Bacillus subtilis zhaochanglin000439 and application thereof

Abstract

The application provides a bacillus subtilis zhaochanglin000439 and application thereof, and relates to the technical field of microbial prevention and treatment. The preservation number of the bacillus subtilis zhaochanglin000439 is CGMCC No. 34828, and the bacillus subtilis zhaochanglin000439 is applied to prevention and treatment of roxburgh rose fruit rot caused by fusarium decemcellulare. The application overcomes the defects of the prior art, the bacillus subtilis zhaochanglin000439 can have a good inhibiting effect on the pathogenic bacteria fusarium decemcellulare of roxburgh rose fruit rot, and plays a good enlightenment role for microbial prevention and treatment of roxburgh rose fruit rot.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial prevention and control, and particularly relates to a bacillus subtilis zhaochanglin000439 and application thereof. BACKGROUND

[0002] Rosa roxburghii is a perennial deciduous shrub of Rosaceae-Rosa, also known as R. roxburghii Tratt, R. roxburghii Tratt, and R. roxburghii Tratt. This species has both medicinal and edible values. Its fruits are rich in various bioactive components, including amino acids, vitamins, and mineral elements. In terms of vitamin C content, R. roxburghii is significantly higher than other fruits and vegetables, ranking first in the plant kingdom. Meanwhile, its rutin and superoxide dismutase (SOD) contents are also among the highest levels in fruits, thus it is praised as the “three-king fruit” by the academic community. The medicinal value of R. roxburghii is not only reflected in its fruits, but also in its flowers, leaves, and seeds, which have significant medicinal effects. Studies have shown that R. roxburghii has good effects on anti-tumor, free radical scavenging, and radiation damage reduction.

[0003] However, the control of R. roxburghii fruit rot, one of the main diseases restricting the development of the R. roxburghii industry, is particularly important. This disease is caused by multiple pathogenic fungi, leading to water spot-like lesions on the surface of the fruit, which rapidly expand into brown to black-brown rot, accompanied by a foul odor, and even causing fruit drop in severe cases. Investigation results show that the incidence of R. roxburghii fruit rot in some areas can reach 30% to 50%, resulting in a 20% to 40% reduction in fruit yield, with direct economic losses exceeding tens of millions of yuan per year. In addition, this disease also reduces the commodity value of the fruit, further affecting the quality and market competitiveness of R. roxburghii processing products.

[0004] During the cultivation of R. roxburghii, there are many types of pathogenic fungi causing fruit rot, and the pathogenic mechanisms and control methods of different pathogenic fungi are significantly different. R. roxburghii fruit rot can be caused by Guignardia bidwellii, Fusarium spp., Penicillium spp., and Alternaria spp. These pathogenic fungi invade the fruit interior through wounds, stomata, and other pathways under suitable temperature and humidity conditions, and then cause diseases. However, the pathogens of R. roxburghii fruit rot may differ in different regions, and the specific pathogen types need to be accurately identified based on local actual conditions. Therefore, it is necessary to further explore the diversity of pathogenic fungi of R. roxburghii fruit rot and comprehensively use ecological control, chemical control, and biological control, etc. to develop scientific and reasonable comprehensive control strategies, so as to ensure the sustainable development of the R. roxburghii industry. SUMMARY

[0005] In view of the deficiencies in the prior art, the present application provides a bacillus subtilis zhaochanglin000439 and application thereof, which can have good antagonistic effect on the pathogenic bacteria of roxburgh rose fruit rot, effectively inhibit the growth of the bacteria, and play a microbial control effect.

[0006] To achieve the above object, the present application is realized by the following technical solutions:

[0007] A bacillus subtilis zhaochanglin000439, characterized in that the preservation number of the bacillus subtilis zhaochanglin000439 is CGMCC No. 34828.

[0008] The bacillus subtilis zhaochanglin000439 is applied to prevent and treat roxburgh rose fruit rot.

[0009] Preferably, the pathogenic bacteria of roxburgh rose fruit rot is fusarium decemcellulare. Fusarium annulatum ).

[0010] The present application provides a bacillus subtilis zhaochanglin000439 and application thereof, which has the following advantages compared with the prior art:

[0011] The newly preserved bacillus subtilis zhaochanglin000439 can have good bacteriostatic effect on the pathogenic bacteria fusarium decemcellulare of roxburgh rose fruit rot, effectively inhibit the growth of the fusarium decemcellulare, so as to achieve the effect of preventing and treating roxburgh rose fruit rot, and play a good enlightenment role for microbial control of roxburgh rose fruit rot. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 It is a morphological feature diagram of the bacillus subtilis zhaochanglin000439 in the embodiments of the present application.

[0013] Figure 2 It is a phylogenetic tree of the bacillus subtilis zhaochanglin000439 in the embodiments of the present application.

[0014] Figure 3 It is a schematic diagram of the plate confrontation experiment results of the bacillus subtilis zhaochanglin000439 and the pathogenic bacteria fusarium decemcellulare in the embodiments of the present application.

[0015] Figure 4 It is a schematic diagram of the surface lesion situation of roxburgh rose inoculated with pathogenic bacteria after 7 days in the control group and the experimental group in the embodiment 3 of the present application. DETAILED DESCRIPTION

[0016] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the embodiments of the present application. Obviously, the described embodiments are 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 of ordinary skill in the art without creative efforts belong to the scope of the present application.

[0017] The Bacillus subtilis zhaochanglin000439 provided below is deposited in the China General Microbiological Culture Collection Center on June 10, 2025, and the recommended classification name is Bacillus subtilis. Bacillus subtilis The deposit number is CGMCC No. 34828. Embodiment 1

[0018] Identification of Bacillus subtilis zhaochanglin000439:

[0019] The Bacillus subtilis zhaochanglin000439 isolated from soil was identified:

[0020] 1. Morphological identification:

[0021] Liquid medium: glucose 22.2 g / L, yeast extract powder 6.55 g / L, proteose peptone 7.15 g / L and potassium chloride 1.95 g / L, constant volume to 1000 mL with distilled water, 121℃ sterilization for 20 min, standby (control the initial medium pH to 6.0);

[0022] LB solid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar 15 g / L, constant volume to 1000 mL with distilled water, 121℃ sterilization for 20 min, standby;

[0023] Inoculate Bacillus subtilis zhaochanglin000439 into the liquid medium, control the culture temperature to 31℃, the shaking speed to 180 r / min, and culture for 3d to obtain the Bacillus subtilis zhaochanglin000439 suspension;

[0024] Dip the Bacillus subtilis zhaochanglin000439 suspension in the solid medium and activate by streaking, and place it in a constant temperature incubator at 28℃ for 24 h; when single colonies are formed, observe and record the morphological characteristics of the colonies, such as the color, transparency, texture, size and other characteristics of the colonies, as shown on the left side of Figure 1

[0025] 2. Gram staining:​

[0026] Smear: First, the B. subtilis zhaochanglin000439 suspension was smeared on 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 min to color the bacteria. Iodine treatment: Iodine solution was added to combine with the crystal violet dye to form a crystal violet-iodine complex.

[0027] Decolorization: The smear was gently washed with alcohol to remove excess dye. Gram-positive bacteria can maintain the crystal violet-iodine complex, while Gram-negative bacteria have a thin peptidoglycan layer and an outer membrane, causing the complex to be washed off.

[0028] Safranin staining: Finally, red dye was used for counterstaining. Gram-positive bacteria had been stained with the crystal violet-iodine complex before, so they remained purple in this step; while Gram-negative bacteria were stained red or pink in this step.

[0029] The specific staining results are shown on the right side of Figure 1 B. subtilis zhaochanglin000439 is Gram-positive.

[0030] 3. Phylogenetic tree:

[0031] The 16S rDNA gene fragment was used for analysis to construct a phylogenetic tree, and the specific phylogenetic tree is shown in Figure 2 . Example 2:

[0032] Verify the inhibition rate of B. subtilis zhaochanglin000439 on the pathogen of Rosaceae fruit rot disease Fusarium decemcellulare (F. decemcellulare) ; Fusarium annulatum

[0033] The pathogen F. decemcellulare was isolated and extracted from Rosaceae fruits suffering from Rosaceae fruit rot disease and used as the test bacteria to verify the inhibition effect of B. subtilis zhaochanglin000439 on F. decemcellulare by using the plate confrontation culture method.

[0034] 1. PDA medium: 200 g potato broth (200 g peeled potatoes boiled for 30 min and then filtered) + 20 g glucose + 20 g agar, add distilled water to 1 L, pH 6.0±0.2; sterilize at 121℃ for 15 min for standby;

[0035] ​2, inoculate the ring-shaped fusarium into the PDA medium, cultivate at 25℃ for 3d, use the puncher to take the ring-shaped fusarium cake (about 5mm in diameter) and place it in the center of the LB solid medium plate, for the experimental group, use the bacillus subtilis zhaochanglin000439 suspension to streak around the LB solid medium plate, set up the control group only inoculating the ring-shaped fusarium cake without other treatment, cultivate at 25℃ for 3d, then calculate the inhibition rate (inhibition rate (%)=(pathogen colony diameter of the control group-pathogen colony diameter of the experimental group) / pathogen colony diameter of the control group x 100%);

[0036] The specific inhibition rate effect is shown in Figure 3 , wherein the left side is the control group and the right side is the experimental group, and the inhibition rate is 77.5%. Example 3:

[0037] Verify the prevention and treatment effect of bacillus subtilis zhaochanglin000439 on the fruit rot disease of roxburgh rose;

[0038] 1, select fresh roxburgh rose mature fruits, the surface is intact without damage, and soak in 75% alcohol solution for 30 seconds before the experiment, then dry at room temperature.

[0039] 2, obtain the test bacteria:

[0040] 2.1, pathogenic bacteria: extract and separate the pathogenic bacteria fusarium solani from the roxburgh rose fruits with fruit rot disease as pathogenic bacteria; the pathogenic strain is cultured on PDA medium at 28℃ for 7d for activation, ready for use;

[0041] 2.2, inoculate bacillus subtilis zhaochanglin000439 into liquid medium (glucose 22.2 g / L, yeast extract powder 6.55 g / L, peptone 7.15 g / L and potassium chloride 1.95 g / L, constant volume to 1000ml with distilled water) to prepare spore suspension, count the concentration to 1x10 7 / ml with a hemocytometer before use, obtain the antagonistic bacteria liquid for standby.

[0042] 3, inoculation experiment:

[0043] Divide the roxburgh rose mature fruits into two groups, one group is the control group and the other group is the experimental group, both the control group and the experimental group use sterile inoculation needle to prick two wounds on the fruit surface, ensure the size of each wound is consistent and inoculate pathogenic bacteria cake (2mm) on the wound.

[0044] For the control group, spray physiological saline after inoculating the cake, for the experimental group, spray the antagonistic bacteria liquid on the roxburgh rose after inoculating the cake, observe the fruit rot disease of roxburgh rose after 7d, specifically asFigure 4 As shown in the figure, a is the control group, the disease spot of the roxburgh rose is larger, and the fruit rot develops more seriously; and Figure 4 b is the experimental group, the surface of the roxburgh rose is basically unchanged, and the whole is basically free of disease spots.

[0045] The above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can be modified, or some technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

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 control of prickly pear fruit rot, characterized in that: The pathogen causing the prickly pear fruit rot is Fusarium rings (Fusarium moniliforme). Fusarium annulatum ).

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