CN107523524B

CN107523524B - A kind of composite bacteria agent and its preparation method and application of prevention tomato verticillium wilt

Info

Publication number: CN107523524B
Application number: CN201710947727.4A
Authority: CN
Inventors: 孙海远; 赵钢勇; 顾欣燕; 刘亚利
Original assignee: Tianjin Kunhe Biological Group Co ltd
Current assignee: Tianjin Development Zone Kunhe Biotechnology Co ltd
Priority date: 2017-10-12
Filing date: 2017-10-12
Publication date: 2018-08-14
Anticipated expiration: 2037-10-12
Also published as: CN107523524A

Abstract

The present invention relates to a kind of composite bacteria agent and its preparation method and application of prevention tomato verticillium wilt, the composite bacteria agent by the tunning of bacillus amyloliquefaciens MES812, bacillus subtilis MES810 tunnings and bacillusmusilaginosiengineering MES803 tunning by volume 1:1‑3:15 mix.The preparation method of strain is as follows in the composite bacteria agent：Actication of culture；The preparation of primary seed solution；The preparation of secondary seed solution：Ferment tank, discharging obtain tunning.The present invention utilizes the large area of main effect microorganism in the soil in composite bacteria agent to breed, the growth of antagonism pathogen, effectively inhibit or kill harmful microorganism in soil, it is provided with force environment for beneficial microorganism breeding and development, passes through the continuous generation for forming effectively prevention tomato verticillium wilt of beneficial microbe metabolite.Soil is improved simultaneously, soil fertility is improved, increases tomato yield and quality.

Description

A kind of composite bacteria agent and its preparation method and application of prevention tomato verticillium wilt

Technical field

The invention belongs to field of agricultural biotechnology, be related to a kind of composite bacteria agent of prevention tomato verticillium wilt and preparation method thereof and Using.

Background technology

Tomato is the herbaceous plant in Solanaceae tomato genus, the value with food and medicament dual-purpose.Recently as tomato cultivation face Product constantly expands, in addition plant year after year, the rotation of crops is difficult, and tomato verticillium wilt disease is made to aggravate year by year, and morbidity field is caused significantly to subtract Production also seriously reduces the quality of tamato fruit and its is made to lose commodity.Verticillium wilt has become most important illness in greenhouse tomato One of, it is the bottleneck for limiting tomato production and commercialization.Verticillium wilt is by verticillium dahliae (Verticillium dahlia Kieb) cause soil-borne vascular bundle disease, mostly occur in the tomato growth middle and later periods, first xanthelasma occur between lower blade lateral vein Refute, rear blade gradually turns yellow from bottom to top, plant occur blade noon wilt, morning reply the phenomenon that after it is slowly withered.Vertical profile Stalk, middle and upper part is hollow, and vascular bundle white, lower part stalk xylem is brown, and vascular bundle is green or light green, and diseased plant is pulled out It is brown to go out visible part root.At present prevention tomato verticillium wilt method be mainly crop rotation and apply chemical agent.Crop rotation for Industrialized agriculture feasibility is small, though and can effectively prevent the generation of tomato verticillium wilt using chemical agent, it is more toxic, sternly Heavily contaminated soil, long-time service can also make pathogenic bacteria develop immunity to drugs, and seriously endanger the ecological balance.Through inquiry, profit there is no at present The report of tomato verticillium wilt is prevented with composite bacillus microbial bacterial agent.

Invention content

The deficiency that the purpose of the present invention is prevented for tomato verticillium wilt in the prior art, provides a kind of prevention tomato yellow The composite bacteria agent and its preparation method and application for disease of withering, the method for the present invention is easy to operate, and prevention tomato verticillium wilt is with obvious effects, shows Writing reduces the generation of tomato verticillium wilt, at the same time it can also improve the yield and quality of tomato.The present invention utilizes main in composite bacteria agent The large area breeding of microorganism in the soil is imitated, the growth of antagonism pathogen effectively inhibits or kills harmful microorganism in soil, It is provided with force environment for beneficial microorganism breeding and development, passes through the continuous formation effectively prevention of beneficial microbe metabolite The generation of tomato verticillium wilt.Meanwhile complex micro organism fungicide can also promote the release of soil nutrient, promote soil aggregate structure It is formed, soil is improved, in short, the method for the present invention has been repaired soil microenvironment, carried while preventing tomato soil borne disease High soil fertility, increases tomato yield and quality.

To achieve the above object, the present invention adopts the following technical scheme that：

A kind of composite bacteria agent of prevention tomato verticillium wilt, the composite bacteria agent is by bacillus amyloliquefaciens (Bacillus Amyloliquefaciens) the hair of the tunning of MES812, bacillus subtilis (Bacillus subtilis) MES810 The tunning of ferment product and bacillusmusilaginosiengineering (Bacillus mucilaginosus) MES803 by volume 1:1-3: 1-5 is mixed；

Bacillus subtilis (Bacillus subtilis) MES810 is deposited in China General Microbiological strain guarantor Administrative center is hidden, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and the deposit date is Augusts in 2017 10 days, protect It is CGMCCNo.14514 to hide number；

Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) MES812 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 2017 8 The moon 10, deposit number CGMCCNo.14515；

Bacillusmusilaginosiengineering (Bacillus mucilaginosus) MES803, 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 Augusts 10 in 2017 Day, deposit number CGMCCNo.14513.

The B. subtilis cell is rod-shaped, Gram-positive, and size is 0.6-0.8 μm of * 2.0-3.5 μm, gemma Middle life, ellipse, sporangium are not expanded.On broth bouillon, 48h bacterium colonies are round, and edge is irregular, dry tack free, flat, White.Positive reaction：Catalase；Oxidizing ferment；Hydrolyze starch.Negative reaction：Anaerobic growth, lecithinase；Propionate utilizes, breast Sugared fermentation and acid.

Multiplexed PCR amplification is carried out using special primer, which generates unique amplified production, stripe size and withered grass bud Spore bacillus (Bacillus subtilis) is identical.

Bacillus subtilis MES810 is that my company research staff detaches in the potato field of Zhangjiakou, is passed through Plate streaking is cultivated 24 hours, bevel strain at 32 DEG C, 4 DEG C of refrigerations.Then picking single bacterium colony is sliced, crystallized purple Dyeing, microscopy are determined as bacillus subtilis.Using the matrimony vine pine root fungus sickle-like bacteria of separation as target, with bacillus to it Carry out tablet face-off experiment, 30 DEG C of cultures find that the bacillus can significantly inhibit growth of pathogenic bacteria after 4 days, occur compared with Wide antibacterial band, is named as MES810, identifies that MES810 is bacillus subtilis by identification mechanism of the Ministry of Agriculture.

The bacillus amyloliquefaciens morphological feature：Bacillus amyloliquefaciens are rod-shaped, Gram-positive, and size is 0.6-0.8 μm of * 2.0-4.5 μm, raw in gemma, ellipse, sporangium is not expanded.On broth bouillon, 48h bacterium colonies are round, Micro- protuberance, edge is irregular, and color is dark.Positive reaction：Catalase；Oxidizing ferment；Hydrolyze starch.Negative reaction：Anaerobic growth, lecithin Lipase；Propionate utilizes, lactose fermentation production acid.Multiplexed PCR amplification is carried out using special primer, which generates unique amplification Product, stripe size are identical as bacillus amyloliquefaciens (Bacillus amyloliquefaciens).

Bacillus amyloliquefaciens MES812 is that my company research staff detaches in the matrimony vine ground of peaceful Xia Zhongning city Zhongning County , it by plate streaking, is cultivated 24 hours at 32 DEG C, bevel strain, 4 DEG C of refrigerations.Then picking single bacterium colony is sliced, Crystallized purple dyeing, microscopy are determined as bacillus amyloliquefaciens.Using the matrimony vine pine root fungus sickle-like bacteria of separation as target, with solution Bacillus amyloliquefaciens carry out it tablet face-off experiment, and 30 DEG C of cultures find that the bacillus can significantly inhibit after 4 days There is wider antibacterial band, is named as MES812 in growth of pathogenic bacteria, is identified by identification mechanism of the Ministry of Agriculture, and MES812 is Bacillus amyloliquefaciens.

The morphological feature of this plant of bacillusmusilaginosiengineering and identification：Bacillusmusilaginosiengineering cell column, Gram's staining Feminine gender, size are 0.9-1.0 μm of * 2.5-4.0 μm.Gemma ellipse, middle life, sporangium is micro- to expand.Poly- β-hydroxyl is generated into the cell Base butyrate (PHB) particle.The well-grown on silicate bacteria culture medium generates thick-walled pod film, and bacterium colony is big and justifies, protrusion, thoroughly Bright, surface is smooth, and quality is sticky full of elasticity, is not easy to provoke.Positive reaction：Catalase.Negative reaction：Anaerobic growth, lecithin Enzyme；Oxidizing ferment, nutrient growth.

16S rDNA sequence analyses：Genomic DNA is extracted from thalline, 16S r DNA cloning is carried out using universal primer, Direct Sequencing after PCR product is purified.The 16S rDNA sequences surveyed are related to GenBank databases after proofreading, splicing The sequence of kind carries out BLAST comparisons, the results showed that, the 16S rDNA sequences and bacillusmusilaginosiengineering of the bacterial strain The sequence homology of (Bacillus mucilaginosus) is 10 0%.

This plant of bacillusmusilaginosiengineering MES803 is that my company research staff detaches in the farm tomato ground of the Wuqing village great Meng , it by plate streaking, is cultivated 24 hours at 35 DEG C, bevel strain, 4 DEG C of refrigerations.Then picking single bacterium colony is sliced, Crystallized purple dyeing, microscopy are determined as bacillusmusilaginosiengineering.Using the tomato verticillium wilt of separation as target, with gel-shaped gemma Bacillus carries out it tablet face-off experiment, and 30 DEG C of cultures find that the bacillus can significantly inhibit pathogen life after 4 days It is long, there is wider antibacterial band, be named as MES803, identified by identification mechanism of the Ministry of Agriculture, MES803 is gel-shaped bud Spore bacillus.

Preferably, withered grass gemma in the tunning of bacillus subtilis (Bacillus subtilis) MES810 The viable count of bacillus (Bacillus subtilis) MES810 is no less than 2.0*1010cFu/g；

Starch is solved in the tunning of described solution starch bud bacillus (Bacillus amyloliquefaciens) MES812 The viable count of bacillus (Bacillus amyloliquefaciens) MES812 is no less than 2.0*10¹⁰cFu/g；

In the tunning of bacillusmusilaginosiengineering (Bacillus mucilaginosus) MES803, gel-shaped bud The viable count of spore bacillus (Bacillus mucilaginosus) MES803 is no less than 2.0*10¹⁰cFu/g。

Preferably, the composite bacteria agent of the prevention tomato verticillium wilt is liquid bacterial agent.

As preferred technical solution：

The present invention also provides the preparation methods of the composite bacteria agent of above-mentioned prevention tomato verticillium wilt, specifically include following step Suddenly：

1, the preparation of bacillus subtilis (Ehrenberg) Cohn fermented product：

(1) actication of culture：In 250mL conical flasks be packed into 50mL sterile waters, picking bacillus subtilis slant strains in It in conical flask, shakes up, takes in 1-2mL liquid to 500mL Kolle flasks, be packed into the training of 100mL beef extract-peptones in Kolle flask in advance Base is supported, Kolle flask is placed under the conditions of 32 DEG C -36 DEG C and cultivates 24-36h, must activate by 116 DEG C of -121 DEG C of moist heat sterilization 20-35min Strain；

(2) preparation of primary seed solution：The Kolle flask for filling activated spawn above is taken out, it is sterile to pour into 50-100mL Bacteria suspension is made in water, scraping bacterium tire, its whole is poured into the seed bottle equipped with 100-300mL and temperature for 4 DEG C of sterile water, Obtain primary seed solution；

(3) preparation of secondary seed solution：150L secondary seed mediums are housed in 300L fermentation tanks, by 100-300mL Primary seed solution is inoculated in secondary seed medium；In 30 DEG C of -38 DEG C of culture 12-24h；Obtain secondary seed culture solution；

(4) ferment tank：The bacillus subtilis hair of fermenter volume 50%-60% is packed into 3000L fermentation tanks 150L or 200L secondary seed solutions are all inoculated in withered grass bud and embraced by ferment culture medium, 115 DEG C of -122 DEG C of moist heat sterilization 20-40min In bacillus viable bacteria fermentation culture medium, control tank presses 0.02MPa-0.07MPa, after 30 DEG C of -38 DEG C of culture 24-36h, discharges To the tunning of bacillus subtilis；

2, the preparation of bacillus amyloliquefaciens tunning：

(1) actication of culture：50mL sterile waters, picking bacillus amyloliquefaciens slant strains are packed into 250mL conical flasks It in conical flask, shakes up, takes in 1-2mL liquid to 500mL Kolle flasks, be packed into 100mL beef extract-peptones in Kolle flask in advance Culture medium, 116 DEG C of -121 DEG C of moist heat sterilization 20-35min, Kolle flask is placed under the conditions of 32 DEG C -36 DEG C and cultivates 24-36h, obtains work Change strain；

(4) ferment tank：The bacillus subtilis hair of fermenter volume 50%-60% is packed into 3000L fermentation tanks 150L or 200L secondary seed solutions are all inoculated in solution starch bud by ferment culture medium, 115 DEG C of -122 DEG C of moist heat sterilization 20-40min In spore bacillus viable bacteria fermentation culture medium, control tank presses 0.02MPa-0.07MPa, after 30 DEG C of -38 DEG C of culture 24-36h, discharges To bacillus amyloliquefaciens tunning；

3, the preparation of bacillusmusilaginosiengineering tunning：

(1) shaking flask culture：Inoculation through slant activation is subjected to seed flask culture in Shake flask medium, is cultivated Condition is：Rotating speed 180-200r/min, 30 DEG C -33 DEG C of temperature cultivate 10-12h, and shake flask culture is made；

(2) first order seed culture：100mL primary-seed mediums are housed, 116 DEG C -121 DEG C damp and hot in 500mL eggplant bottles Sterilize 20-35min, above-mentioned gel-shaped bud pole bacterium shake flask culture is all inoculated in primary-seed medium, 30 DEG C- 33 DEG C of culture 36-48h, obtain primary seed solution；

(3) secondary seed culture：150L secondary seed mediums are housed in 300L fermentation tanks, 115 DEG C -122 DEG C damp and hot Sterilize 20-40min, and 100-300mL primary seed solutions are inoculated in secondary seed medium；In 30 DEG C of -33 DEG C of culture 8- 14h obtains secondary seed solution；

(4) it ferments：The bacillusmusilaginosiengineering fermentation training of fermenter volume 50%-60% is packed into 3000L fermentation tanks Base is supported, the secondary seed solution of 150L or 200L are inoculated in bacillusmusilaginosiengineering by 115 DEG C of -122 DEG C of moist heat sterilization 20-40min In viable bacteria fermentation culture medium, control tank presses 0.02MPa-0.07MPa, and after 30 DEG C of -33 DEG C of culture 36-48h, discharging is fermented Product；

4, by the tunning of bacillus amyloliquefaciens, the tunning of bacillus subtilis and bacillusmusilaginosiengineering Tunning by volume 1:1-3:1-5 is mixed, and obtains the composite bacteria agent of the prevention tomato verticillium wilt.

Preferably, beef-protein medium phase used by the bacillus amyloliquefaciens and bacillus subtilis Together, specifically, as mass fraction：Beef extract 0.5%-3%, peptone 0.5%-3%, sodium chloride 0.2-1%, nutrition fine jade Fat 1.5%-2.0%, surplus are water, medium pH 6.0-7.5.

Preferably, secondary seed medium used by the bacillus amyloliquefaciens and bacillus subtilis at split-phase Together, specifically, as mass fraction：Glucose 0.3%-2%, corn flour 1%-5%, bean cake powder 1%-5%, phosphoric acid hydrogen two Sodium 0.1 ‰ -0.5 ‰, sodium dihydrogen phosphate 0.5 ‰ -5 ‰, antifoaming agent 0.2 ‰ -2 ‰, surplus is water, and medium pH is adjusted to 6.0- 7.5, and it is spare in 115 DEG C of -122 DEG C of moist heat sterilization 20-40min.

Preferably, the bacillus amyloliquefaciens are identical with the ingredient of fermentation medium used by bacillus subtilis, Specifically, as mass fraction：Glucose 0.3%-2%, corn flour 1%-5%, bean cake powder 1%-5%, disodium hydrogen phosphate 0.1 ‰ -0.5 ‰, sodium dihydrogen phosphate 0.5 ‰ -5 ‰, antifoaming agent 0.2 ‰ -2 ‰, surplus is water, and the pH of culture medium is adjusted to 6.0- 7.5, and in 115 DEG C of -122 DEG C of moist heat sterilization 20-40min.

Preferably, the Shake flask medium, as mass fraction for：Cornstarch 0.5%-2%, bean cake powder 0.2%- 2%, white sugar 0.5%-3%, wheat bran 1%-3%, potassium dihydrogen phosphate 0.5 ‰ -2 ‰, ferric trichloride 0.2%-2%, magnesium sulfate 0.5 ‰ -2 ‰, surplus is water, and 116 DEG C of -121 DEG C of moist heat sterilization 20-35min are spare；

The primary-seed medium, as mass fraction for：Cornstarch 0.5%-2%, bean cake powder 0.2%-2%, White sugar 0.5%-3%, wheat bran 1%-3%, potassium dihydrogen phosphate 0.5 ‰ -2 ‰, ferric trichloride 0.2%-2%, magnesium sulfate 0.5 ‰ - 2 ‰, surplus is water；

The secondary seed medium, as mass fraction for：Cornstarch 0.5%-2%, bean cake powder 0.2%-2%, White sugar 0.5%-3%, wheat bran 1%-3%, potassium dihydrogen phosphate 0.5 ‰ -2 ‰, ferric trichloride 0.2%-2%, magnesium sulfate 0.5 ‰ - 2 ‰, antifoaming agent 0.2 ‰ -2 ‰, surplus is water；

The fermentation medium, as mass fraction for：Cornstarch 0.5%-2%, bean cake powder 0.2%-2%, white sugar 0.5%-3%, wheat bran 1%-3%, potassium dihydrogen phosphate 0.5 ‰ -2 ‰, ferric trichloride 0.2%-2%, magnesium sulfate 0.5 ‰ -2 ‰, Surplus is water.

The present invention also provides the applications of composite bacteria agent, and the composite bacteria agent is diluted with water after 300 times with base manure together Apply soil in, composite bacteria agent dilution after additive amount be 5-10kg/ per acre.

Advantageous effect：

The preparation method of composite bacteria agent of the present invention is easy to operate, and prevention tomato verticillium wilt is with obvious effects, significantly reduces tomato The generation of verticillium wilt, at the same time it can also improve the yield and quality of tomato.

The present invention utilizes the large area of main effect microorganism in the soil in composite bacteria agent to breed, the growth of antagonism pathogen, Effectively inhibit or kill harmful microorganism in soil, force environment is provided with for beneficial microorganism breeding and development, by beneficial The continuous generation for forming effectively prevention tomato verticillium wilt of microbial metabolic products.Meanwhile complex micro organism fungicide can also promote Into the release of soil nutrient, soil aggregate structure is promoted to be formed, improve soil, in short, the method for the present invention is passed in prevention tomato soil While disease, soil microenvironment has been repaired, has improved soil fertility, has increased tomato yield and quality.

Specific implementation mode

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 composite bacteria agent of prevention tomato verticillium wilt, the composite bacteria agent is by bacillus amyloliquefaciens (Bacillus Amyloliquefaciens) the hair of the tunning of MES812, bacillus subtilis (Bacillus subtilis) MES810 The tunning of ferment product and bacillusmusilaginosiengineering (Bacillus mucilaginosus) MES803 by volume 1:3:4 is mixed It closes；

16S rDNA sequence analyses：Genomic DNA is extracted from thalline, 16S rDNA amplifications are carried out using universal primer, Direct Sequencing after PCR product is purified.The 16S rDNA sequences surveyed are related to GenBank databases after proofreading, splicing The sequence of kind carries out BLAST comparisons, the results showed that, the 16S rDNA sequences and bacillusmusilaginosiengineering of the bacterial strain The sequence homology of (Bacillus mucilaginosus) is 100%.

1, the preparation of bacillus subtilis (Ehrenberg) Cohn fermented product：

(1) actication of culture：50mL sterile waters are packed into 250mL conical flasks, picking slant strains are shaken in conical flask It is even, it takes in 1mL liquid to 500mL Kolle flasks, is packed into 100mL beef-protein mediums in Kolle flask in advance, 120 DEG C are damp and hot Sterilize 30min, and Kolle flask is placed under the conditions of 34 DEG C and cultivates 30h, obtains activated spawn；

(2) preparation of primary seed solution：The Kolle flask for filling activated spawn above is taken out, 80mL sterile waters is poured into, scrapes It takes bacterium tire that bacteria suspension is made, its whole is poured into the seed bottle equipped with 200mL and temperature for 4 DEG C of sterile water, level-one kind is obtained Sub- liquid；

(3) preparation of secondary seed solution：150L secondary seed mediums are housed in 300L fermentation tanks, by 200mL level-ones Seed liquor is inoculated in secondary seed medium；20h is cultivated at 35 DEG C；Obtain secondary seed culture solution；

(4) ferment tank：The fermentation of bacillus subtilis culture of fermenter volume 55% is packed into 3000L fermentation tanks 200L secondary seed solutions are all inoculated in bacillus subtilis viable bacteria fermentation culture medium by base, and control tank presses 0.05MPa, 35 DEG C of culture 30h；Obtain bacillus subtilis (Ehrenberg) Cohn fermented product.In fermentation a period of time, such as after 24 hours, every 40 minutes from three Angle bottle in sampling carry out microscopy, in the visual field gemma and total thalline number count, and calculate gemma rate (gemma rate (%)= Grown spore number/(grown spore number+thalline number) × 100)；Gemma rate stops fermented and cultured when reaching 90%, discharging obtains withered Careless bacillus liquid preparation.

Wherein, beef-protein medium is specifically, as mass fraction：Beef extract 3%, peptone 0.5%, chlorination Sodium 1%, nutrient agar 1.5%, surplus are water, medium pH 7.0.

Wherein, the ingredient of secondary seed medium is, as mass fraction：Glucose 1.5%, corn flour 3%, bean cake powder 3%, disodium hydrogen phosphate 0.25 ‰, sodium dihydrogen phosphate 2 ‰, antifoaming agent 1 ‰, surplus is water, and medium pH is adjusted to 7.0, and 118 DEG C moist heat sterilization 30min is spare.

Wherein, the ingredient of fermentation medium is, as mass fraction：Glucose 1%, corn flour 4%, bean cake powder 3%, phosphorus Sour disodium hydrogen 0.3 ‰, sodium dihydrogen phosphate 4 ‰, antifoaming agent 1.5 ‰, surplus are water, and the pH of culture medium is adjusted to 7.1, and at 118 DEG C Moist heat sterilization 35min.

2, the preparation of bacillus amyloliquefaciens tunning：

(1) actication of culture：50mL sterile waters are packed into 250mL conical flasks, picking slant strains are shaken in conical flask It is even, it takes in 1mL liquid to 500mL Kolle flasks, is packed into 100mL beef-protein mediums in Kolle flask in advance, 116 DEG C are damp and hot Sterilize 35min, and Kolle flask is placed under the conditions of 32 DEG C and cultivates 36h, obtains activated spawn；

(2) preparation of primary seed solution：The Kolle flask for filling activated spawn above is taken out, 50mL sterile waters is poured into, scrapes It takes bacterium tire that bacteria suspension is made, its whole is poured into the seed bottle equipped with 100mL and temperature for 4 DEG C of sterile water, level-one kind is obtained Sub- liquid；

(3) preparation of secondary seed solution：150L secondary seed mediums are housed in 300L fermentation tanks, by 100 level-one kinds Sub- liquid is inoculated in secondary seed medium；It is cultivated for 24 hours at 30 DEG C；Obtain secondary seed culture solution；

(4) ferment tank：The fermentation of bacillus subtilis culture of fermenter volume 50% is packed into 3000L fermentation tanks 150L secondary seed solutions are all inoculated in bacillus amyloliquefaciens viable bacteria fermentation culture medium by base, and control tank presses 0.02MPa, 36h is cultivated at 30 DEG C；Discharging obtains bacillus amyloliquefaciens tunning.

Wherein, the ingredient of beef-protein medium is, as mass fraction：Beef extract 0.5%, peptone 3%, chlorine Change sodium 0.2%, nutrient agar 1.5%%, surplus is water, and the pH of culture medium is 6.0, and standby in 116 DEG C of moist heat sterilization 20min With.

Wherein, the ingredient of secondary seed medium is, as mass fraction：Glucose 2%, corn flour 1%, bean cake powder 5%, disodium hydrogen phosphate 0.5 ‰, sodium dihydrogen phosphate 0.5 ‰, antifoaming agent 0.2 ‰, surplus is water, and medium pH is adjusted to 7.5, and 122 DEG C of moist heat sterilization 40min are spare.

Wherein, the ingredient of fermentation medium is, as mass fraction：Glucose 0.3%, corn flour 5%, bean cake powder 5%, Disodium hydrogen phosphate 0.1 ‰, sodium dihydrogen phosphate 5 ‰, antifoaming agent 2 ‰, surplus are water, and the pH of culture medium is adjusted to 6.0, and at 115 DEG C Moist heat sterilization 40min.

3, the preparation of bacillusmusilaginosiengineering tunning：

(1) shaking flask culture：Inoculation through slant activation is subjected to seed flask culture in Shake flask medium, is cultivated Condition is：Rotating speed 180r/min, 30 DEG C of temperature cultivate 10h, and shake flask culture is made；

(2) first order seed culture：100mL primary-seed mediums, 116 DEG C of moist heat sterilizations are housed in 500mL eggplant bottles Above-mentioned gel-shaped bud pole bacterium shake flask culture is all inoculated in primary-seed medium by 20min, and 36h is cultivated at 30 DEG C, Obtain primary seed solution；

(3) secondary seed culture：150L secondary seed mediums, 122 DEG C of moist heat sterilizations are housed in 300L fermentation tanks 100mL primary seed solutions are inoculated in secondary seed medium by 40min；In 30 DEG C of -33 DEG C of culture 8-14h, secondary seed is obtained Liquid；

(4) it ferments：The bacillusmusilaginosiengineering fermentation training of fermenter volume 50%-60% is packed into 3000L fermentation tanks Base is supported, the secondary seed solution of 150L or 200L is inoculated in bacillusmusilaginosiengineering viable bacteria fermentation by 115 DEG C of moist heat sterilization 20min In culture medium, control tank presses 0.02MPa-0.07MPa, and after cultivating 48h at 33 DEG C, discharging obtains tunning；

4, by the tunning of bacillus amyloliquefaciens, the tunning of bacillus subtilis and bacillusmusilaginosiengineering Tunning by volume 1:3:5 mixing, obtain the composite bacteria agent of prevention tomato verticillium wilt.

Embodiment 2

A kind of composite bacteria agent of prevention tomato verticillium wilt, the composite bacteria agent is by bacillus amyloliquefaciens (Bacillus Amyloliquefaciens) the hair of the tunning of MES812, bacillus subtilis (Bacillus subtilis) MES810 The tunning of ferment product and bacillusmusilaginosiengineering (Bacillus mucilaginosus) MES803 by volume 1:2:3 is mixed It closes；

1, the preparation of bacillus subtilis (Ehrenberg) Cohn fermented product：

(4) ferment tank：The fermentation of bacillus subtilis culture of fermenter volume 50% is packed into 3000L fermentation tanks 150L secondary seed solutions are all inoculated in bacillus subtilis viable bacteria fermentation culture medium by base, and control tank presses 0.02MPa, 30 DEG C of culture 36h；Discharging obtains bacillus subtilis liquid fermentation production.

Wherein, the ingredient of beef-protein medium is, as mass fraction：Beef extract 0.5%, peptone 3%, chlorine Change sodium 0.2%, nutrient agar 1.5%, surplus is water, and the pH of culture medium is 6.0, and spare in 116 DEG C of moist heat sterilization 20min.

2, the preparation of bacillus amyloliquefaciens tunning：

(4) ferment tank：The fermentation of bacillus subtilis culture of fermenter volume 55% is packed into 3000L fermentation tanks 200L secondary seed solutions are all inoculated in bacillus amyloliquefaciens viable bacteria fermentation culture medium by base, and control tank presses 0.05MPa, 30h is cultivated at 35 DEG C；Obtain zymotic fluid, i.e. bacillus amyloliquefaciens tunning.In fermentation a period of time, such as after 24 hours, Every 40 minutes from triangular flask sampling carry out microscopy, in the visual field gemma and total thalline number count, and calculate gemma Rate (gemma rate (%)=grown spore number/(grown spore number+thalline number) × 100)；Gemma rate stops fermentation when reaching 90% Culture, discharging obtain bacillus amyloliquefaciens tunning.

3, the preparation of bacillusmusilaginosiengineering tunning：

(1) shaking flask culture：Inoculation through slant activation is subjected to seed flask culture in Shake flask medium, is cultivated Condition is：Rotating speed 200r/min, 33 DEG C of temperature cultivate 12h, and shake flask culture is made；

(2) first order seed culture：100mL primary-seed mediums, 121 DEG C of moist heat sterilizations are housed in 500mL eggplant bottles Above-mentioned gel-shaped bud pole bacterium shake flask culture is all inoculated in primary-seed medium by 35min, and 48h is cultivated at 33 DEG C, Obtain primary seed solution；

(3) secondary seed culture：150L secondary seed mediums, 115 DEG C of moist heat sterilizations are housed in 300L fermentation tanks 100mL primary seed solutions are inoculated in secondary seed medium by 20min；14h is cultivated at 33 DEG C, obtains secondary seed solution；

(4) it ferments：The bacillusmusilaginosiengineering fermentation medium of fermenter volume 60% is packed into 3000L fermentation tanks, The secondary seed solution of 150L is inoculated in bacillusmusilaginosiengineering viable bacteria fermentation culture medium by 115 DEG C of moist heat sterilization 40min, control Tank processed presses 0.07MPa, and after cultivating 36h at 30 DEG C, discharging obtains tunning；

4, by the tunning of bacillus amyloliquefaciens, the tunning of bacillus subtilis and bacillusmusilaginosiengineering Tunning by volume 1:2:3 mixing obtain the composite bacteria agent of the prevention tomato verticillium wilt.

Embodiment 3

A kind of composite bacteria agent of prevention tomato verticillium wilt, the composite bacteria agent is by bacillus amyloliquefaciens (Bacillus Amyloliquefaciens) the hair of the tunning of MES812, bacillus subtilis (Bacillus subtilis) MES810 The tunning of ferment product and bacillusmusilaginosiengineering (Bacillus mucilaginosus) MES803 by volume 1:2:4 is mixed It closes；

1, the preparation of bacillus subtilis (Ehrenberg) Cohn fermented product：

(1) actication of culture：50mL sterile waters are packed into 250mL conical flasks, picking slant strains are shaken in conical flask It is even, it takes in 2mL liquid to 500mL Kolle flasks, is packed into 100mL beef-protein mediums in Kolle flask in advance, 121 DEG C are damp and hot Sterilize 20min, and Kolle flask is placed under the conditions of 36 DEG C and is cultivated for 24 hours, activated spawn is obtained；

(2) preparation of primary seed solution：The Kolle flask for filling activated spawn above is taken out, 100mL sterile waters are poured into, Bacteria suspension is made in scraping bacterium tire, its whole is poured into the seed bottle equipped with 300mL and temperature for 4 DEG C of sterile water, level-one is obtained Seed liquor；

(3) preparation of secondary seed solution：150L secondary seed mediums are housed in 300L fermentation tanks, by 300mL level-ones Seed liquor is inoculated in secondary seed medium；12h is cultivated at 38 DEG C；Obtain secondary seed culture solution；

(4) ferment tank：The fermentation of bacillus subtilis culture of fermenter volume 60% is packed into 3000L fermentation tanks 200L secondary seed solutions are all inoculated in bacillus subtilis viable bacteria fermentation culture medium by base, and control tank presses 0.07MPa, 38 DEG C of cultures are for 24 hours；Discharging obtains bacillus subtilis (Ehrenberg) Cohn fermented product.

Wherein, the ingredient of beef-protein medium is, as mass fraction：Beef extract 3%, peptone 0.5%, chlorine Change sodium 1%, nutrient agar 2.0%, surplus is water, and the pH of culture medium is 7.5, and spare in 121 DEG C of moist heat sterilization 35min.

Wherein, the ingredient of secondary seed medium is, as mass fraction：Glucose 0.3%, corn flour 5%, bean cake powder 1%, disodium hydrogen phosphate 0.5 ‰, sodium dihydrogen phosphate 5 ‰, antifoaming agent 2 ‰, surplus is water, and medium pH is adjusted to 7.5, and 122 DEG C moist heat sterilization 40min is spare.

Wherein, the ingredient of fermentation medium is, as mass fraction：Glucose 2%, corn flour 1%, bean cake powder 1%, phosphorus Sour disodium hydrogen 0.5 ‰, sodium dihydrogen phosphate 5 ‰, antifoaming agent 2 ‰, surplus are water, and the pH of culture medium is adjusted to 6.0, and wet at 122 DEG C Heat sterilization 20min.

2, the preparation of bacillus amyloliquefaciens tunning：

(1) actication of culture：50mL sterile waters are packed into 250mL conical flasks, picking slant strains are shaken in conical flask It is even, it takes in 1.5mL liquid to 500mL Kolle flasks, is packed into 100mL beef-protein mediums in Kolle flask in advance, 117 DEG C Kolle flask is placed under the conditions of 33 DEG C and cultivates 28h, obtains activated spawn by moist heat sterilization 25min；

(2) preparation of primary seed solution：The Kolle flask for filling activated spawn above is taken out, 60mL sterile waters is poured into, scrapes It takes bacterium tire that bacteria suspension is made, its whole is poured into the seed bottle equipped with 150mL and temperature for 4 DEG C of sterile water, level-one kind is obtained Sub- liquid；

(3) preparation of secondary seed solution：150L secondary seed mediums are housed in 300L fermentation tanks, by 250mL level-ones Seed liquor is inoculated in secondary seed medium；20h is cultivated at 35 DEG C；Obtain secondary seed culture solution；

(4) ferment tank：The fermentation of bacillus subtilis culture of fermenter volume 58% is packed into 3000L fermentation tanks 200L secondary seed solutions are all inoculated in bacillus amyloliquefaciens viable bacteria fermentation culture medium by base, 116 DEG C of moist heat sterilization 30min In, control tank presses 0.05MPa, and 28h is cultivated at 34 DEG C；Discharging obtains bacillus amyloliquefaciens tunning.

Wherein, the ingredient of beef-protein medium is, as mass fraction：Beef extract 1.5%, peptone 0.25%, sodium chloride 0.8%, nutrient agar 1.8%, surplus is water, and the pH of culture medium is 6.8, and in 120 DEG C of moist heat sterilizations 24min is spare.

Wherein, the ingredient of secondary seed medium is, as mass fraction：Glucose 1.5%, corn flour 3%, bean cake powder 2.5%, disodium hydrogen phosphate 0.4 ‰, sodium dihydrogen phosphate 3.4 ‰, antifoaming agent 1.5 ‰, surplus is water, and medium pH is adjusted to 7.2, and It is spare in 118 DEG C of moist heat sterilization 35min.

Wherein, the ingredient of fermentation medium is, as mass fraction：Glucose 1.4%, corn flour 2.7%, bean cake powder 2.5%, disodium hydrogen phosphate 0.2 ‰, sodium dihydrogen phosphate 2.4 ‰, antifoaming agent 1.4 ‰, surplus is water, and the pH of culture medium is adjusted to 6.9, And in 118 DEG C of moist heat sterilization 35min.

3, the preparation of bacillusmusilaginosiengineering tunning：

(1) shaking flask culture：Inoculation through slant activation is subjected to seed flask culture in Shake flask medium, is cultivated Condition is：Rotating speed 190r/min, 32 DEG C of temperature cultivate 11h, and shake flask culture is made；

(2) first order seed culture：100mL primary-seed mediums, 118 DEG C of moist heat sterilizations are housed in 500mL eggplant bottles Above-mentioned gel-shaped bud pole bacterium shake flask culture is all inoculated in primary-seed medium by 25min, and 44h is cultivated at 32 DEG C, Obtain primary seed solution；

(3) secondary seed culture：150L secondary seed mediums, 118 DEG C of moist heat sterilizations are housed in 300L fermentation tanks 200mL primary seed solutions are inoculated in secondary seed medium by 30min；10h is cultivated at 32 DEG C, obtains secondary seed solution；

(4) it ferments：The bacillusmusilaginosiengineering fermentation medium of fermenter volume 55% is packed into 3000L fermentation tanks, The secondary seed solution of 200L is inoculated in bacillusmusilaginosiengineering viable bacteria fermentation culture medium by 118 DEG C of moist heat sterilization 30min, control Tank processed presses 0.05MPa, and after cultivating 42h at 31 DEG C, discharging obtains tunning；

4, by the tunning of bacillus amyloliquefaciens, the tunning of bacillus subtilis and bacillusmusilaginosiengineering Tunning by volume 1:2:4 mixing obtain the composite bacteria agent of the prevention tomato verticillium wilt.

It is no less than 2.0* by the content of bacillus subtilis in bacillus subtilis microbial inoculum product made from the above method 10¹⁰cFu/g。

Embodiment 4

A kind of composite bacteria agent of prevention tomato verticillium wilt, the composite bacteria agent is by bacillus amyloliquefaciens (Bacillus Amyloliquefaciens) the hair of the tunning of MES812, bacillus subtilis (Bacillus subtilis) MES810 The tunning of ferment product and bacillusmusilaginosiengineering (Bacillus mucilaginosus) MES803 by volume 1:1:1 is mixed It closes；

Bacillus amyloliquefaciens MES812 is that my company research staff detaches in the matrimony vine ground of peaceful Xia Zhongning city Zhongning County , it by plate streaking, is cultivated 24 hours at 32 DEG C, bevel strain, 4 DEG C of refrigerations.Then picking single bacterium colony is sliced, Crystallized purple dyeing, microscopy are determined as bacillus amyloliquefaciens.Using the matrimony vine pine root fungus sickle-like bacteria of separation as target, with solution Bacillus amyloliquefaciens carry out it tablet face-off experiment, and 30 DEG C of cultures find that the bacillus can significantly inhibit disease after 4 days Opportunistic pathogen is grown, and wider antibacterial band is occurred, is named as MES812, is identified by identification mechanism of the Ministry of Agriculture, and MES812 is solution Bacillus amyloliquefaciens.

1, the preparation of bacillus subtilis (Ehrenberg) Cohn fermented product：

(4) ferment tank：The fermentation of bacillus subtilis culture of fermenter volume 58% is packed into 3000L fermentation tanks 200L secondary seed solutions are all inoculated in bacillus subtilis viable bacteria fermentation culture medium by base, 116 DEG C of moist heat sterilization 30min, It controls tank and presses 0.05MPa, 28h is cultivated at 34 DEG C；Discharging obtains bacillus subtilis liquid preparation.

2, the preparation of bacillus amyloliquefaciens tunning：

(4) ferment tank：The fermentation of bacillus subtilis culture of fermenter volume 60% is packed into 3000L fermentation tanks 200L secondary seed solutions are all inoculated in bacillus amyloliquefaciens viable bacteria fermentation culture medium by base, and control tank presses 0.07MPa, It is cultivated for 24 hours at 38 DEG C；Discharging obtains bacillus amyloliquefaciens tunning.

Wherein, the ingredient of beef-protein medium is, as mass fraction：Beef extract 3%, peptone 0.5%, Sodium chloride 1%, nutrient agar 2.0%, surplus are water, and the pH of culture medium is 7.5, and spare in 121 DEG C of moist heat sterilization 35min.

3, the preparation of bacillusmusilaginosiengineering tunning：

(1) shaking flask culture：Inoculation through slant activation is subjected to seed flask culture in Shake flask medium, is cultivated Condition is：Rotating speed 185r/min, 31 DEG C of temperature cultivate 11.5h, and shake flask culture is made；

(2) first order seed culture：100mL primary-seed mediums, 119 DEG C of moist heat sterilizations are housed in 500mL eggplant bottles Above-mentioned gel-shaped bud pole bacterium shake flask culture is all inoculated in primary-seed medium by 29min, and 39h is cultivated at 31 DEG C, Obtain primary seed solution；

(3) secondary seed culture：150L secondary seed mediums, 120 DEG C of moist heat sterilizations are housed in 300L fermentation tanks 200mL primary seed solutions are inoculated in secondary seed medium by 30min；12h is cultivated at 32 DEG C, obtains secondary seed solution；

(4) it ferments：The bacillusmusilaginosiengineering fermentation medium of fermenter volume 53% is packed into 3000L fermentation tanks, The secondary seed solution of 200L is inoculated in bacillusmusilaginosiengineering viable bacteria fermentation culture medium by 119 DEG C of moist heat sterilization 35min, control Tank processed presses 0.05MPa, and after cultivating 40h at 32 DEG C, discharging obtains tunning；

4, by the tunning of bacillus amyloliquefaciens, the tunning of bacillus subtilis and bacillusmusilaginosiengineering Tunning by volume 1:1:1 mixing obtains the composite bacteria agent of the prevention tomato verticillium wilt.

Two, Function detection

1, antagonism test of the complex micro organism fungicide produced by the present invention to tomato verticillium wilt

For examination tomato verticillium wilt germ source：The Tianjin Wuqing District village great Meng farm is picked up from the germ strain of tomato verticillium wilt, is passed through Company researcher isolates and purifies, and is accredited as verticillium dahliae (Verticillium dahlia Kieb), Pathogenic Tests table It is now High pathogenicity.

(1) tablet of bacillus subtilis MES810, bacillus amyloliquefaciens MES812, bacillusmusilaginosiengineering MES803 Face-off experiment：

Tomato verticillium wilt germ is coated in beef extract-peptone tablet center first, respectively by the withered grass after activation obtained Bacillus MES810, bacillus amyloliquefaciens MES812, bacillusmusilaginosiengineering MES803 points are connected on away from indicator bacteria bacterium piece 2.0 centimeters, if blank control.33 degrees Celsius of constant temperature incubations measure tomato when blank control will cover with entire culture dish The control increment (colony radius) of verticillium wilt germ and the (inhibition after inoculation MES810, MES812, MES803 of processing increment Grow radius), with antagonism bacteriostasis rate (bacteriostasis rate (%)=(control increment-processing increment)/control increment * 100) fungistatic effect is characterized, shown in table 1 specific as follows.

1 bacillus subtilis MES810 of table, bacillus amyloliquefaciens MES812, bacillusmusilaginosiengineering MES803 to kind The antagonistic effect result of eggplant verticillium wilt

Strain name	Compare increment (mm)	Handle increment (mm)	Bacteriostasis rate (%)
				MES810	32.0	5.0	84.38
MES812	32.0	5.0	84.38
				MES803	32.0	4.0	87.50

Experimental result：Table 1 the result shows that bacillus subtilis MES810 and bacillus amyloliquefaciens MES812 to tomato The inhibiting rate of verticillium wilt is up to 84.38%, 11.0 millimeters of transparent antibacterial bandwidth；Bacillusmusilaginosiengineering MES803 withers to tomato yellow The inhibiting rate of disease is up to 87.5%, 10.0 millimeters of transparent antibacterial bandwidth.Illustrate bacillus subtilis MES810, solution starch gemma bar Bacterium MES812, bacillusmusilaginosiengineering MES803 significantly inhibit tomato verticillium wilt, and there is prevention tomato yellow to wither The Biocontrol Potential of disease.

2. the application and control effect of the method for the present invention

The control effect experiment of 2.1 pairs of complex micro organism fungicides of the invention

(1) test medicine

1. complex micro organism fungicide：The complex micro organism fungicide (being prepared by the method for embodiment 4) prepared using 3.2, is used Water dilutes 300 times；

2. chemical agent：50% carbendazol wettable powder is diluted with water 500 times.

3. blank control：Clear water.

(2) test method：Seedling Stage tomato seedling is transplanted in the plastic flowerpot for being 30cm to sowing bore, each handles 5 Basin (per 3 plants of basin), in triplicate.It is placed using random district's groups aligning method.Take long kind consistent to 4-5 pieces true leaf, growth potential Complex micro organism fungicide prepared by embodiment 4 is divided 2 times and is uniformly sprayed at for examination plant leaf and plants stems, chemistry by eggplant seedling Medicament compares and the processing of blank control clear water is same as above.24 as a child spray inoculation tomato verticillium wilt germ (by above-mentioned big beautiful wheel Branch bacterium (Verticillium dahlia Kieb) germ is inoculated on tomato leaf and breeds and obtain in plants stems), inoculation is dense Degree is every plant of 300-500mL；After inoculation in 25-28 degree incubators moisturizing culture 8-10 days, when blank control is fully fallen ill Incidence is investigated, and calculates disease index and control effect, specific experiment the results are shown in Table 2.

2 bacillus subtilis MES810 of table is to tomato verticillium wilt preventive effect comparative test result

Processing	Disease index	Preventive effect (%)
			Complex micro organism fungicide	9.46b	91.36
Chemical agent	7.85bc	95.88
			Blank control	90.19a	0.00

(3) experimental result：After it can be seen that complex micro organism fungicide processing in the result of table 2, disease index (9.46) Substantially less than blank control treated disease index (90.19), and it is poor with the disease index (7.85) after Chemical treatment Different not notable, MES810, MES812, MES803 complex micro organism fungicide up to 91.36%, say the control effect of tomato verticillium wilt The complex micro organism fungicide that three kinds of bright bacillus subtilis, bacillus amyloliquefaciens, bacillusmusilaginosiengineering bacterium form is to tomato Verticillium wilt control effect is preferable.

The application effect experiment of the complex micro organism fungicide of 2.2 present invention

In the Tianjin Wuqing District village great Meng farm serious three pieces of the plot of selection tomato verticillium wilt, each Parcel division is 3 1 check plot, 4 treatment regions are respectively set in cell.

Control 1：It levels land, waters according to local general planting method, basal dressing；

Processing 1：It levels land, waters according to local general planting method, basal dressing；Addition is diluted with water per acre in base manure 300 times of complex micro organism fungicide (effective viable bacteria ＞ 2.0*10¹⁰CFU/g)5kg；

Processing 2：It levels land, waters according to local general planting method, basal dressing；Addition is diluted with water per acre in base manure 100 times of bacillus subtilis microbial agent (effective viable bacteria ＞ 2.0*10¹⁰CFU/g)5kg；

Processing 3：It levels land, waters according to local general planting method, basal dressing；Addition is diluted with water per acre in base manure 100 times of bacillus amyloliquefaciens microbial inoculum (effective viable bacteria ＞ 2.0*10¹⁰CFU/g)5kg；

Processing 4：It levels land, waters according to local general planting method, basal dressing；Addition is diluted with water per acre in base manure 100 times of bacillusmusilaginosiengineering microbial inoculum (effective viable bacteria ＞ 2.0*10¹⁰CFU/g)5kg；

Various microbial inoculums are made according to the method for above-described embodiment 4.April 15 came into effect experiment, and tomato is planted after implementation, And field management is carried out according to conventional process, tomato breeding time investigates tomato verticillium wilt disease index, and August part tomato is united after receiving Count yield.As a result it see the table below：

Comparative test result of 3 complex micro organism fungicide of table to tomato verticillium wilt control effect and yield effect

Processing	Average sick seedling rate (%)	Control effect (%)	Yield (control is 100%)
				Control 1	35.2	~	100%
Processing 1	5.23	85.14	256%
				Processing 2	9.87	71.96	158%
Processing 3	10.36	70.57	152%
				Processing 4	9.53	72.93	164%

Control effect (%)=(control sick seedling rate-processing sick seedling rate)/control sick seedling rate.It can be with from above-mentioned comparing result Find out, complex micro organism fungicide of the present invention, as under base fertilizer, the pathogen in antagonism soil not only effectively controls native biography The generation of disease, while there is increasing yield and improving quality effect.Use the with obvious effects higher than each of 300 times of complex micro organism fungicide of dilution Kind bacterium dilutes 100 times of exclusive use effect, therefore, bacillus subtilis, bacillus amyloliquefaciens, bacillusmusilaginosiengineering three The complex micro organism fungicide of kind bacterium composition mutually cooperates with, best to the control effect of tomato verticillium wilt.

Microbial bacterial agent of the present invention has the characteristics that nontoxic, noresidue, does not inhibit growth and dosage few, this micro- life It during object microbial inoculum is manured into soil, can effectively inhibit growth of pathogenic bacteria, promote beneficial microorganism breeding, soil is made to remain good Microbiota, rehabilitating soil micro-ecological environment.Improve soil aggregate structure simultaneously, promotes absorption of the plant to nutrient, reduce Fertilizer and pesticide usage amount effectively reduces the effect that crop agriculture is residual, and environmental protection while reaches prevention soil-borne disease, increases matter volume increase Fruit.

The foregoing is only a preferred embodiment of the present invention, but being not limited to embodiment of above.The guarantor of the present invention Shield range is not limited thereto, any to be familiar with those skilled in the art in the technical scope of present disclosure, according to this hair Bright technical solution and its inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.

Claims

1. a kind of composite bacteria agent of prevention tomato verticillium wilt, it is characterized in that：The composite bacteria agent is by bacillus amyloliquefaciens (Bacillus amyloliquefaciens) tunning of MES812, bacillus subtilis (Bacillus subtilis) MES810 tunning and bacillusmusilaginosiengineering (Bacillus mucilaginosus) tunning of MES803 presses body Product ratio 1:1-3:1-5 is mixed；

The bacillus subtilis (Bacillus subtilis) MES810, it is deposited in China General Microbiological culture presevation pipe Reason center, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3s, and the deposit date is Augusts in 2017 10 days, and preservation is compiled Number be CGMCCNo.14514；

The bacillus amyloliquefaciens (Bacillus amyloliquefaciens) MES812, 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 Augusts 10 in 2017 Day, deposit number CGMCCNo.14515；

The bacillusmusilaginosiengineering (Bacillus mucilaginosus) MES803, it is deposited in China General Microbiological strain Preservation administrative center, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3s, the deposit date is August in 2017 10 days, Deposit number is CGMCCNo.14513；

The bacillus subtilis (Bacillus subtilis) MES810 tunning in bacillus subtilis (Bacillus subtilis) viable count of MES810 is no less than 2.0*10¹⁰cFu/g；

The solution starch bud bacillus (Bacillus amyloliquefaciens) MES812 tunning in solve starch gemma Bacillus (Bacillus amyloliquefaciens) viable count of MES812 is no less than 2.0*10¹⁰cFu/g；

The bacillusmusilaginosiengineering (Bacillus mucilaginosus) MES803 tunning in, gel-shaped gemma bar Bacterium (Bacillus mucilaginosus) viable count of MES803 is no less than 2.0*10¹⁰cFu/g。

2. a kind of composite bacteria agent of prevention tomato verticillium wilt according to claim 1, it is characterized in that：The prevention tomato yellow The composite bacteria agent for disease of withering is liquid bacterial agent.

3. according to the preparation method of the composite bacteria agent of any prevention tomato verticillium wilt of claim 1-2, which is characterized in that Specifically include following steps：

One, the preparation of bacillus subtilis (Ehrenberg) Cohn fermented product：

(1) actication of culture：50mL sterile waters are packed into 250mL conical flasks, picking bacillus subtilis slant strains are in taper In bottle, shakes up, takes in 1-2mL liquid to 500mL Kolle flasks, be packed into 100mL beef-protein mediums in Kolle flask in advance, 116 DEG C of -121 DEG C of moist heat sterilization 20-35min, Kolle flask is placed under the conditions of 32 DEG C -36 DEG C and cultivates 24-36h, obtains activated spawn；

(2) preparation of primary seed solution：The Kolle flask for filling activated spawn above is taken out, 50-100mL sterile waters is poured into, scrapes It takes bacterium tire that bacteria suspension is made, its whole is poured into the seed bottle equipped with 100-300mL and temperature for 4 DEG C of sterile water, obtains one Grade seed liquor；

(3) preparation of secondary seed solution：150L secondary seed mediums are housed in 300L fermentation tanks, by 100-300mL level-ones Seed liquor is inoculated in secondary seed medium；In 30 DEG C of -38 DEG C of culture 12-24h；Obtain secondary seed culture solution；

(4) ferment tank：The fermentation of bacillus subtilis training of fermenter volume 50%-60% is packed into 3000L fermentation tanks Base is supported, 150L or 200L secondary seed solutions are all inoculated in bacillus subtilis by 115 DEG C of -122 DEG C of moist heat sterilization 20-40min In viable bacteria fermentation culture medium, control tank presses 0.02MPa-0.07MPa, and after 30 DEG C of -38 DEG C of culture 24-36h, discharging obtains withered grass The tunning of bacillus；

Two, the preparation of bacillus amyloliquefaciens tunning：

(1) actication of culture：50mL sterile waters are packed into 250mL conical flasks, picking bacillus amyloliquefaciens slant strains are in cone It in shape bottle, shakes up, takes in 1-2mL liquid to 500mL Kolle flasks, be packed into 100mL beef extract-peptone cultures in Kolle flask in advance Base, 116 DEG C of -121 DEG C of moist heat sterilization 20-35min, Kolle flask is placed under the conditions of 32 DEG C -36 DEG C and cultivates 24-36h, must activate bacterium Kind；

(4) ferment tank：The fermentation of bacillus subtilis training of fermenter volume 50%-60% is packed into 3000L fermentation tanks Base is supported, 150L or 200L secondary seed solutions are all inoculated in solution starch gemma bar by 115 DEG C of -122 DEG C of moist heat sterilization 20-40min In bacterium viable bacteria fermentation culture medium, control tank presses 0.02MPa-0.07MPa, and after 30 DEG C of -38 DEG C of culture 24-36h, discharging is solved Bacillus amyloliquefaciens tunning；

Three, the preparation of bacillusmusilaginosiengineering tunning：

(1) shaking flask culture：Inoculation through slant activation is subjected to seed flask culture, condition of culture in Shake flask medium For：Rotating speed 180-200r/min, 30 DEG C -33 DEG C of temperature cultivate 10-12h, and shake flask culture is made；

(2) first order seed culture：100mL primary-seed mediums, 116 DEG C of -121 DEG C of moist heat sterilizations are housed in 500mL eggplant bottles Above-mentioned gel-shaped bud pole bacterium shake flask culture is all inoculated in primary-seed medium, at 30 DEG C -33 DEG C by 20-35min 36-48h is cultivated, primary seed solution is obtained；

(3) secondary seed culture：150L secondary seed mediums, 115 DEG C of -122 DEG C of moist heat sterilizations are housed in 300L fermentation tanks 100-300mL primary seed solutions are inoculated in secondary seed medium by 20-40min；In 30 DEG C of -33 DEG C of culture 8-14h, obtain Secondary seed solution；

(4) it ferments：The bacillusmusilaginosiengineering fermentation medium of fermenter volume 50%-60% is packed into 3000L fermentation tanks, 115 DEG C of -122 DEG C of moist heat sterilization 20-40min, bacillusmusilaginosiengineering viable bacteria is inoculated in by the secondary seed solution of 150L or 200L In fermentation medium, control tank presses 0.02MPa-0.07MPa, and after 30 DEG C of -33 DEG C of culture 36-48h, discharging obtains fermentation production Object；

Four, by the tunning of bacillus amyloliquefaciens, the hair of the tunning of bacillus subtilis and bacillusmusilaginosiengineering Ferment product by volume 1:1-3:1-5 is mixed, and obtains the composite bacteria agent of the prevention tomato verticillium wilt；

The bacillus amyloliquefaciens are identical with beef-protein medium used by bacillus subtilis, specifically, pressing Mass fraction meter：Beef extract 0.5%-3%, peptone 0.5%-3%, sodium chloride 0.2-1%, nutrient agar 1.5%-2.0%, Surplus is water, medium pH 6.0-7.5；

The bacillus amyloliquefaciens are identical with the ingredient of secondary seed medium used by bacillus subtilis, specifically, As mass fraction：Glucose 0.3%-2%, corn flour 1%-5%, bean cake powder 1%-5%, disodium hydrogen phosphate 0.1 ‰- 0.5 ‰, sodium dihydrogen phosphate 0.5 ‰ -5 ‰, antifoaming agent 0.2 ‰ -2 ‰, surplus is water, and medium pH is adjusted to 6.0-7.5, and 115 DEG C of -122 DEG C of moist heat sterilization 20-40min are spare；

The bacillus amyloliquefaciens are identical with the ingredient of fermentation medium used by bacillus subtilis, specifically, pressing matter Measure score meter：Glucose 0.3%-2%, corn flour 1%-5%, bean cake powder 1%-5%, disodium hydrogen phosphate 0.1 ‰ -0.5 ‰, phosphorus Acid dihydride sodium 0.5 ‰ -5 ‰, antifoaming agent 0.2 ‰ -2 ‰, surplus are water, and the pH of culture medium is adjusted to 6.0-7.5, and 115 DEG C - 122 DEG C of moist heat sterilization 20-40min；

Shake flask medium when the bacillusmusilaginosiengineering culture, as mass fraction for：Cornstarch 0.5%-2%, beans The dregs of rice powder 0.2%-2%, white sugar 0.5%-3%, wheat bran 1%-3%, potassium dihydrogen phosphate 0.5 ‰ -2 ‰, ferric trichloride 0.2%- 2%, magnesium sulfate 0.5 ‰ -2 ‰, surplus is water, and 116 DEG C of -121 DEG C of moist heat sterilization 20-35min are spare；

Primary-seed medium when the bacillusmusilaginosiengineering culture, as mass fraction for：Cornstarch 0.5%- 2%, bean cake powder 0.2%-2%, white sugar 0.5%-3%, wheat bran 1%-3%, potassium dihydrogen phosphate 0.5 ‰ -2 ‰, ferric trichloride 0.2%-2%, magnesium sulfate 0.5 ‰ -2 ‰, surplus is water；

Secondary seed medium when the bacillusmusilaginosiengineering culture, as mass fraction for：Cornstarch 0.5%- 2%, bean cake powder 0.2%-2%, white sugar 0.5%-3%, wheat bran 1%-3%, potassium dihydrogen phosphate 0.5 ‰ -2 ‰, ferric trichloride 0.2%-2%, magnesium sulfate 0.5 ‰ -2 ‰, antifoaming agent 0.2 ‰ -2 ‰, surplus is water；

Fermentation medium when the bacillusmusilaginosiengineering culture, as mass fraction for：Cornstarch 0.5%-2%, beans The dregs of rice powder 0.2%-2%, white sugar 0.5%-3%, wheat bran 1%-3%, potassium dihydrogen phosphate 0.5 ‰ -2 ‰, ferric trichloride 0.2%- 2%, magnesium sulfate 0.5 ‰ -2 ‰, surplus is water.

4. the application of the composite bacteria agent of prevention tomato verticillium wilt according to claim 1, which is characterized in that will be described compound Microbial inoculum applies in soil after being diluted with water 300 times with base manure together, composite bacteria agent dilution after additive amount be 5-10kg/ per acre.