CN115181693B

CN115181693B - Bacillus bailii and application thereof

Info

Publication number: CN115181693B
Application number: CN202210717943.0A
Authority: CN
Inventors: 赵磊; 葛同玲; 程庭峰
Original assignee: Tianjin Institute of Industrial Biotechnology of CAS
Current assignee: Tianjin Institute of Industrial Biotechnology of CAS
Priority date: 2022-06-23
Filing date: 2022-06-23
Publication date: 2023-08-22
Anticipated expiration: 2042-06-23
Also published as: CN115181693A

Abstract

The invention discloses bacillus bailii 2021TIBBST23, which is classified and named: bacillus bailiiBacillus velezensisIs preserved in China Center for Type Culture Collection (CCTCC), and has the preservation number of: cctccc NO: m2022307, the preservation time is: 2022, 03 and 24. The invention also discloses application of the bacillus beijerinus 2021TIBBST23 strain. The strain has broad-spectrum antibacterial effect, provides good biocontrol resources for preventing and controlling diseases on solanaceous crops such as tomatoes, especially tomato/tobacco gray mold, and has important significance in the agricultural field.

Description

Bacillus bailii and application thereof

Technical Field

The invention belongs to the technical fields of microbiology, biotechnology and biological control, and relates to bacillus beijerinckii and application thereof.

Background

In recent years, with the increase of the demand of China on vegetables, the continuous cropping obstacle problem of a plurality of vegetable planting areas is serious, and the diseases and insect pests of vegetables are aggravated year by year due to the lack of soil organic fertilizer caused by the addition of a large amount of applied fertilizers, especially the serious soil-borne diseases such as damping off, root rot, fusarium wilt, gray mold and the like.

At present, the coverage rate of the green prevention and control technology for plant diseases and insect pests in China is still low, chemical prevention and control are mainly used as main prevention and control measures, however, long-term abuse of chemical pesticides can not only generate drug-resistant pathogens to increase the difficulty of disease prevention and control, but also pollute the ecological environment, endanger human health and prevent sustainable development of agriculture. In order to reduce the usage amount of chemical pesticides, develop a green prevention and control utilization technology for plant diseases and insect pests, protect the quality safety of agricultural products and promote the sustainable development of agriculture, the research and development of biological pesticides (live bacteria preparations produced by microorganisms or microbial metabolites) have been paid attention to for many years in China.

The biological control agent not only can directly act on pathogenic bacteria, but also can induce plants to generate drug resistance, thereby improving the resistance of the plants to the pathogenic bacteria and achieving the purpose of controlling or relieving diseases. At present, various microorganisms such as trichoderma, bacillus subtilis, serratia, streptomyces and the like are used for controlling gray mold. Therefore, microorganisms capable of antagonizing gray mold bacteria are screened from different agricultural ecological environments, and the development of the microorganisms into microbial agents is gradually becoming an important point of gray mold pest control research.

Disclosure of Invention

It is an object of the present invention to address at least the above problems and/or disadvantages and to provide at least the advantages described below.

It is a further object of the present invention to provide a Bacillus bailii 2021TIBBST23.

Another object of the present invention is to provide a method for culturing Bacillus bailii.

It is a further object of the present invention to provide the use of Bacillus bailii 2021TIBBST23 strain.

For this purpose, the technical scheme provided by the invention is as follows:

bacillus bailii 2021TIBBST23 (hereinafter abbreviated as BST 23), which is classified and named: bacillus bailiiBacillus velezensisBacterial strain bacillus bailiiBacillus velezensis2021TIBBST23 was deposited at the China Center for Type Culture Collection (CCTCC), accession number: cctccc NO: m2022307, the preservation time is: 2022, 24 d 03, deposit unit address: chinese university of Wuhan and Wuhan.

The bacillus belicus culturing process includes the following steps:

the bacillus beijerinus 2021TIBBST23 strain is inoculated in an LB culture medium for culture, and fermentation broth is obtained.

Preferably, in the method for culturing bacillus belicus, the culturing temperature is 37 ℃ and the culturing time is more than 72 hours.

The application of the bacillus belicus 2021TIBBST23 strain is any one of the following applications:

use in inhibiting plant pathogenic fungal activity;

use in inhibiting the activity of a phytopathogenic bacterium;

use in promoting plant side root count;

use in increasing plant biomass accumulation.

Preferably, in said application, said agricultural field comprises the field of biological control of crop diseases.

Preferably, in the application, the application is application in preparation of a biocontrol microbial agent.

Preferably, in said application, said application comprises inhibiting the growth of pathogenic bacteria: fusarium oxysporum, botrytis cinerea, rhizoctonia solani, xanthomonas campestris pepper spot disease pathotype, ralstonia solanacearum and Dike.

Preferably, in the application, the crop is a solanaceae crop.

Preferably, in the application, the solanaceous crop is tomato.

Preferably, among the applications, the application includes application in promoting the improvement of germination rate and rooting ability of crop seeds.

The invention at least comprises the following beneficial effects:

the invention provides bacillus beijerinus 2021TIBBST23 and application thereof in agriculture. The strain is preserved in China center for type culture collection (China) for 3 months and 24 days in 2022, the sample name is 2021TIBBST23, and the registration number is CCTCC NO: m2022307. The strain 2021TIBBST23 has the function of generating antibacterial active substances and volatile substances, has remarkable inhibition effects on bacterial pathogens such as tomato scab pathogen, tomato bacterial wilt pathogen, black shank pathogen and the like, and also has remarkable inhibition effects on fungal pathogens including botrytis cinerea, fusarium wilt pathogen and rhizoctonia solani. The result of the invention shows that the strain has broad-spectrum antibacterial effect, provides good biocontrol resources for preventing and controlling diseases on solanaceae crops such as tomatoes, in particular to tomato/tobacco gray mold, and has important significance in the agricultural field.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 shows two plate antagonism tests (antagonistic bacteria vs Xcv) with antibacterial effect in the examples of the present invention.

FIG. 2 is a phylogenetic analysis based on 16S rDNA according to the example of the present invention.

FIG. 3 is a time-screening chart of the bacterial strain BST23 with the best antibacterial effect in the embodiment of the invention.

Fig. 4 is a diagram showing the bacteriostatic effect of the strain BST23 having the optimal bacteriostatic effect in the embodiment of the invention.

FIG. 5 is a graph showing the inhibitory effect of strain BST23 on three fungi and three bacterial pathogens in the examples of the present invention.

Fig. 6 is a graph of control effects (botrytis cinerea) on tobacco leaves in an embodiment of the present invention.

Detailed Description

The present invention is described in further detail below with reference to the drawings to enable those skilled in the art to practice the invention by referring to the description.

It will be understood that terms, such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

The experimental methods described in the following embodiments are conventional methods unless otherwise indicated, and the reagents and materials are commercially available.

For a better understanding of the technical solution of the present invention, the following examples are now provided for illustration:

the strains and sources involved in the following examples are as follows:

TABLE 1 pathogenic bacteria and sources thereof

Example 1 isolation screening and identification of Strain BST23

1. Isolation and screening of Strain BST23

Soil was collected from the Yangqing district of Beijing in 2021. Mixing 1 g soil with 99 mL 1 XPBS buffer solution (dilution)

10 ² ) Shaking culture is carried out for 30 min at 200 rpm and 37 ℃. Standing for 20 min, collecting supernatant of the soil suspension, performing gradient dilution, and respectively collecting dilution factors of 10 ² 、10 ³ 、10 ⁴ 100 mu L of the dilution liquid of (2) is evenly coated on a LB solid culture medium (yeast extract 5 g/L, tryptone 10 g/L, naCl 10 g/L and agar 15 g/L), a 37 ℃ incubator is used for inverted culture of 24 h-48 h, a sterile pipette tip is used for picking bacterial single bacteria with different forms, the bacterial single bacteria are streaked and purified on the LB solid culture medium, and then are propagated in the LB liquid culture medium, and mixed sterilized glycerol (the working concentration is 25%) is stored in a refrigerator at the temperature of minus 80 ℃ for standby.

Detecting biocontrol effect of the separated strain by adopting a flat-plate counter culture method, and selecting antagonism object as the pathogenic type of xanthomonas campestris pepper spot disease capable of causing tomato bacterial scabXanthomonas campestris pv. vesicatoriaXcv). Suspension of pathogen Xcv (concentration 10) ⁹ CFU/mL) is uniformly mixed into LB solid culture medium according to the proportion of 1:100, 2 mu L of bacterial suspension of the strain to be detected is sucked (the concentration is 10 ⁹ CFU/mL) was added dropwise to the center of the plate of the solid medium with LB, and after 24-h incubation at 37 ℃, the diameter of the zone of inhibition was measured. 3 replicates of each test strain were obtained.

31 monoclonal pure culture strains are separated and stored in 5 soil samples, and 2 bacillus strains with inhibition effect on tomato scab are initially screened out through a plate counter experiment, and the inhibition effect is shown in figure 1. Then, the bacterial strain BST23 with obvious antibacterial effect is screened out again for subsequent experiments.

2. Identification of strains

Molecular biology identification was performed on 31 monoclonal culture strains including BST23, and preliminary identification was performed by amplifying 16S rDNA sequences (base sequences shown in SEQ ID NO: 1) thereof using a bacterial universal primer pair 27F/1492R (Table 2). 25. mu.L of the reaction system is shown in Table 3. The PCR reaction conditions were: pre-denaturation at 94℃for 5 min; 94. denaturation at 30 s, annealing at 56℃for 30 s, extension at 72℃for 1 min,30 cycles; extending at 72℃for 5 min.

The strain BST23 was subjected to whole genome sequencing analysis. DNA extraction is carried out on BST23 bacterial suspension, and then Meger genes are sent for sequencing construction of bacterial frame map. The main flow comprises three parts: DNA library construction, high throughput sequencing (Illumina) and genome assembly and subsequent analysis. The assembled bacterial frame map further identifies bacterial genome relatedness by average nucleotide similarity (ANI) analysis, indicating that both genomes belong to the same species when ANI > 95%. ANI analysis results for the genome versus the reference group Table 4.

TABLE 2 primers used for identification of strains

TABLE 3 polymerase chain reaction System

TABLE 4 average nucleotide similarity analysis results

QueryID	ReferenceID	ANI	Mapped_fragment	Query_fragment	Taxon
						BST23	GCA_006965525.1	99.305	1244	1293	Bacillus velezensis

Sequencing amplified product in agarose gel electrophoresis to obtain sequencing result, analyzing with Snapgene viewer and MEGA11, BLAST comparison on NCBI website to determine strain classification, and displaying the strain BST23 and Bacillus bailiiBacillusvelezensis) The similarity is as high as 99%, and finally, a phylogenetic tree is constructed for the obtained monoclonal strains, and the result is shown in fig. 2.

According to the morphological characteristics of the thalli, combining the results of evolution analysis and average nucleotide similarity analysis, the strain BST23 is identified as bacillus belicusBacillus velezensis). The strain BST23 is in a shape of irregular round with saw-toothed edges on LB solid medium, and the surface of the strain BST23 is slightly wrinkled and is not raised.

Example 2 time measurement of optimal bacteriostatic Effect of Strain BST23

The strain BST23 was inoculated in LB liquid medium and cultured overnight at 37℃and 200 rpm. Transferring 2 μl of the bacterial suspension, dripping the bacterial suspension in the center of the LB solid medium, and airing for later use. Respectively are provided withSpraying pathogenic bacteria suspension (1 ml 10) of 0, 1, 2, 3, 4 days after dripping antagonistic bacteria ⁹ CFU/ml+19 ml sterilized water). Culturing is based on dark culture in a 37 ℃ incubator, and bacteriostasis circle measurement is carried out 1 day after inoculation of pathogenic bacteria. And drawing a curve for the culture time and the diameter of the inhibition zone.

As shown in FIG. 3 and FIG. 4, the bacterial strain was cultured for 72 hours or less, the antibacterial effect was in positive correlation with the culture time, and after 72 hours, the antibacterial effect was gradually smoothed.

Example 3 determination of antibacterial spectrum

The bacteriostasis spectrum of the strain BST23 is detected by adopting a counter culture method. The method is used for respectively measuring the fungal pathogenic bacteria Botrytis cinerea of three kindsBotrytis cinerea) Fusarium oxysporum (F.oxysporum)Fusarium oxysporum) Rhizoctonia solani (wall.) kuntzeRhizoctonia solani) Three bacterial pathogens of Xanthomonas campestris pepper spot disease pathotypeXanthomonas campestris pv. vesicatoria) Lei's bacterial strainRalstonia solanacearum) The Dirichia solani is a speciesDickeyasolani). For the fungal challenge, the pathogenic bacteria were first activated on PDA plates, the pathogenic bacterial disease was produced with a 5 mm punch, inoculated in the center of the PDA plates, and 2. Mu.L of the strain BST23 suspension (10 ⁹ CFU/mL) was cultured at a distance of 2 cm to mycosis at 25℃for 4-7 days, the pathogenic bacteria diameters of the control group and the treatment group were measured, and the inhibition ratio was calculated as inhibition ratio= (control colony diameter-treatment group colony antagonistic bacteria direction diameter)/control colony diameter. The counter method was followed for the primary screening of antagonistic bacteria according to example 1.

The result shows that the strain BST23 has broad-spectrum bacteriostasis and can resist three fungal pathogens of Botrytis cinereaBotrytis cinerea) Fusarium oxysporum (F.oxysporum)Fusarium oxysporum) Rhizoctonia solani (wall.) kuntzeRhizoctonia solani) The inhibition rates of (a) are 84.28%, 63.64% and 84.31% (Table 5), respectively, and the antibacterial effect is shown in FIG. 5.

TABLE 5 antibacterial spectrum of BST23

EXAMPLE 4 inhibition of BST23 on Botrytis cinerea infection of tobacco leaves

Healthy tobacco leaves with the same leaf age and basically consistent leaf size are selected, and are cleaned by sterile water and dried in the air, and then are subjected to subsequent treatment by using fermentation liquor of the strain for 48 hours. The preparation method of the fermentation liquor comprises the following steps: the original strain stored at-80 ℃ is activated in LB solid medium, after overnight culture at 37 ℃, single colony is selected to be inoculated in 5 mL LB liquid medium, shaking culture propagation is carried out in a shaking table at 200 rpm at 37 ℃ to be used as seed fermentation broth, and then the seed fermentation broth is inoculated in 500 mL LB liquid medium at a ratio of 1:100 to be shaking cultured in a shaking table at 200 rpm at 37 ℃ to be 48 h. When the fermentation liquor is used, the viable count of the strain fermentation liquor is diluted to 10 ⁸ CFU/mL and uniformly sprayed on the leaves, and the situation that the leaves just overflow is taken as the criterion; after air drying, 1 gray mold pathogenic bacteria cake with the diameter of 3 mm is inoculated in the middle of each leaf, and the leaf is subjected to moisture preservation and culture for 3 days at 25 ℃. The fermentation liquor of the Zuorun effective strain QST713 is used as a positive control, sterile water is used as a negative control, and the treatment mode and the dosage are the same as those of the BST23 microbial inoculum. Each treatment contained 6 leaves and was repeated 3 times. And counting the disease area of each capsicum leaf. And calculating the control effect on the gray mold according to a formula.

Area of spread of lesions (mm) ² ) Total area of lesions-area of patties

The results show that BST23 has good control effect on tobacco gray mold on in vitro leaves (figure 6). There was no significant difference compared to the commercial strain QST713 (table 5), and BST23 completely inhibited the occurrence of tobacco leaf gray mold by up to 100% compared to the blank control.

TABLE 6 control effect of BST23 on Botrytis cinerea on tobacco leaves

Treatment of	Area of onset (cm) ² ）	Preventing effect (%)
			Control	4.95±1.81	-
Zhuo Run QST713	0.03±0.05	99.4%
			BST23	0	100%

Example 5 BST23 capability test

Preparing an MS+LB spliced culture medium, which is prepared by the following specific steps of; MS and LB solid culture media are respectively prepared according to the formula (MS culture media: MS 4.44 g/L, sucrose 30 g/L, agar 7 g/L and KOH regulating pH to 5.6-5.8; LB solid culture media: yeast extract 5 g/L, sodium chloride 10 g/L, tryptone 10 g/L and agar 15 g/L), after sterilization for 20 min at 121 ℃, the LB solid culture media are poured into a 10 cm multiplied by 10 cm square culture dish, after the LB solid culture media are solidified, half of the LB solid culture media are removed, and are poured into the MS culture media for filling, and after the MS culture media are solidified, an MS+LB spliced culture medium is prepared, and the culture media can simultaneously meet the growth of arabidopsis thaliana and strains in the same space. After the arabidopsis seeds are vernalized at 4 ℃ for 48 h, the arabidopsis seeds are sterilized by using absolute ethyl alcohol for 3 min and are washed by using sterile water for 3 times, after the sterilization, the arabidopsis seeds are transferred into an MS culture medium part of an MS+LB splicing culture medium, and the LB culture medium part is inoculated with a strain BST23, so that the germination rate is calculated. Meanwhile, the arabidopsis seeds are transplanted into an MS culture medium to accelerate germination for the influence of subsequent strains on the growth condition of the arabidopsis. The specific method comprises the following steps: after the seeds germinate and root, transferring the arabidopsis seedlings which grow consistently when the main roots stretch by about 1 cm to an MS culture medium part of an MS+LB splicing culture medium, placing an arabidopsis at intervals of 4 mm in the direction of root elongation towards the strain, and inoculating the strain BST23 at the LB culture medium part. QST713 was inoculated as a positive control and LB broth was inoculated as a negative control. 20 Arabidopsis plants were treated each. After inoculation, the root system change of Arabidopsis thaliana was observed daily, and after 7 days, the length of main roots, the number of lateral roots and biomass were measured.

The results show that BST23 increased germination rate and the number of lateral roots and biomass were significantly increased relative to the negative control and commercial product strain QST713 (Table 7)

TABLE 7 Arabidopsis physiological change index

	Root length	Number of side roots	Biomass mass	Germination percentage
					CK	2.13±0.26	2.12±1.36	2.27±0.40	82.61
BST23	2.2±0.22	3.39±1.48	2.78±0.90	89.47
					QST713	2.05±0.18	1.82±1.13	1.13±0.39	81.82

The number of modules and the scale of processing described herein are intended to simplify the description of the present invention. Applications, modifications and variations of the present invention will be readily apparent to those skilled in the art.

Although embodiments of the present invention have been disclosed above, it is not limited to the details and embodiments shown and described, it is well suited to various fields of use for which the invention would be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the specific details and illustrations shown and described herein, without departing from the general concepts defined in the claims and their equivalents.

SEQUENCE LISTING

<110> institute of Tianjin Industrial biotechnology, national academy of sciences

<120> Bacillus bailii strain and application thereof

<130> 2021

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<170> PatentIn version 3.5

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<213> Bacillus bailii Bacillus velezensis

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Claims

1. Bacillus bailii 2021TIBBST23, classified and named: bacillus bailii @ and method of producing the sameBacillus velezensis) Bacterial strain bacillus bailii @Bacillus velezensis) 2021TIBBST23 was deposited with the chinese collection of typical cultures under the accession number: cctccc NO: m2022307, the preservation time is: 2022, 24 d 03, deposit unit address: chinese university of Wuhan and Wuhan.

2. The bacillus beijerinus culture method is characterized by comprising the following steps:

the bacillus beijerinus 2021TIBBST23 strain according to claim 1 is inoculated in an LB culture medium for culture to obtain a fermentation broth.

3. The method according to claim 2, wherein the culturing temperature is 37℃and the culturing time is 72 hours or longer.

4. The use of bacillus belgium 2021TIBBST23 strain according to claim 1, wherein the use is any one of the following:

application in inhibiting activity of plant pathogenic fungi, namely Botrytis cinereaBotrytis cinerea) Fusarium oxysporum (F.oxysporum)Fusarium oxysporum) And rhizoctonia solani @Rhizoctonia solani）；

Application in inhibiting activity of plant pathogenic bacteria, namely Xanthomonas campestris pepper spot disease pathotypeXanthomonas campestris pv. vesicatoria) Lei's bacterial strainRalstonia solanacearum) And Solanum diradicum (Dirichlet) of Solanum torvumDickeyasolani）；

Use in promoting plant side root count;

use in increasing plant biomass accumulation.

5. The use according to claim 4, wherein the plant is a plant of the solanaceae class.

6. The use according to claim 5, wherein the solanaceous crop is tomato.

7. The use according to claim 4, wherein the use comprises use in promoting an increase in germination and rooting of arabidopsis seeds.