CN115181693A

CN115181693A - Bacillus beleisi and application thereof

Info

Publication number: CN115181693A
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: 2022-10-14
Anticipated expiration: 2042-06-23
Also published as: CN115181693B

Abstract

The invention discloses Bacillus belgii 2021TIBBST23, which is named in classification: bacillus belgiiBacillus velezensisIs preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of: CCTCC NO: m2022307, deposit time: 24/03/2022. The invention also discloses application of the Bacillus belgii 2021TIBBST23 strain. The strain has broad-spectrum antibacterial effect, is used for preventing and treating diseases of solanaceous crops such as tomatoes and the like, and particularly is used for preventing and treating botrytis cinereaProvides good biocontrol resources and has important significance in the agricultural field.

Description

Bacillus beleisi and application thereof

Technical Field

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

Background

In recent years, with the increase of the demand of our country for vegetables, the problem of continuous cropping obstacles in a plurality of vegetable planting areas is serious, and in addition, a large amount of fertilizer is applied, the organic fertilizer in soil is lost, the phenomena of plant diseases and insect pests of the vegetables are aggravated year by year, and especially the soil-borne diseases such as damping off, root rot, blight, gray mold and the like are serious.

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 taken as main prevention and control measures, however, long-term abuse of chemical pesticides not only can generate drug-resistant pathogens to increase the difficulty of disease prevention and control, but also pollutes the ecological environment, endangers the health of human beings and hinders the sustainable development of agriculture. In order to reduce the usage amount of chemical pesticides, develop a green prevention, control and utilization technology of plant diseases and insect pests, protect the quality and safety of agricultural products, and promote the sustainable development of agriculture, china has been paying attention to the research and development of biological pesticides (viable bacteria preparations produced by microorganisms or microbial metabolites) for many years.

The biological control medicament 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 lightening diseases. At present, various microorganisms such as trichoderma, bacillus subtilis, serratia, streptomycete and the like are used for preventing and treating gray mold. Therefore, screening microorganisms capable of antagonizing botrytis cinerea from different agricultural ecological environments and developing the microorganisms into microbial agents become important for the research of preventing and treating botrytis cinerea.

Disclosure of Invention

An object of the present invention is to solve at least the above problems and/or disadvantages and to provide at least the advantages described hereinafter.

Still another object of the present invention is to provide a strain of Bacillus beiLeisi 2021TIBBST23.

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

Still another object of the present invention is to provide the use of the strain Bacillus belgii 2021TIBBST23.

Therefore, the technical scheme provided by the invention is as follows:

bacillus belgii 2021TIBBST23 (hereinafter abbreviated as BST 23), which is classified under the name: bacillus belgiiBacillus velezensisBacterial strain Bacillus belgiiBacillus velezensis2021TIBBST23 is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of: CCTCC NO: m2022307, deposit time: 24/03/2022, the address of the depository is: wuhan, wuhan university.

The method for culturing the Bacillus belgii comprises the following steps:

the Bacillus belgii 2021TIBBST23 strain is inoculated in an LB culture medium for culture to obtain fermentation liquor.

Preferably, in the method for culturing Bacillus belgii, the culturing temperature is 37 ℃ and the culturing time is 72 hours or more.

The application of the Bacillus belgii 2021TIBBST23 strain is any one of the following applications:

the application in inhibiting the activity of plant pathogenic fungi;

the application of inhibiting the activity of plant pathogenic bacteria;

the application in promoting the root number of the plant;

application in increasing the biomass accumulation of plants.

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

Preferably, in the application, the application is the application in the preparation of the biocontrol microbial inoculum.

Preferably, in said use, said use comprises inhibiting the growth of: fusarium oxysporum, botrytis cinerea, rhizoctonia solani, xanthomonas campestris pepper spot disease pathotype, ralstonia solani and Ditaceae solani.

Preferably, in the application, the crops are solanaceae crops.

Preferably, in the application, the solanaceous crops are tomatoes.

Preferably, the application comprises application of promoting the germination rate and rooting capacity of crop seeds.

The invention at least comprises the following beneficial effects:

the invention provides a bacillus belgii 2021TIBBST23 strain and application thereof in agriculture. The strain is preserved in China Center for Type Culture Collection (CCTCC) 24.3.2022, 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 obvious inhibiting effect on bacterial pathogenic bacteria such as tomato scab, tomato ralstonia solanacearum and phytophthora parasitica, and also has obvious inhibiting effect on fungal pathogenic bacteria including tomato botrytis cinerea, fusarium oxysporum and rhizoctonia solani. The result of the invention shows that the strain has broad-spectrum antibacterial effect, provides good biocontrol resources for preventing and treating diseases of solanaceae crops such as tomatoes and the like, particularly 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 bacteriostatic effect according to the examples of the present invention.

FIG. 2 is a diagram of a 16S rDNA-based phylogenetic analysis in the examples of the present invention.

FIG. 3 is a time screening chart showing that the strain BST23 has the best bacteriostatic effect in the example of the present invention.

FIG. 4 is a diagram of the bacteriostatic effect of the BST23 strain with the best bacteriostatic effect in the embodiment of the invention.

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

FIG. 6 is a graph showing the control effect (Botrytis cinerea) on tobacco leaves in an example of the present invention.

Detailed Description

The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.

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.

It is to be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified.

In order to make the technical scheme of the invention better understood by those skilled in the art, the following examples are now provided for illustration:

the strains and sources referred to 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

In 2021, soil was collected from Yanqing district of Beijing City. 1 g of soil was mixed with 99 mL of 1 XPBS buffer solution (dilution factor)

10 ² ) Shaking at 200 rpm and 37 deg.C for 30 min. Standing for 20 min, taking supernatant of the soil suspension for gradient dilution, and respectively taking dilution times of 10 ² 、10 ³ 、10 ⁴ Uniformly coating 100 mu L of the diluent on an LB solid culture medium (yeast extract powder is 5 g/L, tryptone is 10 g/L, naCl is 10 g/L, and agar powder is 15 g/L) flat plate, carrying out inverted culture in a 37 ℃ incubator for 24-48 h, picking out single bacterial colonies with different forms by using a sterile pipette tip, carrying out streak purification on the bacterial colonies on the LB solid culture medium flat plate, carrying out propagation in an LB liquid culture medium, and storing mixed sterilized glycerol (the working concentration is 25%) in a refrigerator at-80 ℃ for later use.

Detecting biocontrol effect of separated strain by plate confronting culture method, and selecting antagonistic objectIs a xanthomonas campestris pepper spot disease pathotype which can cause tomato bacterial scab (Xanthomonas campestris pv. vesicatoriaXcv). Suspending pathogenic bacteria Xcv bacteria at the concentration of 10 ⁹ CFU/mL) is uniformly mixed into an LB solid culture medium according to the proportion of 1 ⁹ CFU/mL) was added to the center of the LB solid medium plate with bacteria, and after culturing at 37 ℃ for 24 hours, the diameter of the zone of inhibition was measured. Each test strain was 3 replicates.

31 single clone pure culture strains are separated and preserved from 5 soil samples, 2 bacillus with the inhibiting effect on tomato scab are primarily screened out through a plate confrontation experiment, and the bacteriostatic effect is shown in figure 1. And then, re-screening out the strain BST23 with remarkable bacteriostatic effect for subsequent experiments.

2. Identification of strains

The molecular biology identification of 31 monoclonally cultured strains including BST23 was performed, and the preliminary identification was performed by amplifying the 16S rDNA sequence (base sequence shown in SEQ ID NO: 1) using the bacterial universal primer pair 27F/1492R (Table 2). 25. The reaction system is shown in Table 3. The PCR reaction conditions are as follows: pre-denaturation at 94 deg.C for 5 min; 94. denaturation at 56 deg.C for 30 s, annealing at 56 deg.C for 30 s, extension at 72 deg.C for 1 min, and 30 cycles; extension at 72 ℃ for 5 min.

The strain BST23 was subjected to whole genome sequencing analysis. And (3) carrying out DNA extraction on the BST23 bacterial suspension, and then sending the Meger gene to carry out sequencing construction on a bacterial frame map. The main process comprises three parts: DNA library construction, high throughput sequencing (Illumina) and genome assembly and subsequent analysis. The assembled bacterial frame maps were further characterized by mean nucleotide similarity (ANI) analysis to identify bacterial genome relatedness, indicating that two genomes belong to the same species when ANI > 95%. The results of ANI analysis of the genome with the reference group are shown in 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

Detecting a target band of the amplified product by agarose gel electrophoresis, sending the target band to Beijing optimak Biotechnology Co., ltd for sequencing, analyzing the obtained sequencing result by using Snapgene viewer and MEGA11, simultaneously carrying out BLAST comparison on an NCBI website to determine the strain classification, and displaying the result that the strain BST23 and the Bacillus belgii (Bacillus belgii)Bacillusvelezensis) The similarity reaches up to 99 percent, and finally a phylogenetic tree is constructed for the obtained monoclonal strainsAs shown in fig. 2.

According to the morphological characteristics of the bacteria, the result of evolutionary analysis and average nucleotide similarity analysis is combined, and the strain BST23 is identified as the bacillus beiLeisi (B.Bacillus velezensis). The shape of the strain BST23 on an LB solid culture medium is irregular and round, the edge of the strain is saw-toothed, and the surface of the strain is slightly wrinkled and does not bulge.

Example 2 time determination of the best bacteriostatic Effect of the Strain BST23

The strain BST23 was inoculated in LB liquid medium and cultured overnight at 37 ℃ and 200 rpm. And transferring 2 mul of bacterial suspension, dropwise adding the bacterial suspension into the center of an LB solid culture medium, and airing for later use. Spraying pathogenic bacteria suspension (1 ml 10 ml) 0, 1, 2, 3, 4 days after adding antagonist respectively ⁹ CFU/ml + 19 ml sterile water). The culture is carried out in a dark culture at 37 ℃ in an incubator, and the inhibition zone is measured 1 day after the inoculation of pathogenic bacteria. And curves are drawn for the culture time and the diameter of the zone of inhibition.

As shown in FIGS. 3 and 4, the bacteriostatic effect was positively correlated with the culture time within 72 hours of the strain culture, and gradually stabilized after 72 hours.

EXAMPLE 3 determination of the bacteriostatic spectra

The bacterial inhibition spectrum of the strain BST23 is detected by adopting a confrontation culture method. Separately determining the three kinds of pathogenic bacteria (Botrytis cinerea) of the fungusBotrytis cinerea) Fusarium oxysporum (F.), (Fusarium oxysporum) Rhizoctonia solani bacteria (A), (B)Rhizoctonia solani) Three bacterial pathogens of wild rape Xanthomonas pepper spot disease pathotype: (Xanthomonas campestris pv. vesicatoria) "Qingkuo" medicine for curing diabetes Ralstonia (A), (B) and (C)Ralstonia solanacearum) Eggplant disease treating medicine Koehrla (C)Dickeyasolani). Aiming at the confronting experiment of fungi, firstly, pathogenic bacteria are activated on a PDA plate, a 5 mm hole puncher is used for preparing pathogenic mycosis, the pathogenic mycosis is inoculated in the center of a PDA plate, and 2 mu L of strain BST23 bacterial suspension (10 mu L) is transferred by a liquid transfer gun ⁹ CFU/mL) at a distance of 2 cm from the mycosis, culturing at 25 deg.C for 4-7 days, measuring the diameters of pathogenic bacteria in control group and treatment group, respectively, and calculating the inhibition rate, wherein the calculation formula is inhibition rate = (diameter of control colony-diameter of direction of antagonistic bacteria in treatment group)/coupleAccording to the colony diameter. The antagonistic method against the bacterial pathogens was performed according to the primary screening method for antagonistic bacteria in example 1.

The result shows that the strain BST23 has broad-spectrum antibacterial activity and can kill three fungi pathogenic bacteria Botrytis cinerea (A.cinerea) (B.cinerea)Botrytis cinerea) Fusarium oxysporum (F.), (Fusarium oxysporum) Rhizoctonia solani (F.), (Rhizoctonia solani) The inhibition rates of (A) and (B) were 84.28%, 63.64% and 84.31%, respectively (Table 5), and the bacteriostatic effect is shown in FIG. 5.

TABLE 5 bacterial inhibition spectra of BST23

Example 4 Effect of BST23 on inhibition of Botrytis cinerea infection of tobacco leaves

Healthy tobacco leaves with the same leaf age and the same leaf size are selected, cleaned by sterile water, aired and subjected to subsequent treatment by using a strain fermentation liquor for 48 hours. The preparation method of the fermentation liquor comprises the following steps: activating an original strain preserved at the temperature of-80 ℃ in an LB solid medium, culturing at the temperature of 37 ℃ overnight, selecting a single colony to be inoculated in 5 mL of LB liquid medium, performing shake propagation in a shaker at the temperature of 37 ℃ and 200 rpm to obtain a seed fermentation liquid, and then inoculating the seed fermentation liquid in 500 mL of LB liquid medium at the ratio of 1. When the fermentation liquor is used, firstly, the viable count of the strain fermentation liquor is diluted to 10 ⁸ CFU/mL, and uniformly spraying on the blades, wherein the water overflow on the blade surface is just the standard; after air drying, 1 botrytis cinerea germ 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 at 25 ℃ for 3 days. Taking fermentation liquor of Zuorun effective strain QST713 strain as positive control, taking sterile water as negative control, and using the same treatment mode and amount as BST23 microbial inoculum. Each treatment contained 6 leaves, which were repeated 3 times. And (4) counting the disease area of each pepper leaf. And calculating the prevention effect on the gray mold according to a formula.

Extended area of lesion (mm) ² ) = total lesion area-area of fungus cake

The results show that BST23 has good control effect on tobacco gray mold on the detached leaves (figure 6). Compared with the commercialized strain QST713, the strain QST713 has no significant difference (Table 5), and compared with a blank control, the BST23 completely inhibits the gray mold of tobacco leaves, and the control effect is as high as 100%.

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

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

Example 5 detection of growth-promoting ability of BST23

Preparing an MS + LB splicing culture medium, which comprises the following specific steps of; respectively preparing MS and LB solid culture media according to a formula (the MS culture medium is MS 4.44 g/L, sucrose 30 g/L, agar 7 g/L, and the pH value is regulated by KOH to 5.6-5.8), after sterilizing for 20 min at 121 ℃, pouring the LB solid culture medium into a square culture dish of 10 cm multiplied by 10 cm, removing half after the LB solid culture medium is solidified, pouring the LB solid culture medium into an MS culture medium for filling, and preparing the MS and LB splicing culture medium after the MS culture medium is solidified, wherein the culture medium can simultaneously meet the growth of arabidopsis thaliana and strains in the same space. Vernalizing the arabidopsis seeds at 4 ℃ for 48 h, disinfecting for 3 min by using absolute ethyl alcohol and washing for 3 times by using sterile water, transferring the arabidopsis seeds into an MS culture medium part of an MS + LB splicing culture medium after disinfection, inoculating a strain BST23 into the LB culture medium part, and calculating the germination rate. Meanwhile, the arabidopsis seeds are transplanted into an MS culture medium for accelerating germination so as to be used for influencing the growth condition of the arabidopsis by subsequent strains. The specific method comprises the following steps: after the seeds germinate and root, when the main root is elongated by about 1 cm, the arabidopsis seedlings with consistent growth are transferred to the MS culture medium part of the MS + LB splicing culture medium, the root elongation direction faces to the strain, one arabidopsis is placed every 4 mm, and the strain BST23 is inoculated on the LB culture medium part. The inoculated QST713 is used as a positive control, and the inoculated LB liquid culture medium is used as a negative control. Each of 20 Arabidopsis thaliana plants was treated. After inoculation, root changes of Arabidopsis thaliana were observed daily, and the length of the main root, the number of lateral roots and biomass were measured 7 days later.

The results showed that BST23 increased germination relative to the negative control and commercial product strain QST713, and that the lateral root count and biomass increased significantly (Table 7)

TABLE 7. Arabidopsis thaliana physiological change index

	Root length of	Number of side elements	Biomass	Germination rate
					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 the process described herein are intended to simplify the description of the invention. Applications, modifications and variations of the present invention will be apparent to those skilled in the art.

While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.

SEQUENCE LISTING

<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences

<120> Bacillus belgii and application thereof

<130> 2021

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 1385

<212> DNA

<213> Bacillus velezensis

<400> 1

aaggttacct caccgacttc gggtgttaca aactctcgtg gtgtgacggg cggtgtgtac 60

aaggcccggg aacgtattca ccgcggcatg ctgatccgcg attactagcg attccagctt 120

cacgcagtcg agttgcagac tgcgatccga actgagaaca gatttgtggg attggcttaa 180

cctcgcggtt tcgctgccct ttgttctgtc cattgtagca cgtgtgtagc ccaggtcata 240

aggggcatga tgatttgacg tcatccccac cttcctccgg tttgtcaccg gcagtcacct 300

tagagtgccc aactgaatgc tggcaactaa gatcaagggt tgcgctcgtt gcgggactta 360

acccaacatc tcacgacacg agctgacgac aaccatgcac cacctgtcac tctgcccccg 420

aaggggacgt cctatctcta ggattgtcag aggatgtcaa gacctggtaa ggttcttcgc 480

gttgcttcga attaaaccac atgctccacc gcttgtgcgg gcccccgtca attcctttga 540

gtttcagtct tgcgaccgta ctccccaggc ggagtgctta atgcgttagc tgcagcacta 600

aggggcggaa accccctaac acttagcact catcgtttac ggcgtggact accagggtat 660

ctaatcctgt tcgctcccca cgctttcgct cctcagcgtc agttacagac cagagagtcg 720

ccttcgccac tggtgttcct ccacatctct acgcatttca ccgctacacg tggaattcca 780

ctctcctctt ctgcactcaa gttccccagt ttccaatgac cctccccggt tgagccgggg 840

gctttcacat cagacttaag aaaccgcctg cgagcccttt acgcccaata attccggaca 900

acgcttgcca cctacgtatt accgcggctg ctggcacgta gttagccgtg gctttctggt 960

taggtaccgt caaggtgccg ccctatttga acggcacttg ttcttcccta acaacagagc 1020

tttacgatcc gaaaaccttc atcactcacg cggcgttgct ccgtcagact ttcgtccatt 1080

gcggaagatt ccctactgct gcctcccgta ggagtctggg ccgtgtctca gtcccagtgt 1140

ggccgatcac cctctcaggt cggctacgca tcgtcgcctt ggtgagccgt tacctcacca 1200

actagctaat gcgccgcggg tccatctgta agtggtagcc gaagccacct tttatgtctg 1260

aaccatgcgg ttcagacaac catccggtat tagccccggt ttcccggagt tatcccagtc 1320

ttacaggcag gttacccacg tgttactcac ccgtccgccg ctaacatcag ggagcaagct 1380

cccat 1385

<210> 2

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> 27F-16S rDNA upstream primer

<400> 2

agagtttgat cctggtcaga acgaacgct 29

<210> 3

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> 1492R-16S rDNA downstream primer

<400> 3

tacggctacc ttgttacgac ttcacccc 28

Claims

1. Bacillus belgii 2021TIBBST23, assigned to the taxonomic nomenclature: bacillus belgiiBacillus velezensisBacterial strain Bacillus belgiiBacillus velezensis2021TIBBST23 is deposited at the chinese type culture collection under accession number: CCTCC NO: m2022307, deposit time: 24/03/2022, the address of the depository is: wuhan, wuhan university.

2. The method for culturing the Bacillus belgii is characterized by comprising the following steps of:

the Bacillus beiLeisi 2021TIBBST23 strain of claim 1 is inoculated to LB culture medium and cultured to obtain fermentation liquid.

3. The method for culturing Bacillus belgii according to claim 2, wherein the culturing is carried out at 37 ℃ for 72 hours or more.

4. Use of a strain of bacillus beilesiensis 2021TIBBST23 according to claim 1, for any one of the following applications:

the application in inhibiting the activity of plant pathogenic fungi;

the application of the compound in inhibiting the activity of plant pathogenic bacteria;

application in promoting plant root number;

application in increasing the biomass accumulation of plants.

5. The use according to claim 4, wherein the agricultural field comprises the field of biological control of crop diseases.

6. The use according to claim 4, in the preparation of a biocontrol agent.

7. The use of claim 4, wherein said use comprises inhibiting the growth of a pathogen selected from the group consisting of: fusarium oxysporum, botrytis cinerea, rhizoctonia solani, ralstonia solani, and Ditaceae solani.

8. Use according to claim 5, wherein the crop plant is a solanaceous crop plant.

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

10. Use according to claim 4, wherein said use comprises use in promoting increased germination and improved rooting of crop seeds.