CN113846039A

CN113846039A - Bacillus belgii and application thereof

Info

Publication number: CN113846039A
Application number: CN202111381184.7A
Authority: CN
Inventors: 杨统一; 翟德丽; 黄锡春; 胡毅翀; 刘典三; 张正杨; 谌剑; 许明敏; 杨庆根; 邱荣俊; 徐信超; 赵玉媛; 李静
Original assignee: Jiangsu University of Science and Technology
Current assignee: Jiangsu University of Science and Technology
Priority date: 2021-11-20
Filing date: 2021-11-20
Publication date: 2021-12-28
Anticipated expiration: 2041-11-20
Also published as: CN113846039B

Abstract

The invention discloses Bacillus belgii and application thereof. The strain is named as Bacillus belgii (B.) (Bacillus velezensis) ZJK2, deposited in China general microbiological culture Collection center on 8 th 6 th 2020 with the preservation number of CGMCC No. 20037. The strain and thalli and sterile fermentation liquor prepared by liquid fermentation have good antagonistic effect on gray mold or anthracnose; the biological agent can achieve remarkable control effect on gray mold and anthracnose of mulberry, grape, strawberry and the like, has the advantages of greenness, safety, environmental friendliness and the like compared with the traditional chemical agent, and has wide application prospect in the biological control aspect of gray mold and anthracnose.

Description

Bacillus belgii and application thereof

Technical Field

The invention belongs to the field of microbiology, and particularly relates to bacillus beiLeisi and application thereof.

Background

At present, the prevention and treatment of gray mold and anthracnose are mainly carried out by chemical means, and the gray mold and the anthracnose are considered to be easy to generate drug resistance. The control effect is better by adopting the imported medicine spray of the prochloraz and the like, but under the condition of continuous use, the control effect is reduced, the botrytis cinerea and the gray mold are easy to generate drug resistance, even generate multiple drug resistance, and the thunberg gradually loses the control capability on the gray mold and the anthracnose. And when the chemical pesticides are used, environmental pollution and ecological damage are caused. Meanwhile, the chemical pesticides have certain toxicity, and if people eat the fruits and vegetables with the residual chemical pesticides for a long time, the human health can be seriously damaged.

Currently, biological agent control gradually draws attention and attention of people. Compared with chemical bactericides, the microbial control can reduce the probability of drug resistance of pathogenic bacteria, reduce environmental pollution, be beneficial to ecological balance and have simple production process. Bacillus belgii is a newly discovered species of Bacillus. Widely distributed in nature, can be separated from marine and river sediments, soil, plant rhizosphere and plant tissues, is harmless to human and livestock, has no pollution to environment, and can generate secondary metabolites with antagonistic action on various pathogenic microorganisms. In recent years, research on bacillus belgii has been mainly focused in the fields of feed, medicine, textile, aquatic products, sewage treatment, plant protection, and the like. The microbial agent control is an environment-friendly way for controlling agricultural diseases in protected areas, combines microbial control with comprehensive treatment of the diseases, and has important significance and profound influence on the sustainable development of green agriculture. Therefore, the method for screening the Bacillus belgii with excellent biocontrol ability has wide application prospect.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides the Bacillus belgii and the application thereof, and the screened Bacillus belgii provides good application prospects for prevention and treatment of gray mold or anthracnose of mulberry, strawberry and grape.

In order to solve the problems of the prior art, the invention adopts the technical scheme that:

a Bacillus belgii, named as Bacillus velezensis ZJK2, is preserved in China general microbiological culture Collection center (CGMCC) at 6-8 th of 2020 with the preservation number of CGMCC No. 20037.

The improvement is that the colony of the Bacillus belgii is light yellow, is round in shape and small in diameter, has smooth and moist surface, is provided with bulges and is transparent, has neat edges and can form spores; can grow in the environment with the pH value of 7-7.5 and the growth temperature of 28-37 ℃.

In a refinement, the Bacillus belgii is a gram positive bacterium.

As an improvement, the Bacillus belgii has antagonistic effect on gray mold of mulberry, strawberry or grape.

As an improvement, the Bacillus belgii has antagonistic effect on anthracnose of mulberry, strawberry or grape.

The Bacillus belgii is applied to resisting bacteria for gray mold of mulberry, strawberry or grape.

The Bacillus belgii is applied to gray mold anthracnose of mulberry, strawberry or grape.

Has the advantages that:

the separated Bacillus velezensis ZJK2 has better control effect on gray mold and anthracnose of mulberry, strawberry or grape; the bacterium has the advantages of high propagation speed, strong stress resistance, safety, no toxicity and no pollution to the environment, and is an ideal biological bactericide; the preparation method of the microbial inoculum is simple, can be used for artificial large-scale culture, is easy to realize industrial production, and has good development and application prospects.

Drawings

FIG. 1 is a colony morphology of Bacillus belgii (Bacillus velezensis) ZJK2 strain;

FIG. 2 is a phylogenetic tree of Bacillus belgii (Bacillus velezensis) ZJK2 based on the 16S rRNA gene sequence;

FIG. 3 shows the control effect of diluted bacterial liquid on gray mold of strawberry leaves.

Detailed Description

The present invention will be described in detail with reference to specific examples.

Example 1

(1) Cutting the non-diseased part of the strawberry fruit with gray mold from the strawberry gray mold disease fruits collected from Zhenjiang, Tanshu, white rabbits, sterilizing the surface of the strawberry fruit with 0.1% mercuric chloride solution for 3min, and grinding under aseptic condition; screening in LB culture medium by gradient dilution method;

(2) after single colonies appear on the flat plate, different colonies are distinguished according to the differences of the colony forms and colors, and different colonies are respectively picked by inoculating loops and streaked on a PDA solid culture medium flat plate;

(3) carrying out primary screening of antagonistic bacteria: placing Botrytis cinerea blocks with basically consistent sizes in the center of a PDA flat plate, drawing a short line around the Botrytis cinerea blocks by using an inoculating loop to draw out bacteria cultured by lineation, drawing lines of four bacteria on the flat plate, placing the flat plate in a constant-temperature incubator at 30 ℃ for 5 days, and selecting bacteria which have an inhibition effect on the growth of pathogenic bacteria, are parallel to the edges of hypha of the inhibited pathogenic bacteria and have a lasting antagonistic effect;

(4) entering antagonistic bacteria rescreening: adopting a confrontation culture method, placing mould fungus blocks with consistent sizes in the center of a PDA (personal digital Assistant) flat plate, picking bacteria with good antagonism by using an inoculating loop, inoculating the bacteria on 4 angular points 3cm away from the center of the flat plate, placing the flat plate in a 30 ℃ incubator for culturing for 3 days, and observing the size of a bacteriostatic circle (from the center of a bacterial colony to the edge of hypha of pathogenic bacteria);

(5) purifying antagonistic bacteria with a larger antibacterial zone by a flat plate line drawing method, drawing a line on a flat plate, burning off residual bacteria on an inoculating loop, drawing a line from the tail end of the previous line after the inoculating loop is cooled, continuously drawing 4-5 lines according to the steps, inversely placing the lines in a constant-temperature incubator at 37 ℃ for culturing for 1-2 days, and performing a confrontation experiment according to the confrontation culture method after a single bacterium grows out;

(6) repeating the step (5) to culture the next generation of bacteria, and continuously subculturing for 3-4 generations to obtain the strain with better antagonistic performance.

The Bacillus velezensis ZJK2 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of West Lu No. 1 of Beijing Korean area, the microorganism research institute of Chinese academy of sciences, the preservation number is CGMCC NO.20037, and the preservation date is 6 months and 8 days in 2020.

Example 2

The Bacillus belgii (Bacillus velezensis) ZJK2 separated in example 1 is cultured on LB culture medium at constant temperature, the colony color is light yellow, the shape is round, the diameter is small, the surface is smooth and moist, the surface has protrusions, the transparency is high, and the edge is neat, as shown in figure 1.

Physiological and biochemical experiments show that catalase test, VP test and citrate utilizationAll tests were positive, MR test, H₂The S production tests were all negative and are detailed in Table 1.

TABLE 1 Biochemical test results of biocontrol bacteria ZJK2

Note "+" indicates positive; "-" indicates negative

Extracting DNA of Bacillus velezensis ZJK2, determining fragments through PCR reaction, performing 16S rRNA gene sequencing, comparing the sequences at NCBI website Blast to construct phylogenetic tree (figure 2), and determining the antagonistic bacterium ZJK2 as Bacillus velezensis from the results of the phylogenetic tree by combining morphological observation and physiological and biochemical experiments.

Example 3

Firstly, the leaves on the collected strawberry plants are pretreated, the leaves are fully soaked in water to remove impurities on the surfaces of the leaves, and the surfaces of the leaves are lightly wiped by alcohol cotton balls with the concentration of 75 percent to disinfect the surfaces of the leaves. Inoculating Botrytis cinerea to strawberry leaf to infect gray mold, and making 5 cuts with length of 1cm on two sides of main vein of leaf with sterile needle, soaking leaf in prepared 1 × 10⁶Taking out the seeds per mL of the botrytis cinerea suspension after 5s, and naturally airing for 2 h. And finally, soaking the aired strawberry leaves in 10-time diluted fermentation liquor for 15s respectively, taking out, and repeating three leaves in the test. The same treatment as above was performed with sterile water as a blank control group, which was also repeated for three leaves. All treated strawberry leaves were inserted into a small beaker containing water, the mouth of the beaker was sealed with a preservative film, and cultured at 25 ℃ under 90% RH for 13 days. And observing the diseased condition of the leaves on the 14 th day, counting the area proportion of the diseased spots of the leaves and calculating the prevention and treatment effect. The prevention and treatment effect calculation formula is as follows:

the results are shown in FIG. 3, and the control effect of 10-fold diluted bacterial liquid is the best and reaches 99%.

The above description is only a partial embodiment of the present invention, and the protection scope of the present invention is not limited thereto, and any simple changes or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the protection scope of the present invention.

Claims

1. A Bacillus belgii strain, which is named as Bacillus belgii (B.), (B.belgii)Bacillus velezensis) ZJK2, deposited in China general microbiological culture Collection center on 8 th 6 th 2020 with the preservation number of CGMCC 20037.

2. The bacillus belgii according to claim 1, wherein the bacillus belgii colony is light yellow, is round in shape and small in diameter, is smooth and moist in surface, has protrusions, is relatively transparent, has regular edges, and can form spores; can grow in the environment with the pH value of 7-7.5 and the growth temperature of 28-37 ℃.

3. The Bacillus belgii of claim 1, wherein the Bacillus belgii is a gram-positive bacterium.

4. The Bacillus belgii of claim 1, wherein the Bacillus belgii is antagonistic to gray mold in mulberry, strawberry or grape.

5. The Bacillus belgii of claim 1, wherein the Bacillus belgii is antagonistic to anthracnose of mulberry, strawberry or grape.

6. Use of the bacillus belgii according to claims 1 to 5 as an antagonist of gray mold in mulberry, strawberry or grape.

7. Use of a bacillus belgii according to claims 1 to 5 as an anthrax antagonist of mulberry, strawberry or grape.