 1. PDA culture medium: 200g potato decoction (200g peeled potatoes boiled for 30 minutes and then filtered) + 20g glucose + 20g agar, add distilled water to make up to 1L, pH 6.0±0.2; sterilize at 121℃ for 15min for later use; 2. Inoculate Fusarium circumflexures into PDA medium and incubate at 25°C for 3 days. Use a punch to take Fusarium circumflexures mycelial cakes (approximately 5 mm in diameter) and place them in the center of LB solid medium plates. For the experimental group, streak Bacillus subtilis suspension around the perimeter of the LB solid medium plates. A control group was set up where only Fusarium circumflexures mycelial cakes were inoculated without any further treatment. After incubation at 25°C for 3 days, calculate the inhibition rate (inhibition rate (%) = (colon diameter of pathogen in control group - colony diameter of pathogen in experimental group) / colony diameter of pathogen in control group × 100%). Specific antibacterial effects are as follows: Figure 3 As shown, the left side is the control group and the right side is the experimental group, with an antibacterial rate of 77.5%. Example 3:

[0019] Verification of the control effect of Bacillus subtilis zhaochanglin000439 on prickly pear fruit rot: 1. Selection of test fruits: Select fresh, ripe prickly pear fruits with intact and undamaged surfaces. Before the experiment, soak them in a 75% alcohol solution for 30 seconds and then dry them at room temperature.

[0020] 2. Obtaining the test bacteria: 2.1 Pathogen: Fusarium rings was extracted and isolated from prickly pear fruits suffering from fruit rot as the pathogen; the pathogenic strain was activated by culturing on PDA medium at 28 ℃ for 7 days and then ready for use. 2.2. Bacillus subtilis (zhaochanglin000439) was inoculated onto liquid culture medium (22.2 g / L glucose, 6.55 g / L yeast extract, 7.15 g / L peptone, and 1.95 g / L potassium chloride, diluted to 1000 mL with distilled water) to prepare a spore suspension. Before use, the concentration was adjusted to 1 × 10⁻⁶ using a hemocytometer. 7 The antagonistic bacterial solution was prepared by adding cells per mL.

[0021] 3. Inoculation experiment: The ripe prickly pear fruits were divided into two groups: a control group and an experimental group. Two wounds were made on the surface of the ripe prickly pear fruits in both groups using a sterile inoculation needle to ensure that each wound was the same size and to inoculate the wounds with pathogenic fungal blocks (2 mm).

[0022] For the control group, physiological saline was sprayed after inoculation of the fungal blocks. For the experimental group, antagonistic bacterial solution was sprayed on the prickly pear after inoculation of the fungal blocks. The disease incidence of prickly pear fruit rot was observed 7 days later. Details are as follows: Figure 4 As shown, 'a' represents the control group, where the prickly pear lesions are larger and the fruit rot is more severe; while... Figure 4Group b was the experimental group. The surface of the prickly pear showed almost no changes, and there were basically no lesions.

[0023] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of Bacillus subtilis ( Bacillus subtilis zhaochanglin000439, characterized in that, The Bacillus subtilis zhaochanglin000439 has the accession number CGMCC No. 34828.

2. The application of Bacillus subtilis zhaochanglin000439 as described in claim 1 in the prevention and control of prickly pear fruit rot.

3. The application according to claim 2, characterized in that: The pathogen causing the prickly pear fruit rot is Fusarium rings (Fusarium moniliforme). Fusarium annulatum ).

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