CN109628345A

CN109628345A - A kind of prevention and treatment succulent brown rot composite bacteria agent and its preparation method and application

Info

Publication number: CN109628345A
Application number: CN201811628263.1A
Authority: CN
Inventors: 赵钢勇; 刘金龙; 顾欣燕; 刘刚; 肖培英; 赵晓琛
Original assignee: Tianjin Development Zone Kunhe Biotechnology Co ltd
Current assignee: Tianjin Development Zone Kunhe Biotechnology Co ltd
Priority date: 2018-12-28
Filing date: 2018-12-28
Publication date: 2019-04-16
Anticipated expiration: 2038-12-28
Also published as: CN109628345B

Abstract

The present invention relates to a kind of prevention and treatment succulent brown rot composite bacteria agent and its preparation method and application, composite bacteria agent by bacillus pumilus (Bacillus Pumilus) tunning of MES828, Brevibacillus laterosporus (Brevibacillus laterosporus) 1-4:1-4 is sufficiently mixed by volume for the tunning of MES818, then 5000rpm-10000rpm is centrifuged 15min-30min and obtains thallus, breaks up thallus resuspension with sterile water and is configured to.Prevention and treatment Crassulaceae succulent brown rot composite bacteria agent produced by the present invention can effectively prevent Crassulaceae succulent brown rot, it can promote absorption and utilization of the Crassulaceae succulent to nutrient, the form for improving Crassulaceae succulent has important positive effect to the ornamental value of enhancing Crassulaceae succulent.

Description

A kind of prevention and treatment succulent brown rot composite bacteria agent and its preparation method and application

Technical field

The invention belongs to field of biotechnology, and in particular to a kind of prevention and treatment succulent brown rot composite bacteria agent and its preparation Methods and applications.

Background technique

Succulent also known as succulent plant or succulent.Because its is rich in color, have a full figure, texture multiplicity, adaptability The features such as stronger, and liked deeply by consuming public.It is sent out with the development of economy with social progress, the consumption idea of people Raw biggish variation, more and more people start to pursue the life of high-quality, and the sales volume of Crassulaceae succulent is promoted to increase sharply, As Crassulaceae succulent area constantly expands, Crassulaceae succulent brown rot disease incidence rises year after year, and becomes increasingly conspicuous, The income for having seriously affected farmer causes serious influence to entire industry.Therefore control Crassulaceae succulent danger Evil has become the upper urgent problem to be solved of production.

One of brown rot system fungal disease.The adaptability and survival ability of brown rot are all very strong, both can be from work It absorbs nourishment, can also be survived in the plant tissue of body in dead residuum.The pathogenecity of brown rot is superpower, once condition The more Crassulaceae succulent organs of Crassulaceae, which can properly be invaded, to breed rapidly.Blade is susceptible, is just in yellow or yellowish-brown Dot is gradually expanded and is round or oval scab, bronzing；Petal is aggrieved, is just in water stain shape brown spot, gradually extends, Entire petal becomes withered, withers sagging；Bulb is aggrieved, and appearance generates irregular blackspot.Under wet condition, one is generated on susceptible cauline leaf The layer mould layer of grey；In leaf sheath, black sclerotium is generated in bulb surface or soil.Crassulaceae succulent nonreactive brown rot at present Kind, control brown rot is still based on chemical prevention in production.Chemical prevention leads to pathogen drug resistance, is difficult from the root It is effectively prevent the generation again of disease, and environmental pollution is larger, endanger the ecological balance, and uses microbial control plant disease It is one of most effective biological and ecological methods to prevent plant disease, pests, and erosion means, but phase there is no for the research in terms of Crassulaceae succulent brown rot about biocontrol agent Close report.

Summary of the invention

The technical problem to be solved by the present invention is to prevent and treat succulent brown rot composite bacteria agent and preparation method thereof and answer With.Prevention and treatment succulent brown rot composite bacteria agent produced by the present invention can effectively prevent Crassulaceae succulent brown rot, can promote Absorption and utilization into Crassulaceae succulent to nutrient improve the form of Crassulaceae succulent, to enhancing Crassulaceae fleshiness The ornamental value of plant has important positive effect.

To solve the above problems, the present invention adopts the following technical scheme:

A kind of preparation method preventing and treating succulent brown rot composite bacteria agent provided by the invention, the composite bacteria agent is by short Tunning, the Brevibacillus laterosporus (Brevibacillus of bacillus pumilus (Bacillus Pumilus) MES828 Laterosporus) 1-4:1-4 is sufficiently mixed the tunning of MES818 by volume, then 5000rpm-10000rpm from Heart 15min-30min obtains thallus, breaks up thallus resuspension with sterile water and is configured to, makes viable bacteria in composite bacteria agent product Sum is 2.5 × 10⁹~2.0 × 10¹⁰In range.

Bacillus pumilus (Bacillus Pumilus) the MES828 tunning and Brevibacillus laterosporus (Brevibacillus laterosporus) MES818 tunning is all made of the preparation of liquid high-density fermentation technique；It is wherein short Viable count in bacillus pumilus (Bacillus Pumilus) MES828 tunning is no less than 2.5 × 10⁹CFu/g, side spore Viable count in bacillus brevis (Brevibacillus laterosporus) MES818 tunning is no less than 2.5 × 10⁹cFu/g。

A kind of bacterial strain for preventing and treating Crassulaceae succulent brown rot, the bacterial strain are bacillus pumilus (Bacillus Pumilus) MES828, is deposited in China General Microbiological culture presevation administrative center, and preservation address is Chaoyang District, Beijing City north The institute 3 of occasion West Road 1, the deposit date is on December 03rd, 2018, deposit number CGMCCNo.16858.

Brevibacillus laterosporus (Brevibacillus laterosporus) MES818 is deposited in Chinese common micro- Biological inoculum preservation administrative center, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3s, and the deposit date is 2018 December 03, deposit number CGMCCNo.16859.

The bacillus pumilus thallus is presented thin rod shape, Gram-positive, size be 0.6~0.7 μ m 2.0~ 3.0 μm, gemma ellipse, middle life or close middle raw, sporangium is unobvious to expand, and on nutrient agar, bacterium colony is circle, It is faint yellow, opaque, flat, surface wettability, neat in edge.Positive reaction: catalase；Oxidizing ferment；V-P measurement；Gelatin liquefaction； Citrate utilizes.Negative reaction: propionate utilizes；Starch Hydrolysis；Nitrate reduction；Gelatin liquefaction；Anaerobic growth.Using spy Different primer carries out multiplexed PCR amplification, which generates unique amplified production, stripe size and bacillus pumilus (Bacillus Pumilus) is identical.

The Brevibacillus laterosporus thallus is presented thin rod shape, Gram-positive, size be 1.0~1.2 μ ms 2.5~ 3.0 μm, there is only navicular side spore, ellipse, sporangiocyst expands, and on nutrient agar, bacterium colony is round, smaller, canescence, Neat in edge, surface wettability is smooth, translucent.Positive reaction: catalase；Oxidizing ferment；Gelatin liquefaction；Nitrate reduction；Anaerobism Growth.Negative reaction: V-P measurement；Citrate utilizes；Starch Hydrolysis.Multiplexed PCR amplification, the bacterium are carried out using special primer Strain generates unique amplified production, stripe size and Brevibacillus laterosporus (Brevibacillus laterosporus) phase Together.

Preferably, the fermentation process in high density of the bacillus pumilus is, comprising the following steps:

(1) preparation of strain；Bacillus pumilus (Bacillus Pumilus) MES828 that separation obtains is forwarded to Eggplant bottle culture medium cultivates 48h~72h, obtains activated strains, (2.0% glucose is molten by 30ml protective agent in 35 DEG C~37 DEG C + 20% glycerite of+3.5% skimmed milk power solution of liquid) it is added in eggplant bottle, it is scraped with sterile spatula, ground device grinding is uniform Afterwards, it saves to -80 DEG C of refrigerators, it is spare；

(2) preparation of seed liquor: drawing the strain of -80 DEG C of preservations, accesses 300mL liquid according to 5%~15% inoculum concentration In culture medium, in 35 DEG C~37 DEG C, 220r/min cultivates 72h, obtains seed liquor；

(3) seeding tank ferments: the seed liquor of above-mentioned preparation is carried out with 5%~15% inoculum concentration access 50L seeding tank Fermentation, seeding tank coefficient 45%~55%, 35 DEG C~37 DEG C of fermentation temperature, revolving speed 220rpm~250rpm, tank pressure is kept Between 0.05Mpa~0.08Mpa, ventilating ratio is 1:0.5~1, seed culture medium initial pH value 6.5~7.0, fermentation period For 24 hours~36h, stream plus 5M~10M NaOH in fermentation process make the control of fermentation liquid pH value 6.5~7.0；

(4) ferment tank: after fermentation to seeding tank, according to 5%~15% inoculum concentration culture transferring to 5000L ferment Tank progress deep fermentation, fermentor coefficient 45%~55%, 35 DEG C~37 DEG C of fermentation temperature, revolving speed 220rpm~ 250rpm, tank pressure are maintained between 0.05Mpa~0.08Mpa, and ventilating ratio is 1:0.5~1, fermentation medium initial pH value 6.5 ~7.0, wait ferment for 24 hours~36h when, exogenous nutrition object (0.5%~1% glucose and 1%~4% big is added with feed profile stream The mixed liquor of beans polypeptide), continuing the 10h~12h that ferments, to can be obtained bacillus pumilus MES828 liquid after fermentation Tunning.

Preferably, the fermentation process in high density of the Brevibacillus laterosporus is, comprising the following steps:

(1) preparation of strain；The Brevibacillus laterosporus (Brevibacillus laterosporus) that separation is obtained MES818 is forwarded to eggplant bottle culture medium and cultivates 48h~72h in 35 DEG C~37 DEG C, activated strains are obtained, by 30ml protective agent (+20% glycerite of+3.5% skimmed milk power solution of 2.0% glucose solution) is added in eggplant bottle, is scraped, is ground with sterile spatula After grinder grinding uniformly, save to -80 DEG C of refrigerators, it is spare；

(4) ferment tank: after fermentation to seeding tank, according to 5%~15% inoculum concentration culture transferring to 5000L ferment Tank carries out liquid fermentation, fermentor coefficient 45%~55%, and 35 DEG C~37 DEG C of fermentation temperature, revolving speed 220rpm~ 250rpm, tank pressure are maintained between 0.05Mpa~0.08Mpa, and ventilating ratio is 1:0.5~1, fermentation medium initial pH value 6.5 ~7.0, wait ferment for 24 hours~36h when, exogenous nutrition object (0.5%~1% glucose and 1%~4% big is added with feed profile stream The mixed liquor of beans polypeptide), continuing the 10h~12h that ferments, to can be obtained Brevibacillus laterosporus MES818 liquid after fermentation Body tunning.

Preferably, the eggplant bottle culture medium when bacillus pumilus MES828 is cultivated, specifically, as mass fraction: Tryptone 0.5~2%, beef extract 0.5%~2%, sodium chloride 0.2%~1%, agar 1.5%~2%, surplus be go from Sub- water, pH are controlled in 7.0~7.3,121 DEG C of sterilizing 30min；

Seed liquid culture medium when the bacillus pumilus MES828 is cultivated, specifically, as mass fraction: Fish protein Peptone 0.5%~2%, soy peptone 0.5%~2%, yeast powder 0.5%~2%, NaCl 0.2%~1%, natural ph, 121 DEG C of sterilizing 30min；

Seed tank culture base when the bacillus pumilus MES828 is cultivated, specifically, as mass fraction: molasses 2%~4%, soybean powder 0.5%~3%, the peanut meal powder 0.5%~2% of enzymatic hydrolysis, yeast extract 0.5%~1%, sodium chloride 0.2%~2%, magnesium sulfate 0.5 ‰~2 ‰, citric acid 0.2%~2%, KH₂PO₄0.05%~0.10%, surplus be go from Sub- water, pH are controlled in 7.0~7.3,121 DEG C of sterilizing 30min；

The peanut meal powder of the enzymatic hydrolysis is compounded with neutral proteinase according to 1:3~3:1 using flavor protease, enzyme concentration 0.5%~1% (E/S, in terms of substrate quality), 40 DEG C~55 DEG C of hydrolysis temperature, enzymolysis time 4h~6h, through being filtered by vacuum, 80 DEG C drying after obtain；

Fermentor liquid culture medium when the bacillus pumilus MES828 is cultivated, specifically, as mass fraction: white sugar 1%~3%, molasses 1%~3%, groundnut meal 0.5%~2%, yeast extract 0.5%~1%, sodium chloride 0.2%~2%, Magnesium sulfate 0.5 ‰~2 ‰, citric acid 0.2%~2%, KH₂PO₄0.05~0.1%, neutral proteinase 0.5 ‰~2 ‰, wind Taste proteinase-10 .5 ‰~2 ‰, GPE 0.2 ‰~1.5 ‰, surplus are deionized water, 6.0~7.0,121 DEG C of medium pH sterilizings 30min。

Preferably, the eggplant bottle culture medium when Brevibacillus laterosporus MES818 is cultivated, specifically, pressing mass fraction Meter: tryptone 0.5%~2%, beef extract 0.5%~2%, sodium chloride 0.2%~1%, agar 1.5%~2%, surplus are Deionized water, pH are controlled in 7.0~7.3,121 DEG C of sterilizing 30min；

Seed liquid culture medium when the Brevibacillus laterosporus MES818 is cultivated, specifically, as mass fraction: fish egg White peptone 0.5%~2%, soy peptone 0.5%~2%, yeast powder 0.5%~2%, NaCl 0.2%~1%, natural pH Value, 121 DEG C of sterilizing 30min；

Seed tank culture base when the Brevibacillus laterosporus MES818 is cultivated, specifically, as mass fraction: yeast Soak powder 0.5%~2%, molasses 1%~3%, the peanut meal powder 1%~4% of enzymatic hydrolysis, potassium dihydrogen phosphate 0.5%~1%, sulfuric acid Magnesium 0.03%~0.05%, calcium carbonate 0.03%~0.05%, sodium chloride 0.5%~1%, surplus are deionized water, pH naturally, 121 DEG C of sterilizing 30min；

Fermentor liquid culture medium when the Brevibacillus laterosporus MES818 is cultivated, specifically, as mass fraction: Portugal Grape sugar 2%~4%, molasses 1%~3%, yeast extract 1%~3%, peanut meal 2%~4%, sodium chloride 1%~3%, sulphur Sour magnesium 1%~2%, neutral proteinase 0.5 ‰~2 ‰, flavor protease 0.5 ‰~2 ‰, sodium citrate 0.5%~3%, bubble 0.2 ‰~1.5 ‰ are opposed, surplus is deionized water, 6.0~7.0,121 DEG C of sterilizing 30min of medium pH.

The present invention also provides a kind of applications for preventing and treating succulent brown rot composite bacteria agent, are characterized in that, by compound bacteria Agent is diluted by 1:1000~1:500 water, is then administered to succulent blade using foliage-spray mode or is filled mode using root It is administered to succulent root.

Beneficial effect

The composite bacteria agent of prevention and treatment Crassulaceae fleshiness brown rot of the invention, effect test show to Crassulaceae succulent Brown rot pathogen prevents and treats efficiency up to 84% or more.

The composite bacteria agent of prevention and treatment Crassulaceae fleshiness brown rot of the invention, can promote suction of the Crassulaceae succulent to nutrient It receives and utilizes, improve the form of Crassulaceae succulent, have important product to the ornamental value of enhancing Crassulaceae succulent Pole meaning.

The composite bacteria agent of prevention and treatment Crassulaceae fleshiness brown rot of the invention, viable count are up to 2,500,000,000/mL or more, and microorganism is living Power is stablized, and pH is between 4.0~7.5.

The composite bacteria agent of prevention and treatment Crassulaceae succulent brown rot of the invention, is combined using multi-cultur es, can be maintained Crassulaceae succulent root microflora balance improves rhizosphere microorganism Bacterial community.

The composite bacteria agent of prevention and treatment Crassulaceae succulent brown rot of the invention, during strain fermentation, seeding tank training Base is supported using the peanut meal powder of enzymatic hydrolysis as one of raw material, this is because the peanut meal powder of enzymatic hydrolysis is rich in a large amount of soluble protein With the substances such as micromolecule polypeptide class, be conducive to the quick utilization of strain, so as to shorten growth cycle.

The composite bacteria agent of prevention and treatment Crassulaceae succulent brown rot of the invention, it is short with single bacillus pumilus and side spore Bacillus is compared, and the antagonism of composite bacteria agent is stronger, and two plants of compound bacterium mutually cooperate with, can be in Crassulaceae fleshiness Rhizosphere, Gen Biao and the field planting in vivo of plant are bred and are shifted, and have stronger antagonistic effect to Crassulaceae succulent brown rot, The product has larger application potential in the prevention and treatment of brown rot.

Detailed description of the invention

Fig. 1 is the bacterium colony and thallus photo of MES828 bacillus pumilus；

Fig. 2 is the bacterium colony and thallus photo of MES818 Brevibacillus laterosporus.

Specific embodiment

The invention will be further elucidated with reference to specific embodiments.It should be understood that these embodiments are merely to illustrate this hair It is bright rather than limit the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, art technology Personnel can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited Fixed range.

Embodiment 1

A kind of bacterial strain for preventing and treating Crassulaceae succulent brown rot, including bacillus pumilus (Bacillus Pumilus), The bacterial strain is bacillus pumilus (Bacillus Pumilus) MES828, is deposited in China General Microbiological culture presevation management Center, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3s, and the deposit date is on December 03rd, 2018, deposit numbers For CGMCCNo.16858.The bacterium colony and thallus photo of the thallus are as shown in Figure 1.

It further include Brevibacillus laterosporus, which is Brevibacillus laterosporus (Brevibacillus Laterosporus) MES818, is deposited in China General Microbiological culture presevation administrative center, and preservation address is court, Beijing The institute 3 of positive area's North Star West Road 1, the deposit date is on December 03rd, 2018, deposit number CGMCCNo.16859.The thallus Bacterium colony and thallus photo it is as shown in Figure 2.

Bacillus pumilus produced by the present invention, Brevibacillus laterosporus imitate the antagonism of Crassulaceae succulent brown rot Fruit test:

It is big that the Wuqing District town Chen Zui Mei Shi highway Pang Zhuan greenhouse base is picked up from for examination Crassulaceae succulent brown rot pathogen Beneficial farm.

The preparation of Crassulaceae succulent brown rot pathogen:

With sterile tweezers clamping morbidity fleshiness blade, about 5mm fleshiness blade is invaded and is handled 2~3 seconds in 75% alcohol, It is transferred in 3% liquor natrii hypochloritis 3~5min of processing, sterile water is put into and changes clothes 3 times, every time 2~3min, then with sterile filter Paper blots surface moisture, and finally leaf tissue is put on PDA culture medium plate, 28 DEG C cultivate 3~5 days, mycelia to be grown or After spore, a little lawn scribing line of picking is transferred into PDA slant medium culture, spare after 28 DEG C are cultivated 3~6 days.

Pathogenic investigation:

By the pathogen slant tube to have grown, 15ml sterile water is added, scraping lawn is made bacteria suspension, takes healthy growth Crassulaceae succulent, Crassulaceae succulent blade is gently scratched with transfer needle, pathogen is accessed, to access sterile water For control group.Routine Management in greenhouse, each processing are inoculated with 5 plants of fleshiness plant, and each processing is repeated 5 times, observation morbidity feelings Condition.The result shows that the Disease symptoms and disease Crassulaceae that cause after the Crassulaceae succulent blade of separation bacterium access health are more Meat Symptoms are similar, the soft corruption of brown occur, and late stage of culture causes integrated plate blade to rot, and Pathogenic Tests are shown as relatively by force, It is accredited as the general bacterium of pineapple (Pantoea ananatis) through identification mechanism, the Ministry of Agriculture, number is KHSWBYJ-012.

Function detection

The plate dual test of bacillus pumilus MES828, Brevibacillus laterosporus MES818:

The Crassulaceae succulent brown rot lawn isolated and purified with oese picking, it is then that Crassulaceae succulent is brown Maize ear rot lawn is applied to sterile nutrient agar panel center, respectively will culture to logarithmic phase bacillus pumilus MES828, Brevibacillus laterosporus MES818, point connects at away from indicator bacteria bacterium piece 2.0cm, if blank control.30 DEG C of cultures, found 2 after 4 days Kind bacillus can significantly inhibit growth of pathogenic bacteria, wider antibacterial band occur.30 DEG C of constant temperature incubations are to blank control When will cover with entire culture dish, the control group increment (colony radius) and experiment of Crassulaceae fleshiness plant brown rot pathogen are measured Group increment (inhibition after inoculation MES828, MES818 grows radius), with antagonism bacteriostasis rate (bacteriostasis rate=(control group Increment-experimental group increment)/control group increment × 100%) indicate inhibitory effect, shown in table 1 specific as follows.

1 bacillus pumilus MES828 of table, Brevibacillus laterosporus MES818 are short of money to Crassulaceae succulent brown rot Anti- test result.

The results showed that table 1 the result shows that bacillus pumilus MES828 to Crassulaceae succulent brown rot Inhibiting rate is up to 85.7%, transparent antibacterial bandwidth 10.3mm；Brevibacillus laterosporus MES818 is to Crassulaceae succulent brown rot The inhibiting rate of disease is up to 85.4%, transparent antibacterial bandwidth 10.0mm.Illustrate bacillus pumilus MES828, Brevibacillus laterosporus MES818 significantly inhibits Crassulaceae succulent brown rot, has prevention and treatment Crassulaceae succulent brown rot Biocontrol Potential.

Embodiment 2

The present invention also provides a kind of prevention and treatment Crassulaceae succulent brown rot composite bacteria agents, and composite bacteria agent is by short and small gemma Tunning, the Brevibacillus laterosporus (Brevibacillus of bacillus (Bacillus Pumilus) MES828 Laterosporus) 1:4 is sufficiently mixed the tunning of MES818 by volume, and then 5000rpm is centrifuged 30min and obtains bacterium Thallus resuspension is broken up and is configured to sterile water by body, makes in composite bacteria agent product total viable count 2.5 × 10⁹~1.0 × 10¹⁰In range.

The bacillus pumilus MES828 tunning and Brevibacillus laterosporus MES818 tunning are all made of liquid Prepared by body high-density fermentation technology, wherein the viable bacteria in bacillus pumilus (Bacillus Pumilus) MES828 tunning Number no less than 2.5 × 10⁹cFu/g；Brevibacillus laterosporus (Brevibacillus laterosporus) the MES818 hair Viable count in ferment product is no less than 2.5 × 10⁹cFu/g。

(1) preparation of strain；Bacillus pumilus (Bacillus Pumilus) MES828 that separation obtains is forwarded to Eggplant bottle culture cultivates 72h, activated strains is obtained, by 30ml protective agent (+3.5% defatted milk of 2.0% glucose solution in 37 DEG C + 20% glycerite of powder solution) it is added in eggplant bottle, it is scraped with sterile spatula, after ground device grinding uniformly, is saved to -80 DEG C Refrigerator, it is spare；

(2) preparation of seed liquor: drawing the strain of -80 DEG C of preservations, accesses 300mL fluid nutrient medium by 5% inoculum concentration In, in 37 DEG C, 220r/min cultivates 72h, obtains seed liquor；

(3) seeding tank ferments: the seed liquor of above-mentioned preparation being fermented with 10% inoculum concentration access 50L seeding tank, is planted Sub- tank coefficient 45%, 37 DEG C of fermentation temperature, revolving speed 220rpm, tank pressure is maintained between 0.05Mpa, and ventilating ratio is 1:1, kind Sub- initial pH value of medium 7.0, fermentation period 36h, stream plus 5M NaOH in fermentation process make the control of fermentation liquid pH value 6.8；

(4) ferment tank: after fermentation to seeding tank, according to 15% inoculum concentration culture transferring to 5000L fermentor into Row deep fermentation, fermentor coefficient 50%, 37 DEG C of fermentation temperature, revolving speed 250rpm, tank pressure be maintained at 0.05Mpa it Between, ventilating ratio is 1:0.5, and fermentation medium initial pH value 7.0 adds exogenous nutrition object wait ferment for 24 hours with feed profile stream (0.5% glucose and 1% soya-bean polypeptides mixed liquor) is continuing the 12h that ferments, to can be obtained short and small gemma bar after fermentation Bacterium MES828 liquid fermentation production.

(1) preparation of strain；The Brevibacillus laterosporus (Brevibacillus laterosporus) that separation is obtained MES818 bacterial strain is forwarded to eggplant bottle culture medium and cultivates 72h in 35 DEG C, activated strains are obtained, by 30ml protective agent (2.0% Portugal + 20% glycerite of+3.5% skimmed milk power solution of grape sugar juice) it is added in eggplant bottle, it is scraped with sterile spatula, dismembyator grinding After uniformly, save to -80 DEG C of refrigerators, it is spare；

(2) preparation of seed liquor: drawing the strain of -80 DEG C of preservations, accesses 300mL fluid nutrient medium by 10% inoculum concentration In, in 35 DEG C, 220r/min cultivates 72h, obtains seed liquor；

(3) seeding tank ferments: the seed liquor of above-mentioned preparation is fermented with 15% inoculum concentration access 50L seeding tank, Seeding tank coefficient 50%, 35 DEG C of fermentation temperature, revolving speed 220rpm, tank pressure is maintained between 0.05Mpa, and ventilating ratio is 1: 0.5~1, seed culture medium initial pH value 7.0, fermentation period 36h, stream plus 5M NaOH, make fermentation liquid pH value in fermentation process Control is 6.8；

(4) ferment tank: after fermentation to seeding tank, according to 15% inoculum concentration culture transferring to 5000L fermentor into Row liquid fermentation, fermentor coefficient 55%, 35 DEG C of fermentation temperature, revolving speed 250rpm, tank pressure is maintained between 0.05Mpa, Ventilating ratio is 1:1, fermentation medium initial pH value 7.0, and 36h to be fermented adds exogenous nutrition object (1% grape with feed profile stream Sugar juice and 1% soya-bean polypeptides mixed liquor), continuing the 10h that ferments, to can be obtained Brevibacillus laterosporus after fermentation MES818 liquid fermentation production.

Preferably, the eggplant bottle culture medium when bacillus pumilus MES828 is cultivated, specifically, as mass fraction: Tryptone 1%, beef extract 0.5%, sodium chloride 0.5%, agar 2%, surplus are deionized water, and pH is controlled at 7.0,121 DEG C Sterilize 30min；

Seed liquid culture medium when the bacillus pumilus MES828 is cultivated, specifically, as mass fraction: Fish protein Peptone 2%, soy peptone 0.5%, yeast powder 1%, NaCl 0.5%, natural ph, 121 DEG C of sterilizing 30min；

Seed tank culture base when the bacillus pumilus MES828 is cultivated, specifically, as mass fraction: molasses 3%, soybean powder 0.5%, the peanut meal powder 2% of enzymatic hydrolysis, yeast extract 1%, sodium chloride 0.3%, magnesium sulfate 2 ‰, citric acid 0.5%, KH₂PO₄0.10%, surplus is deionized water, and pH is controlled in 7.0,121 DEG C of sterilizing 30min；

The peanut meal powder of the enzymatic hydrolysis is compounded with neutral proteinase according to 1:3 using flavor protease, enzyme concentration 1% (E/S, in terms of substrate quality), through being filtered by vacuum, is obtained after 80 DEG C of drying by 40 DEG C of hydrolysis temperature, enzymolysis time 6h；

Fermentor liquid culture medium when the bacillus pumilus MES828 is cultivated, specifically, as mass fraction: white sugar 2%, molasses 2%, groundnut meal 2%, yeast extract 0.5%, sodium chloride 0.5%, magnesium sulfate 0.5 ‰, citric acid 0.5%, KH₂PO₄0.1%, neutral proteinase 2 ‰, flavor protease 2 ‰, GPE 0.5 ‰, surplus is deionized water, medium pH 7.0, 121 DEG C of sterilizing 30min.

Preferably, the eggplant bottle culture medium when Brevibacillus laterosporus MES818 is cultivated, specifically, pressing mass fraction Meter: tryptone 1%, beef extract 1%, sodium chloride 0.5%, agar 2%, surplus are deionized water, and pH is controlled at 7.0,121 DEG C Sterilize 30min；

Seed liquid culture medium when the Brevibacillus laterosporus MES818 is cultivated, specifically, as mass fraction: fish egg White peptone 2%, soy peptone 0.5%, yeast powder 1%, NaCl 0.5%, natural ph, 121 DEG C of sterilizing 30min；

Seed tank culture base when the Brevibacillus laterosporus MES818 is cultivated, specifically, as mass fraction: yeast Leaching powder 1%, molasses 2%, the peanut meal powder 4% of enzymatic hydrolysis, potassium dihydrogen phosphate 0.5%, magnesium sulfate 0.05%, calcium carbonate 0.05%, Sodium chloride 0.5%, surplus are deionized water, and pH is naturally, 121 DEG C of sterilizing 30min；

The peanut meal powder of the enzymatic hydrolysis is compounded with neutral proteinase according to 1:1 using flavor protease, enzyme concentration 1% (E/S, in terms of substrate quality), through being filtered by vacuum, is obtained after 80 DEG C of drying by 50 DEG C of hydrolysis temperature, enzymolysis time 4h；

Fermentor liquid culture medium when the Brevibacillus laterosporus MES818 is cultivated, specifically, as mass fraction: Portugal Grape sugar 3%, molasses 2%, yeast extract 1%, peanut meal 4%, sodium chloride 1%, magnesium sulfate 1%, neutral proteinase 2 ‰, flavor Protease 2 ‰, sodium citrate 1%, GPE 0.5 ‰, surplus are deionized water, 7.0,121 DEG C of sterilizing 30min of medium pH.

Embodiment 3

The present invention also provides a kind of prevention and treatment Crassulaceae succulent brown rot composite bacteria agents, and composite bacteria agent is by short and small gemma Tunning, the Brevibacillus laterosporus (Brevibacillus of bacillus (Bacillus Pumilus) MES828 Laterosporus) 3:2 is sufficiently mixed the tunning of MES818 by volume, and then 8000rpm is centrifuged 20min and obtains bacterium Thallus resuspension is broken up and is configured to sterile water by body, makes in composite bacteria agent product total viable count 2.5 × 10⁹~2.0 × 10¹⁰In range.

The bacillus pumilus MES828 liquid fermentation production and Brevibacillus laterosporus MES818 tunning are adopted It is prepared with liquid high-density fermentation technique, wherein in bacillus pumilus (Bacillus Pumilus) MES828 tunning Viable count is no less than 2.5 × 10⁹cFu/g；The Brevibacillus laterosporus (Brevibacillus laterosporus) Viable count in MES818 tunning is no less than 2.5 × 10⁹cFu/g。

(1) preparation of strain；Bacillus pumilus (Bacillus Pumilus) MES828 that separation obtains is forwarded to Eggplant bottle culture medium cultivates 48h, activated strains is obtained, by (+3.5% degreasing of 2.0% glucose solution of 30ml protective agent in 35 DEG C + 20% glycerite of milk power solution) it is added in eggplant bottle, it is scraped with sterile spatula, after ground device grinding uniformly, is saved to -80 DEG C refrigerator, it is spare；

(2) preparation of seed liquor: drawing the strain of -80 DEG C of preservations, accesses 300mL fluid nutrient medium by 15% inoculum concentration In, in 35 DEG C, 220r/min cultivates 72h, obtains seed liquor；

(3) seeding tank ferments: the seed liquor of above-mentioned preparation is fermented with 15% inoculum concentration access 50L seeding tank, Seeding tank coefficient 55%, 35 DEG C of fermentation temperature, revolving speed 220rpm, tank pressure is maintained between 0.08Mpa, and ventilating ratio is 1: 0.5~1, seed culture medium initial pH value 6.8, for 24 hours, stream plus 5M NaOH, make fermentation liquid pH value control to fermentation period in fermentation process System is 7.0；

(4) ferment tank: after fermentation to seeding tank, according to 10% inoculum concentration culture transferring to 5000L fermentor into Row deep fermentation, fermentor coefficient 45%, 35 DEG C of fermentation temperature, revolving speed 220rpm, tank pressure be maintained at 0.08Mpa it Between, ventilating ratio is 1:0.5~1, seed culture medium initial pH value 6.8,36h to be fermented, with feed profile fed-batch medium (1% Glucose and 3% soya-bean polypeptides mixed liquor), continuing the 10h that ferments, to can be obtained bacillus pumilus after fermentation MES828 liquid fermentation production.

(1) preparation of strain；The Brevibacillus laterosporus (Brevibacillus laterosporus) that separation is obtained MES818 bacterial strain is forwarded to eggplant bottle culture medium and cultivates 48h in 37 DEG C, activated strains are obtained, by 30ml protective agent (2.0% Portugal + 20% glycerite of+3.5% skimmed milk power solution of grape sugar juice) it is added in eggplant bottle, it is scraped with sterile spatula, dismembyator grinding After uniformly, save to -80 DEG C of refrigerators, it is spare；

(2) preparation of seed liquor: drawing the strain of -80 DEG C of preservations, accesses 300mL fluid nutrient medium by 8% inoculum concentration In, in 36 DEG C, 220r/min cultivates 72h, obtains seed liquor；

(3) seeding tank ferments: the seed liquor of above-mentioned preparation being fermented with 5% inoculum concentration access 50L seeding tank, is planted Sub- tank coefficient 45%, 37 DEG C of fermentation temperature, revolving speed 250rpm, tank pressure is maintained between 0.08Mpa, and ventilating ratio is 1:0.5 ~1, seed culture medium initial pH value 6.5, for 24 hours, stream plus 10M NaOH, make fermentation liquid pH value control to fermentation period in fermentation process System is 7.0；

(4) ferment tank: after fermentation to seeding tank, according to 10% inoculum concentration culture transferring to 5000L fermentor into Row liquid fermentation, fermentor coefficient 45%, 37 DEG C of fermentation temperature, revolving speed 230rpm, tank pressure is maintained between 0.05Mpa, Ventilating ratio is 1:0.5~1, fermentation medium initial pH value 6.5, wait ferment for 24 hours, with feed profile fed-batch medium (0.5% Portugal Grape sugar and 1% soya-bean polypeptides mixed liquor), continuing the 12h that ferments, to can be obtained Brevibacillus laterosporus after fermentation MES818 liquid fermentation production.

Preferably, the eggplant bottle culture medium when bacillus pumilus MES828 is cultivated, specifically, as mass fraction: Tryptone 2%, beef extract 2%, sodium chloride 1%, agar 1.5%, surplus are deionized water, and pH control is gone out at 7.3,121 DEG C Bacterium 30min；

Seed liquid culture medium when the bacillus pumilus MES828 is cultivated, specifically, as mass fraction: Fish protein Peptone 0.5%, soy peptone 2%, yeast powder 2%, NaCl 0.5%, natural ph, 121 DEG C of sterilizing 30min；

Seed tank culture base when the bacillus pumilus MES828 is cultivated, specifically, as mass fraction: molasses 2%, soybean powder 3%, the peanut meal powder 1% of enzymatic hydrolysis, yeast extract 0.5%, sodium chloride 0.2%, magnesium sulfate 1 ‰, citric acid 0.2%, KH₂PO₄0.05%, surplus is deionized water, and pH is controlled in 7.3,121 DEG C of sterilizing 30min；

The peanut meal powder of the enzymatic hydrolysis is compounded with neutral proteinase according to 1:1 using flavor protease, enzyme concentration 0.5% (E/S, in terms of substrate quality), through being filtered by vacuum, is obtained after 80 DEG C of drying by 50 DEG C of hydrolysis temperature, enzymolysis time 4h；

Fermentor liquid culture medium when the bacillus pumilus MES828 is cultivated, specifically, as mass fraction: white sugar 1%, molasses 3%, groundnut meal 1%, yeast extract 1%, sodium chloride 2%, magnesium sulfate 1 ‰, citric acid 0.2%, KH₂PO₄ 0.05%, neutral proteinase 1 ‰, flavor protease 2 ‰, GPE 1.5 ‰, surplus is deionized water, 6.5,121 DEG C of medium pH Sterilize 30min.

Preferably, the eggplant bottle culture medium when Brevibacillus laterosporus MES818 is cultivated, specifically, pressing mass fraction Meter: tryptone 2%, beef extract 0.5%, sodium chloride 0.2%, agar 1.5%, surplus are deionized water, pH control 7.3, 121 DEG C of sterilizing 30min；

Seed liquid culture medium when the Brevibacillus laterosporus MES818 is cultivated, specifically, as mass fraction: fish egg White peptone 0.5%, soy peptone 2%, yeast powder 0.5%, NaCl 1%, natural ph, 121 DEG C of sterilizing 30min；

Seed tank culture base when the Brevibacillus laterosporus MES818 is cultivated, specifically, as mass fraction: yeast Soak powder 2%, molasses 3%, the peanut meal powder 2% of enzymatic hydrolysis, potassium dihydrogen phosphate 1%, magnesium sulfate 0.03%, calcium carbonate 0.03%, chlorine Change sodium 1%, surplus is deionized water, and pH is naturally, 121 DEG C of sterilizing 30min；

The peanut meal powder of the enzymatic hydrolysis is compounded with neutral proteinase according to 1:3 using flavor protease, enzyme concentration 0.5% (E/S, in terms of substrate quality), through being filtered by vacuum, is obtained after 80 DEG C of drying by 55 DEG C of hydrolysis temperature, enzymolysis time 4h；

Fermentor liquid culture medium when the Brevibacillus laterosporus MES818 is cultivated, specifically, as mass fraction: Portugal Grape sugar 2%, molasses 1%, yeast extract 3%, peanut meal 2%, sodium chloride 3%, magnesium sulfate 2%, neutral proteinase 2 ‰, flavor Proteinase-10 .5 ‰, sodium citrate 0.5%, GPE 1.5 ‰, surplus are deionized water, 6.8,121 DEG C of medium pH sterilizings 30min。

Embodiment 4

The present invention also provides a kind of prevention and treatment Crassulaceae succulent brown rot composite bacteria agents, and composite bacteria agent is by short and small gemma Tunning, the Brevibacillus laterosporus (Brevibacillus of bacillus (Bacillus Pumilus) MES828 Laterosporus) 4:1 is sufficiently mixed the tunning of MES818 by volume, and then 10000rpm is centrifuged 15min and obtains bacterium Body is broken up and is configured to sterile water resuspension, makes in composite bacteria agent product total viable count 2.5 × 10⁹~2.0 × 10¹⁰Model In enclosing.

(1) preparation of strain；Bacillus pumilus (Bacillus Pumilus) MES828 that separation obtains is forwarded to Eggplant bottle culture cultivates 60h, activated strains is obtained, by 30ml protective agent (+3.5% defatted milk of 2.0% glucose solution in 36 DEG C + 20% glycerite of powder solution) it is added in eggplant bottle, it is scraped with sterile spatula, after ground device grinding uniformly, is saved to -80 DEG C Refrigerator, it is spare；

(2) preparation of seed liquor: drawing the strain of -80 DEG C of preservations, accesses 300mL fluid nutrient medium by 10% inoculum concentration In, in 36 DEG C, 220r/min cultivates 72h, obtains seed liquor；

(3) seeding tank ferments: the seed liquor of above-mentioned preparation being fermented with 5% inoculum concentration access 50L seeding tank, is planted Sub- tank coefficient 50%, 36 DEG C of fermentation temperature, revolving speed 230rpm, tank pressure is maintained between 0.07Mpa, and ventilating ratio is 1:0.5 ~1, seed culture medium initial pH value 6.5, fermentation period 30h, stream plus 5M NaOH in fermentation process control fermentation liquid pH value 6.5；

(4) it ferment tank: after fermentation to seeding tank, is carried out according to 5% inoculum concentration culture transferring to 5000L fermentor Deep fermentation, fermentor coefficient 55%, 36 DEG C of fermentation temperature, revolving speed 230rpm, tank pressure be maintained at 0.07Mpa it Between, ventilating ratio is 1:0.5~1, fermentation medium initial pH value 6.5, and 30h to be fermented adds exogenous nutrition object with feed profile stream (0.8% glucose and 2% soya-bean polypeptides mixed liquor) is continuing the 11h that ferments, to can be obtained short and small gemma bar after fermentation Bacterium MES828 liquid fermentation production.

(1) preparation of strain；The Brevibacillus laterosporus (Brevibacillus laterosporus) that separation is obtained MES818 is forwarded to eggplant bottle culture medium and cultivates 63h in 36 DEG C, activated strains are obtained, by 30ml protective agent (2.0% glucose + 20% glycerite of+3.5% skimmed milk power solution of solution) it is added in eggplant bottle, it is scraped with sterile spatula, dismembyator grinding is uniform Afterwards, it saves to -80 DEG C of refrigerators, it is spare；

(2) preparation of seed liquor: drawing the strain of -80 DEG C of preservations, accesses 300mL fluid nutrient medium by 15% inoculum concentration In, in 36 DEG C, 220r/min cultivates 72h, obtains seed liquor；

(3) seeding tank ferments: the seed liquor of above-mentioned preparation is fermented with 10% inoculum concentration access 50L seeding tank, Seeding tank coefficient 55%, 36 DEG C of fermentation temperature, revolving speed 240rpm, tank pressure is maintained between 0.07Mpa, and ventilating ratio is 1: 0.5~1, seed culture medium initial pH value 6.8, fermentation period 33h, stream plus 5M NaOH, make fermentation liquid pH value control in fermentation process System is 6.5；

(4) it ferment tank: after fermentation to seeding tank, is carried out according to 5% inoculum concentration culture transferring to 5000L fermentor Liquid fermentation, fermentor coefficient 50%, 36 DEG C of fermentation temperature, revolving speed 220rpm, tank pressure is maintained between 0.08Mpa, is led to Wind ratio is 1:0.5~1, fermentation medium initial pH value 6.8, wait ferment 30h when, exogenous nutrition object is added with feed profile stream (0.8% glucose and 4% soya-bean polypeptides mixed liquor) is continuing the 11h that ferments, to can be obtained the short gemma of side spore after fermentation Bacillus MES818 liquid fermentation production.

Preferably, the eggplant bottle culture medium when bacillus pumilus MES828 is cultivated, specifically, as mass fraction: Tryptone 0.5%, beef extract 1%, sodium chloride 0.2%, agar 1.7%, surplus are deionized water, and pH is controlled 7.2,121 DEG C sterilizing 30min；

Seed liquid culture medium when the bacillus pumilus MES828 is cultivated, specifically, as mass fraction: Fish protein Peptone 1%, soy peptone 1%, yeast powder 0.5%, NaCl 2%, natural ph, 121 DEG C of sterilizing 30min；

Seed tank culture base when the bacillus pumilus MES828 is cultivated, specifically, as mass fraction: molasses 4%, soybean powder 2%, the peanut meal powder 0.5% of enzymatic hydrolysis, yeast extract 0.8%, sodium chloride 2%, magnesium sulfate 0.5 ‰, citric acid 2%, KH₂PO₄0.08%, surplus is deionized water, and pH is controlled in 7.2,121 DEG C of sterilizing 30min；

The peanut meal powder of the enzymatic hydrolysis is compounded with neutral proteinase according to 3:1 using flavor protease, enzyme concentration 0.8% (E/S, in terms of substrate quality), through being filtered by vacuum, is obtained after 80 DEG C of drying by 55 DEG C of hydrolysis temperature, enzymolysis time 6h；

Fermentor liquid culture medium when the bacillus pumilus MES828 is cultivated, specifically, as mass fraction: white sugar 3%, molasses 1%, groundnut meal 0.5%, yeast extract 0.8%, sodium chloride 0.2%, magnesium sulfate 2 ‰, citric acid 2%, KH₂PO₄ 0.08%, neutral proteinase 0.5 ‰, flavor protease 0.5 ‰, GPE 0.2 ‰, surplus is deionized water, medium pH 6.0, 121 DEG C of sterilizing 30min.

Preferably, the eggplant bottle culture medium when Brevibacillus laterosporus MES818 is cultivated, specifically, pressing mass fraction Meter: tryptone 0.5%, beef extract 2%, sodium chloride 1%, agar 1.7%, surplus are deionized water, and pH is controlled 7.2,121 DEG C sterilizing 30min；

Seed liquid culture medium when the Brevibacillus laterosporus MES818 is cultivated, specifically, as mass fraction: fish egg White peptone 1%, soy peptone 1%, yeast powder 2%, NaCl 0.2%, natural ph, 121 DEG C of sterilizing 30min；

Seed tank culture base when the Brevibacillus laterosporus MES818 is cultivated, specifically, as mass fraction: yeast Soak powder 0.5%, molasses 1%, the peanut meal powder 1% of enzymatic hydrolysis, potassium dihydrogen phosphate 0.7%, magnesium sulfate 0.05%, calcium carbonate 0.05%, sodium chloride 1%, surplus is deionized water, and pH is naturally, 121 DEG C of sterilizing 30min；

The peanut meal powder of the enzymatic hydrolysis is compounded with neutral proteinase according to 3:1 using flavor protease, enzyme concentration 1% (E/S, in terms of substrate quality), through being filtered by vacuum, is obtained after 80 DEG C of drying by 55 DEG C of hydrolysis temperature, enzymolysis time 4h；

The fermentor liquid culture medium of the Brevibacillus laterosporus MES818 are as follows: glucose 4%, molasses 3%, yeast extract 1%, peanut meal 4%, sodium chloride 2%, magnesium sulfate 1%, neutral proteinase 0.5 ‰, flavor protease 2 ‰, sodium citrate 3%, GPE 0.2 ‰, surplus are deionized water, 6.0,121 DEG C of sterilizing 30min of medium pH.

Embodiment 5

The present invention also provides a kind of prevention and treatment Crassulaceae succulent brown rot composite bacteria agents, and composite bacteria agent is by short and small gemma Tunning, the Brevibacillus laterosporus (Brevibacillus of bacillus (Bacillus Pumilus) MES828 Laterosporus) 3:2 is sufficiently mixed the tunning of MES818 by volume, and then 10000rpm is centrifuged 30min and obtains bacterium Body is broken up and is configured to sterile water resuspension, makes in composite bacteria agent product total viable count 2.5 × 10⁹~2.0 × 10¹⁰Model In enclosing.

(2) preparation of seed liquor: drawing the strain of -80 DEG C of preservations, accesses 300mL fluid nutrient medium by 10% inoculum concentration In, in 37 DEG C, 220r/min cultivates 72h, obtains seed liquor；

(3) seeding tank ferments: the seed liquor of above-mentioned preparation is fermented with 10% inoculum concentration access 50L seeding tank, Seeding tank coefficient 50%, 37 DEG C of fermentation temperature, revolving speed 230rpm, tank pressure is maintained between 0.05Mpa, and ventilating ratio is 1: 0.5~1, seed culture medium initial pH value 6.8, fermentation period 48h, stream plus 5M NaOH, make fermentation liquid pH value in fermentation process Control is 6.8；

(4) ferment tank: after fermentation to seeding tank, according to 10% inoculum concentration culture transferring to 5000L fermentor into Row deep fermentation, fermentor coefficient 50%, 37 DEG C of fermentation temperature, revolving speed 230rpm, tank pressure be maintained at 0.05Mpa it Between, ventilating ratio is 1:0.5~1, and fermentation medium initial pH value 6.8 adds exogenous nutrition object wait ferment for 24 hours with feed profile stream (1% glucose and 1% soya-bean polypeptides mixed liquor) is continuing the 10h that ferments, to can be obtained bacillus pumilus after fermentation MES828 liquid fermentation production.

(1) preparation of strain；The Brevibacillus laterosporus (Brevibacillus laterosporus) that separation is obtained MES818 is forwarded to eggplant bottle culture medium, and in 37 DEG C, culture 72h obtains activated strains, and by 30ml protective agent, (2.0% glucose is molten + 20% glycerite of+3.5% skimmed milk power solution of liquid) it is added in eggplant bottle, it is scraped with sterile spatula, after dismembyator grinding uniformly, It saves to -80 DEG C of refrigerators, it is spare；

(3) seeding tank ferments: the seed liquor of above-mentioned preparation is fermented with 10% inoculum concentration access 50L seeding tank, Seeding tank coefficient 45%, 37 DEG C of fermentation temperature, revolving speed 220rpm, tank pressure is maintained between 0.05Mpa, and ventilating ratio is 1: 0.5~1, seed culture medium initial pH value 7.0, fermentation period 33h, stream plus 5M NaOH, make fermentation liquid pH value in fermentation process Control is 7.0；

(4) ferment tank: after fermentation to seeding tank, according to 15% inoculum concentration culture transferring to 5000L fermentor into Row liquid fermentation, fermentor coefficient 50%, 37 DEG C of fermentation temperature, revolving speed 230rpm, tank pressure is maintained between 0.05Mpa, Ventilating ratio is 1:0.5~1, fermentation medium initial pH value 6.8, wait ferment 30h when, exogenous nutrition object is added with feed profile stream (1% glucose and 3% soya-bean polypeptides mixed liquor) is continuing the 11h that ferments, to can be obtained the short gemma bar of side spore after fermentation Bacterium MES818 liquid fermentation production.

Preferably, the eggplant bottle culture medium when bacillus pumilus MES828 is cultivated, specifically, as mass fraction: Tryptone 1%, beef extract 0.8%, sodium chloride 0.5%, agar 2%, surplus are deionized water, and pH is controlled at 7.0,121 DEG C Sterilize 30min；

Seed liquid culture medium when the bacillus pumilus MES828 is cultivated, specifically, as mass fraction: Fish protein Peptone 0.8%, soy peptone 0.8%, yeast powder 0.5%, NaCl 1%, natural ph, 121 DEG C of sterilizing 30min；

Seed tank culture base when the bacillus pumilus MES828 is cultivated, specifically, as mass fraction: molasses 2%, soybean powder 1%, the peanut meal powder 1% of enzymatic hydrolysis, yeast extract 0.8%, sodium chloride 1%, magnesium sulfate 0.5 ‰, citric acid 2%, KH₂PO₄0.08%, surplus is deionized water, and pH is controlled in 7.0,121 DEG C of sterilizing 30min；

The peanut meal powder of the enzymatic hydrolysis is compounded with neutral proteinase according to 3:1 using flavor protease, enzyme concentration 1% (E/S, in terms of substrate quality), through being filtered by vacuum, is obtained after 80 DEG C of drying by 55 DEG C of hydrolysis temperature, enzymolysis time 5h；

The fermentor liquid culture medium of the bacillus pumilus MES828 are as follows: white sugar 2%, molasses 1%, groundnut meal 1%, Yeast extract 0.8%, sodium chloride 0.5%, magnesium sulfate 1 ‰, citric acid 1%, KH₂PO₄0.08%, neutral proteinase 0.5 ‰, flavor Proteinase-10 .5 ‰, GPE 0.5 ‰, surplus are deionized water, 6.0,121 DEG C of sterilizing 30min of medium pH.

Preferably, the eggplant bottle culture medium when Brevibacillus laterosporus MES818 is cultivated, specifically, pressing mass fraction Meter: tryptone 0.5%, beef extract 1%, sodium chloride 0.5%, agar 2%, surplus are deionized water, and pH is controlled 7.0,121 DEG C sterilizing 30min；

Seed liquid culture medium when the Brevibacillus laterosporus MES818 is cultivated, specifically, as mass fraction: fish egg White peptone 0.8%, soy peptone 0.8%, yeast powder 1%, NaCl 0.5%, natural ph, 121 DEG C of sterilizing 30min；

Seed tank culture base when the Brevibacillus laterosporus MES818 is cultivated, specifically, as mass fraction: yeast Soak powder 0.8%, molasses 1%, the peanut meal powder 2% of enzymatic hydrolysis, potassium dihydrogen phosphate 0.7%, magnesium sulfate 0.05%, calcium carbonate 0.05%, sodium chloride 0.5%, surplus is deionized water, and pH is naturally, 121 DEG C of sterilizing 30min；

The peanut meal powder of the enzymatic hydrolysis is compounded with neutral proteinase according to 1:1 using flavor protease, enzyme concentration 1% (E/S, in terms of substrate quality), through being filtered by vacuum, is obtained after 80 DEG C of drying by 40 DEG C of hydrolysis temperature, enzymolysis time 6h；

The fermentor liquid culture medium of the Brevibacillus laterosporus MES818 are as follows: glucose 2%, molasses 1%, yeast extract 1%, peanut meal 2%, sodium chloride 1%, magnesium sulfate 0.8%, neutral proteinase 1 ‰, flavor protease 2 ‰, sodium citrate 1%, GPE 0.2 ‰, surplus are deionized water, 7.0,121 DEG C of sterilizing 30min of medium pH.

Embodiment 6

Composite bacteria agent is pressed 1:1000~1:500 by a kind of application for preventing and treating Crassulaceae succulent brown rot composite bacteria agent Then water dilution is administered to succulent blade by the way of foliage spray or is administered to succulent by the way of root filling Root.

Composite bacteria agent (composite bacteria agent of embodiment 2-5 preparation) is applied by the way of foliage-spray.

Bacillus pumilus MES828, Brevibacillus laterosporus MES818 are subjected to activation culture respectively according to the method described above To logarithmic growth phase, by the fermentation of the tunning, Brevibacillus laterosporus MES818 of the bacillus pumilus MES828 of activation Product 5000rpm-10000rpm is centrifuged 15min-30min and obtains thallus, breaks up thallus resuspension with sterile water and is configured to bacterium Agent makes single culture living bacteria count 1.0 × 10⁹~1.0 × 10¹⁰In range；Then by the bacillus pumilus of activation The tunning of MES828, the tunning of Brevibacillus laterosporus MES818 are mixed according to volume ratio 1-4:1-4, then 5000rpm-10000rpm is centrifuged 15min-30min and obtains thallus, breaks up thallus resuspension with sterile water and is configured to microbial inoculum, makes Total viable count is 2.5 × 10 in composite bacteria agent product⁹~2 × 10¹⁰In range.

Application method: by bacillus pumilus agent, Brevibacillus laterosporus microbial inoculum, composite bacteria agent and 70% thiophanate methyl After wettable powder is diluted according to 1:500 water, foliage-spray is in Crassulaceae succulent leaf on piece.

Sample plot point is selected from the town Wuqing District Chen Zui Mei Shi highway Pang Zhuan greenhouse base Wei Yi farm greenhouse and carries out.

Experimental design: it chooses the almost the same brown rot succulent saussurea involucrata seed seedling of growing way and carries out application effect research. Seedling medium chooses turf and vermiculite, mixes according to 2:1 ratio, by seed transplantation of seedlings to fleshiness flowerpot (bore 17cm, height It spends in 14).Test setting is as shown in table 2 below.20 basins are arranged in each processing, are repeated 3 times.It sprays single treatment liquid within every 7 days, sprays The amount of applying is stained with treatment fluid with blade face completely to be advisable, normal management, and calculates disease index and control efficiency, shown in table 2 specific as follows.

Experimental design

Preventive effect comparative test result of the different foliage-spray processing modes of table 2 to Crassulaceae succulent brown rot

Group	Processing mode	Disease index	Preventive effect/%
				Test 1 group	Foliage-spray bacillus pumilus agent	16.14b	66.4
Test 2 groups	Foliage-spray Brevibacillus laterosporus microbial inoculum	15.31b	68.9
				Test 3 groups	Foliage-spray composite bacteria agent	10.10bc	84.4
Test 4 groups	70% thiophanate methyl wettable powder of foliage-spray	9.11c	92.6
				Control group	Foliage-spray sterile water	85.21a	—

Experimental result: 2 test result of table shows disease index 16.14 of 1 group of the test to Crassulaceae succulent brown rot, Control efficiency is 66.4%；2 groups of disease indexs 15.31 to Crassulaceae succulent brown rot are tested, control efficiency is 68.9%；3 groups of disease indexs 10.10 to Crassulaceae succulent brown rot are tested, control efficiency 84.4% tests 4 groups To the disease index 9.11 of Crassulaceae succulent brown rot, control efficiency 92.6%.

Embodiment 7

Bacillus pumilus MES828, Brevibacillus laterosporus MES818 are subjected to activation culture respectively according to the method described above To logarithmic growth phase, by the fermentation of the tunning, Brevibacillus laterosporus MES818 of the bacillus pumilus MES828 of activation Product 5000rpm-10000rpm is centrifuged 15min-30min and obtains thallus, breaks up thallus resuspension with sterile water and is configured to bacterium Agent makes single culture living bacteria count 1.0 × 10⁹~1.0 × 10¹⁰In range；Then by the bacillus pumilus of activation The tunning of MES828, the tunning of Brevibacillus laterosporus MES818 are mixed according to volume ratio 1-4:1-4, then 5000rpm-10000rpm is centrifuged 15min-30min and obtains thallus, breaks up thallus resuspension with sterile water and is configured to microbial inoculum, makes Total viable count is 2.5 × 10 in composite bacteria agent product⁹~2.0 × 10¹⁰In range.

Application method: by bacillus pumilus agent, Brevibacillus laterosporus microbial inoculum, composite bacteria agent and 70% thiophanate methyl After wettable powder is diluted according to 1:1000 water, foliage-spray is in Crassulaceae succulent leaf on piece.

Experimental design: it chooses the almost the same brown rot succulent saussurea involucrata seed seedling of growing way and carries out application effect research. Seedling medium chooses turf and vermiculite, mixes according to 2:1 ratio, by seed transplantation of seedlings to fleshiness flowerpot (bore 17cm, height It spends in 14).Test setting is as shown in table 2 below.20 basins are arranged in each processing, are repeated 3 times.It sprays single treatment liquid within every 10 days, sprays The amount of applying is stained with treatment fluid with blade face completely to be advisable, normal management, and calculates disease index and control efficiency, shown in table 3 specific as follows.

Experimental design

Preventive effect comparative test result of the different foliage-spray processing modes of table 3 to Crassulaceae succulent brown rot

Group	Processing mode	Disease index	Preventive effect/%
				Test 1 group	Foliage-spray bacillus pumilus agent	15.33b	68.8
Test 2 groups	Foliage-spray Brevibacillus laterosporus microbial inoculum	14.97b	70.6
				Test 3 groups	Foliage-spray composite bacteria agent	10.94bc	86.8
Test 4 groups	70% thiophanate methyl wettable powder of foliage-spray	8.96c	95.6
				Control group	Foliage-spray sterile water	85.21a	—

Experimental result: 3 test result of table shows disease index 15.33 of 1 group of the test to Crassulaceae succulent brown rot, Control efficiency is 68.8%；2 groups of disease indexs 14.97 to Crassulaceae succulent brown rot are tested, control efficiency is 70.6%；3 groups of disease indexs 10.94 to Crassulaceae succulent brown rot are tested, control efficiency 86.8% tests 4 groups To the disease index 8.96 of Crassulaceae succulent brown rot, control efficiency 95.6%.

Embodiment 8

Composite bacteria agent (composite bacteria agent of embodiment 2-5 preparation) is applied by the way of tank root.

Bacillus pumilus MES828, Brevibacillus laterosporus MES818 are subjected to activation culture respectively according to the method described above To logarithmic growth phase, by the fermentation of the tunning, Brevibacillus laterosporus MES818 of the bacillus pumilus MES828 of activation Product 5000rpm-10000rpm is centrifuged 15min-30min and obtains thallus, breaks up thallus resuspension with sterile water and is configured to microbial inoculum, Make single culture living bacteria count 1.0 × 10⁹~1.0 × 10¹⁰In range；Then by the bacillus pumilus MES828 of activation Tunning, Brevibacillus laterosporus MES818 tunning mixed according to volume ratio 1-4:1-4, then 5000rpm- 10000rpm is centrifuged 15min-30min and obtains thallus, breaks up thallus resuspension with sterile water and is configured to microbial inoculum, makes composite bacteria agent Total viable count is 2.5 × 10 in product⁹~2.0 × 10¹⁰In range.

Application method: by bacillus pumilus agent, Brevibacillus laterosporus microbial inoculum, composite bacteria agent and 70% thiophanate methyl After wettable powder is diluted according to 1:500 water, root filling imposes on Crassulaceae succulent root.

Experimental design: it chooses the almost the same brown rot succulent saussurea involucrata seed seedling of growing way and carries out application effect research. Seedling medium chooses turf and vermiculite, mixes according to 2:1 ratio, by seed transplantation of seedlings to fleshiness flowerpot (bore 17cm, height It spends in 14).Test setting is as shown in table 2 below.20 basins are arranged in each processing, are repeated 3 times.Every basin pouring root 30ml treatment fluid, every 7 Its pouring root is primary, normal management, and calculates disease index and control efficiency, shown in table 4 specific as follows.

Experimental design

The different roots of table 4 fill processing mode to the preventive effect comparative test result of Crassulaceae succulent brown rot

Group	Processing mode	Disease index	Preventive effect/%
				Test 1 group	Pouring root bacillus pumilus agent	15.71b	68.2
Test 2 groups	Pouring root Brevibacillus laterosporus agent	14.82b	70.1
				Test 3 groups	Pouring root composite bacteria agent	11.10bc	84.2
Test 4 groups	70% thiophanate methyl wettable powder of pouring root	8.82c	96.2
				Control group	Pouring root sterile water	85.21a	—

Experimental result: 4 test result of table shows disease index 15.71 of 1 group of the test to Crassulaceae succulent brown rot, Control efficiency is 68.2%；2 groups of disease indexs 14.82 to Crassulaceae succulent brown rot are tested, control efficiency is 70.1%；3 groups of disease indexs 11.10 to Crassulaceae succulent brown rot are tested, control efficiency 84.2% tests 4 groups To the disease index 8.82 of Crassulaceae succulent brown rot, control efficiency is and 96.2%.

Embodiment 9

Bacillus pumilus MES828, Brevibacillus laterosporus MES818 are subjected to activation culture respectively according to the method described above To logarithmic growth phase, by the fermentation of the tunning, Brevibacillus laterosporus MES818 of the bacillus pumilus MES828 of activation Product 5000rpm-10000rpm is centrifuged 15min-30min and obtains thallus, breaks up thallus resuspension with sterile water and is configured to bacterium Agent makes single culture living bacteria count 1.0 × 10⁹~1.0 × 10¹⁰In range；Then by the bacillus pumilus of activation The tunning of MES828, the tunning of Brevibacillus laterosporus MES818 are mixed according to volume ratio 1-4:1-4, then 5000rpm-10000rpm is centrifuged 15min-30min and obtains, and breaks up thallus resuspension with sterile water and is configured to microbial inoculum, makes compound Total viable count is 2.5 × 10 in microbial inoculum product⁹~2.0 × 10¹⁰In range.

Application method: by bacillus pumilus agent, Brevibacillus laterosporus microbial inoculum, composite bacteria agent and 70% thiophanate methyl After wettable powder is diluted according to 1:1000 water, root filling imposes on Crassulaceae succulent root.

Experimental design: it chooses the almost the same brown rot succulent saussurea involucrata seed seedling of growing way and carries out application effect research. Seedling medium chooses turf and vermiculite, mixes according to 2:1 ratio, by seed transplantation of seedlings to fleshiness flowerpot (bore 17cm, height It spends in 14).Test setting is as shown in table 2 below.20 basins are arranged in each processing, are repeated 3 times.Every basin pouring root 30ml treatment fluid, every 10 Its pouring root is primary, normal management, and calculates disease index and control efficiency, shown in table 5 specific as follows.

Experimental design

The different roots of table 5 fill processing mode to the preventive effect comparative test result of Crassulaceae succulent brown rot

Group	Processing mode	Disease index	Preventive effect/%
				Test 1 group	Pouring root bacillus pumilus agent	15.21b	74.8
Test 2 groups	Pouring root Brevibacillus laterosporus agent	14.71b	75.2
				Test 3 groups	Pouring root composite bacteria agent	10.11bc	88.3
Test 4 groups	70% thiophanate methyl wettable powder of pouring root	8.71c	96.4
				Control group	Pouring root sterile water	85.21a	—

Experimental result: 5 test result of table shows disease index 15.21 of 1 group of the test to Crassulaceae succulent brown rot, Control efficiency is 74.8%；Test 2 groups of disease indexs 14.71 to Crassulaceae succulent brown rot, control efficiency be and 75.2%；3 groups of disease indexs 10.11 to Crassulaceae succulent brown rot are tested, control efficiency 88.3% tests 4 groups To the disease index 8.71 of Crassulaceae succulent brown rot, control efficiency 96.4%.

A kind of composite bacteria agent of prevention and treatment Crassulaceae succulent brown rot of the invention, can from embodiment 6-9 test result Know, composite bacteria agent control efficiency is slightly inferior to chemical agent.But in the long run chronic administration chemical agent to the growth of plant not Benefit, while also will affect plant rhizosphere microbe Bacterial community, and composite bacteria agent has nontoxic, noresidue, does not inhibit raw Long and few dosage feature；Prevention and treatment Crassulaceae succulent brown rot composite bacteria agent produced by the present invention can effectively prevent red-spotted stonecrop Section's succulent brown rot can promote absorption and utilization of the Crassulaceae succulent to nutrient, improve Crassulaceae succulent Form has important positive effect to the ornamental value of enhancing Crassulaceae succulent.With single administration bacillus pumilus It is compared with Brevibacillus laterosporus, the antagonism of composite bacteria agent is stronger, and two plants of compound bacterium mutually cooperate with, can be in Crassulaceae Rhizosphere, Gen Biao and the field planting in vivo of succulent are bred and are shifted, and have stronger antagonism to imitate Crassulaceae succulent brown rot Fruit.Therefore, composite bacteria agent is preferably chosen for preventing and treating Crassulaceae succulent brown rot.

SEQUENCE LISTING

<110>Tianjin Development Zone Kun He Bioisystech Co., Ltd

<120>a kind of prevention and treatment succulent brown rot composite bacteria agent and its preparation method and application

<140> 201811628263.1

<160> 2

<210> 1

<211> 1453

<212> DNA

<213> Bacillus Pumilus

<400> 1

gcgggggggg gctatactgc agtcgagcgg acagaaggga gcttgctccc ggatgttagc 60

ggcggacggg tgagtaacac gtgggtaacc tgcctgtaag actgggataa ctccgggaaa 120

ccggagctaa taccggatag ttccttgaac cgcatggttc aaggatgaaa gacggtttcg 180

gctgtcactt acagatggac ccgcggcgca ttagctagtt ggtggggtaa tggctcacca 240

aggcgacgat gcgtagccga cctgagaggg tgatcggcca cactgggact gagacacggc 300

ccagactcct acgggaggca gcagtaggga atcttccgca atggacgaaa gtctgacgga 360

gcaacgccgc gtgagtgatg aaggttttcg gatcgtaaag ctctgttgtt agggaagaac 420

aagtgcgaga gtaactgctc gcaccttgac ggtacctaac cagaaagcca cggctaacta 480

cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg tccggaatta ttgggcgtaa 540

agggctcgca ggcggtttct taagtctgat gtgaaagccc ccggctcaac cggggagggt 600

cattggaaac tgggaaactt gagtgcagaa gaggagagtg gaattccacg tgtagcggtg 660

aaatgcgtag agatgtggag gaacaccagt ggcgaaggcg actctctggt ctgtaactga 720

cgctgaggag cgaaagcgtg gggagcgaac aggattagat accctggtag tccacgccgt 780

aaacgatgag tgctaagtgt tagggggttt ccgcccctta gtgctgcagc taacgcatta 840

agcactccgc ctggggagta cggtcgcaag actgaaactc aaaggaattg acgggggccc 900

gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accaggtctt 960

gacatcctct gacaacccta gagatagggc tttcccttcg gggacagagt gacaggtggt 1020

gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac 1080

ccttgatctt agttgccagc atttagttgg gcactctaag gtgactgccg gtgacaaacc 1140

ggaggaaggt ggggatgacg tcaaatcatc atgcccctta tgacctgggc tacacacgtg 1200

ctacaatgga cagaacaaag ggctgcgaga ccgcaaggtt tagcgaatcc cataaatctg 1260

ttctcagttc ggatcgcagt ctgcaactcg actgcgtgaa gctggaatcg ctagtaatcg 1320

cggatcagca tgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacaa 1380

tacacgagag tttgcaacac ccgaagtcgg tgaggtaacc tttatggagc cagccgccga 1440

agctgacaga gag 1453

<210> 2

<211> 1437

<212> DNA

<213> Brevibacillus laterosporu

<400> 1

gtaggggggg gtctataatg cagtcgagcg agggttttcg gaccctagcg gcggacgggt 60

gagtaacacg taggcaacct gcctgtaaga ctgggataac atagggaaac ttatgctaat 120

accggataga gttttgcttc gcatgaagcg aaacggaaag atggcgcaag ctatcacttg 180

cagatgggcc tgcggcgcat tagctagttg gtgaggtaaa ggctcaccaa ggcgacgatg 240

cgtagccgac ctgagagggt gaccggccac actgggactg agacacggcc cagactccta 300

cgggaggcag cagtagggaa ttttccacaa tggacgaaag tctgatggag caacgccgcg 360

tgaacgatga aggctttcgg gtcgtaaagt tctgttgtta gggaagaaac agtgccattt 420

aaataaggtg gcaccttgac ggtacctaac gagaaagcca cggctaacta cgtgccagca 480

gccgcggtaa tacgtaggtg gcaagcgttg tccggaatta ttgggcgtaa agcgcgcgca 540

ggtggctatg taagtctgat gttaaagccc ggggctcaac ctcggttcgc attggaaact 600

gcgtagcttg agtgcaggag aggaaagtgg tattccacgt gtagcggtga aatgcgtaga 660

gatgtggagg aacaccagtg gcgaaggcga ctttctggcc tgtaactgac actgaggcgc 720

gaaagcgtgg ggagcaaaca ggattagata ccctggtagt ccacgccgta aacgatgagt 780

gctaggtgtt aggggtttca atacccttag tgccgcagct aacgcaataa gcactccgcc 840

tggggagtac gctcgcaaga gtgaaactca aaggaattga cgggggcccg cacaagcggt 900

ggagcatgtg gtttaattcg aagcaacgcg aagaacctta ccaggtcttg acatcccact 960

gaccgctcta gagatagagc ttcccttcgg ggcagtggtg acaggtggtg catggttgtc 1020

gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttatcttta 1080

gttgccagca ttcagttggg cactctagag agactgccgt cgacaagacg gaggaaggcg 1140

gggatgacgt caaatcatca tgccccttat gacctgggct acacacgtgc tacaatggtt 1200

ggtacaacgg gatgctactt cgcgagaaga tgctaatctc ttaaaaccaa tctcagttcg 1260

gattgtaggc tgcaactcgc ctacatgaag tcggaatcgc tagtaatcgc ggatcagcat 1320

gccgcggtga atacgttccc gggccttgta cacaccgccc gtcacaccac gggagtttgc 1380

aacacccgaa gtcggtgagg taaccgcaag gagccagccg ccgaaggtgg agatccg 1437

Claims

1. a kind of prevention and treatment Crassulaceae succulent brown rot composite bacteria agent, is characterized in that, the composite bacteria agent includes short and small gemma Bacillus (Bacillus Pumilus) MES828 tunning and Brevibacillus laterosporus (Brevibacillus laterosporus) MES818 tunning, the bacillus pumilus (Bacillus Pumilus) MES828 and side spore Bacillus brevis (Brevibacillus laterosporus) MES818 tunning volume ratio be 1-4:1-4, it is described Total viable count is 2.5 × 10 in composite bacteria agent product⁹~2.0 × 10¹⁰In range.

2. the preparation method of prevention and treatment Crassulaceae succulent brown rot composite bacteria agent according to claim 1, feature exist In, the bacillus pumilus (Bacillus Pumilus) MES828 tunning and Brevibacillus laterosporus (Brevibacillus laterosporus) MES818 tunning be all made of liquid high-density fermentation technique preparation, wherein short Bacillus pumilus (Bacillus Pumilus) viable count in MES828 tunning is no less than 2.5 × 10⁹CFu/g, side spore Bacillus brevis (Brevibacillus laterosporus) viable count in MES818 tunning is no less than 2.5 × 10⁹cFu/g；

The bacillus pumilus (Bacillus Pumilus) MES828, it is deposited in China General Microbiological culture presevation management Center, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3s, and the deposit date is on December 03rd, 2018, deposit numbers For CGMCCNo.16858；

The Brevibacillus laterosporus (Brevibacillus laterosporus) MES818, it is deposited in China General Microbiological Culture presevation administrative center, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3s, and the deposit date is in December, 2018 03 day, deposit number CGMCCNo.16859.

3. the preparation method of prevention and treatment Crassulaceae succulent brown rot composite bacteria agent according to claim 2, feature exist In, by bacillus pumilus (Bacillus Pumilus) MES828 tunning, Brevibacillus laterosporus (Brevibacillus laterosporus) 1-4:1-4 is sufficiently mixed by volume for the tunning of MES818, then 5000rpm-10000rpm is centrifuged 15min-30min and obtains thallus, breaks up thallus resuspension with sterile water and is configured to, and makes multiple Total viable count is 2.5 × 10 in combined bacteria agent product⁹~2.0 × 10¹⁰In range.

4. the preparation method of prevention and treatment Crassulaceae succulent brown rot composite bacteria agent according to claim 2, feature exist In the fermentation process in high density of the bacillus pumilus, comprising the following steps:

(1) preparation of strain；By separation obtain bacillus pumilus (Bacillus Pumilus) MES828 be forwarded to eggplant bottle Culture medium cultivates 48h~72h in 35 DEG C~37 DEG C, obtains activated strains, and protective agent is added and saves backup；

(2) preparation of seed liquor: according in 5%~15% inoculum concentration access fluid nutrient medium, in 35 DEG C~37 DEG C, 220r/min is trained 72h is supported, seed liquor is obtained；

(3) seeding tank ferments: the seed liquor of above-mentioned preparation is fermented with 5%~15% inoculum concentration access seeding tank, seed Tank coefficient 45%~55%, 35 DEG C~37 DEG C of fermentation temperature, revolving speed 220rpm~250rpm, tank pressure be maintained at 0.05Mpa~ Between 0.08Mpa, ventilating ratio is 1:0.5~1, seed culture medium initial pH value 6.5~7.0, fermentation period for 24 hours~36h, fermentation Stream plus 5M~10M NaOH in the process make the control of fermentation liquid pH value 6.5~7.0；

(4) ferment tank: after fermentation to seeding tank, deep layer is carried out according to 5%~15% inoculum concentration culture transferring to fermentor Liquid fermentation, fermentor coefficient 45%~55%, 35 DEG C~37 DEG C of fermentation temperature, revolving speed 220rpm~250rpm, tank pressure is protected It holds between 0.05Mpa~0.08Mpa, ventilating ratio is 1:0.5~1, fermentation medium initial pH value 6.5~7.0, wait ferment For 24 hours~36h when, exogenous nutrition object is added with feed profile stream, continuing ferment 10h~12h, it is short to can be obtained after fermentation Bacillus pumilus MES828 liquid fermentation production.

5. the preparation method of prevention and treatment succulent brown rot composite bacteria agent according to claim 2, which is characterized in that described The fermentation process in high density of Brevibacillus laterosporus is, comprising the following steps:

(1) preparation of strain；By separation obtain Brevibacillus laterosporus (Brevibacillus laterosporus) MES818 is forwarded to eggplant bottle culture medium and cultivates 48h~72h in 35 DEG C~37 DEG C, obtains activated strains, protective agent is added and saves It is spare；

(2) preparation of seed liquor: according in 5%~15% inoculum concentration access 300mL fluid nutrient medium, in 35 DEG C~37 DEG C, 220r/ Min cultivates 72h, obtains seed liquor；

(4) ferment tank: after fermentation to seeding tank, liquid is carried out according to 5%~15% inoculum concentration culture transferring to fermentor Fermentation, fermentor coefficient 45%~55%, 35 DEG C~37 DEG C of fermentation temperature, revolving speed 220rpm~250rpm, tank pressure is maintained at Between 0.05Mpa~0.08Mpa, ventilating ratio is 1:0.5~1, fermentation medium initial pH value 6.5~7.0, wait ferment for 24 hours~ When 36h, exogenous nutrition object is added with feed profile stream, is continuing the 10h~12h that ferments, it is short to can be obtained side spore after fermentation Bacillus MES818 liquid fermentation production.

6. the preparation method of prevention and treatment succulent brown rot composite bacteria agent according to claim 3, which is characterized in that described Eggplant bottle medium component when bacillus pumilus MES828 is cultivated, specifically, as mass fraction: tryptone 0.5%~ 2%, beef extract 0.5%~2%, sodium chloride 0.2%~1%, agar 1.5%~2%, surplus is deionized water, pH control 7.0~ 7.3,121 DEG C of sterilizing 30min；

Seed liquor medium component when the bacillus pumilus MES828 is cultivated, specifically, as mass fraction: Fish protein Peptone 0.5%~2%, soy peptone 0.5%~2%, yeast powder 0.5%~2%, NaCl 0.2%~1%, natural ph, 121 DEG C of sterilizings 30min；

Seed tank culture based component when the bacillus pumilus MES828 is cultivated, specifically, as mass fraction: molasses 2% ~4%, soybean powder 0.5%~3%, the peanut meal powder 0.5%~2% of enzymatic hydrolysis, yeast extract 0.5%~1%, sodium chloride 0.2%~2%, sulfuric acid Magnesium 0.5 ‰~2 ‰, citric acid 0.2%~2%, KH₂PO₄0.05%~0.10%, surplus is deionized water, pH control 7.0~ 7.3,121 DEG C of sterilizing 30min；

The peanut meal powder of the enzymatic hydrolysis is compounded with neutral proteinase according to 1:3~3:1 using flavor protease, enzyme concentration 0.5% ~1%(E/S, in terms of substrate quality), 40 DEG C~55 DEG C of hydrolysis temperature, enzymolysis time 4h~6h, through being filtered by vacuum, 80 DEG C of drying After obtain；

Fermentor liquid culture medium ingredient when the bacillus pumilus MES828 is cultivated, specifically, as mass fraction: white sugar 1%~3%, molasses 1%~3%, groundnut meal 0.5%~2%, yeast extract 0.5%~1%, sodium chloride 0.2%~2%, magnesium sulfate 0.5 ‰ ~2 ‰, citric acid 0.2%~2%, KH₂PO₄0.05~0.1%, neutral proteinase 0.5 ‰~2 ‰, flavor protease 0.5 ‰~ 2 ‰, GPE 0.2 ‰~1.5 ‰, surplus is deionized water, 6.0~7.0,121 DEG C of sterilizing 30min of medium pH.

7. the preparation method of prevention and treatment succulent brown rot composite bacteria agent according to claim 4, which is characterized in that described Eggplant bottle medium component when Brevibacillus laterosporus MES818 is cultivated, specifically, as mass fraction: tryptone 0.5%~ 2%, beef extract 0.5%~2%, sodium chloride 0.2%~1%, agar 1.5%~2%, surplus is deionized water, pH control 7.0~ 7.3,121 DEG C of sterilizing 30min；

Seed liquor medium component when the Brevibacillus laterosporus MES818 is cultivated, specifically, as mass fraction: fish egg White peptone 0.5%~2%, soy peptone 0.5%~2%, yeast powder 0.5%~2%, NaCl 0.2%~1%, natural ph, 121 DEG C go out Bacterium 30min；

Seed tank culture based component when the Brevibacillus laterosporus MES818 is cultivated, specifically, as mass fraction: yeast Leaching powder 0.5%~2%, molasses 1%~3%, the peanut meal powder 1%~4% of enzymatic hydrolysis, potassium dihydrogen phosphate 0.5%~1%, magnesium sulfate 0.03%~ 0.05%, calcium carbonate 0.03%~0.05%, sodium chloride 0.5%~1%, surplus is deionized water, and pH is naturally, 121 DEG C of sterilizing 30min；

Fermentor liquid culture medium ingredient when the Brevibacillus laterosporus MES818 is cultivated, specifically, as mass fraction: Portugal Grape sugar 2%~4%, molasses 1%~3%, yeast extract 1%~3%, peanut meal 2%~4%, sodium chloride 1%~3%, magnesium sulfate 1%~2%, Neutral proteinase 0.5 ‰~2 ‰, flavor protease 0.5 ‰~2 ‰, sodium citrate 0.5%~3%, GPE 0.2 ‰~1.5 ‰, Surplus is deionized water, 6.0~7.0,121 DEG C of sterilizing 30min of medium pH.

8. a kind of application for preventing and treating succulent brown rot composite bacteria agent according to claim 1, is characterized in that, will be compound Microbial inoculum is diluted by 1:1000~1:500 water, is then administered to succulent blade using foliage-spray mode or is used root filling side Formula is administered to succulent root.