CN113846039B

CN113846039B - Bacillus bailii and application thereof

Info

Publication number: CN113846039B
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: 2023-10-03
Anticipated expiration: 2041-11-20
Also published as: CN113846039A

Abstract

The invention discloses bacillus belicus and application thereof. The strain is named as bacillus belicusBacillus velezensis) ZJK2 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.20037 in the year 6 and 8 of 2020. The strain and the thallus and aseptic thallus fermentation liquor prepared by liquid fermentation have good antagonism effect on gray mold or anthracnose; the biological agent can have remarkable control effects on gray mold and anthracnose of mulberry, grape, strawberry and the like, has the advantages of green safety, environmental friendliness and the like compared with the traditional chemical agent, and has wide application prospect in the aspect of biological control of gray mold and anthracnose.

Description

Bacillus bailii and application thereof

Technical Field

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

Background

At present, the control of gray mold and anthracnose is mainly chemical means, and gray mold and anthracnose are considered to easily generate drug resistance. In the prevention and treatment, imported medicines such as fast keling and the like are mostly adopted to spray, so that a better prevention and treatment effect is achieved, but under the condition of continuous use, the prevention effect is reduced, the botrytis cinerea and the botrytis cinerea are easy to generate drug resistance, even multiple drug resistance is generated, and the control capability of the botrytis cinerea and anthracnose is gradually lost. And when using these chemical pesticides, environmental pollution and ecological damage are often caused. Meanwhile, the chemical pesticides have certain toxicity, and if people eat the fruits and vegetables with the chemical pesticides left for a long time, the health of human bodies can be seriously damaged.

Current biological agent control is attracting attention and importance. Obviously different from chemical bactericides, the microbial control can reduce the probability of pathogenic bacteria drug resistance, reduce environmental pollution, be beneficial to ecological balance and have simple production process. Bacillus belicus is a newly discovered species in Bacillus. Is widely distributed in nature, can be separated from sea and river sediment, soil, plant rhizosphere and plant tissue, is harmless to human and livestock, has no pollution to the environment, and can generate secondary metabolites with antagonism to various pathogenic microorganisms. In recent years, researches on bacillus belicus have been mainly focused on the fields of feed, medicine, textiles, 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, and the microbial agent control is connected with the comprehensive treatment of the diseases, so that the microbial agent control has important significance and profound influence on the sustainable development of green agriculture. Therefore, the bacillus beleiensis with excellent biocontrol capability is screened, and the bacillus beleiensis has wide application prospect.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides bacillus beljalis and application thereof, and the screened bacillus beljalis provides good application prospects for preventing and treating gray mold or anthracnose of mulberry, strawberry and grape.

In order to solve the problems in the prior art, the invention adopts the following technical scheme:

bacillus belicus, named Bacillus belicus (Bacillus velezensis) ZJK2, is preserved in China general microbiological culture Collection center (CGMCC) No.20037, at 6/8/2020.

As an improvement, the bacterial colony of the bacillus belicus is light yellow, is round in shape and smaller in diameter, has smooth and moist surface and bulges, is transparent and 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 ℃.

As an improvement, the bacillus belicus is a gram positive bacillus.

As an improvement, the bacillus belicus has antagonism to gray mold of mulberry, strawberry or grape.

As an improvement, the bacillus belicus has antagonism to anthracnose of mulberry, strawberry or grape.

The bacillus belicus is applied to gray mold antagonistic bacteria of mulberries, strawberries or grapes.

The bacillus belicus is applied to gray mold anthracnose of mulberry, strawberry or grape.

The beneficial effects are that:

the separated bacillus belicus (Bacillus velezensis) ZJK2 has good control effect on gray mold and anthracnose of mulberry, strawberry or grape; the strain has high propagation speed, strong stress resistance, safety and no toxicity, does not pollute 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 shows colony morphology of Bacillus belicus (Bacillus velezensis) ZJK2 strain;

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

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

Detailed Description

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

Example 1

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

(2) After single bacterial colonies appear on the plate, different bacterial colonies are distinguished according to the differences of the morphology and the color of the bacterial colonies, and different bacterial colonies are respectively picked by an inoculating loop and streaked on a PDA solid culture medium plate for culture;

(3) Performing antagonistic bacteria primary screening: placing gray mold blocks with basically consistent sizes in the center of a PDA flat plate, drawing a short line around the mold blocks by using an inoculating loop to draw bacteria cultivated by the line, making four lines of bacteria on the flat plate, placing the flat plate in a constant temperature incubator at 30 ℃ for cultivation for 5 days, and selecting bacteria with inhibitory effect on the growth of pathogenic bacteria, flush edge of the hypha of the inhibited pathogenic bacteria and lasting antagonism;

(4) Entering an antagonistic bacteria rescreening: placing mould fungus blocks with the same size in the center of a PDA flat plate by adopting a counter culture method, picking bacteria with good antagonism by using an inoculating loop, inoculating the bacteria on 4 corner 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 bacteria inhibition zone (from the center of a bacterial colony to the edge of pathogenic bacteria hypha);

(5) Purifying antagonistic bacteria with a large inhibition zone by using a flat plate line drawing method, firstly drawing a line on a flat plate, then burning residual bacteria on an inoculating loop, drawing a line again from the tail end of the line after the inoculating loop is cooled, continuously drawing 4-5 lines according to the step, culturing in a constant temperature incubator at 37 ℃ for 1-2 days after inversion, and carrying out a counter experiment according to the counter culturing method after a single bacterial colony is grown;

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

Bacillus belicus (Bacillus velezensis) ZJK2 used in the invention is preserved in China general microbiological culture Collection center (China Committee for culture Collection), the preservation address is North Chen Silu No. 1 and 3 of the Korean area of Beijing, the preservation number is CGMCC No.20037, and the preservation date is 2020, 6 and 8 days.

Example 2

Bacillus belicus (Bacillus velezensis) ZJK2 obtained in example 1 was cultured at constant temperature on LB medium, and the colony was pale yellow, round and small in diameter, smooth and moist in surface, raised, transparent, and regular in edge, as shown in FIG. 1.

The physiological and biochemical experiments show that the contact enzyme test, the VP test and the citrate utilization test are positive, and the MR test and the H test are all carried out ₂ The S-production tests were negative and are detailed in Table 1.

TABLE 1 physiological and biochemical test results of biocontrol strain ZJK2

The "+" indicates positive; "-" indicates negative

Extracting DNA of bacillus bailii (Bacillus velezensis) ZJK2, determining fragments by PCR reaction, performing 16S rRNA gene sequencing, performing Blast comparison on the NCBI website to construct a phylogenetic tree (figure 2), and determining that the antagonistic bacteria ZJK2 are bacillus bailii from the result of the phylogenetic tree in combination with morphological observation and physiological and biochemical experiments.

Example 3

Firstly, preprocessing the collected leaves on the strawberry plants, fully soaking the leaves with water to remove some impurities on the surfaces of the leaves, gently wiping the surfaces of the leaves with 75% alcohol cotton balls, and sterilizing the surfaces of the leaves. Inoculating Botrytis cinerea on strawberry leaf to infect Botrytis cinerea, by making 5 times of 1cm long wound on both sides of main vein of leaf with sterile needle, soaking leaf in 1×10 prepared in advance ⁶ Taking out the grape spores in the volume of each/mL of the gray grape spore suspension after 5s, and naturally airing for 2h. And finally, respectively soaking the dried strawberry leaves in 10-time diluted fermentation liquid for 15 seconds, taking out, and repeating three leaves during the test. The same treatment as above was performed with sterile water, and as a blank group, three leaves were repeated. All the treated strawberry leaves were inserted into small beakers containing water, the mouths of the beakers were sealed with a preservative film, and cultured at 25℃for 13 days under 90% RH. And observing the disease condition of the leaf on the 14 th day, counting the area proportion of leaf spots and calculating the control effect. The calculation formula of the control effect is as follows:

as shown in FIG. 3, the prevention and treatment effect of the 10-fold diluted bacterial liquid is best and reaches 99%.

In the foregoing, only some embodiments of the present invention are described, and the scope of the present invention is not limited thereto, but any simple changes or equivalent substitutions of the technical solutions that are obvious to those skilled in the art within the technical scope of the present invention disclosed in the present invention fall within the scope of the present invention.

Claims

1. A bacillus beleiensis is characterized in that the bacillus beleiensis is named as bacillus beleiensisBacillus velezensis) ZJK2 is preserved in China general microbiological culture Collection center (CGMCC) at 6 and 8 days in 2020, and has the preservation number of CGMCC No.20037, the bacillus beleiensis has antagonism to gray mold of strawberries, and bacterial colonies of the bacillus beleiensis are light yellow, have a circular shape and smaller diameter, have smooth and moist surfaces and bulges, are transparent and have regular edges, and can form spores; the bacillus belicus can grow in an environment with the pH value of 7-7.5 and the growth temperature of 28-37 ℃, and the bacillus belicus is gram-positive bacillus.

2. Use of bacillus belgium according to claim 1 as a gray mold antagonistic bacterium for strawberries.