CN115725471A

CN115725471A - Bacillus safensis strain 05-2101 and application, product and method thereof

Info

Publication number: CN115725471A
Application number: CN202211510222.9A
Authority: CN
Inventors: 卢灿华; 夏振远; 李倩; 姜宁; 许银莲; 王继明; 蒯雁; 钱雷; 马俊红; 盖晓彤; 李军营; 晋艳
Original assignee: Yunnan Academy of Tobacco Agricultural Sciences
Current assignee: Yunnan Academy of Tobacco Agricultural Sciences
Priority date: 2022-11-29
Filing date: 2022-11-29
Publication date: 2023-03-03
Anticipated expiration: 2042-11-29
Also published as: CN115725471B

Abstract

The invention discloses a bacillus safensis strain 05-2101 and application, a product and a method thereof, belonging to the technical field of microorganisms. The invention provides a Bacillus safensis (Bacillus safensis) strain 05-2101, the preservation number of which is CCTCC No: m2022547. The invention also provides application of the Bacillus safensis (Bacillus safensis) strain 05-2101 in the aspects of bacterial wilt prevention and control or bacteriostasis. The invention also provides a Bacillus safensis (Bacillus safensis) strain 05-2101-based microbial inoculum, a bacteriostatic product, a seed germination promoter or a plant growth promoter, a method for promoting seed germination or plant growth, a method for preventing and treating bacterial wilt and a bacteriostatic method. Experiments prove that the Bacillus safensis (Bacillus safensis) strain 05-2101 can effectively maintain the disease index of a tobacco plant with bacterial wilt at a lower level, has an inhibition and antagonism effect on various plant pathogenic bacteria, and also has a remarkable promoting effect on seed germination and growth of plants.

Description

Bacillus safensis strain 05-2101 and application, product and method thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a bacillus safensis strain 05-2101, and an application, a product and a method thereof.

Background

Bacterial wilt is caused by infection of Ralstonia (Ralstonia) and is an important soil-borne disease of crops. The ralstonia host has wide range, can infect more than 400 crops, and is seriously harmful to bananas, tomatoes, potatoes and tobaccos. Tobacco bacterial wilt is mainly caused by infection of ralstonia pseudosolanacearum, which commonly occurs in Yangtze river basin and various tobacco regions in south China. Luspanhua, liujunying, majunhong are equal to the "preliminary investigation of pathogen diversity of tobacco bacterial wilt disease in Yunnan province" published in 2019 (C. Proceedings of academic annual meeting in 2019 of Chinese plant pathologist, 2019). Although the tobacco bacterial wilt is serious, the disease is always an important factor for restricting the improvement of the yield and the quality of the tobacco due to the lack of disease-resistant resources and the absence of specific chemical prevention and control medicines.

Biological control has received much attention because it is environmentally friendly. For example, 9 biocontrol bacteria are developed and registered for tobacco bacterial wilt in China, the biocontrol bacteria mainly comprise 3 pseudomonas fluorescens, 2 salofusbacillus amyloliquefaciens, 3 polymyxa salofusbacillus and 1 bacillus subtilis, and most of the bacteria have the capability of antagonizing ralstonia bacteria. The related patents of tobacco bacterial wilt biocontrol spore bacteria reported in China mainly comprise: bacillus amyloliquefaciens (B. Amyloliquefaciens) TBA03 (CN 111117936A), paenibacillus polymyxa NX1-05-2101 (CN 107365729A) and Bacillus subtilis (B. Safensis) biocontrol strain Trb3 (CN 102747013A). The researches show that the biocontrol microbial inoculum for the tobacco bacterial wilt is mostly bacillus at present, and the main reason is that the bacillus has stronger stress resistance and is easy to create the microbial inoculum and prolong the shelf life. But the requirements of tobacco bacterial wilt in southern and southwest tobacco planting areas of China that the tobacco bacterial wilt is generated all the year round and the green tobacco production is urgently needed cannot be met due to the large difference of regional adaptability of different biocontrol bacteria, high trans-regional transportation cost, few varieties of spore bacteria products and the like.

The Bacillus safensis can be applied to agricultural production to a certain extent. For example, bacillus safensis S21 controls bacterial domestic blight of melons (CN 114958665A), bacillus safensis T1-5 controls potato scab (CN 114836335A), bacillus safensis HMD9204 controls eggplant and banana blight (CN 108865934A), bacillus safensis HCX-01 controls cotton verticillium wilt (CN 108587969A), bacillus safensis YJC-4 controls tobacco black shank, bacillus safensis SD23 controls rice sheath blight, and Bacillus safensis strain ZDC-02 controls crop diseases caused by colletotrichum gloeosporioides (CN 106119146A). However, the application of the bacillus safensis in the control of the crop bacterial wilt is not reported, and the application of the bacillus safensis in the control of the tobacco bacterial wilt is not found.

Disclosure of Invention

Based on the blank existing in the prior art in the field, the invention provides Bacillus safensis (Bacillus safensis) 05-2101 capable of preventing and treating tobacco bacterial wilt and an application, a product and a method thereof.

The technical scheme of the invention is as follows:

a Bacillus safensis (Bacillus safensis) strain 05-2101 is characterized in that the preservation number is CCTCC No: m2022547.

The preservation number is CCTCC No: the application of the Bacillus safensis (Bacillus safensis) strain 05-2101 of M2022547 in the aspects of bacterial wilt prevention and control, bacteriostasis and/or seed germination or plant growth promotion.

The bacterial wilt refers to tobacco bacterial wilt and is a disease caused by infection of Ralstonia solanacearum (Ralstonia solanacearum) or Ralstonia pseudosolanacearum (Ralstonia pseudolycolareum) or Ralstonia syzygii (Ralstonia syzygii);

preferably, bacillus safensis (Bacillus safensis) strain 05-2101 does not control bacterial wilt by antagonizing or inhibiting Ralstonia solanacearum or Ralstonia pseudosolanacearum or Ralstonia syzygosaceum;

preferably, the bacteriostatic bacteria are selected from: rhizopus arrhizus (Rhizopus arrhizus) and/or Alternaria alternata (Alternaria alternata).

Preferably, said promoting seed germination or plant growth is selected from the group consisting of: the method comprises the steps of increasing the fresh weight of the plant, increasing the maximum leaf area of the plant and increasing the germination rate.

A microbial inoculum, comprising: the preservation number is CCTCC No: m2022547 Bacillus safensis (Bacillus safensis) strain 05-2101.

Preferably, the microbial inoculum further comprises: an auxiliary material;

preferably, the formulation of the microbial inoculum is selected from: fermentation liquor, powder and suspoemulsion.

A seed germination or plant growth promoter comprising: the active ingredient for promoting seed germination or plant growth is characterized by comprising the following active ingredients: the preservation number is CCTCC No: m2022547 Bacillus safensis (Bacillus safensis) strain 05-2101.

Preferably, said seed germination or plant growth promoter further comprises: an auxiliary material;

preferably, the seed germination or plant growth promoter is in a dosage form selected from the group consisting of: fermentation liquor, powder and suspoemulsion.

A method for promoting seed germination or plant growth is characterized in that the method adopts a method with a preservation number of CCTCC No: m2022547 Bacillus safensis (Bacillus safensis) strain 05-2101 treats seed germination or plant growth substrates.

Preferably, said promoting seed germination or plant growth is selected from the group consisting of: a group consisting of increasing the fresh weight of the plant, increasing the maximum leaf area of the plant, and increasing the germination rate;

preferably, the treatment concentration of the strain 05-2101 is 10 ⁸ CFU/mL；

Preferably, the plant is tobacco.

The method for preventing and treating bacterial wilt is characterized in that the preservation number is CCTCC No: m2022547, bacillus safensis strain 05-2101 for controlling bacterial wilt.

The concentration of the adopted bacteria is 10 ⁷ -10 ⁹ C, irrigating roots of diseased plants by CFU/mL fermentation diluent of Bacillus safensis (Bacillus safensis) strains 05-2101;

and/or, the diseased plant refers to: the tobacco plants which initiate bacterial wilt are infected with Ralstonia solanacearum (Ralstonia solanacearum) or Ralstonia pseudosolanacearum (Ralstonia pseudomonacea) or Ralstonia prunanensis (Ralstonia syzygii).

A bacteriostatic article, comprising: bacteriostatic active ingredients, characterized in that the bacteriostatic active ingredients comprise: the preservation number is CCTCC No: m2022547 Bacillus safensis (Bacillus safensis) strain 05-2101.

Preferably, the bacteriostatic product further comprises: an auxiliary material;

preferably, the formulation of the bacteriostatic product is selected from: fermentation liquor, powder, suspoemulsion;

The bacteriostasis method is characterized in that the antibacterial agent adopts a medium with the preservation number of CCTCC No: m2022547, bacillus safensis strain 05-2101.

The invention has the following beneficial effects:

1. the biocontrol bacterium is easy to commercialize: the bacillus safensis has better stress resistance, can create powder which is easy to transport, reduces the production and transportation cost, delays the shelf life of a biocontrol product, increases the effective viable count, has strong tolerance and adaptability of biocontrol bacteria in soil, is easy to colonize in soil and rhizosphere, and is beneficial to exerting the biocontrol effect.

2. No drug resistance is generated: most of the bacillus safensis has good bacteriostatic action on crop pathogenic bacteria, but the bacillus safensis 05-2101 has no antagonistic action on a plurality of tobacco important disease pathogenic bacteria, which indicates that the bacterial strain 05-2101 does not mainly produce antibiotics to play a role in controlling diseases. Therefore, the tolerance of the ralstonia to the bacterial strain 05-2101 or a bacterial strain metabolite cannot be enhanced when the bacterial strain 05-2101 is applied to control the tobacco bacterial wilt in the field, and the applied bacterial strain 05-2101 has better safety;

3. and (3) enriching biocontrol resources: the tobacco bacterial wilt biological control Bacillus safensis takes Bacillus subtilis, bacillus amyloliquefaciens and Bacillus polymyxa as main materials, and Bacillus safensis (Bacillus safensis) 05-2101 related to the invention provides a new biological control resource for the control of crop bacterial wilt.

The preservation information of Bacillus safensis (Bacillus safensis) 05-2101 of the present invention is as follows:

the preservation number is: CCTCC NO: M2022547;

and (3) classification and naming: bacillus safensis 05-2101;

the preservation date is as follows: 2022, 5 months and 3 days;

the preservation unit: china center for type culture Collection;

and (4) storage address: china, wuhan and Wuhan university.

The deposit information of ralstonia pseudosolanacearum (r. Pseudolycolariacaceum) RS is as follows:

the preservation number is: GDMCC 1.3533;

and (3) classification and naming: ralstonia sp;

the preservation date is as follows: 7 months and 14 days 2022;

the preservation unit: guangdong province microbial strain preservation center;

and (4) storage address: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5.

Drawings

FIG. 1 is a colony morphology and a cell morphology of strain 05-2101 of Experimental example 1 of the present invention cultured for 48 hours.

FIG. 2 is a comparison chart of the colony morphology of strains 05-2101 of Experimental example 2 of the present invention cultured against main disease pathogens of tobacco

FIG. 3 is a phylogenetic tree diagram of Bacillus safensis 05-2101 and related Bacillus closely based on whole genome construction according to Experimental example 3 of the present invention.

FIG. 4 shows the effect of the microbial inoculum 05-2101 of Experimental example 4 of the present invention on promoting seed germination and tobacco seedling growth by mixing with a medium.

FIG. 5 shows the effect of the powders 05 to 2101 of Experimental example 5 of the present invention in controlling tobacco bacterial wilt in a greenhouse at 28 ℃.

FIG. 6 shows the effect of the powders 05 to 2101 of Experimental example 6 of the present invention on controlling tobacco bacterial wilt in greenhouses.

Detailed Description

The following detailed description will explain the technical solutions of the present invention in detail with reference to specific examples, experimental examples, and drawings, but the present invention is not limited to the following technical solutions.

Sources of biological material

1. The tobacco material used in experimental example 4 was a known and publicly available tobacco variety Yunyan 87, which was stored in the laboratory of the applicant or commercially available; the tobacco materials used in experimental examples 5 and 6 were known tobacco varieties, honghuadajinyuan, which are commonly used and stored in the laboratory of the applicant, or commercially available.

2. Pathogenic bacteria used in experimental example 2:

pseudomonas solanacearum (r. Pseudosolanacearum) RS has completed genome sequencing, and the sequence is submitted to GenBank database, bioproject Number PRJNA594457, genBank association access Number GCA _018243235.1, which has been deposited at the guangdong province collection of microorganisms at 7/14/2022 with a accession Number GDMCC 1.3533.

Rosemophilus persicae (Ralstonia syzygii) LLRS-1 is the LLRS-1 Strain described in "Can-Hua Lu, jun-Ying Li, meng-Ge Mi, et al. Complete Genome Sequence of Ralstonia syzygii subsp. Indenesis Strain LLRS-1, isolated from wild Tobacco in Phytopathology,2021, 111.

Pseudosolanaceae Ralstonia pseudosolani (Ralstonia pseudosolanacearum) BSRS-1 and QBSRS-1 are strains preserved in the applicant's laboratory, and the applicant promises to release the strains to the public for verification of the effect of the invention within 20 years from the application date of the invention.

The Sclerotinia sclerotiorum (sclerotiorum) HP-1 of the Tobacco Sclerotinia sclerotiorum is "Lu C. -H., liu J. -Y., lin Z. -L., et al.first Report of sclerotia sclerotiorum on organic Tobacco in the Yunann Provision of China.plant Disease,2020,104 (6): 1867" HP-1 described herein.

The tobacco gray mold pathogen Botrytis cinerea (Botrytis cinerea) HM-1 is ' Lulau Hua, lima Luzhong, yan ' an Zheng, and the like, the identification of pathogens of the tobacco gray mold and sclerotinia rot and the screening of indoor medicaments [ J ]. Chinese tobacco science, 2020,41 (6): 68-75 '.

Identifying pathogenic bacteria of flue-cured tobacco mildew and screening the pathogenic bacteria thereof [ J ] China tobacco science, 2022,43 (2): 45-51.

Phytophthora nicotianae (Phytophthora nicotiana) No. 0 physioraces strain 19HJ130 and No. 1 physioraces strain 19HJ80, alternaria alternata (Alternata) cx-1, cerrena curatodes (Thanatephorus cumericis) LCTS-6, fusarium oxysporum (Fusarium oxysporum) xw03 are respectively ' Lupinhua ', xiyaosheng, jinyan ', etc.. One strain of Bacillus sphaericus 05-1205, an acquisition method and application thereof.

3. The ralstonia used in experimental examples 5 and 6 was ralstonia pseudosolanacearum (r. Pseudosolanaceae) RS, whose genome sequencing was completed and whose sequence was submitted to GenBank database with Bioproject Number PRJNA594457 and GenBank assignment access Number GCA _018243235.1, which was deposited at the guangdong province collection of microorganisms at 7/14 d 2022 under GDMCC 1.3533.

Culture medium and culture seedling method adopted in experimental example of the invention

1. The culture medium adopted

The separation medium contained 0.5% (w/v) peptone, 0.3% beef extract 3.0, 0.8% sodium chloride, 1.5% agar;

LB liquid medium contained 1% (w/v, the same applies below) tryptone, 0.5% yeast extract, 1% sodium chloride and 0.5% sucrose;

CG medium contained 0.1% acid hydrolyzed casein, 0.5% glucose, and 2% peptone;

CGA contains 0.1% acid hydrolyzed casein, 0.5% glucose, 2% peptone, and 1.5% agar;

oligotrophic medium CN containing 0.1% casamino acids, 0.1% nutrient broth, 1.5% agar;

the TZC culture medium contains 1% of peptone, 1% of glucose, 0.1% of casein hydrolysate, 1.5% of agar and 0.005% of triphenyltetrazolium chloride (TTC);

PDA medium contains 20% potato, 2% glucose, and 1.5% agar.

2. Float seedling raising

The method takes the Honghuadajinyuan of the infected tobacco variety as a test object, and the tobacco seedlings are cultivated by floating seedling to 4-5 leaf stages.

3. Cultivation of pathogenic bacteria

Activating the ralstonia sp RS from an ultra-low temperature refrigerator at minus 80 ℃ on the surface of a TZC culture medium [1% peptone, 1% glucose, 0.1% casein hydrolysate, 1.5% agar and 0.005% triphenyltetrazolium chloride (TTC) ], and culturing for 36-48 h in a constant-temperature incubator at 28 ℃;

selecting a typical bacterial colony which has a wider white edge and stronger fluidity and is pink or light red dilute liquid in the middle, inoculating the typical bacterial colony into a triangular flask containing 100mL of CG liquid culture medium (1% peptone, 1% glucose and 0.1% casein hydrolysate), and placing the triangular flask in a constant-temperature shaking culture at 28 ℃ and 225r/min for 24 hours;

diluting 100 μ L to 10 ^-7 Taking 100 μ L of 10 ^-5 、10 ^-6 、10 ^-7 And coating the TZC plate with the diluent, observing colony morphology and counting the number of colonies after 48 hours, and calculating the amount of the bacteria contained in the cultured bacteria liquid.

Group 1 examples, strains 05-2101 of the invention

The group of embodiments provides a Bacillus safensis (Bacillus safensis) strain 05-2101, which is characterized in that the preservation number is CCTCC No: m2022547.

Any utilization, use, sale, promissory sale, production, preparation, culture, propagation, cloning and fermentation preservation number is CCTCC No: the behavior of the M2022547 Bacillus safensis (Bacillus safensis) strain 05-2101 falls within the scope of the present invention.

According to the teaching and inspiration of the invention, the skilled in the art selects and mixes the proper auxiliary materials by combining the common technical means in the field of microbial technology according to the actual production needs, and the preservation number of the invention is CCTCC No: m2022547, bacillus safensis strain 05-2101 can be made into various dosage forms meeting the requirements of technological production, such as powder, tablet, liquid, etc.

In this context, the biocontrol bacteria, bacillus safensis 05-2101, the invention described in the summary of the invention, the detailed description of the invention, refer to: the preservation number of the invention is CCTCC No: m2022547 Bacillus safensis (Bacillus safensis) strain 05-2101.

Group 2 examples, applications of strains 05-2101 of the invention

The embodiment of the group provides a preservation number of CCTCC No: application of Bacillus safensis (Bacillus safensis) strain 05-2101 of M2022547 in bacterial wilt prevention and control, bacteriostasis and/or seed germination or plant growth promotion.

In some embodiments, the bacterial wilt is tobacco bacterial wilt, a disease caused by infection with Ralstonia solanacearum (Ralstonia solanacearum) or Ralstonia pseudosolanacearum (Ralstonia pseudosolanacearum) or Ralstonia persicae (Ralstonia syzygii).

In other embodiments, bacillus safensis (Bacillus safensis) strain 05-2101 does not control bacterial wilt by antagonizing or inhibiting Ralstonia solanacearum (Ralstonia solanacearum) or Ralstonia pseudosolanacearum (Ralstonia pseudosolanacearum) or Ralstonia syzygii (Ralstonia syzygii).

In particular embodiments, the bacteriostatic bacteria are selected from: rhizopus arrhizus (Rhizopus arrhizus) and/or Alternaria alternata (Alternaria alternata).

In specific embodiments, the promoting seed germination or plant growth is selected from the group consisting of: the method comprises the steps of increasing the fresh weight of the plant, increasing the maximum leaf area of the plant and increasing the germination rate.

Based on the description of the invention, the technicians in the field have motivation to use the preservation number of the invention as CCTCC No: the Bacillus safensis (Bacillus safensis) strain 05-2101 of M2022547 can be used for preventing and treating other plant diseases or symptoms except bacterial wilt, such as plant tissue decay caused by rhizopus arrhizus, plant blight caused by fusarium oxysporum, sclerotinia sclerotiorum caused by sclerotinia sclerotiorum, stem rot caused by panoraria necator, gray mold caused by botrytis cinerea, leaf blight caused by alternaria alternata, fruit rot caused by phytophthora nicotianae and the like, and the inhibition antagonism of the strain 05-2101 in various tobacco pathogenic bacteria can be expected to have similar preventing and treating effects to the bacterial wilt in various tobacco diseases. For the sake of space, the control data for various plant diseases are not listed herein.

Any utilization, use, sale, promissory sale, production, preparation, culture, propagation, cloning, fermentation and preservation number is CCTCC No: the M2022547 Bacillus safensis (Bacillus safensis) strain 05-2101 is used for preventing and controlling bacterial wilt, plant tissue decay, plant fusarium wilt, sclerotinia, stem rot, gray mold, leaf blight and fruit rot, and falls into the protection scope of the invention.

Group 3 examples, microbial Agents of the invention

The embodiment of the group provides a microbial inoculum. All embodiments of this group share the following common features: the microbial inoculum comprises: the preservation number is CCTCC No: m2022547 Bacillus safensis (Bacillus safensis) strain 05-2101.

In a further embodiment, the microbial inoculum further comprises: and (5) auxiliary materials.

In more specific embodiments, the pharmaceutical excipient is selected from the group consisting of: solvents, propellants, solubilizers, solubilizing agents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integration agents, permeation enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickeners, encapsulation agents, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retardants, and the like.

In a particular embodiment, the dosage form is a powder.

The preparation form of the microbial inoculum is not limited to powder, and the technicians in the field select appropriate auxiliary materials for blending according to the teaching and inspiration of the invention and the common technical means in the field of microbial technology (for example, encyclopedia of preparation technology, pharmaceutical preparation technology and the like) in combination with the actual production requirements, wherein the preservation number of the microbial inoculum is CCTCC No: m2022547 Bacillus safensis (Bacillus safensis) strain 05-2101 can be made into various products in other dosage forms, such as tablet, liquid, spray, granule, etc., meeting the production requirements.

Group 4 examples of the method for controlling bacterial wilt disease according to the present invention

The embodiments of the group provide a method for controlling bacterial wilt. All embodiments of this group share the following common features: the preservation number is CCTCC No: m2022547, bacillus safensis strain 05-2101 for controlling bacterial wilt.

In a preferred embodiment, a bacteria concentration of 10 is used ⁷ -10 ⁸ The root irrigation treatment is carried out on the diseased plants by CFU/mL fermentation diluent of Bacillus safensis (Bacillus safensis) strain 05-2101.

In specific embodiments, the diseased plant refers to: the tobacco plants which initiate bacterial wilt are infected with Ralstonia solanacearum (Ralstonia solanacearum) or Ralstonia pseudosolanacearum (Ralstonia pseudomonacea) or Ralstonia prunanensis (Ralstonia syzygii).

Group 5 examples, bacteriostatic articles of the invention

The present group of embodiments provides a bacteriostatic article. All embodiments of this group share the following common features: the bacteriostatic product comprises: a bacteriostatic active ingredient; the bacteriostatic active ingredients comprise: the preservation number is CCTCC No: m2022547 Bacillus safensis (Bacillus safensis) strain 05-2101.

In a further embodiment, the bacteriostatic agent further comprises: an auxiliary material;

in more specific embodiments, the adjuvant is selected from: solvents, propellants, solubilizers, solubilizing agents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integration agents, permeation enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickeners, encapsulation agents, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retardants, and the like.

according to the teaching and inspiration of the invention, the skilled in the art selects appropriate auxiliary materials to mix according to the actual production needs by combining the common technical means in the field of microbial technology (for example, encyclopedia of preparation technology, pharmaceutical preparation technology and the like), and the preservation number of the invention is CCTCC No: m2022547 Bacillus safensis strain 05-2101 can be made into various other dosage forms such as tablet, liquid, spray, granule, etc.

Preferably, the bacteriostatic bacteria are selected from: one or two of Rhizopus arrhizus and Alternaria alternata.

EXAMPLE 6 group 6 method of inhibiting bacteria according to the present invention

The present group of embodiments provides a method of inhibiting bacteria. All embodiments of this group share the following common features: the preservation number is CCTCC No: m2022547, bacillus safensis strain 05-2101.

Preferably, the bacteriostatic bacteria are selected from: rhizopus arrhizus (Rhzopus arrhizus) and/or Alternaria (Alternaria alternata);

preferably, the bacteriostatic concentration of the strain 05-2101 is 10 ⁸ CFU/mL。

Group 7 examples of seed Germination promoters or plant growth promoters of the present invention

The present group of embodiments provides a seed germination or plant growth promoter. All embodiments of this group share the following common features: the seed germination or plant growth promoter includes: the active ingredients for promoting seed germination or plant growth comprise: the preservation number is CCTCC No: m2022547 Bacillus safensis (Bacillus safensis) strain 05-2101.

in specific embodiments, the adjuvant is selected from: solvents, propellants, solubilizers, solubilizing agents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integration agents, permeation enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickeners, encapsulation agents, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retardants, and the like.

Group 8 examples, methods of the invention for promoting seed germination or plant growth

The present set of embodiments provide a method of promoting seed germination or plant growth. All embodiments of this group share the following common features: the preservation number is CCTCC No: m2022547 Bacillus safensis (Bacillus safensis) strain 05-2101 treats seed germination or plant growth substrates.

Preferably, the plant is tobacco.

Experimental example 1 Strain acquisition

1. Isolation and culture

0.4g of stems of tobacco plants which are thoroughly sterilized by the surface inspection are taken and put into a 2mL sterilizing centrifuge tube together with sterilizing steel balls, 600 mu L of sterile water is added, and the mixture is ground for 3min by a tissue grinder with the frequency of 30r/s. Diluting the ground tissue fluid to 10 ^-4 200. Mu.L of each homogenate per gradient was applied to a separation medium (peptone 5.0g/L, beef extract 3.0g/L, sodium chloride 8.0g/L, agar 15.0g/L, pH 7.05) and repeated 4 times per treatment. And after dark culture at 28 ℃ for 48h, selecting single colonies according to the differences of colony morphology, color and the like, streaking and purifying on the separation culture medium again, and moving the single colonies subjected to streaking culture into the test tube inclined plane of the separation culture medium for preservation.

2. Results of the experiment

Through the experiment, the pure bacteria 05-2101 is obtained by selecting the isolated culture medium and streaking the isolated culture medium again; single colonies of the strain 05-2101 were picked and inoculated into a tube containing 2.5mL of LB liquid medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride and 0.5% sucrose), and cultured with shaking at 28 ℃ and 225r/min for 48h, and the colony morphology is shown in FIG. 1.

Experimental example 2 Effect of the strains 05 to 2101 on inhibiting the growth of pathogenic bacteria of tobacco major diseases

1. Antagonizing ralstonia: CG medium (0.1% acid hydrolyzed casein, 0.5% glucose and 2% peptone) was cultured for 24h respectively with Ralstonia ralstoni LLRS-1, RS, BSRS-1, QBSRS-1 (bacteria concentration 10) ⁹ CFU/mL) gradient dilution to 10 ^-4 (ii) a Placing 100 μ L of the diluted solution on the surface of CGA (0.1% acid hydrolyzed casein, 0.5% glucose, 2% peptone, and 1.5% agar) culture medium, and coating with the strainEvenly coating, and drying in ultra-clean work; picking out a single colony of the bacteria 05-2101 to be tested by using a sterile toothpick, inoculating the single colony on the surface of the screening culture medium at a distance of 2.50cm from the center of the culture dish, wherein the concentration of the inoculated bacteria 05-2101 is 10 ⁸ CFU/mL, each dish was inoculated with 4 spots symmetrically, air dried, and then cultured in an incubator at 28 ℃ for 48h, and the zone and band of inhibition of the strain were measured (FIG. 2).

2. Antagonistic pathogenic fungi: the bacteriostatic ability of the strains 05 to 2101 on tobacco Sclerotinia sclerotiorum (Sclerotinia sclerotiorum) HP-1, tobacco gray mold pathogen Botrytis cinerea (Botrytis cinerea) HM-1, flue-cured tobacco mildew pathogen Rhizopus arrhizus (Rhizopus arrhizus) DL8, phytophthora nicotianae (Phytophthora nicotianae) No. 0 physiorace strain 19HJ130 and No. 1 physiorace strain 19HJ80, alternaria alternata (Alternaria alternata) cx-1, coriolus versicolor (Thanatephorus cumaris) LCTS-6 and Fusarium oxysporum (Fusarium oxysporum) xw03 are respectively used as indicator bacteria to detect the bacteriostatic ability of the strains 05 to 2101 on the pathogens. The experiment adopts a plate opposing method, indicator bacterium blocks are attached to the center of a plate of a PDA culture medium (200 g of potato, 20g of glucose, 15g of agar, distilled water is added to 1000mL, and the pH is 7.2) by a puncher with the diameter of 0.8cm, endophytic bacteria are symmetrically streaked on two sides 3.5cm away from the center of a bacterium cake, and the concentration of the inoculated bacteria in 05-2101 is 10 ⁸ CFU/mL, control treatment with pathogen only mycelium block (FIG. 2). And (3) carrying out constant-temperature culture at 28 ℃, checking the opposing culture result when the total culture dish is full of contrast, recording the width of a bacteriostasis zone, judging whether the separated endophytic bacteria have antagonistic action on pathogenic bacteria according to the growth speed of two bacteria, the existence of the bacteriostasis zone between bacterial colonies, whether hyphae at the edges of the bacterial colonies are sparse and withered and the like, and repeating the treatment for 3 times.

And (3) test results: as can be seen from table 2 and fig. 2 and 3, the strain 05-2101 has the strongest ability to inhibit the growth of alternaria alternate (a.alternata) cx-1; the strain 05-2101 has limited ability to inhibit the growth of rhizopus arrhizus DL 8; the strains 05 to 2101 have no inhibition ability on the growth of Phytophthora nicotianae (P.nicotianae) No. 0 strain 19HJ130, phytophthora nicotianae (P.nicotianae) No. 1 strain 19HJ80, deuteroceras cucumeris (T.cucumeris) LCTS-6, fusarium oxysporum (F.oxysporum) xw03, sclerotinia sclerotiorum (S.sclerotiorum) HP-1, botrytis cinerea (B.cinerea) HM-1 and 4 Ralstonia rosea (R.pseudolactocerarum). The results show that the antagonistic action of the strain 05-2101 on most of main disease pathogenic bacteria of tobacco is weak, and the strain 05-2101 can prevent and treat diseases through a non-antagonistic action mechanism.

TABLE 1 determination of the ability of strains 05-2101 to antagonize pathogenic bacteria of major diseases of tobacco

Experimental example 3 identification and preservation of Strain

1. Identification of strains

Conventional bacteria identification is described in the reference handbook of identification of common bacteria systems (eastern X. Bead et al, science publishers, 2001).

The Biolog GEN III plate was used to analyze the metabolic characteristics of the compounds of strain 05-2101.

The molecular identification method comprises the following steps: the kit for extracting the bacterial genome DNA, and the method is referred to the kit instruction. The 16S rDNA sequence is subjected to PCR amplification by using a universal primer F27/R1492, the amplification is carried out under the conventional condition, an amplification product is connected to a pEAZY-T5 Zero vector after being recovered by glue, escherichia coli competent cell DH5 alpha is transformed by heat shock, and a colony is picked to carry out colony PCR identification by using M13F/M13R as a primer. Positive clones were sent to Shanghai Yingjun Biotech Co., ltd for sequencing. And analyzing the model species similar to the strain 05-2101 by using an Ezbiocloud database (https:// www.ezbiocloud. Net /), and preliminarily confirming the primary classification status of the strain.

Further, the whole genome of the strain is obtained by using a genome sequencing technology. And (3) comparing the strains by using a TYPE database, calculating the DNA molecular hybridization value (dDDH) of the strains 05-2101 and the species with the closest genetic relationship, and finally determining the molecular classification status of the strains, as shown in figure 3 and table 2.

And determining the classification status of the biocontrol bacteria by combining morphological and molecular biological characteristics.

2. The results of the experiment are reported below:

1. the culture characteristics are as follows: the strain 05-2101 is cultured on an NA plate culture medium at 28 ℃, and the colony is light milky, purulent, round and neat in edge at the initial culture stage; the bacterial colony at the later culture stage is milky white and flat, has no bulge in the center, irregular edges, dry surface and folds on the surface; and (4) performing static culture in a liquid culture medium to form a biological film on the surface. The morphological characteristics are basically consistent with those of the bacillus described in the handbook of identification of common bacteria systems (Dongxu bead et al, science Press, 2001), and the strain 05-2101 is preliminarily judged to be bacillus.

2. Morphological characteristics: the strain 05-2101 is cultured at 28 ℃, under a microscope, spores are mesogenic or terminal, and the strain is oval, does not expand and has spores. Can move and the flagellum is grown. The average cell size is 0.52-0.76 μm × 2.0-2.8 μm, and gram-positive staining is observed.

3. The adaptability characteristic is as follows: the strain 05-2101 can grow in a culture medium containing 0-8 g/L NaCl and pH 5.0-9.0 at a temperature of 4-60 deg.C, preferably 28-35 deg.C, and preferably 7.0-7.2.

4. Metabolic characteristics of the compounds:

gen III identification results show that the carbon sources which can be utilized by the strains 05-2101 comprise citric acid, D-cellobiose, L-serine, gentiobiose, glyconic acid, L-fructose, N-acetyl-beta-D-mannosamine, D-arabitol, N-acetylneuraminic acid, D-serine, gelatin, gamma-amino-butyric acid, 3-formylglucose, D-fructose, D-sorbitol, D-fructose, L-arginine, acetoacetic acid and beta-hydroxy-D, L-butyric acid, L-histamine, D-gluconic acid, N-acetyl-D-glucosamine, aminoacetyl-L-proline, melibiose, β -formyl-D-glucoside, pectin, D-mannitol, D-glucose-6-phosphate, α -D-lactose, D-galacturonic acid, L-galacturonolactone, tween 40, α -hydroxy-butyric acid, methyl pyruvate, bromo-succinic acid, L-malic acid, D-methyl lactate, p-hydroxy-phenylacetic acid, non-utilizable carbon sources including quinic acid, propionic acid, inositol, formic acid, α -keto-butyric acid, L-lactic acid, acetic acid, α -keto-glutaric acid, sucrose, D-malic acid, D-trehalose, D-galactose, L-rhamnose, glucuronamide, N-acetyl-D-galactosamine, D-glucuronic acid, L-glutamic acid, mucic acid; mucic acid, L-pyroglutamic acid, L-aspartic acid, alpha-D-glucose, D-maltose, D-fructose-6-phosphate, D-aspartic acid, L-alanine, dextrin, D-turanose, D-salicin, inosine, glycerol, D-mannose, stachyose, raffinose; the strain 05-2101 can grow under the condition of containing sodium bromate, sodium tetradecyl sulfate, oleandomycin acetate, tetrazole violet, 1% sodium lactate, aztreonam, minocycline, 1% NaCl, nalidixic acid and sodium butyrate, but can not grow under the condition of containing potassium tellurite, 4% NaCl, 8% NaCl, lithium chloride, lincomycin, D-serine, fusidic acid, guanidine hydrochloride, tetrazole blue, vancomycin and rifamycin SV.

5. Molecular identification of the strain 05-2101:

16S rDNA sequence analysis shows that the strain 05-2101 and the Bacillus safensis (B.safensis) NCIB 3610 ^T The similarity is highest and is 99.93 percent; and Bacillus tequilensis (B.tequilensis) KCTC 13622 ^T Bacillus calvaria Safossi (B.cabrialisii) TE3 ^T Bacillus safensis (b. Inaquosorum) KCTC 13429 ^T Bacillus safensis (b. Stercoris) JCM 30051 compost ^T The 16S rDNA sequence similarity of the two sequences is 99.86 percent. The above results indicate that the strain 05-2101 belongs to the genus Bacillus and has a closest relationship to Bacillus safensis. Genome sequencing to obtain Genome sketch of the Strain 05-2101, and analyzing with Type (Strain) Genome Server (https:// type.dsmz.de /) database to show that the Strain 05-2101 and Bacillus safensis (B.safensis) ATCC 6051 ^T And NCIB 3610 ^T The similarity is highest, namely 89.3 percent and 89.1 (dDDH 4) which are respectively larger than the identification threshold value of the new species by 70.0 percent, which indicates that the strain 05-2101 is bacillus safensis; strain 05-2101 and compost bacillus safensis (B.sternoris) D7XPN1 ^T A dDDH4 value of 62.1%; the dDDH4 value of the strain 05-2101 and other standard strains of Bacillus safensis is less than or equal to 50.5 percent. It is noteworthy that the strain 05-2101 is related to Micromonospora novocataris MT25 ^T A ddddh 4 value of 81.6%, greater than 70% of the new species identification threshold; however, the difference of G + C composition reaches 28.2%, and the two belong to different bacteria of the same genus. Construction of phylogenetic trees based on bacterial strain 05-2101 and related bacillus safensis genome, result tableTwo sequenced standard strains ATCC 6051 of Ming strain 05-2101 and Bacillus safensis (B.safensis) ^T And NCIB 3610 ^T Grouped into one branch (fig. 2). The above results indicate that strain 05-2101 is bacillus safensis (b.

TABLE 2 comparative analysis of genome similarity of Strain 05-2101 and Bacillus

(2) Preservation of Strain 05-2101

By the above identification, it was confirmed that the strain 05-2101 was a strain of bacillus safensis (b.safensis), named 05-2101, and the strain was sent for storage, and the storage information is as follows:

the preservation number is: CCTCC NO: M2022547;

and (3) classification and naming: bacillus safensis 05-2101;

the preservation date is as follows: 2022, 5 months and 10 days;

the preservation unit: china center for type culture Collection;

and (4) storage address: china, wuhan and Wuhan university.

Experimental example 4, application of the strain 05-2101 in promotion of seed germination and tobacco seedling growth

1) Selecting single colony of strain 05-2101, culturing in 100mL culture solution (LB +0.5% sucrose) at 28 deg.C for 48h with a concentration of 10 ⁸ CFU/mL；

2) Diluting the microbial inoculum by 10 times by using sterile water, adding 90mL of the microbial inoculum into a proper amount of matrix, uniformly mixing, and subpackaging into 3 floating trays with 63 holes;

3) 1 seed is sowed in each hole, and all the seeds are covered by the matrix;

4) Taking the tray, injecting 2L of sterile water into the tray, and then putting the tray into a floating tray;

5) Observing the water absorption condition of each hole after the floating disc is placed, if the hole cannot absorb the water in the disc to the substrate, taking sterile water by using a liquid shifter, and adding the sterile water into the hole which does not absorb the water to wet the substrate;

6) The experimental setup was repeated 3 times, and the substrate treated with 10-fold dilution of the culture medium (LB +0.5% sucrose) was used as a control;

7) And (4) beginning investigation and recording the budding condition at 7d after sowing, continuously investigating for 5d once a day, selecting tobacco plants at 30d after sowing by adopting a 5-point sampling method, measuring the plant height, fresh weight and leaf area of the largest leaf, and analyzing the difference of each treatment.

And (3) test results: the result shows that at 7d after sowing, after the microbial inoculum 05-2101 treats the matrix, the seed germination rate is 87.30 +/-4.20%, while the control group is only 46.03 +/-10.41%; at 11d after sowing, when the germination rates of the seeds of the treatment group treated by the microbial inoculum 05-2101 and the control group treated by the culture solution are not increased, the germination rate of the seeds of the treatment group reaches 98.41 +/-1.59 percent, and the germination rate of the seeds of the control group is 95.77 +/-0.92 percent, which is improved by about 2.7 percent. At 30 days after sowing, the fresh weight and the maximum leaf area of the tobacco seedlings in the treatment group are both significantly higher than those in the control group (fig. 4). The results show that the microbial inoculum 05-2101 has the functions of promoting seed germination, improving germination rate and promoting tobacco seedling growth.

Experimental example 5, evaluation of greenhouse control effect of biocontrol microbial inoculum 05-2101 on tobacco bacterial wilt

The experiment was carried out in a constant temperature greenhouse at 28 ℃.

1. Seed liquid culture: separately selecting and inoculating the one-ring biocontrol bacterium into 500mL of a centrifugal tube containing 500mL of seed culture solution (10.0 g/L of peptone, 3.0g/L of beef extract, 5.0g/L of sodium chloride, 15.0g/L of agar and pH 7.0), and placing the centrifugal tube in a constant-temperature shaking table at 28 ℃ for 15 hours.

2. Fermentation: and (3) mixing the seed liquid of the biocontrol bacterium according to the proportion of 1:100 portions of the powder were transferred to a medium containing 150L of fermentation medium for fermentation (20 g/L glucose, 25g/L soybean meal, 4g/L yeast extract, 1.5g/L dipotassium phosphate, pH 7.3). The fermentation process parameters are as follows: (1) sterilizing: the air elimination conditions are as follows: 0.15MPa,123 ℃,1h; the actual elimination conditions are as follows: 0.15MPa,123 ℃,35min; (2) inoculation: after sterilization, when the temperature of the culture solution is reduced to 37 ℃, starting inoculation, wherein the inoculation ratio is that the strain/culture solution is 1; (3) fermentation: rotating at a speed of 120r/min until fermentation is finished; tong (Chinese character of 'tong')Gas quantity: hold 15m ³ Keeping the reaction time at the end of fermentation; the whole process keeps the tank pressure at 0.05Mpa; the ventilation is allowed to float for short periods of time for equipment reasons. Temperature: the temperature is kept at 37 ℃ in the whole fermentation process, and the temperature can float up and down by 0.5 ℃. Fermentation time: the fermentation is run for 24h.

3. Powder preparation: centrifuging fermentation liquor (about 130.63 hundred million CFU/mL), collecting centrifugal thalli as a raw material, mixing the raw material with diatomite according to a mass ratio of 1.

4. And (3) preventing and treating: applying 2.5g of microbial inoculum to each tobacco seedling, adding 50mL of water to obtain a bacterial powder suspension with the bacterial concentration of 9.1 hundred million CFU/mL, and irrigating roots, wherein 15 plants are repeated for each time, 3 times of repetition is set, and clear water is set as a reference; inoculating 0.1OD of Ralstonia RS on the next day; disease grade was investigated once a week and disease index was calculated.

5. Survey and statistics: disease index was investigated every 7 days after inoculation, 3-6 times in total. The morbidity, disease index and prevention and treatment effect of the tobacco bacterial wilt are calculated according to the following formula: morbidity = diseased plants/total number of investigated plants x 100%; disease index = [ Σ (number of disease stages × number of strains at this stage)/(highest stage × number of total strains) ] × 100; the prevention and treatment effect = (contrast disease index-treatment disease index)/contrast disease index x 100%. The disease index is investigated according to the tobacco disease classification and investigation method of the tobacco industry standard of the people's republic of China (GB/T23222-2008), and the disease classification is as follows: the disease-free state of the whole plant is 0 grade, the withering of the leaves below one half of the diseased side is 1 grade, the withering of the leaves from one half to two thirds of the diseased side is 3 grade, the withering of the leaves above two thirds of the diseased side is 5 grade, the complete withering of the leaves of the diseased plant is 7 grade, and the basic withering of the diseased plant is 9 grade.

And (3) test results: as shown in FIG. 5, the disease condition of the control group gradually worsened with the increase of the inoculation days, while the tobacco strains treated by the inocula 05-2101 had a lighter disease at 24 days after inoculation, and the area under the disease index curve was 10.00 + -17.32, while the control group was 473.00 + -48.72. The control effect of the treatment group of the microbial inoculum 05-2101 is 97.89 percent by calculating the area under the disease index curve, and the control effect is better.

Experimental example 6 application of the Strain in tobacco bacterial wilt prevention and control

Experimental example 6 the same procedure as in Experimental example 5 was repeated except that the following tests and results were different.

And (3) experimental setting: performing in a greenhouse, and setting a Ralstonia treatment group and a control group (root irrigation equal volume LB culture medium and tap water) in the test, wherein the treatment is repeated for 3 times and 10 tobacco plants are repeated for each time;

transplanting tobacco seedlings: tobacco seedlings cultivated in a floating seedling cultivation mode are cultivated for about 50d, and the tobacco seedlings are transplanted after red soil and organic matters are uniformly mixed according to a ratio of 3;

inoculation: taking 2.5g of powder 05-2101 from each tobacco seedling after transplanting, diluting the powder to 200mL by adding water, and treating a control group by using an equal volume of LB diluent; the next day, the culture medium CG was shaken and incubated for 24 hours for 100-fold dilution of Ralstonia RS, and 100mL of a Ralstonia dilution (inoculation concentration about 10) was inoculated per cigarette ⁷ CFU/mL)。

And (3) test results: the results show that the disease indexes of the tobacco plants treated by the microbial inoculum 05-2101 are all lower than those of chemical agent of the blue vitriol and clear water contrast in the observation period. At 35dpi, the area under the curve of the disease index of the tobacco plant treated by the microbial inoculum 05-2101 is 401.20 +/-105.10 percent, which is lower than the area under the curve of the tobacco plant treated by the blue copperas, 452.40 +/-140.50 percent. The control effect is calculated by the area under the disease index curve, and the result shows that the control effect of the treatment group of the microbial inoculum 05-2101 is 73.50 percent, while the control effect of the treatment group of the chlorourea copper sulfate is 70.12 percent, and the control effects are equivalent. The results show that the microbial inoculum has better control effect on tobacco bacterial wilt caused by ralstonia QBSR-1 (figure 6).

Claims

1. A Bacillus safensis (Bacillus safensis) strain 05-2101 is characterized in that the preservation number is CCTCC No: m2022547.

2. The preservation number is CCTCC No: application of Bacillus safensis (Bacillus safensis) strain 05-2101 of M2022547 in bacterial wilt prevention and control, bacteriostasis and/or seed germination or plant growth promotion.

3. The preservation number of claim 2 is CCTCC No: use of Bacillus safensis (Bacillus safensis) strain 05-2101 of M2022547 for controlling and/or inhibiting bacterial wilt, and/or promoting seed germination or plant growth, characterized in that the bacterial wilt refers to tobacco bacterial wilt, a disease caused by infection by Ralstonia solanacearum (Ralstonia solanacearum) or Ralstonia pseudosolanacearum (Ralstonia pseudo-pseudomonas pseudomonanaceae) or Ralstonia syzygii (Ralstonia syzygii);

and/or, bacillus safensis (Bacillus safensis) strain 05-2101 does not control bacterial wilt by antagonizing or inhibiting Ralstonia solanacearum (Ralstonia solanacearum) or Ralstonia pseudosolanacearum (Ralstonia pseudosolanacearum) or Ralstonia syzygii (Ralstonia syzygii);

and/or, the bacteriostatic bacteria are selected from: rhizopus arrhizus and/or Alternaria alternata

And/or, the promoting seed germination or plant growth is selected from: the method comprises the steps of increasing the fresh weight of the plant, increasing the maximum leaf area of the plant and increasing the germination rate.

4. A microbial inoculum, comprising: the preservation number is CCTCC No: m2022547 Bacillus safensis (Bacillus safensis) strain 05-2101.

And/or, the microbial inoculum further comprises: an auxiliary material;

and/or the formulation of the microbial inoculum is selected from the following compositions: fermentation liquor, powder and suspoemulsion.

5. A seed germination or plant growth promoter comprising: the active ingredients for promoting seed germination or plant growth, wherein the active ingredients for promoting seed germination or plant growth comprise: the preservation number is CCTCC No: m2022547 Bacillus safensis (Bacillus safensis) strain 05-2101.

And/or, the seed germination or plant growth promoter further comprises: an auxiliary material;

and/or, the dosage form of the seed germination or plant growth promoter is selected from the following: fermentation liquor, powder and suspoemulsion.

6. A method for promoting seed germination or plant growth is characterized in that the method adopts a method with a preservation number of CCTCC No: m2022547 Bacillus safensis (Bacillus safensis) strain 05-2101 treatment of seed germination or plant growth medium.

And/or, the promotion of seed germination or plant growth is selected from: a group consisting of increasing the fresh weight of the plant, increasing the maximum leaf area of the plant, and increasing the germination rate;

and/or the treatment concentration of the strain 05-2101 is 10 ⁸ CFU/mL；

And/or, the plant is tobacco.

7. The method for preventing and treating bacterial wilt is characterized in that the preservation number is CCTCC No: m2022547, bacillus safensis strain 05-2101 for controlling bacterial wilt.

8. The method for controlling bacterial wilt according to claim 7, wherein bacterial concentration is 10 ⁷ -10 ⁹ C, irrigating roots of diseased plants by using CFU/mL fermentation diluent of Bacillus safensis (Bacillus safensis) strain 05-2101;

9. A bacteriostatic article comprising: bacteriostatic active ingredients, characterized in that the bacteriostatic active ingredients comprise: the preservation number is CCTCC No: m2022547 Bacillus safensis (Bacillus safensis) strain 05-2101.

And/or, the bacteriostatic product further comprises: an auxiliary material;

and/or, the formulation of the bacteriostatic product is selected from: fermentation liquor, powder, suspoemulsion;

and/or, the bacteriostatic bacteria are selected from: rhizopus arrhizus (Rhizopus arrhizus) and/or Alternaria alternata (Alternaria alternata).

10. The bacteriostasis method is characterized in that the antibacterial agent adopts a medium-temperature storage medium with a preservation number of CCTCC No: m2022547, bacillus safensis strain 05-2101.