CN110951657A

CN110951657A - Bacterial wilt preventing and growth promoting microbial inoculum and application thereof

Info

Publication number: CN110951657A
Application number: CN202010009707.4A
Authority: CN
Inventors: 魏海雷; 谷医林; 张宏越
Original assignee: Institute of Agricultural Resources and Regional Planning of CAAS
Current assignee: Moon Guangzhou Biotech Co ltd
Priority date: 2019-09-25
Filing date: 2019-09-25
Publication date: 2020-04-03
Anticipated expiration: 2039-09-25
Also published as: CN110951657B; CN110923178B; CN110923178A; CN110408578A; CN110408578B

Abstract

The invention discloses a bacterial wilt prevention and growth promotion microbial inoculum and application thereof. The bacterial wilt preventing and growth promoting microbial inoculum contains pseudomonas aeruginosa S58 with the registration number of CGMCC No.17043 in the common microorganism center of China Committee for culture Collection of microorganisms. The microbial inoculum has inhibiting effect on phytopathogen such as phytophthora sojae, rhizoctonia solani, Pyricularia oryzae and Laurella solani. The bacterial wilt prevention and growth promotion microbial inoculum can generate auxin (IAA), can obviously inhibit the elongation of main roots of plants, promotes the growth of lateral roots and improves the biomass of the plants.

Description

Bacterial wilt preventing and growth promoting microbial inoculum and application thereof

The application is a divisional application with the application number of 201910911436.9, the application date of 2019, 09 and 25, and the invention and creation name of 'one strain of pseudomonas winkle and application thereof'.

Technical Field

The invention relates to a bacterial wilt prevention and growth promotion microbial inoculum and application thereof in the field of microorganisms.

Background

Tobacco is an economic plant in china. With the development of tobacco production, the continuous cropping area is increased, and the occurrence of diseases is becoming more and more serious, which becomes one of the main limiting factors for damaging the tobacco production. Tobacco black shank and bacterial wilt are main and common soil-borne diseases of tobacco, can be infected from seedling stage to adult plant, and are destructive diseases of tobacco. In recent years, due to the enlargement of the area of continuous cropping tobacco fields and the increase of the continuous cropping years, tobacco black shank and bacterial wilt tend to be more prevalent in many places. Because of lack of ideal disease-resistant tobacco cultivars in production, currently, the prevention and treatment of tobacco black shank and bacterial wilt still depend on chemical agents, but the effects are not ideal and a plurality of defects exist. For example, the currently used chemical bactericides cause pollution or damage to soil microorganisms, plants, water sources and the atmosphere, meanwhile, the problem of drug resistance is increasingly prominent, and the residue of the drugs in plants indirectly causes harm to human beings. Therefore, development of a microbial preparation which is environmentally friendly and nontoxic to humans and animals is required.

The method is an effective way to prevent and treat plant diseases and insect pests by using antagonistic bacteria attached to plants, and the prerequisite condition for ensuring the success of biological prevention and treatment is that antagonistic strains with good effect and stable activity are screened. The biocontrol bacteria mainly studied at present are plant root growth-promoting bacteria (PGPR), and biocontrol pseudomonas (A), (B) and (C)Pseudomonasspp.) Bacillus bacteria (b), (b)Bacillusspp.), and the like. The rhizosphere bacteria of plants exist around the root system of the plants in large quantity, and the bacteria can colonize the surface layer of the plants, so that the living environment is stableSome of them are antagonistic to pathogenic bacteria and some of them induce disease resistance in plants. The microorganism is harmless to human and livestock, is environment-friendly, and can be developed into biocontrol bacteria resources for plant diseases.

Disclosure of Invention

The technical problem to be solved by the invention is how to inhibit the tobacco black shank and/or how to inhibit the tobacco bacterial wilt and/or how to inhibit the tobacco wildfire, and/or how to inhibit the plant pathogenic fungi and/or how to inhibit the plant pathogenic bacteria and/or how to improve the biomass of the plant and/or how to improve the soil.

In order to solve the technical problems, the invention firstly provides a strain of pseudomonas aeruginosa for wrinkle.

The pseudomonas winkle provided by the invention is pseudomonas winkle (Pseudomonas winkle)Pseudomonas corrugata) S58, wherein the registration number of the general microbiological center of China Committee for culture Collection of microorganisms is CGMCC No. 17043. The strain is preserved in China general microbiological culture Collection center (CGMCC for short) in 2018, 12 months and 27 days. Hereinafter referred to as Pseudomonas wrinkle S58.

The Pseudomonas winkle S58 has rod-like shape, average size of 0.5-1.5 μm × 1.6-2.5 μm, gram-negative, single-polar single-or multi-flagella, and no fluorescence. The colony of the pseudomonas winkle S58 on the NA culture medium is white, and has raised folds and irregular edges. The growth temperature range of the pseudomonas aeruginosa S58 is 8-45 ℃, the optimum growth temperature is 28-32 ℃, the growth pH value is 6.5-8, and the optimum pH value is 7. The physiological and biochemical characteristics of Pseudomonas aeruginosa S58 are shown in Table 1. The pseudomonas aeruginosa S58 has 16S rDNA shown in a sequence 1 in a sequence table.

Any of the following uses of the metabolites of pseudomonas aeruginosa S58 or/and pseudomonas aeruginosa S58 are also within the scope of the present invention:

u1, Pseudomonas aeruginosa S58 and/or Pseudomonas aeruginosa S58 for preventing and/or treating tobacco black shank,

u2, Pseudomonas aeruginosa S58 and/or Pseudomonas aeruginosa S58 in preventing and/or treating tobacco bacterial wilt,

the application of the metabolites of U3, Pseudomonas aeruginosa S58 and/or Pseudomonas aeruginosa S58 in preventing and/or treating tobacco wildfire,

the application of the metabolites of U4, Pseudomonas winkle S58 and/or Pseudomonas winkle S58 in improving soil,

the application of U5, Pseudomonas aeruginosa S58 or/and Pseudomonas aeruginosa S58 metabolites in hydrolyzing organophosphorus,

the application of metabolites of U6, pseudomonas aeruginosa S58 or/and pseudomonas aeruginosa S58 in inhibiting tobacco wildfire pathogen,

the application of the metabolites of U7, Pseudomonas aeruginosa S58 and/or Pseudomonas aeruginosa S58 in inhibiting phytophthora nicotianae,

the application of the metabolites of U8, Pseudomonas aeruginosa S58 and/or Pseudomonas aeruginosa S58 in inhibiting phytophthora sojae,

the application of the metabolites of U9, Pseudomonas aeruginosa S58 and/or Pseudomonas aeruginosa S58 in inhibiting Rhizoctonia solani,

the application of the metabolites of U10, Pseudomonas aeruginosa S58 and/or Pseudomonas aeruginosa S58 in inhibiting Pyricularia oryzae,

the application of the metabolites of U11, Pseudomonas aeruginosa S58 and/or Pseudomonas aeruginosa S58 in inhibiting acidovorax citrulli,

the application of U12, Pseudomonas aeruginosa S58 and/or Pseudomonas aeruginosa S58 metabolites in inhibiting clavibacterium michiganensis,

the application of the metabolites of U13, Pseudomonas aeruginosa S58 and/or Pseudomonas aeruginosa S58 in inhibiting Xanthomonas oryzae,

u14, Pseudomonas aeruginosa S58 and/or Pseudomonas aeruginosa S58 for inhibiting Lawsonia solanacearum,

use of metabolites of U15, Pseudomonas rhynchophylla S58 or/and Pseudomonas rhynchophylla S58 for the production of auxin (IAA),

the application of U16, Pseudomonas aeruginosa S58 or/and Pseudomonas aeruginosa S58 metabolites in inhibiting the elongation of main roots of plants,

the application of the metabolites of U17, Pseudomonas aeruginosa S58 and/or Pseudomonas aeruginosa S58 in promoting the growth of lateral roots of plants,

the application of U18, Pseudomonas aeruginosa S58 and/or Pseudomonas aeruginosa S58 metabolites in improving plant biomass,

the application of the metabolites of U19, Pseudomonas aeruginosa S58 and/or Pseudomonas aeruginosa S58 in producing amylase,

the application of the metabolites of U20, Pseudomonas rhynchophylla S58 and/or Pseudomonas rhynchophylla S58 in ammonia production,

the application of metabolites of U21, Pseudomonas aeruginosa S58 or/and Pseudomonas aeruginosa S58 in producing siderophins.

In order to solve the technical problems, the invention also provides a microbial inoculum.

The microbial inoculum provided by the invention contains metabolites of pseudomonas aeruginosa S58 or/and pseudomonas aeruginosa S58.

The microbial inoculum can be any one of the following microbial inoculants:

a1, a microbial inoculum for preventing and/or treating tobacco black shank,

a2, bacterial agent for preventing and/or treating bacterial wilt of tobacco,

a3, a microbial inoculum for preventing and/or treating tobacco wildfire,

a4, a microbial inoculum for improving soil,

a5, a microbial inoculum for hydrolyzing organic phosphorus,

a6, a microbial inoculum for inhibiting tobacco wildfire germs,

a7, a bacterial agent for inhibiting phytophthora nicotianae,

a8, a bacterial agent for inhibiting soybean phytophthora,

a9, a bacterial agent for inhibiting rhizoctonia solani,

a10, a bacterial agent for inhibiting Pyricularia oryzae,

a11, a microbial inoculum for inhibiting acidovorax citrulli,

a12, a microbial agent for inhibiting clavibacterium michiganensis,

a13, a bacterial agent for inhibiting xanthomonas oryzae,

a14, bacterial agent for inhibiting Lawsonia solanacearum,

a15, an auxin (IAA) -producing microbial inoculum,

a16, a bacterial agent for inhibiting the elongation of the main root of the plant,

a17, bacterial agent for promoting the growth of lateral roots of plant,

a18, a microbial inoculum for increasing plant biomass,

a19, an amylase-producing microbial inoculum,

a20, an ammonia-producing microbial inoculum,

a21, a siderophore microbial inoculum.

The active ingredients of the microbial inoculum can be metabolites of pseudomonas aeruginosa S58 or/and pseudomonas aeruginosa S58, the active ingredients of the microbial inoculum can also contain other biological ingredients or non-biological ingredients, and the other active ingredients of the microbial inoculum can be determined by the technicians in the field according to the effects of the microbial inoculum.

The microbial inoculum may also include a carrier. The carrier may be a solid carrier or a liquid carrier. The solid carrier is a mineral material or a biological material; the mineral material may be at least one of grass peat, clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the biological material is at least one of straws, pine shells, rice straws, peanut shells, corn flour, bean flour, starch, grass peat and animal manure of various crops; the liquid carrier can be water; in the microbial inoculum, metabolites of pseudomonas aeruginosa S58 or/and pseudomonas aeruginosa S58 can exist in the form of cultured living cells, fermentation broth of living cells, filtrate of cell culture or mixture of cells and filtrate. The preparation formulation of the microbial inoculum can be various preparation formulations, such as liquid, emulsion, suspending agent, powder, granules, wettable powder or water dispersible granules.

According to the requirement, the microbial inoculum can also be added with a surfactant (such as Tween 20, Tween 80 and the like), a binder, a stabilizer (such as an antioxidant), a pH regulator and the like.

Hereinbefore, the metabolite of pseudomonas aeruginosa S58 may be a fermentation broth of pseudomonas aeruginosa S58. The fermentation broth of Pseudomonas winkle S58 can be prepared as follows: culturing the pseudomonas winkle S58 in a liquid fermentation culture medium, and collecting a fermentation liquid (containing the pseudomonas winkle S58 and substances secreted into the liquid culture medium), wherein the fermentation liquid is a metabolite of the pseudomonas winkle S58.

In the above application, the product may be a microbial inoculum, a microbial ecological agent or a biological fertilizer.

Cultures of Pseudomonas winogradskyi S58 are also within the scope of the invention. The culture of Pseudomonas winkle S58 is obtained by culturing Pseudomonas winkle S58 in a microorganism culture medium (such as a fermentation broth containing Pseudomonas winkle S58 and secreted into a liquid culture medium, or such as a solid culture medium containing Pseudomonas winkle S58).

The culture of Pseudomonas aeruginosa S58 has at least one of the following functions B1-B21:

b1, preventing and/or treating tobacco black shank,

b2, preventing and/or treating tobacco bacterial wilt,

b3, preventing and/or treating the tobacco wildfire,

b4, improving the soil,

b5, hydrolyzing organic phosphorus,

b6, inhibiting the pathogenic bacteria of tobacco wildfire,

b7, inhibiting phytophthora nicotianae,

b8, inhibiting the soybean phytophthora,

b9, inhibiting rhizoctonia solani,

b10, inhibiting the rice blast germs,

b11, inhibiting acidovorax citrulli,

b12, inhibiting clavibacter michiganensis,

b13, inhibiting Xanthomonas oryzae,

b14, inhibiting Laurella solanacearum,

b15, producing auxin (IAA),

b16, inhibiting the elongation of the main root of the plant,

b17, promoting the growth of lateral roots of plants,

b18, increasing the biomass of the plant,

b19, producing amylase,

b20, producing ammonia,

b21, producing siderophore.

Any one of the following applications of the microbial inoculum also belongs to the protection scope of the invention:

c1, the application of the microbial inoculum in preventing and/or treating tobacco black shank,

c2, the application of the microbial inoculum in preventing and/or treating tobacco bacterial wilt,

c3, the application of the microbial inoculum in preventing and/or treating tobacco wildfire,

c4, the application of the microbial inoculum in soil improvement,

c5, the application of the microbial inoculum in hydrolyzing organophosphorus,

c6, the application of the microbial inoculum in inhibiting the tobacco wildfire pathogen,

c7, the application of the microbial inoculum in inhibiting phytophthora nicotianae,

c8, the application of the microbial inoculum in inhibiting phytophthora sojae,

c9, the application of the microbial inoculum in inhibiting rhizoctonia solani,

c10, the application of the microbial inoculum in inhibiting rice blast bacteria,

c11, the application of the microbial inoculum in inhibiting acidovorax citrulli,

c12, the application of the microbial inoculum in inhibiting the clavibacterium michiganense,

c13, the application of the microbial inoculum in inhibiting Xanthomonas oryzae,

c14, the application of the bacterial agent in inhibiting the Laurella solanacearum,

c15, the application of the microbial inoculum in the production of auxin,

c16, the application of the microbial inoculum in inhibiting the elongation of the main roots of plants,

c17, the application of the microbial inoculum in promoting the growth of lateral roots of plants,

c18, the application of the microbial inoculum in improving plant biomass,

c19, the application of the microbial inoculum in producing amylase,

c20, the application of the microbial inoculum in ammonia production,

c21, and the application of the microbial inoculum in producing siderophins.

In the present application, the plant may be a monocotyledon or a dicotyledon. The dicotyledonous plant may be a tubular plant of the order florida. The tubular florales plant may be a solanaceae plant. The Solanaceae plant can be plant of Nicotiana. The nicotiana species plant can be tobacco.

In the present application, the dicotyledonous plant may be a plant of the order Capricorales. The plant of order Capparis can be a plant of the family Brassicaceae.

Culturing said Pseudomonas rugosa (M.), (Pseudomonas corrugata) Also belongs to the protection scope of the invention.

The invention provides a method for culturing the pseudomonas winkle (Pseudomonas aeruginosa)Pseudomonas corrugata) The method comprises subjecting said Pseudomonas aeruginosa to a treatment of (A) and (B)Pseudomonas corrugata) A step of culturing in a medium for culturing Pseudomonas bacteria.

The method for preparing the microbial inoculum also belongs to the protection scope of the invention.

The method for preparing the microbial inoculum provided by the invention comprises the step of mixing the pseudomonas winkle (pseudomonas aeruginosa), (b) and (c)Pseudomonas corrugata) And/or said Pseudomonas aeruginosa (Pseudomonas corrugata) The metabolite of (b) is used as a component of the microbial inoculum to obtain the microbial inoculum.

In the above method, the microbial inoculum may be a liquid microbial inoculum. In the above method, the Pseudomonas aeruginosa (A)Pseudomonas corrugata) Can be cultured in a fermentation medium to obtain fermentation liquor, and the fermentation liquor is mixed with a carrier to obtain the liquid microbial inoculum. The fermentation medium may consist of: 1-2% of bean cake powder, 1-2% of soluble starch, 0.5-0.8% of beef extract and NaCNO₃0.02%-0.04%、CaCO₃0.1 to 0.3 percent of the total weight of the composition, and the balance of water, wherein the weight percentages are all the weight percentages. The carrier may be at least one selected from sodium humate, chitosan and chitin. In the microbial inoculum, the volume content of the carrier can be 0.16-0.6%. When the carrier consists of humic acidWhen the microbial inoculum consists of sodium, chitosan and chitin, the volume content of sodium humate in the microbial inoculum can be 0.05-0.2%, the volume content of chitosan can be 0.01-0.1% and the volume content of chitin can be 0.1-0.3%.

Experiments prove that the pseudomonas winkle S58 has stable, efficient and broad-spectrum antibacterial performance and has an inhibiting effect on plant pathogenic fungi such as phytophthora nicotianae, phytophthora sojae, rhizoctonia solani and pyricularia oryzae and plant pathogenic bacteria such as acidovorax citrulli, clavibacterium michiganense, xanthomonas oryzae paddy rice pathogenic varieties and ralstonia solanacearum. The control effect of the pseudomonas aeruginosa S58 on tobacco bacterial wilt reaches 76.2%, and the control effect on tobacco black shank reaches 60.5%. The pseudomonas winkle S58 can produce siderophin and amylase, can cause programmed death of immunoreactive cells on the surface of plants, and can control the occurrence of tobacco wildfire by stimulating immune resistance. The pseudomonas winkle S58 can hydrolyze organophosphorus and can be used for soil improvement. The pseudomonas winkle S58 can produce auxin (IAA), can obviously inhibit the elongation of main roots of plants, promote the growth of lateral roots and improve the biomass of the plants. The pseudomonas winkle S58 is safe to human and livestock, has no problem of environmental pollution, has simple culture condition and easy storage, and is suitable for industrial production.

Biological material preservation instructions.

Classification nomenclature of biological materials: pseudomonas aeruginosa.

Latin literature name of biomaterial:Pseudomonas corrugata。

strain number of biological material: and S58.

The preservation unit is called as follows: china general microbiological culture Collection center.

The preservation unit is abbreviated as: CGMCC.

Address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.

The preservation date is as follows: 12 and 27 days 2018.

The preservation number is: CGMCC No. 17043.

Drawings

FIG. 1 shows the bacterial inhibition spectrum of Pseudomonas aeruginosa S58. Wherein, A is phytophthora nicotianae, B is rhizoctonia solani, C is rice blast, D is phytophthora sojae, E is acidovorax citrulli, F is clavibacterium michiganense subspecies, G is xanthomonas oryzae paddy pathogenic variant, and H is ralstonia solanacearum.

FIG. 2 is a photograph of each treated tobacco leaf 7 days after inoculation with tobacco wildfire pathogen.

FIG. 3 shows that Pseudomonas aeruginosa S58 can stimulate strong expression of immune-related genes.

FIG. 4 is a photograph of Arabidopsis thaliana after inoculation and culture for 10 days. A is inoculated LB culture medium, B is inoculated with Pseudomonas wrinkle S58 fermentation liquid.

FIG. 5 is a photograph showing the results of Salkowski colorimetry. The upper left row is pure auxin, the upper left row is negative control of culture medium, the upper left row is negative control without IAA, and the lower three rows are three replicates of Pseudomonas aeruginosa S58 treatment.

FIG. 6 is a photograph of Pseudomonas aeruginosa S58 cultured in Monkina organophosphorus medium.

FIG. 7 is a photograph of Pseudomonas aeruginosa S58 showing significant ammonia-producing ability. The top left panel is CK, the top left panel and the left panel are controls without any added material, and the bottom three panels are three replicates of Pseudomonas aeruginosa S58 treatment.

FIG. 8 is a photograph of the production of siderophore by Pseudomonas aeruginosa S58.

FIG. 9 shows that the Pseudomonas winkle S58 strain has a remarkable amylase-producing ability.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The pathogenic bacteria public used in the following examples were collected from the field or obtained from the institute of agricultural resources and agricultural divisions of the chinese academy of agricultural sciences to repeat the experiments of the present application:

tobacco black shank pathogen-tobacco phytophthora (A)Phytophthora parasiticavar.nicotianae) (Wang Universal, general school, Xiao Chong Steel. tobacco Phytophthora spore production and inoculation method research. plant protection bulletin 20005, 32(1): 18-22), hereinafter referred to as Phytophthora nicotianae.

Bacterial wilt of tobacco-Laurella solanacearum: (Rastonia solanacearum) (Weihai Reye, Wang Ye, tension group, Tangwenhua. biocontrol strain 2P24 and CPF-10 identification and its biocontrol related characters preliminary analysis. plant pathology report, 2004, 34: 80-85), hereinafter referred to as "Ralstonia solanacearum".

Pseudomonas syringae (a) of tobacco wildfire pathogenPseudomonas syringaepv.tabaci) Leather (leather, Yongguang, Hakka, Liu Lianzi. tobacco wildfire pathogen (Pseudomonas syringae pv. tabaci) has an effect on 5 enzyme defense systems in tobacco cells, proceedings of Shandong university of agriculture (Nature science edition), 2002, 33(1): 28-31), hereinafter referred to as Pseudomonas syringae.

Phytophthora sojae (A.sojae) ((B.))Phytophtora sojae) (overwintering survival rate of phytophthora sojae oospores in soil of black dragon Jiang province. plant protection academy 2015, 42(1): 72-78), which is called phytophthora sojae for short.

Rhizoctonia solani (A), (B), (CRhizoctonia solani) (Weihai Rele, Wang Ye, tension group, Tangwenhua. biocontrol strain 2P24 and CPF-10 identification and its biocontrol related trait preliminary analysis. plant pathology report, 2004, 34: 80-85), hereinafter referred to as Rhizoctonia solani.

Pyricularia oryzae (A)Magnaporthe oryzae) Apoptosis induction and detection of Magnaporthe oryzae (Magnaporthe oryzae) in the east of the morning, Lujianping, Liu Xiao hong, Linfu Zhang, and Phytopathology Proc 2011, 41(4): 361-370), which is hereinafter referred to as Magnaporthe oryzae.

Acidovorax citrulli (a)Acidovorax citrulli) (leather jade allergy, cloud Xiaomin, Liyuwen, Lijiaoqiang, Luo Xin, Bio-PCR method for screening specific primers for detecting citrullus lanatus carrying in cucurbitaceae crop seeds, report of plant pathology, 2018,48(2), 263-270), hereinafter referred to as "acid-producing bacterium of watermelon".

Clavibacter michiganensis subspecies michiganensis (Clavibacter michiganensissubsp.michiganensis) (Xiamengxing, Zhao Wen Jun, Ma Qing, Zhu Shuangfang, tomato bacterial canker germ real-time fluorescence PCR detection, plant pathology reports, 2006, 6(2): 152-.

Xanthomonas oryzae pathopoiesia variant of rice (Xanthomonas oryzaepv.oryzae，Xoo) (Yangyun, Zhang Tongyu, Chengling, Chenyue, Yikui, Jianchun, Xiaosu Qin, Kouchun, Yuteng, Wang, Pajiao, Shiqiao fragrant, Chengongyou, Chenguan-Quan. resistance identification of four main diseases of rice by Yunnan medicinal wild rice. report of plant pathology. 2019, 1:101 wall 112), which is hereinafter referred to as xanthomonas oryzae paddy pathogenic variety.

The configuration of the relevant media in the following examples:

PDA culture medium: 200g of potato, 20g of glucose and 20g of agar, and the volume is set to 1000mL by using distilled water.

LB liquid medium: 5g of yeast extract, 10g of tryptone and 10g of NaCl, and the volume is adjusted to 1000mL by using distilled water, and the pH value is 7.0-7.2.

LB solid medium: 5g of yeast extract, 10g of tryptone, 10g of NaCl and 15g of agar, and the volume is set to 1000mL by using distilled water, and the pH value is 7.0-7.2.

Nutrient agar medium (NA medium): 3g of beef extract, 5g of peptone, 2.5g of glucose, 15g of agar, pH7.0, and distilled water for fixing the volume to 1000 mL.

Nutrient broth medium (NB medium): 3g of beef extract, 5g of peptone, 2.5g of glucose, pH7.0, and distilled water to reach the volume of 1000 mL.

CA culture medium: 23g of peptone, 1g of corn flour, 5g of sodium chloride, 15g of agar, pH 7.3, and distilled water to reach the volume of 1000 mL.

Oat culture solution: 60g of oat flour, and adding distilled water to 1000 mL.

Oat culture medium: 60g of oat flour and 15g of agar, and the volume is set to 1000mL by using distilled water.

Example 1 Pseudomonas aeruginosa (Pseudomonas corrugata) Separation and identification of S58 CGMCC No.17043

First, Pseudomonas pinicola (B)Pseudomonas corrugata) Separation of S58 CGMCC No.17043

Pseudomonas pindovurean (A)Pseudomonas corrugata) S58 CGMCC No.17043 is isolated from the rhizosphere of tobacco. Tobacco rhizosphere samples were collected from the research and town of Yuxi city, Yunnan province, rhizosphere soil samples were taken back to the laboratory for low-temperature storage, 1g of the soil samples were suspended in 9ml of sterile water, after sufficient shaking, serial dilution was applied to NA medium plates, and culture was carried out at 28 ℃ for 48 h. Picking single colony, streaking, purifying and storing on a plate, wherein the flourishing one is named as S58 strain, namely the pseudomonas winklensis of the applicationPseudomonas corrugata）S58 CGMCC No.17043。

II, Pseudomonas pinkeya (II)Pseudomonas corrugata) Identification of S58 CGMCC No.17043

The species of the S58 strain separated above was identified by observing biological characteristics and morphology, and the method was as follows:

the specific method for observing the morphology, size, roughness and edges of bacterial colonies, gram staining and testing physiological and biochemical characteristics refers to the handbook for identifying common bacterial systems. The whole genome sequencing of the strain was conducted by Jinzhi Biotechnology Ltd. The different carbon sources of FIG. 1 were analyzed using a Biolog automated microbiological analysis System in the United states, with water as the negative control.

As a result, the S58 strain had a rod-like shape, an average cell size of 0.5 to 1.5. mu. m.times.1.6 to 2.5. mu.m, a gram-negative, unipolar single-or multiple-flagella, and no fluorescence. The colony of the S58 strain on the NA culture medium is white, and has raised folds and irregular edges. The S58 strain has growth temperature of 8-45 deg.C, optimum growth temperature of 28-32 deg.C, growth pH of 6.5-8, optimum pH of 7, and can grow in 1% NaCl solution and 1% sodium lactate solution. The physiological and biochemical characteristics of the S58 strain are shown in table 1. Sequencing the whole genome of the S58 strain, wherein the genome has the total length of 6481050 bp, the G + C content of 61.06 percent, and no plasmid exists. The S58 strain has a sequence tableThe 16S rDNA shown in the middle sequence 1, the S58 strain, the 16S rDNA sequence, and the strain of Pseudomonas wrinkle have a similarity of 99% to each other, the S58 strain can utilize a carbon source selected from the group consisting of D-trehalose, sucrose, α -D-glucose, D-mannose, D-fructose, D-galactose, 1% sodium lactate, D-mannitol, acelomycin, rifamycin SV, L-alanine, L-glutamic acid, L-pyroglutamic acid, lincomycin, guanidine hydrochloride, tetrasecylte sulfate, pectin, D-gluconic acid, mucic acid, quinic acid, D-saccharic acid, vancomycin, tetrazole violet, tetrazole blue, p-hydroxyphenylacetic acid, L-lactic acid, citric acid, α -ketoglutaric acid, L-malic acid, nalidixic acid, potassium tellurite, gamma-aminobutyric acid, acetic acid, aztreonamide, S58 strain can not utilize a carbon source selected from the group consisting of galactosan, D-maltose, D-cellobiose, D-pinosyl, D-stachyose, D-glucosamine, D-D₄Minocycline, gelatin, glycyl-L-proline, L-arginine, L-histidine, L-serine, L-galactonolactone, glucuronamide, D-methyl lactate, D-malic acid, bromosuccinic acid, lithium chloride, Tween-40, α -hydroxy-butyric acid, β -hydroxy-D, L-butyric acid, α -keto-butyric acid, acetoacetate, formic acid, sodium butyrate, sodium bromate S58 Strain may utilize as a carbon source D-fucose, inositol, D-fructose-6-PO-6₄D-aspartic acid, L-aspartic acid, galacturonic acid, glucuronic acid, methyl pyruvate, propionic acid.

The S58 strain is firmed as pseudomonas wrinkled through the analysis of the strain morphological characteristics, culture characteristics, physiological and biochemical characteristics and Biolog microorganism automatic analysis systemPseudomonas corrugata). Pseudomonas pindovurean (A)Pseudomonas corrugata) S58 has been deposited in China general microbiological culture Collection center (CGMCC) in 2018, 12 months and 27 months, and it is deposited in China general microbiological culture Collection center (CGMCC)The registration number of the general microorganism center of the preservation management committee is CGMCC No. 17043. Hereinafter referred to as Pseudomonas wrinkle S58.

Example 2 Pseudomonas winkles (Pseudomonas corrugata) Determination of bacteriostatic spectrum of S58 CGMCC No.17043

Test for phytopathogenic fungi: phytophthora nicotianae, phytophthora sojae, rhizoctonia solani, and magnaporthe oryzae.

Test for plant pathogenic bacteria: acidovorax citrulli, clavibacterium michiganensis subspecies, xanthomonas oryzae paddy rice pathogenic variant, and ralstonia solanacearum.

And respectively carrying out bacteriostasis experiments on the pathogenic fungi of the plants to be tested by adopting a plate confronting culture method. The specific operation is as follows: pseudomonas rugosa S58 was incubated on LB solid medium at 28 ℃ for 48 h. Respectively culturing the pathogenic fungi of the test plants on a PDA (personal digital assistant) flat plate at a constant temperature of 25-28 ℃ for 3-4 days, and beating agar sheets with pathogenic fungi from the edges of colonies by using a sterile stainless steel puncher with the diameter of 0.7 cm; and (3) picking the pathogenic fungus slice by using an aseptic inoculating needle, inoculating the pathogenic fungus slice at the center of the PDA flat plate with the hypha facing downwards, inoculating sterile inoculating ring lined pseudomonas winkle S58 at a position which is about 3.00cm away from both sides of the pathogenic fungus slice, taking the flat plate only inoculated with the pathogenic fungus as a control, repeating the treatment for three times, culturing at the constant temperature of 25-28 ℃ for 5-7 days, and measuring the width of the antibacterial zone between the edge of the pathogenic fungus and the colony bandwidth center of the pseudomonas winkle S58.

And respectively carrying out bacteriostasis experiments on the plant pathogenic bacteria to be tested by adopting a double-layer culture method. The specific operation is as follows: an LB solid medium (5 g of yeast extract, 10g of tryptone, 10g of NaCl, 10g of agar, volume 1000mL with distilled water, pH 7.0-7.2) containing 1% agar was thawed and incubated at 50 ℃. Preparing activated target pathogenic bacteria into 10⁸cfu/mL of the bacterial suspension, 1mL of the suspension was added to the LB solid medium containing 1% agar, which was incubated, and the plate was inverted. After air drying, 10 mu lLB liquid culture medium activated pseudomonas winkle S58 is spotted on the center of an LB culture medium plate, the culture is carried out for 48h at the temperature of 28 ℃, the bacteriostasis effect is observed, and the diameter of the bacteriostasis zone is measured by a cross method.

The result shows that when the culture is carried out for 4 to 5 days, when hypha on a flat plate only connected with various pathogenic fungi nearly grows over the whole flat plate, the bacteriostatic bandwidths of the pseudomonas winkle S58 on phytophthora nicotianae, phytophthora sojae, rhizoctonia solani and pyricularia oryzae are respectively 7.1mm, 3.2mm, 10.9mm and 4.8mm, and the bacteriostatic bandwidths of the selected 4 plant pathogenic fungi are all between 3 and 11mm, so that the effect of inhibiting fungi is stronger. The diameters of inhibition zones of Pseudomonas winkle S58 against acidovorax citrulli, Clavibacter michiganensis subspecies, Xanthomonas oryzae paddy rice pathovar and Laurella solani are 35.5mm, 56.6 mm, 63.7 mm and 24.7mm, respectively (FIG. 1). The experiment is repeated for many times to obtain the same stable bacteriostasis effect, so that the pseudomonas winkle S58 has stable, efficient and broad-spectrum antibacterial performance.

Example 3 prevention and treatment experiment of bacterial wilt of tobacco by using bacterial agent containing Pseudomonas aeruginosa S58

1. Preparation of pathogen inoculum: and (3) carrying out streak inoculation on the ralstonia solanacearum-ralstonia solanacearum on an NA culture medium, and carrying out inverted culture at 28 ℃ for 3d for later use.

2. Preparation of liquid microbial inoculum containing pseudomonas aeruginosa S58:

the preparation of the liquid microbial inoculum containing the pseudomonas winkle S58 comprises the steps of slant culture, seed culture, fermentation and microbial inoculum preparation, and specifically comprises the following steps:

A. slant culture: inoculating Pseudomonas wrinkle S58 to slant culture medium-nutrient agar culture medium, and culturing at 28-30 deg.C for 48 hr to obtain slant strain;

B. seed culture: inoculating slant strains into liquid culture medium, and culturing at 29-33 deg.C for 18-24 hr to obtain liquid fermented seeds; the liquid culture medium comprises 2-4% of beef extract, 0.4-0.6% of peptone, 0.4-0.6% of sodium chloride and the balance of water by mass percent, and the pH value is 6.5-8.0.

C. Fermentation: inoculating the liquid fermentation seeds into a fermentation tank filled with a fermentation medium according to the volume of 4% of the fermentation medium, culturing at 29-33 ℃ for 42h with the stirring speed of 120-; the fermentation medium comprises the following components: 1% of bean cake powder, 1% of soluble starch, 0.5% of beef extract and NaCNO₃0.02%、CaCO₃0.1 percent of water and the balance of water, wherein the percentages are mass percentages.

D. Preparation of a microbial inoculum: adding 0.2% sodium humate (carrier) into the fermentation liquor, and fully mixing to obtain the liquid microbial inoculum containing the pseudomonas winkle S58. The content of pseudomonas winkle S58 in the liquid microbial inoculum containing pseudomonas winkle S58 is 10⁹cfu/ml。

3. Prevention and control experiment: the flue-cured tobacco variety is Honghuadajinyuan, and after the tobacco seedlings are transplanted and returned for 5 days, a test field is selected. The test adopts a random block design, 2 treatment areas are randomly arranged, and each treatment area is provided with three repetitions (three cells). The 2 treatment zones were blank control treatment zone (CK treatment zone) and S58 treatment zone, respectively. The test field is divided into 6 cells, and 15 flue-cured tobaccos are planted in each cell. And the processing is distributed in a random drawing mode.

S58 processing area: and (5) after the tobacco seedlings are transplanted and returned, inoculating biocontrol bacteria by adopting a root irrigation method. Wetting soil with small amount of clear water before root irrigation, and using the liquid microbial inoculum (containing Pseudomonas aeruginosa S58 content of 10) containing Pseudomonas aeruginosa S58 obtained in step 2⁹cfu/ml) and 10ml of root is irrigated to each plant, and biocontrol bacteria are inoculated. Irrigating the root 1 time every three days, and irrigating the root 3 times in total. Inoculating tobacco ralstonia solanacearum 7 days after 3 rd root irrigation, scratching stem base of tobacco plant with scalpel, and inoculating 10ml of each plant⁷cfu/ml tobacco ralstonia solanacearum suspension, immediately covering soil and moisturizing after inoculation, and watering in the morning and at night each day. The disease condition was investigated after inoculating tobacco ralstonia solanacearum for 10 days. The tobacco bacterial wilt classification standard is implemented according to the tobacco industry standard (GB/T23222-2008) of the people's republic of China.

Blank control treatment zone (CK treatment zone): and (5) after the tobacco seedlings are transplanted and returned, irrigating roots with NB culture medium. Before root irrigation, the soil is moistened by a small amount of clear water, and each plant is irrigated with 10ml of NB medium. Irrigating the root 1 time every three days, and irrigating the root 3 times in total. Irrigating the root 1 time every three days, and irrigating the root 3 times in total. Inoculating tobacco ralstonia solanacearum 7 days after 3 rd root irrigation, scratching stem base of tobacco plant with scalpel, and inoculating 10ml of each plant⁷cfu/ml tobacco ralstonia solanacearum suspension, immediately covering soil and moisturizing after inoculation, and watering in the morning and at night each day. Inoculating tobacco greenThe disease onset was investigated after 10 days of the onset of Bacillus subtilis. The tobacco bacterial wilt classification standard is implemented according to the tobacco industry standard (GB/T23222-2008) of the people's republic of China.

Inoculating ralstonia solanacearum 7 days after inoculating biocontrol bacteria, scratching the stem base of tobacco plant with a scalpel, and then inoculating 10ml of each plant⁷cfu/ml ralstonia solanacearum suspension, immediately covering soil and moisturizing after inoculation, and watering in the morning and at night each day. The disease was investigated 10 days after inoculation for each 15 strains treated. The tobacco bacterial wilt classification standard is implemented according to the tobacco industry standard (GB/T23222-2008) of the people's republic of China.

Disease index = Σ (number of diseased plants at each stage × corresponding stage)/(total number of investigated plants × maximum number of stages) × 100.

The prevention and treatment effect (%) = (disease index of CK treatment area-disease index of S58 treatment area)/disease index of CK treatment area x 100%.

The disease investigation result shows that the treatment is different in disease incidence in 10 days after the inoculation of the pseudomonas solanacearum, the S58 treatment area treated by the pseudomonas aeruginosa S58 microbial inoculum root irrigation shows a strong disease prevention effect, the disease index is 20.3, the disease prevention effect is 76.2 percent (shown in table 1) which is lower than that of the CK treatment area treated by the contrast NB culture solution root irrigation, and the application value is good.

TABLE 1 control effect of Pseudomonas ornithii S58 on tobacco bacterial wilt

Treatment zone	Index of disease condition	Control effect (%)
			CK	85.2±5.9	－
S58	20.3±3.5	76.2

Example 4 prevention and treatment experiment of tobacco black shank by using microbial inoculum containing Pseudomonas aeruginosa S58

1. Preparation of pathogen inoculum: phytophthora nicotianae was transplanted on a CA plate (CA medium), and cultured in an inverted state at 28 ℃ for 7 days. Inoculating a hypha block into an oat culture solution, culturing at 28 ℃ and 180r/min for 7d, and transferring to an oat culture medium for culturing for 21d to obtain the phytophthora nicotianae cereals for later use.

2. Prevention and control experiment: the flue-cured tobacco variety is Honghuadajinyuan, and after the tobacco seedlings are transplanted and returned for 5 days, a test field is selected. The test adopts a random block design, 2 treatment areas are randomly arranged, and each treatment area is provided with three repetitions (three cells). The 2 treatment zones were blank control treatment zone (CK treatment zone) and S58 treatment zone, respectively. The test field is divided into 6 cells, and 15 flue-cured tobaccos are planted in each cell. And the processing is distributed in a random drawing mode.

S58 processing area: and (5) after the tobacco seedlings are transplanted and returned, inoculating biocontrol bacteria by adopting a root irrigation method. Wetting soil with small amount of clear water before root irrigation, and using the liquid microbial inoculum (containing Pseudomonas aeruginosa S58 content of 10) containing Pseudomonas aeruginosa S58 obtained in step 2⁹cfu/ml) and 10ml of root is irrigated to each plant, and biocontrol bacteria are inoculated. Irrigating the root 1 time every three days, and irrigating the root 3 times in total. Inoculating tobacco black shank bacteria 7 days after 3 rd root irrigation, scratching the stem base of a tobacco plant by using a scalpel, then inoculating phytophthora nicotianae strains with the inoculation amount of 2 g/strain, immediately covering soil and moisturizing after inoculation, and watering once a day in the morning and at the evening. The disease condition was investigated after inoculation of ralstonia solanacearum for 14 d. The tobacco black shank grading standard is implemented according to the tobacco industry standard (GB/T23222-2008) of the people's republic of China.

Blank control treatment zone (CK treatment zone): and (5) after the tobacco seedlings are transplanted and returned, irrigating roots with NB culture medium. Before root irrigation, the soil is moistened by a small amount of clear water, and each plant is irrigated with 10ml of NB medium. Irrigating the root 1 time every three days, and irrigating the root 3 times in total. Irrigating the root 1 time every three days, and irrigating the root 3 times in total. Inoculating tobacco black shank bacteria 7 days after 3 rd root irrigation, scratching the stem base of a tobacco plant by using a scalpel, then inoculating phytophthora nicotianae strains with the inoculation amount of 2 g/strain, immediately covering soil and moisturizing after inoculation, and watering once a day in the morning and at the evening. The disease condition was investigated after inoculation of ralstonia solanacearum for 14 d. The tobacco black shank grading standard is implemented according to the tobacco industry standard (GB/T23222-2008) of the people's republic of China.

The experimental results are as follows: the disease investigation result shows that on 14 days after phytophthora nicotianae inoculation, each treatment is attacked to different degrees, the S58 treatment area irrigated by the pseudomonas aeruginosa S58 bacterial liquid shows a strong disease prevention effect, the disease index is 31.7, the disease prevention effect is 60.5 percent (shown in table 2) lower than that of the CK treatment area irrigated by the control NB culture solution, and the application value is good.

TABLE 2 Pseudomonas ornithica S58 control Effect on tobacco Black shank

Treatment zone	Index of disease condition	Control effect (%)
			CK	80.2±6.4	－
S58	31.7±5.8	60.5

Example 5 Pseudomonas wrinkle S58 ability to control the development of tobacco wildfire by eliciting immune resistance

The Pseudomonas aeruginosa S58 was streaked on LB plates for 48h, surface colonies were scraped off, and resuspended in 10mM MgCl₂The obtained Pseudomonas aeruginosa S58 content in the solution is 5 × 10⁸cfu/ml bacterial suspension. The mixture was centrifuged at 13000 rpm at 4 ℃ for 10 min, and the supernatant and the pellet were separated. The bacteria were suspended in 10mM MgCl₂In the solution, the concentration was adjusted to 5X 10⁸cfu/ml, Silwet L-77 (surfactant) was added to a level of 0.05% to give 5X 10⁸cfu/ml of cell suspension. The centrifuged supernatant was subjected to deep sterilization by filtration through a 0.45 μm filter membrane, and Silwet L-77 (surfactant) was added thereto to a content of 0.05% to obtain a metabolic supernatant. This experiment was repeated 3 times, and 4 weeks of Bo-Shi tobacco were taken for each repetition (Nicotiana benthamiana) Set 4 treatments: 10mM MgCl₂+ Pta treatment, S58 super + Pta treatment, S58pellet + Pta treatment, and S58pellet treatment, 5 tobacco plants were treated for each treatment.

10 mM MgCl₂+ Pta treatment: spraying 10ml MgCl on each tobacco plant₂The solution is cultured in an incubator at 25 ℃ and moisturized for 24 hours; then spraying 10ml of each tobacco plant⁷cfu/ml suspension of Pseudomonas syringae, a strain of the tobacco wildfire pathogen, was cultured in an incubator at 25 ℃ for 7 days and recorded by photographing.

S58 super + Pta treatment: spraying 10ml of the metabolism supernatant liquid on each tobacco plant, and keeping moisture for 24 hours in an incubator at 25 ℃; then spraying 10ml of each tobacco plant⁷cfu/ml suspension of Pseudomonas syringae, a strain of the tobacco wildfire pathogen, was cultured in an incubator at 25 ℃ for 7 days and recorded by photographing.

S58pellet + Pta treatment: spraying 10ml of the above 5 × 10 solution to each tobacco plant⁸cfu/ml thallus suspension, an incubator at 25 ℃ and moisture preservation for 24 hours; then spraying 10ml of each tobacco plant⁷cfu/ml suspension of Pseudomonas syringae, a strain of the tobacco wildfire pathogen, was cultured in an incubator at 25 ℃ for 7 days and recorded by photographing.

S58pellet treatment (unvaccinated pathogen control): spraying 10ml of the above 5 × 10 solution to each tobacco plant⁸cfu/ml thallus suspension, an incubator at 25 ℃ and moisture preservation for 24 hours; then 10ml MgCl is sprayed on each tobacco plant₂The solution was incubated at 25 ℃ for 7 days and recorded by photography.

The results show 10mM MgCl₂After 7 days of Pta treatment and inoculation of tobacco wildfire germs, wildfire germs are serious, disease spots are connected into pieces, the edges of the disease spots are burned and spread towards the middle, the area of the disease spots accounts for more than 21% of the area of the leaves, and the severity of the disease reaches 9 grades of GB/T23222-2008; the wild fire disease symptoms are lighter after the S58 supermatant + Pta is used for treating and inoculating the tobacco wildfire pathogen for 7 days, burn symptoms do not exist, infected spots are occult, the area of the infected spots accounts for less than 1% of the area of the leaves, the disease severity reaches 1 level of GB/T23222-2008, the edges of the leaves are slightly burnt after the S58 pellett + Pta is used for treating and inoculating the tobacco wildfire pathogen for 7 days, the area of the infected spots accounts for 2-5% of the area of the leaves, and the disease severity reaches 3 levels of GB/T23222-2008; no disease was observed with the S58pellet treatment (FIG. 2). The pseudomonas aeruginosa S58 can remarkably control the occurrence of the tobacco wildfire by stimulating immune resistance.

Example 6 Pseudomonas winkle S58 is able to stimulate strong expression of immune-related genes

The Pseudomonas aeruginosa S58 was streaked on LB plates for 48h, surface colonies were scraped off, and resuspended in 10mM MgCl₂The solution is prepared by adding 5 × 10 of Pseudomonas wrinkle S58⁸cfu/ml to obtain pseudomonas aeruginosa wrinkle S58 suspension; 10mM MgCl₂Solutions were control, inoculated with 3 replicates of each treatment. After 6h, 3 leaf tissues were taken with 0.5cm diameter punches each and quickly frozen with liquid nitrogen. RNA was extracted using a Plant RNA extraction Kit (Plant RNA Kit (R6827-01), OMEGA), digested with DNase (DNase I RNase-Free (M0303S), NEB) and subjected to reverse transcription of cDNA (Protoscript II first strand cDNA Synthesis Kit (E6560S), NEB). Quantitative PCR assay was performed using SYBR Green (Universal qPCR Master Mix (M3003L), NEB) with Actin gene as reference and 10mM MgCl₂Comparison of the plants inoculated with the solution, resultsIt was shown that Pseudomonas aeruginosa S58 can stimulate the up-regulated expression of the immune-related genes PR1 gene, WRKY7 gene, Acre31 gene, NPR1 gene, ICS1 gene and WRKY40 gene (FIG. 3). The instrument uses the ABI QuantStaudio 6 Flex real-time fluorescent quantitative PCR system. The primers were designed as follows: the primers of PR1 gene are NbPR1-F and NbPR1-R, the primers of WRKY7 gene are NBWRKY7-F and NBWRKY7-R, the primers of Acre31 gene are NbAcre31-F and NbAcre31-R, the primers of NPR1 gene are NbNPR1-F and NbNPR1-R, the primers of ICS1 gene are NbICS1-F and NbICS1-R, the primers of WRKY40 gene are NbWRKY40-93F and NbKY 40-93R, and the primers of Actin gene are NbActin-80F and NbActin-80R.

NbPR1-F：5′-GTAATATCCCACTCTTGCCG-3′，

NbPR1-R：5′-ATGAAATCGCCACTTCCCTC-3′，

NBWRKY7-F：5′-CACAAGGGTACAAACAACACAG-3′，

NBWRKY7-R：5′-GGTTGCATTTGGTTCATGTAAG-3′，

NbAcre31-F：5′-AATTCGGCCATCGTGATCTTGGTC-3′，

NbAcre31-R：5′-GAGAAACTGGGATTGCCTGAAGGA-3′，

NbNPR1-F：5′-AGGACCGGTTATGCATTGAG-3′，

NbNPR1-R：5′-GCTTCTCCTAGCAGTGGATCTC-3′，

NbICS1-F：5′-TTAAACTCATCATCTTCAG-3′，

NbICS1-R：5′-GGCTTCGCCGGCATTCATT-3′，

NbWRKY40-93F：5′-gtgcccagtcaaaaagaagg-3′，

NbWRKY40-93R：5′-gatgggagaggatggttgtg-3′，

NbActin-80F：5′-gtcctggattctggtgatgg-3′，

NbActin-80R：5′-agacggaggatagcatgtgg-3′。

Example 7, Pseudomonas aeruginosa S58 can significantly inhibit elongation of main root of Arabidopsis thaliana, promote growth of lateral root, and increase biomass of Arabidopsis thaliana

The Arabidopsis seeds were sterilized with 75% ethanol for 5 min and washed 3 times with sterile water. Uniformly point-connected to one side of MS flat plate, each plate has about 8-10 seedsThen wrapping with tinfoil paper, and aging at 4 deg.C for 2 d. Taking out, and culturing at 22 deg.C in incubator (light-dark time 16/8h, illumination intensity 100 μmol/m)²S, humidity around 65%) for 5 d. Inoculating the fermentation broth of Pseudomonas winkle S58 at the inoculation position shown in FIG. 4. The inoculum concentration OD600=1.0, inoculum size 5 μ L × 8 drops. The same amount of LB medium was inoculated as a Control (CK), and each treatment was repeated 3 times. Incubate under the same culture conditions for 10d, when CK grows to near the bottom of the plate. The root length of each treated arabidopsis thaliana and the fresh weight of each plant were measured. The result shows that the length of the main root of the arabidopsis treated by inoculating the pseudomonas winkle S58 is 26.9 +/-2.9 mm, and the length of the main root of the arabidopsis of CK is 47.6 +/-6.0 mm; the number of arabidopsis thaliana lateral roots inoculated with the pseudomonas winkle S58 is 11.0 +/-1.5 roots/strain, and the number of the arabidopsis thaliana lateral roots CK is 1.4 +/-0.7 roots/strain; the fresh weight of the Pseudomonas aeruginosa S58-inoculated portion was 12.1. + -. 2.4 mg/strain, and the fresh weight of the CK-inoculated portion on the Arabidopsis thaliana side was 3.9. + -. 0.5 mg/strain. The pseudomonas aeruginosa S58 is shown to be capable of remarkably inhibiting elongation of main root of arabidopsis thaliana, promoting growth of lateral root and improving biomass of arabidopsis thaliana.

The endophytic bacteria secreting plant growth hormone (IAA) is measured by a Salkowski colorimetric method, test strains (pseudomonas wrinkled S58 and negative control strains which do not produce IAA) are respectively inoculated into triangular flasks of LB/KB liquid culture medium (tryptophan needs to be added into the culture medium), each flask contains 50mL of the culture medium, each strain is repeated for 3 times, and the culture medium is placed in a shaking table at 28 ℃ and is shake-cultured at 180rpm for 4 d. 100. mu.L of the bacterial suspension was dropped on a white ceramic plate, and 100. mu.L of Salkowski colorimetric solution was added thereto. For control, 100. mu.L of 100mg/L IAA was added to the colorimetric solution alone. The white ceramic plate was left at room temperature in the dark for 30min and observed, and the plate turned red in color to show that it was able to secrete IAA (FIG. 5). The results show that Pseudomonas winkle S58 reddens the colorimetric fluid and produces auxin (IAA).

Example 8 Pseudomonas winkle S58 ability to hydrolyse organophosphorus

Activated pseudomonas winkle S58 was spotted onto menkina organophosphorous medium, 3 inoculum spots per dish, 3 replicates per strain. Culturing at 30 ℃ for about one week, wherein the larger the diameter of the phosphorus-dissolving ring is, the stronger the phosphorus-dissolving capacity is. The results show that Pseudomonas aeruginosa S58 can generate a phosphate solubilizing loop on Monkina organophosphorus medium (FIG. 6). The pseudomonas winkle S58 can hydrolyze organic phosphorus and can be used for soil improvement.

Wherein, the Monkina organophosphorus culture medium: 10g glucose, 0.5g (NH)₄)₂SO₄，0.3g NaCl，0.3g KCl，0.03g FeSO₄•7H₂O， 0.03g MnSO₄•4H₂O, 0.2g egg yolk lecithin, 5g CaCO₃0.4g of yeast extract, 20g of agar and distilled water to l000mL, and the pH value is 7.0. Sterilizing at 121 deg.C for 30 min.

Example 9, Pseudomonas aeruginosa S58 has significant ammonia-producing ability.

The activated pseudomonas winkle S58 is transferred into peptone ammoniation culture medium and cultured for 48h at 30 ℃. No inoculated peptone ammoniated medium was used as Control (CK). Adding 3-5 drops of Nashin reagent into the culture solution, and indicating that the strain has NH production when yellow or brownish red precipitate appears₃The ability of the cell to perform. The results showed that no yellow or brownish red precipitate appeared after CK was added to the Nahner 'S reagent and yellow or brownish red precipitate appeared after the treatment with Pseudomonas aeruginosa S58 was added to the Nahner' S reagent (FIG. 7). Indicating that the pseudomonas aeruginosa S58 has remarkable ammonia generating capacity.

Example 10, pseudomonas aeruginosa S58 strain had a significant siderophore production capacity.

Media for detecting siderophins (CAS media) (Schwyn B. and Neilands J.B. Universal chemical assay for the detection and determination of siderophores [ J].Analytical Biochemistry,1987,160: 47-56): consisting of 4 solutions (solutions 1-3 and casamimoacid), which were sterilized separately before mixing.

Solution 1 (CAS/HDTMA) ① CAS solution 60.5mg CAS (Chromel) dissolved in 50mL water ② iron solution 1mM FeCl₃·6H₂O in 10mM HCl at pH 2.0, ③ HDTMA solution 72.9mg cetyltrimethylammonium bromide in 40mL water, ① solution and 10mL ② solution mixed and added to ③ solution and stirred well, the resulting blue-black liquid, i.e., CAS/HDTMA solution, was sterilized.

Solution 2 (Salts/Buffer solution): ① Salts solution (750 mL): KH₂PO₄0.3g，NaCl 0.5g，NH₄Dissolving Cl 1.0g in deionized water 750mL, dissolving ② PIPES, piperazine-1, 4-diethylsulfonic acid 30.24g in ① Salts solution, adjusting pH to 6.8 with KOH solution of 50% (W/V), adding agar 15.0g to volume 800mL, sterilizing under high pressure, and cooling to 50 deg.C to obtain Salts/Buffer solution.

Solution 3 (750 mL): 2g of glucose, 2g of mannitol and MgSO₄·7H₂O 493mg，CaCl₂11mg，H₃BO₃1.4mg，ZnSO₄·7H₂O 1.2mg，MnSO₄·2H₂O 1.17mg，Na₂MoO₄·2H₂O 1mg，CuSO ₄40 mug, dissolved in 750mL deionized water and autoclaved.

After the solution 3 is cooled to 50 ℃, the solution 2 is added and mixed with 30 mL of 10% (W/V) casamimo acid which is subjected to filtration sterilization, the solution 1 is added, the mixture is slowly stirred (foam is prevented from being generated), and a flat plate is paved, wherein the flat plate is a siderophin detection plate. The activated pseudomonas winkle S58 is inoculated on a siderophilic assay plate and cultured at 28 ℃ for 24, 48, 60 and 72 hours to observe whether yellow halos are generated on the periphery of colonies. The appearance of a yellow halo around the colony indicates the production of siderophins, since siderophins compete for iron ions chelated by EDTA in the medium, changing the medium from blue to yellow.

The results indicated the appearance of a yellow halo around the colony of Pseudomonas aeruginosa S58, indicating that Pseudomonas aeruginosa S58 is able to produce siderophiles (FIG. 8).

Example 11, Pseudomonas winkle S58 has significant amylase producing ability.

Inoculating activated pseudomonas winkle S58 on an amylase identification culture medium plate, culturing for 2-4d, pouring gram iodine solution to cover the culture medium, pouring the gram iodine solution after 2min to observe whether a transparent ring is generated around the bacterial colony or not, and indicating that the bacterial colony has the capability of secreting amylase if the transparent ring is generated. The results showed that a transparent circle was formed around the colony of Pseudomonas aeruginosa S58 (FIG. 9). Indicating that the pseudomonas winkle S58 has the capability of secreting amylase.

Wherein, the amylase identification culture medium: 1g of soluble starch; 5g of peptone; 5g of glucose; 5g of NaCl; 5g of beef extract; 15g of agar; 1000ml of distilled water.

Sequence listing

<110> institute of agricultural resources and agricultural regionalism of Chinese academy of agricultural sciences

<120> bacterial wilt prevention and growth promoting microbial inoculum and application thereof

<160>1

<170>PatentIn version 3.5

<210>1

<211>1541

<212>DNA

<213> Pseudomonas aeruginosa (Pseudomonas corrugata)

<400>1

attaaggagg tgatccagcc gcaggttccc ctacggctac cttgttacga cttcacccca 60

gtcatgaatc acaccgtggt aaccgtcccc ccgaaggtta gactagctac ttctggtgca 120

acccactccc atggtgtgac gggcggtgtg tacaaggccc gggaacgtat tcaccgcgac 180

attctgattc gcgattacta gcgattccga cttcacgcag tcgagttgca gactgcgatc 240

cggactacga tcggttttgt gggattagct ccacctcgcg gcttggcaac cctctgtacc 300

gaccattgta gcacgtgtgt agcccaggcc gtaagggcca tgatgacttg acgtcatccc 360

caccttcctc cggtttgtca ccggcagtct ccttagagtg cccaccataa tgtgctggta 420

actaaggaca agggttgcgc tcgttacggg acttaaccca acatctcacg acacgagctg 480

acgacagcca tgcagcacct gtctcaatgc tcccgaaggc accaatccat ctctggaaag 540

ttcattggat gtcaaggcct ggtaaggttc ttcgcgttgc ttcgaattaa accacatgct 600

ccaccgcttg tgcgggcccc cgtcaattca tttgagtttt aaccttgcgg ccgtactccc 660

caggcggtca acttaatgcg ttagctgcgc cactaagagc tcaaggctcc caacggctag 720

ttgacatcgt ttacggcgtg gactaccagg gtatctaatc ctgtttgctc cccacgcttt 780

cgcacctcag tgtcagtatc agtccaggtg gtcgccttcg ccactggtgt tccttcctat 840

atctacgcat ttcaccgcta cacaggaaat tccaccaccc tctaccatac tctagctcga 900

cagttttgaa tgcagttccc aggttgagcc cggggctttc acatccaact taacgaacca 960

cctacgcgcg ctttacgccc agtaattccg attaacgctt gcaccctctg tattaccgcg 1020

gctgctggca cagagttagc cggtgcttat tctgtcggta acgtcaaaac catcacgtat 1080

taggtaatgg cccttcctcc caacttaaag tgctttacaa tccgaagacc ttcttcacac 1140

acgcggcatg gctggatcag gctttcgccc attgtccaat attccccact gctgcctccc 1200

gtaggagtct ggaccgtgtc tcagttccag tgtgactgat catcctctca gaccagttac 1260

ggatcgtcgc cttggtgagc cattacctca ccaactagct aatccgacct aggctcatct 1320

gatagcgcaa ggcccgaagg tcccctgctt tctcccgtag gacgtatgcg gtattagcgt 1380

ccgtttccga gcgttatccc ccactaccag gcagattcct aggcattact cacccgtccg 1440

ccgctctcaa gaggtgcaag cacctctcta ccgctcgact tgcatgtgtt aggcctgccg 1500

ccagcgttca atctgagcca tgatcaaact cttcagttca a 1541

Claims

1. The microbial inoculum is characterized in that: the microbial inoculum contains pseudomonas winkle (A)Pseudomonas corrugata) Or/and said Pseudomonas wrinkle (A), (B)Pseudomonas corrugata) The metabolite of (a), the Pseudomonas wrinkle (a)Pseudomonas corrugata) The strain number of (1) is S58, and the registration number of the strain in the China general microbiological culture Collection center is CGMCC No. 17043; the microbial inoculum is any one of the following microbial inocula:

a2, bacterial agent for preventing and/or treating bacterial wilt of tobacco,

a8, a bacterial agent for inhibiting soybean phytophthora,

a9, a bacterial agent for inhibiting rhizoctonia solani,

a10, a bacterial agent for inhibiting Pyricularia oryzae,

a14, bacterial agent for inhibiting Lawsonia solanacearum,

a15, a bacterial agent for producing auxin,

a17, bacterial agent for promoting the growth of lateral roots of plant,

a18, a microbial inoculum for increasing plant biomass,

a19, an amylase-producing microbial inoculum,

a20, an ammonia-producing microbial inoculum,

a21, a siderophore microbial inoculum.

2. The Pseudomonas wrinkle (Pseudomonas aeruginosa) according to claim 1Pseudomonas corrugata) The culture of (A) is prepared by culturing the Pseudomonas wrinkle (Pseudomonas aeruginosa) of claim 1Pseudomonas corrugata) Culturing the resultant material in a microbial culture medium.

3. The culture of claim 2, wherein: the culture has at least one of the following functions:

b2, preventing and/or treating tobacco bacterial wilt,

b4, improving the soil,

b5, hydrolyzing organic phosphorus,

b8, inhibiting the soybean phytophthora,

b9, inhibiting rhizoctonia solani,

b10, inhibiting the rice blast germs,

b14, inhibiting Laurella solanacearum,

b15, producing the auxin,

b16, inhibiting the elongation of the main root of the plant,

b17, promoting the growth of lateral roots of plants,

b18, increasing the biomass of the plant,

b19, producing amylase,

b20, producing ammonia,

b21, producing siderophore.

4. The Pseudomonas wrinkle (Pseudomonas aeruginosa) according to claim 1Pseudomonas corrugata) Or/and said Pseudomonas wrinkle (A), (B)Pseudomonas corrugata) The metabolite of (2) in the prevention and/or treatment of tobacco bacterial wilt.

5. The Pseudomonas wrinkle (Pseudomonas aeruginosa) according to claim 1Pseudomonas corrugata) Or/and said Pseudomonas wrinkle (A), (B)Pseudomonas corrugata) The use of the metabolite in inhibiting phytophthora sojae.

6. The Pseudomonas wrinkle (Pseudomonas aeruginosa) according to claim 1Pseudomonas corrugata) Or/and said Pseudomonas wrinkle (A), (B)Pseudomonas corrugata) The use of the metabolite of (a) in inhibiting rhizoctonia solani.

7. The Pseudomonas wrinkle (Pseudomonas aeruginosa) according to claim 1Pseudomonas corrugata) Or/and said Pseudomonas wrinkle (A), (B)Pseudomonas corrugata) The metabolite of (5) in inhibiting Pyricularia oryzae.

8. The Pseudomonas wrinkle (Pseudomonas aeruginosa) according to claim 1Pseudomonas corrugata) Or/and said Pseudomonas wrinkle (A), (B)Pseudomonas corrugata) The use of the metabolite of (b) in inhibiting Lawsonia solanacearum.

9. Any of the following applications:

u15, Pseudomonas aeruginosa according to claim 1 (1)Pseudomonas corrugata) Or/and said Pseudomonas wrinkle (A), (B)Pseudomonas corrugata) To the use of the metabolites of (a) for the production of auxin (IAA),

u16, Pseudomonas aeruginosa according to claim 1 (1)Pseudomonas corrugata) Or/and said Pseudomonas wrinkle (A), (B)Pseudomonas corrugata) The use of the metabolite of (a) in inhibiting elongation of the main root of a plant,

u17, Pseudomonas aeruginosa according to claim 1 (1)Pseudomonas corrugata) Or/and said Pseudomonas wrinkle (A), (B)Pseudomonas corrugata) The use of the metabolite of (a) in promoting the growth of lateral roots of plants,

u18, Pseudomonas aeruginosa according to claim 1 (1)Pseudomonas corrugata) Or/and said Pseudomonas wrinkle (A), (B)Pseudomonas corrugata) The use of the metabolites of (a) for increasing plant biomass,

u19, Pseudomonas aeruginosa according to claim 1 (1)Pseudomonas corrugata) Or/and said Pseudomonas wrinkle (A), (B)Pseudomonas corrugata) The use of the metabolite of (a) in the production of amylase,

u20, Pseudomonas aeruginosa according to claim 1 (1)Pseudomonas corrugata) Or/and said Pseudomonas wrinkle (A), (B)Pseudomonas corrugata) The use of the metabolite of (a) in the production of ammonia,

u21, Pseudomonas aeruginosa according to claim 1 (1)Pseudomonas corrugata) Or/and said Pseudomonas wrinkle (A), (B)Pseudomonas corrugata) The use of the metabolite of (a) in the production of siderophores.