CN112608860B

CN112608860B - Bacillus amyloliquefaciens YB130 and application thereof

Info

Publication number: CN112608860B
Application number: CN202011502068.1A
Authority: CN
Inventors: 孙润红; 杨丽荣; 陈岩岩; 夏明聪; 张洁; 徐文; 武超
Original assignee: Institute of Plant Protection of Henan Academy of Agricultural Sciences
Current assignee: Institute of Plant Protection of Henan Academy of Agricultural Sciences
Priority date: 2020-12-17
Filing date: 2020-12-17
Publication date: 2022-06-28
Anticipated expiration: 2040-12-17
Also published as: CN112608860A

Abstract

The invention relates to the technical field of microorganisms. The invention provides a Bacillus amyloliquefaciens YB130 and application thereof, wherein the Latin article of the Bacillus amyloliquefaciens YB130 is Bacillus amyloliquefaciens, the strain is preserved in the common microorganism center of the China Committee for culture Collection of microorganisms, and the address is as follows: the preservation date of No. 3 Xilu Beijing Xiyan No.1 Beijing, Chaoyang district is: 7/8/2018, the preservation number is: CGMCC No. 16229. The bacillus amyloliquefaciens YB130 can effectively inhibit the generation of fusarium graminearum cyst shells, thereby reducing the initial infection source of the wheat scab, achieving the purposes of prevention, forward movement and comprehensive prevention and control of the wheat scab, realizing green prevention and control and having good application prospect.

Description

Bacillus amyloliquefaciens YB130 and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to a bacillus amyloliquefaciens YB130 and application thereof.

Background

Fusarium graminearum (Fusarium graminearum) is a filamentous ascomycete fungus and is the main pathogen of wheat scab. The growth and development can be divided into two stages: asexual and sexual periods. Conidia are produced in asexual period; the spores produced during sexual periods are ascospores. Researches show that fusarium graminearum overwinter on disease plant residues such as corn, wheat and rice in the form of saprophytic hypha, and the generation and maturation of fusarium graminearum ascocarp are facilitated in warm and humid days in spring. In the flowering period of wheat, a large amount of viscous ascospores are strongly ejected from ascospores in ascospores mature on the surfaces of plant disease residues and spread to host plants along with media such as wind, rainwater, insects and the like, the ascospores are used as a primary infection source to be infected into ear tissues such as wheat or anthers, lemma or glume of other host plants to be attached to form hypha, and the hypha invades the host tissues and grows and expands in cells. Thus, the number of mature ascospores and their release of ascospores play a crucial role in the cycle of fusarium graminearum infestation.

The method controls the initial infection source of the wheat scab, reduces the base number of bacteria sources, and has important significance for preventing and controlling the occurrence of the wheat scab. However, the traditional control strategy of wheat scab is mainly chemical control, which is mainly carried out in the heading and flowering period of wheat, especially in the severe epidemic years of wheat scab, the chemical control effect and the toxin reducing effect are not ideal, and more importantly, the chemical pesticide is used in successive years, so that the generation of fusarium graminearum resistance is caused, the quality of wheat and the safety of agricultural ecological environment are seriously influenced, and the wheat production faces huge pressure.

Therefore, according to the disease cycle characteristics of wheat scab and the current large background of straw returning, antagonistic microorganisms are utilized to carry out returning straw treatment, the generation and maturation of the ascocarp shell of fusarium graminearum or the release of ascospores are inhibited, and the method becomes an important mode for comprehensive prevention and control of wheat scab by 'standing prevention and active attack'.

Disclosure of Invention

The invention aims to provide a bacillus amyloliquefaciens YB130 and application thereof, which can effectively inhibit the generation of fusarium graminearum ascocarp and reduce the initial infection source of wheat scab.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention provides a Bacillus amyloliquefaciens YB130, wherein the Latin article of the Bacillus amyloliquefaciens YB130 is Bacillus amyloliquefaciens, the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the address is as follows: xilu No.1 Hospital No. 3, Beijing, Chaoyang, with a preservation date of: 7/8/2018, the preservation number is: CGMCC No. 16229.

The invention also provides application of the bacillus amyloliquefaciens YB130 in inhibiting the generation of ascospores by fusarium graminearum.

Preferably, the bacillus amyloliquefaciens YB130 is used in 2-3 months or before straw returning.

The invention provides a Bacillus amyloliquefaciens YB130 and application thereof, wherein the Latin article of the Bacillus amyloliquefaciens YB130 is Bacillus amyloliquefaciens, the strain is preserved in the common microorganism center of the China Committee for culture Collection of microorganisms, and the address is as follows: xilu No.1 Hospital No. 3, Beijing, Chaoyang, with a preservation date of: 7/8/2018, the preservation number is: CGMCC No. 16229. The bacillus amyloliquefaciens YB130 can effectively inhibit the production of fusarium graminearum ascocarp, thereby reducing the initial infection source of wheat scab, achieving the purposes of preventing and controlling wheat scab, realizing green prevention and control and having good application prospect.

Drawings

FIG. 1 is a graph of the inhibitory effect of different biocontrol bacterial strains on Fusarium graminearum subcapsule shells in example 1;

FIG. 2 shows the inhibitory effect of YB130 Bacillus amyloliquefaciens fermentation broth on Fusarium graminearum cyst shell;

FIG. 3 shows the formation of the seed shell of Bacillus amyloliquefaciens YB 130.

Deposit description

Bacillus amyloliquefaciens YB130, Latin is Bacillus amyloliquefaciens, the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, No. 3 of Xilu No.1 of Beijing Kogyo-Yang district, the address of Beijing city, the preservation date is as follows: 7/8/2018, the preservation number is: CGMCC No. 16229.

Detailed Description

In the invention, the bacillus amyloliquefaciens YB130 is preferably used in 2-3 months or before straw returning.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

The preparation method of the culture medium and the reagent related to the embodiment comprises the following steps:

LB liquid medium: weighing 10g of tryptone, 5g of yeast extract and 10g of sodium chloride, adding distilled water to 1L, and carrying out high-temperature moist heat sterilization at 121 ℃ for 30 min.

LB solid medium: weighing 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 15g of agar powder, adding distilled water to 1L, and carrying out moist heat sterilization at 121 ℃ for 30 min.

PDA solid medium: weighing potato 200g, glucose 20g, agar 15g, deionized water 1.0L, and boiling at 115 deg.C for 20 min.

Carrot culture medium: weighing 250g of carrot, cutting into small pieces, adding 500ml of distilled water, sterilizing, crushing with a juicer, adding distilled water to 1L, adding 20g of agar, and performing moist heat sterilization at 121 ℃ for 30 min.

0.1% tween-20: weighing 100 μ L of Tween-20 stock solution, adding distilled water to 100ml, mixing, and sterilizing at 121 deg.C for 20 min.

Example 1 Primary screening of biocontrol bacterial strains

On a carrot culture medium with a diameter of 6cm plastic plate, bio-control bacteria YB128, YB129, YB130, YB131, YB132 separated from field straws and fusarium graminearum are oppositely cultured for 6 days, sterile 0.1 percent of tween-20 is added, a sterilization spoon is used for pressing down aerial hyphae to ensure that the aerial hyphae are attached to the surface of the culture medium, and the aerial hyphae are moved to a 25 ℃ incubator under a black light: 12h for darkness: after 12h of conditioned culture, the number of produced ascochyta graminearum at the boundary was observed after 4 days, and the results are shown in FIG. 1.

Example 2 rescreening experiment of biocontrol bacteria fermentation broth for inhibiting Fusarium graminearum cyst Shell

(1) Culturing biocontrol bacteria at 37 deg.C and 180rpm for 48 hr, filtering with 0.22 μm microporous membrane to obtain fermentation broth, and adding into carrot culture medium at a ratio of 7%.

(2) Selecting 5mm cake blocks from activated culture fusarium graminearum PDA plate, placing in the middle of the culture medium in the step (1), culturing for 7 days in an incubator at 25 ℃, taking out, adding sterile 0.1% tween-20, and pressing down aerial hyphae with a sterilizing spoon to be attached to the surface of the culture medium.

(3) Moving the plate in the step (2) to an incubator at 25 ℃, and using a black light: dark-12 h: culturing for 12h, and observing the generation amount of the ascochyta graminicola shell after 4 days.

Example 3 morphological Observation and molecular biological identification of biocontrol bacterial strains

(1) Morphological characterization of strains

After culturing for 48 hours on an LB solid culture medium in an incubator at 37 ℃, the front side of a YB130 bacillus amyloliquefaciens colony is circular, the side surface is convex, and the surface is provided with folds; the colonies were opaque and milky white.

(2) Molecular characterization of strains

The genome DNA of the biocontrol bacteria is extracted by using the bacterial genome extraction kit, the 16S universal primer is amplified, the amplified fragment is 1500bp, the band is single, the length is correct, the BLAST comparison analysis is carried out on the amplified fragment and an NCBI database, the strain with the highest similarity is YB130 Bacillus amyloliquefaciens FZB42T (16S rDNA Gen Bank accession number is HQ840645), and the sequence similarity reaches 99%. And downloading the kindred sequence from the NCBI database to construct a phylogenetic tree, wherein the result shows that the phylogenetic tree is identified as YB130 Bacillus amyloliquefaciens by combining morphological identification recently with YB130 Bacillus amyloliquefaciens.

Example 4 biocontrol bacterial strains inhibition of Fusarium graminearum cyst Shell production in crop straw

(1) Culturing in liquid LB at 27 deg.C and 175rpm shaking table, detecting OD value of bacterial liquid to 0.8, and taking out;

(2) reference patent "a method for predicting the initial infection source abundance of wheat scab" cultures fusarium graminearum conidia and inoculates crop straws;

(3) after the growth of the step (2) is carried out for 24 hours in an incubator at 25 ℃, spraying the bacterial liquid obtained in the step (1) on the crop straws in the step (2), and continuously culturing for 6 days in the incubator at 25 ℃;

(4) taking out the crop straws in the step (3) and placing the crop straws on the ground surface of the wheat field row in normal field management, and fixing the straws on the ground surface of the wheat field row;

(5) the number of ascochyta shells produced on the inoculated straw was investigated 10 days before wheat blossoms.

The investigation method comprises the following steps:

using a dissecting mirror, 6 points are selected for each straw before counting, and the area of each point is 0.1923cm²Calculating 3 straws in each group, dissecting 30 or 40 times of a mirror, counting the number of the ascocarp shells in the selected area, and then converting the number into the number of the ascocarp shells on the surfaces of the straws;

the results are shown in Table 1.

TABLE 1

The experimental results are as follows: the YB130 bacillus amyloliquefaciens has the effect of efficiently inhibiting the ascocarp of fusarium graminearum, and the inhibition rate is 75-85%.

From the above embodiments, the present invention provides a strain of Bacillus amyloliquefaciens YB130 and applications thereof, wherein latin text of the Bacillus amyloliquefaciens YB130 is Bacillus amyloliquefaciens, and the strain is deposited in the common microorganism center of the china committee for culture collection of microorganisms, address: the preservation date of No. 3 Xilu Beijing Xiyan No.1 Beijing, Chaoyang district is: 7/8/2018, the preservation number is: CGMCC No. 16229. The bacillus amyloliquefaciens YB130 can effectively inhibit the production of fusarium graminearum ascocarp, thereby reducing the initial infection source of wheat scab, achieving the purposes of preventing and controlling wheat scab, realizing green prevention and control and having good application prospect.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. The application of the bacillus amyloliquefaciens YB130 in inhibiting the production of ascospores by fusarium graminearum is characterized in that the bacillus amyloliquefaciens YB130 Latin is Bacillus amyloliquefaciensThe strain is preserved in the China general microbiological culture Collection center, and the address is as follows: xilu No.1 Hospital No. 3, Beijing, Chaoyang, with a preservation date of: and 8, 7 days in 2018, wherein the preservation number is as follows: CGMCC No. 16229.

2. The application of claim 1, wherein the Bacillus amyloliquefaciens YB130 is used in 2-3 months or before straw returning.