CN107523524A

CN107523524A - A kind of composite bacteria agent for preventing and treating tomato verticillium wilt and its preparation method and application

Info

Publication number: CN107523524A
Application number: CN201710947727.4A
Authority: CN
Inventors: 孙海远; 赵钢勇; 顾欣燕; 刘亚利
Original assignee: Tianjin Kunhe Bio Technology Co ltd
Current assignee: Tianjin Development Zone Kunhe Biotechnology Co ltd
Priority date: 2017-10-12
Filing date: 2017-10-12
Publication date: 2017-12-29
Anticipated expiration: 2037-10-12
Also published as: CN107523524B

Abstract

The present invention relates to a kind of composite bacteria agent and its preparation method and application for preventing and treating tomato verticillium wilt, the composite bacteria agent by bacillus amyloliquefaciens MES812 tunning, 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 large area breeding of the main effect microorganism in soil in composite bacteria agent, the growth of antagonism pathogen, effectively suppress or kill harmful microorganism in soil, force environment is provided with for beneficial microorganism breeding and development, the generation of tomato verticillium wilt is effectively prevented and treated by the continuous formation of beneficial microbe metabolite.Improved soil simultaneously, improves soil fertility, adds tomato yield and quality.

Description

A kind of composite bacteria agent for preventing and treating tomato verticillium wilt and its preparation method and application

Technical field

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

Background technology

Tomato is the herbaceous plant in Solanaceae tomato genus, has the value of food and medicament dual-purpose.Recently as tomato cultivation face Long-pending constantly to expand, plus planting year after year, the rotation of crops is difficult, tomato verticillium wilt disease is aggravated year by year, causes morbidity field significantly to subtract Production, also seriously reduce the quality of tamato fruit and its is lost commodity.Verticillium wilt is into 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, occurred 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 phenomenon after it is slowly withered.Vertical profile Stalk, middle and upper part is hollow, and vascular bundle white, bottom 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.The method of preventing and treating tomato verticillium wilt is mainly crop rotation and using chemical agent at present.Crop rotation for Industrialized agriculture feasibility is small, though and can effectively prevent and treat the generation of tomato verticillium wilt using chemical agent, its toxicity is larger, sternly Heavily contaminated soil, long-term use can also make pathogenic bacteria develop immunity to drugs, and seriously endanger the ecological balance.Through inquiry, profit is there is no at present With the report of composite bacillus microbial bacterial agent preventing and treating tomato verticillium wilt.

The content of the invention

The purpose of the present invention is the deficiency for tomato verticillium wilt of the prior art preventing and treating, there is provided one kind preventing and treating tomato yellow Composite bacteria agent for disease of withering and its preparation method and application, the inventive method is simple to operate, prevents and treats tomato verticillium wilt positive effect, 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 Large area breeding of the microorganism in soil is imitated, the growth of antagonism pathogen, effectively suppresses or kills harmful microorganism in soil, Force environment is provided with for beneficial microorganism breeding and development, is effectively prevented and treated by the continuous formation 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 granular structure Formed, improved soil, in a word, the inventive method have been repaired soil microenvironment, carried while tomato soil borne disease is prevented and treated High soil fertility, adds tomato yield and quality.

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

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

Bacillus subtilis (Bacillus subtilis) MES810, it 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 preservation date is August in 2017 10, protects It is CGMCCNo.14514 to hide numbering；

Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) MES812, it 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 preservation 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 preservation date is August 10 in 2017 Day, deposit number CGMCCNo.14513.

The B. subtilis cell is shaft-like, 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 circular, and edge is irregular, dry tack free, flat, White.Positive reaction：Catalase；Oxidizing ferment；Hydrolysis starch.Negative reaction：Anaerobic growth, lecithinase；Propionate utilizes, breast Sugared fermentation and acid.

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

Bacillus subtilis MES810 is that my company research staff separates in the potato field of Zhangjiakou, is passed through Plate streaking, cultivated 24 hours at 32 DEG C, bevel strain, 4 DEG C of refrigerations.Then picking single bacterium colony, cut into slices, crystallized purple Dyeing, microscopy, is defined as bacillus subtilis.Using the matrimony vine pine root fungus sickle-like bacteria of separation as target, with bacillus to it Carry out flat board face-off experiment, 30 DEG C culture, 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 shaft-like, Gram-positive, and size is 0.8 μm of * 2.0-4.5 μm of 0.6-, raw in gemma, ellipse, sporangium is not expanded.On broth bouillon, 48h bacterium colonies are circular, Micro- protuberance, edge is irregular, and color is dark.Positive reaction：Catalase；Oxidizing ferment；Hydrolysis starch.Negative reaction：Anaerobic growth, lecithin Lipase；Propionate utilizes, lactose fermentation production acid.Multiplexed PCR amplification is carried out using special primer, the bacterial strain produces unique amplification Product, stripe size are identical with bacillus amyloliquefaciens (Bacillus amyloliquefaciens).

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

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 produced into the cell Base butyrate (PHB) particle.The well-grown on silicate bacteria culture medium, thick-walled pod film is produced, bacterium colony is big and justifies, raised, 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 rDNA amplifications, P are carried out using universal primer Direct Sequencing after CR products are purified.The 16S rDNA sequences surveyed kind related to GenBank databases after proofreading, splicing The sequence of category carries out BLAST comparisons, the results showed that, 16S rDNA sequences and bacillusmusilaginosiengineering (the B acillus of the bacterial strain Mucilaginosus sequence homology) is 100%.

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

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

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

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

Preferably, the composite bacteria agent of the preventing and treating tomato verticillium wilt is liquid bacterial agent.

As preferable technical scheme：

Present invention also offers the preparation method of the composite bacteria agent of above-mentioned preventing and treating tomato verticillium wilt, following step is specifically included Suddenly：

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

(1) actication of culture：In 250mL conical flasks load 50mL sterilized waters, picking bacillus subtilis slant strains in In conical flask, shake up, take 1-2mL liquid into 500mL Kolle flasks, load the training of 100mL beef extract-peptones in Kolle flask in advance Base is supported, 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 be activated Strain；

(2) preparation of primary seed solution：The Kolle flask that activated spawn is filled by more than takes out, and it is sterile to pour into 50-100mL Bacteria suspension is made in water, scraping bacterium tire, and its whole is poured into the seed bottle equipped with the sterilized water that 100-300mL and temperature are 4 DEG C, 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 nutrient solution；

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

2nd, the preparation of bacillus amyloliquefaciens tunning：

(1) actication of culture：Load 50mL sterilized waters, picking bacillus amyloliquefaciens slant strains in 250mL conical flasks In conical flask, shake up, take 1-2mL liquid into 500mL Kolle flasks, load 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：Load fermenter volume 50%-60% bacillus subtilis hair in 3000L fermentation tanks Ferment culture medium, 115 DEG C of -122 DEG C of moist heat sterilization 20-40min, 150L or 200L secondary seed solutions are all inoculated in solution starch bud In spore bacillus viable bacteria fermentation culture medium, control tank pressure 0.02MPa-0.07MPa, after 30 DEG C of -38 DEG C of culture 24-36h, discharge To bacillus amyloliquefaciens tunning；

3rd, the preparation of bacillusmusilaginosiengineering tunning：

(1) Shaking culture：Inoculation through slant activation is subjected to seed flask culture in Shake flask medium, cultivated Condition is：Rotating speed 180-200r/min, 30 DEG C -33 DEG C of temperature, 10-12h is cultivated, 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 Sterilized 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, obtain secondary seed solution；

(4) ferment：Load fermenter volume 50%-60% bacillusmusilaginosiengineering fermentation training in 3000L fermentation tanks Base is supported, 115 DEG C of -122 DEG C of moist heat sterilization 20-40min, 150L or 200L secondary seed solution is inoculated in gel-shaped gemma bar In bacterium viable bacteria fermentation culture medium, control tank pressure 0.02MPa-0.07MPa, after 30 DEG C of -33 DEG C of culture 36-48h, discharging is sent out Ferment product；

4th, 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 preventing and treating 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%, nutrient agar 1.5%-2.0%, surplus are water, medium pH 6.0-7.5.

Preferably, used by the bacillus amyloliquefaciens and bacillus subtilis secondary seed medium into split-phase Together, 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 ‰, defoamer 0.2 ‰ -2 ‰, surplus is water, and medium pH is adjusted to 6.0-7.5, It is and standby in 115 DEG C of -122 DEG C of moist heat sterilization 20-40min.

Preferably, the composition of fermentation medium is identical used by the bacillus amyloliquefaciens and 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 ‰, defoamer 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 standby；

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 ‰, defoamer 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.

Present invention also offers the application of composite bacteria agent, and the composite bacteria agent is diluted with water after 300 times with base manure together Apply in soil, the addition after composite bacteria agent dilution is every mu of 5-10kg/.

Beneficial effect：

The preparation method of composite bacteria agent of the present invention is simple to operate, prevents and treats tomato verticillium wilt positive effect, 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 large area of the main effect microorganism in soil in composite bacteria agent to breed, the growth of antagonism pathogen, Effectively suppress or kill harmful microorganism in soil, force environment is provided with for beneficial microorganism breeding and development, by beneficial The continuous formation of microbial metabolic products effectively prevents and treats the generation of tomato verticillium wilt.Meanwhile complex micro organism fungicide can also promote Enter the release of soil nutrient, promote soil granular structure to be formed, improved soil, in a word, the inventive method pass in preventing and treating tomato soil While disease, soil microenvironment has been repaired, has improved soil fertility, has added tomato yield and quality.

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 Bright rather than limitation the scope of the present invention.In addition, it is to be understood that after the content of the invention lectured has been read, art technology Personnel can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited Fixed scope.

Embodiment 1

A kind of composite bacteria agent for preventing and treating tomato verticillium wilt, the composite bacteria agent is by bacillus amyloliquefaciens (Bacillus Amyloliquefaciens) MES812 tunning, bacillus subtilis (Bacillus subtilis) MES810 hair Ferment product and bacillusmusilaginosiengineering (Bacillus mucilaginosus) MES803 tunning by volume 1:3:4 is mixed Conjunction forms；

The bacillus amyloliquefaciens morphological feature：Bacillus amyloliquefaciens are shaft-like, 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 circular, Micro- protuberance, edge is irregular, and color is dark.Positive reaction：Catalase；Oxidizing ferment；Hydrolysis starch.Negative reaction：Anaerobic growth, lecithin Lipase；Propionate utilizes, lactose fermentation production acid.Multiplexed PCR amplification is carried out using special primer, the bacterial strain produces unique amplification Product, stripe size are identical with bacillus amyloliquefaciens (Bacillus amyloliquefaciens).

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

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

(1) actication of culture：Load 50mL sterilized waters in 250mL conical flasks, picking slant strains are shaken in conical flask It is even, 1mL liquid is taken into 500mL Kolle flasks, loads 100mL beef-protein mediums in Kolle flask in advance, 120 DEG C are damp and hot Sterilized 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 that activated spawn is filled by more than takes out, and pours into 80mL sterilized waters, scrapes Take bacterium tire that bacteria suspension is made, its whole is poured into equipped with the seed bottle of 200mL and temperature for 4 DEG C of sterilized water, obtains one-level kind Sub- liquid；

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

(4) ferment tank：Load the fermentation of bacillus subtilis culture of fermenter volume 55% in 3000L fermentation tanks Base, 200L secondary seed solutions are all inoculated in bacillus subtilis viable bacteria fermentation culture medium, control tank pressure 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 In the bottle of angle sampling carry out microscopy, the gemma in the visual field and total thalline number are counted, 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 composition 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 ‰, defoamer 1 ‰, surplus is water, and medium pH is adjusted to 7.0, and 118 DEG C moist heat sterilization 30min is standby.

Wherein, the composition 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 ‰, defoamer 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.

2nd, the preparation of bacillus amyloliquefaciens tunning：

(1) actication of culture：Load 50mL sterilized waters in 250mL conical flasks, picking slant strains are shaken in conical flask It is even, 1mL liquid is taken into 500mL Kolle flasks, loads 100mL beef-protein mediums in Kolle flask in advance, 116 DEG C are damp and hot Sterilized 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 that activated spawn is filled by more than takes out, and pours into 50mL sterilized waters, scrapes Take bacterium tire that bacteria suspension is made, its whole is poured into equipped with the seed bottle of 100mL and temperature for 4 DEG C of sterilized water, obtains one-level kind Sub- liquid；

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

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

Wherein, the composition 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 composition 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 ‰, defoamer 0.2 ‰, surplus is water, and medium pH is adjusted to 7.5, and 122 DEG C of moist heat sterilization 40min are standby.

Wherein, the composition 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 ‰, defoamer 2 ‰, surplus are water, and the pH of culture medium is adjusted to 6.0, and at 115 DEG C Moist heat sterilization 40min.

3rd, the preparation of bacillusmusilaginosiengineering tunning：

(1) Shaking culture：Inoculation through slant activation is subjected to seed flask culture in Shake flask medium, cultivated Condition is：Rotating speed 180r/min, 30 DEG C of temperature, 10h is cultivated, 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 20min, above-mentioned gel-shaped bud pole bacterium shake flask culture is all inoculated in primary-seed medium, 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 40min, 100mL primary seed solutions are inoculated in secondary seed medium；In 30 DEG C of -33 DEG C of culture 8-14h, secondary seed is obtained Liquid；

(4) ferment：Load fermenter volume 50%-60% bacillusmusilaginosiengineering fermentation training in 3000L fermentation tanks Base is supported, 115 DEG C of moist heat sterilization 20min, 150L or 200L secondary seed solution is inoculated in bacillusmusilaginosiengineering viable bacteria fermentation In culture medium, control tank pressure 0.02MPa-0.07MPa, after 33 DEG C are cultivated 48h, discharging obtains tunning；

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

Embodiment 2

A kind of composite bacteria agent for preventing and treating tomato verticillium wilt, the composite bacteria agent is by bacillus amyloliquefaciens (Bacillus Amyloliquefaciens) MES812 tunning, bacillus subtilis (Bacillus subtilis) MES810 hair Ferment product and bacillusmusilaginosiengineering (Bacillus mucilaginosus) MES803 tunning by volume 1:2:3 is mixed Conjunction forms；

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

(4) ferment tank：Load the fermentation of bacillus subtilis culture of fermenter volume 50% in 3000L fermentation tanks Base, 150L secondary seed solutions are all inoculated in bacillus subtilis viable bacteria fermentation culture medium, control tank pressure 0.02MPa, 30 DEG C of culture 36h；Discharging obtains bacillus subtilis liquid fermentation production.

2nd, the preparation of bacillus amyloliquefaciens tunning：

(4) ferment tank：Load the fermentation of bacillus subtilis culture of fermenter volume 55% in 3000L fermentation tanks Base, 200L secondary seed solutions are all inoculated in bacillus amyloliquefaciens viable bacteria fermentation culture medium, control tank pressure 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 sampling progress microscopies from triangular flask, the gemma in the visual field and total thalline number are counted, 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.

3rd, the preparation of bacillusmusilaginosiengineering tunning：

(1) Shaking culture：Inoculation through slant activation is subjected to seed flask culture in Shake flask medium, cultivated Condition is：Rotating speed 200r/min, 33 DEG C of temperature, 12h is cultivated, 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 35min, above-mentioned gel-shaped bud pole bacterium shake flask culture is all inoculated in primary-seed medium, 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 20min, 100mL primary seed solutions are inoculated in secondary seed medium；14h is cultivated at 33 DEG C, obtains secondary seed solution；

(4) ferment：Load the bacillusmusilaginosiengineering fermentation medium of fermenter volume 60% in 3000L fermentation tanks, 115 DEG C of moist heat sterilization 40min, 150L secondary seed solution is inoculated in bacillusmusilaginosiengineering viable bacteria fermentation culture medium, controlled Tank processed presses 0.07MPa, and after 30 DEG C are cultivated 36h, discharging obtains tunning；

4th, 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 preventing and treating tomato verticillium wilt.

Embodiment 3

A kind of composite bacteria agent for preventing and treating tomato verticillium wilt, the composite bacteria agent is by bacillus amyloliquefaciens (Bacillus Amyloliquefaciens) MES812 tunning, bacillus subtilis (Bacillus subtilis) MES810 hair Ferment product and bacillusmusilaginosiengineering (Bacillus mucilaginosus) MES803 tunning by volume 1:2:4 is mixed Conjunction forms；

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

(1) actication of culture：Load 50mL sterilized waters in 250mL conical flasks, picking slant strains are shaken in conical flask It is even, 2mL liquid is taken into 500mL Kolle flasks, loads 100mL beef-protein mediums in Kolle flask in advance, 121 DEG C are damp and hot Sterilized 20min, and Kolle flask is placed under the conditions of 36 DEG C and cultivates 24h, obtains activated spawn；

(2) preparation of primary seed solution：The Kolle flask that activated spawn is filled by more than takes out, and pours into 100mL sterilized waters, scrapes Take bacterium tire that bacteria suspension is made, its whole is poured into equipped with the seed bottle of 300mL and temperature for 4 DEG C of sterilized water, obtains one-level kind Sub- liquid；

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

(4) ferment tank：Load the fermentation of bacillus subtilis culture of fermenter volume 60% in 3000L fermentation tanks Base, 200L secondary seed solutions are all inoculated in bacillus subtilis viable bacteria fermentation culture medium, control tank pressure 0.07MPa, 38 DEG C of culture 24h；Discharging obtains bacillus subtilis (Ehrenberg) Cohn fermented product.

Wherein, the composition 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 standby in 121 DEG C of moist heat sterilization 35min.

Wherein, the composition 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 ‰, defoamer 2 ‰, surplus is water, and medium pH is adjusted to 7.5, and 122 DEG C moist heat sterilization 40min is standby.

Wherein, the composition 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 ‰, defoamer 2 ‰, surplus are water, and the pH of culture medium is adjusted to 6.0, and wet at 122 DEG C Heat sterilization 20min.

2nd, the preparation of bacillus amyloliquefaciens tunning：

(1) actication of culture：Load 50mL sterilized waters in 250mL conical flasks, picking slant strains are shaken in conical flask It is even, 1.5mL liquid is taken into 500mL Kolle flasks, loads 100mL beef-protein mediums in Kolle flask in advance, 117 DEG C are wet Heat sterilization 25min, Kolle flask is placed under the conditions of 33 DEG C and cultivates 28h, obtains activated spawn；

(2) preparation of primary seed solution：The Kolle flask that activated spawn is filled by more than takes out, and pours into 60mL sterilized waters, scrapes Take bacterium tire that bacteria suspension is made, its whole is poured into equipped with the seed bottle of 150mL and temperature for 4 DEG C of sterilized water, obtains one-level kind Sub- liquid；

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

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

Wherein, the composition 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 standby.

Wherein, the composition 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 ‰, defoamer 1.5 ‰, surplus is water, and medium pH is adjusted to 7.2, and It is standby in 118 DEG C of moist heat sterilization 35min.

Wherein, the composition 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 ‰, defoamer 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.

3rd, the preparation of bacillusmusilaginosiengineering tunning：

(1) Shaking culture：Inoculation through slant activation is subjected to seed flask culture in Shake flask medium, cultivated Condition is：Rotating speed 190r/min, 32 DEG C of temperature, 11h is cultivated, 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 25min, above-mentioned gel-shaped bud pole bacterium shake flask culture is all inoculated in primary-seed medium, 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 30min, 200mL primary seed solutions are inoculated in secondary seed medium；10h is cultivated at 32 DEG C, obtains secondary seed solution；

(4) ferment：Load the bacillusmusilaginosiengineering fermentation medium of fermenter volume 55% in 3000L fermentation tanks, 118 DEG C of moist heat sterilization 30min, 200L secondary seed solution is inoculated in bacillusmusilaginosiengineering viable bacteria fermentation culture medium, controlled Tank processed presses 0.05MPa, and after 31 DEG C are cultivated 42h, discharging obtains tunning；

4th, 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 preventing and treating tomato verticillium wilt.

2.0* is no less than 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 for preventing and treating tomato verticillium wilt, the composite bacteria agent is by bacillus amyloliquefaciens (Bacillus Amyloliquefaciens) MES812 tunning, bacillus subtilis (Bacillus subtilis) MES810 hair Ferment product and bacillusmusilaginosiengineering (Bacillus mucilaginosus) MES803 tunning by volume 1:1:1 is mixed Conjunction forms；

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

(4) ferment tank：Load the fermentation of bacillus subtilis culture of fermenter volume 58% in 3000L fermentation tanks Base, 116 DEG C of moist heat sterilization 30min, 200L secondary seed solutions are all inoculated in bacillus subtilis viable bacteria fermentation culture medium In, control tank pressure 0.05MPa, 28h is cultivated at 34 DEG C；Discharging obtains bacillus subtilis liquid preparation.

2nd, the preparation of bacillus amyloliquefaciens tunning：

(4) ferment tank：Load the fermentation of bacillus subtilis culture of fermenter volume 60% in 3000L fermentation tanks Base, 200L secondary seed solutions are all inoculated in bacillus amyloliquefaciens viable bacteria fermentation culture medium, control tank pressure 0.07MPa, 24h is cultivated at 38 DEG C；Discharging obtains bacillus amyloliquefaciens tunning.

3rd, the preparation of bacillusmusilaginosiengineering tunning：

(1) Shaking culture：Inoculation through slant activation is subjected to seed flask culture in Shake flask medium, cultivated Condition is：Rotating speed 185r/min, 31 DEG C of temperature, 11.5h is cultivated, 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 29min, above-mentioned gel-shaped bud pole bacterium shake flask culture is all inoculated in primary-seed medium, 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 30min, 200mL primary seed solutions are inoculated in secondary seed medium；12h is cultivated at 32 DEG C, obtains secondary seed solution；

(4) ferment：Load the bacillusmusilaginosiengineering fermentation medium of fermenter volume 53% in 3000L fermentation tanks, 119 DEG C of moist heat sterilization 35min, 200L secondary seed solution is inoculated in bacillusmusilaginosiengineering viable bacteria fermentation culture medium, controlled Tank processed presses 0.05MPa, and after 32 DEG C are cultivated 40h, discharging obtains tunning；

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

2nd, Function detection

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

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

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

Tomato verticillium wilt germ is coated in beef extract-peptone flat board center first, respectively by the withered grass after obtained activation 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 incubated, when blank control will cover with whole culture dish, measures tomato yellow Control increment (colony radius) and processing increment (the suppression life after inoculation MES810, MES812, MES803 of disease of withering germ Major 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.

The bacillus subtilis MES810 of table 1, 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：The result of table 1 shows 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 have obvious inhibitory action to tomato verticillium wilt, and there is preventing and treating tomato yellow to wither The Biocontrol Potential of disease.

2. the application and prevention effect of the inventive method

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

(1) test medicine

1. complex micro organism fungicide：Using 3.2 complex micro organism fungicides (being prepared by the method for embodiment 4) prepared, use Water dilutes 300 times；

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

3. blank control：Clear water.

(2) test method：It is in 30cm plastic flowerpot that Seedling Stage tomato seedling, which is transplanted to sowing bore, each handles 5 Basin (per 3 plants of basin), in triplicate.Placed using random district's groups aligning method.Take long kind consistent to 4-5 pieces true leaf, growth potential Eggplant seedling, 2 uniform are sprayed at of complex micro organism fungicide prepared by embodiment 4 point supply examination plant leaf and plants stems, chemistry Medicament is compareed 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 Spend for 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 prevention effect, specific experiment the results are shown in Table 2.

The bacillus subtilis MES810 of table 2 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：From the result of table 2 as can be seen that after complex micro organism fungicide processing, disease index (9.46) Disease index (90.19) substantially less than after blank control processing, 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%, are said to the prevention effect of tomato verticillium wilt Bright bacillus subtilis, bacillus amyloliquefaciens, the complex micro organism fungicide of three kinds of bacterium compositions of bacillusmusilaginosiengineering are to tomato Verticillium wilt prevention effect is preferable.

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

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

Control 1：Level land, water according to local general planting method, basal dressing；

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

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

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

Processing 4：Level land, water according to local general planting method, basal dressing；Every mu of addition is diluted with water 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, tomato breeding time investigation tomato verticillium wilt disease index are carried out according to conventional process, August part tomato is united after receiving Count yield.As a result see the table below：

The complex micro organism fungicide of table 3 is to tomato verticillium wilt prevention effect and the comparative test result of yield effect

Processing	Average sick seedling rate (%)	Prevention 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%

Prevention effect (%)=(control sick seedling rate-processing sick seedling rate)/control sick seedling rate.Can be with from above-mentioned comparing result Find out, complex micro organism fungicide of the present invention, the pathogen in antagonism soil, not only effectively control native biography as under base fertilizer The generation of disease, while there is increasing yield and improving quality effect.Using the positive effect of 300 times of complex micro organism fungicide of dilution higher than each 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, optimal to the prevention effect of tomato verticillium wilt.

Microbial bacterial agent of the present invention has nontoxic, noresidue, does not suppress the characteristics of growth and dosage are few, this micro- life It during thing microbial inoculum is manured into soil, can effectively suppress growth of pathogenic bacteria, promote beneficial microorganism breeding, soil is maintained well Microbiota, rehabilitating soil micro-ecological environment.Improve soil granular structure simultaneously, promote absorption of the plant to nutrient, reduce Fertilizer and pesticide usage amount, effectively reduces that crop agriculture is residual, and preventing and treating soil-borne disease is reached while environmental protection, increases the effect of matter volume increase Fruit.

The foregoing is only a preferred embodiment of the present invention, but it is not limited to embodiment of above.The guarantor of the present invention Shield scope 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 scheme and its inventive concept is subject to equivalent substitution or change, should all be included within the scope of the present invention.

Claims

1. a kind of composite bacteria agent for preventing and treating tomato verticillium wilt, it is characterized in that：The composite bacteria agent is by bacillus amyloliquefaciens （Bacillus amyloliquefaciens）MES812 tunning, bacillus subtilis (Bacillus subtilis) MES810 tunning and bacillusmusilaginosiengineering（Paenibacillus mucilaginosus）MES803 tunning By volume 1:1-3:1-5 is mixed；

Bacillus subtilis (Bacillus subtilis) MES810, 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 preservation date is August in 2017 10, and preservation is compiled Number it is 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 preservation date is August 10 in 2017 Day, deposit number CGMCCNo.14515；

The bacillusmusilaginosiengineering（Paenibacillus mucilaginosus）MES803, it is deposited in Chinese common micro- life Thing culture presevation administrative center, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3s, and preservation date is in August, 2017 10 days, deposit number CGMCCNo.14513.

2. a kind of composite bacteria agent for preventing and treating tomato verticillium wilt according to claim 1, it is characterized in that：The bacillus subtilis Bacillus subtilis (Bacillus subtilis) in bacterium (Bacillus subtilis) MES810 tunning MES810 viable count is no less than 2.0*10¹⁰cFu/g；

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

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

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

4. according to the preparation method of the composite bacteria agent of any described preventing and treating tomato verticillium wilt of claim 1-3, it is characterised in that Specifically include following steps：

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

（1）Actication of culture：Load 50mL sterilized waters in 250mL conical flasks, picking bacillus subtilis slant strains are in taper In bottle, shake up, take 1-2mL liquid into 500mL Kolle flasks, load 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）The preparation of primary seed solution：The Kolle flask that activated spawn is filled by more than takes out, and pours into 50-100mL sterilized waters, scrapes Take bacterium tire that bacteria suspension is made, its whole is poured into equipped with the seed bottle of 100-300mL and temperature for 4 DEG C of sterilized water, obtains one Level seed liquor；

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

（4）Ferment tank：Load fermenter volume 50%-60% fermentation of bacillus subtilis culture in 3000L fermentation tanks Base, 115 DEG C of -122 DEG C of moist heat sterilization 20-40min, 150L or 200L secondary seed solutions are all inoculated in bacillus subtilis and lived In bacteria fermentation culture medium, control tank presses 0.02 MPa-0.07 MPa, 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：Load 50mL sterilized waters in 250mL conical flasks, picking bacillus amyloliquefaciens slant strains are in cone In shape bottle, shake up, take 1-2mL liquid into 500mL Kolle flasks, load 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：Load fermenter volume 50%-60% fermentation of bacillus subtilis culture in 3000L fermentation tanks Base, 115 DEG C of -122 DEG C of moist heat sterilization 20-40min, bacillus amyloliquefaciens are all inoculated in by 150L or 200L secondary seed solutions In viable bacteria fermentation culture medium, control tank presses 0.02 MPa-0.07 MPa, and after 30 DEG C of -38 DEG C of culture 24-36h, discharging is solved Bacillus amyloliquefacienses tunning；

（Three）The preparation of bacillusmusilaginosiengineering tunning：

（1）Shaking 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, 10-12 h are cultivated, 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 20-35min, above-mentioned gel-shaped bud pole bacterium shake flask culture is all inoculated in primary-seed medium, at 30 DEG C -33 DEG C 36-48h is cultivated, obtains primary seed solution；

（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 20-40min, 100-300mL primary seed solutions are inoculated in secondary seed medium；In 30 DEG C of -33 DEG C of culture 8-14h, obtain Secondary seed solution；

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

（Four）By the hair of the tunning of bacillus amyloliquefaciens, 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 preventing and treating tomato verticillium wilt.

5. the preparation method of the composite bacteria agent of preventing and treating tomato verticillium wilt according to claim 4, it is characterised in that the solution Beef-protein medium is identical used by bacillus amyloliquefacienses and bacillus subtilis, specifically, as mass fraction： Beef extract 0.5%-3%, peptone 0.5%-3%, sodium chloride 0.2-1%, nutrient agar 1.5%-2.0%, surplus are water, medium pH For 6.0-7.5.

6. the preparation method of the composite bacteria agent of preventing and treating tomato verticillium wilt according to claim 4, it is characterised in that the solution The composition of secondary seed medium is identical used by bacillus amyloliquefacienses and bacillus subtilis, specifically, pressing mass fraction Meter：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 ‰, defoamer 0.2 ‰ -2 ‰, surplus is water, and medium pH is adjusted to 6.0-7.5, and damp and hot is gone out at 115 DEG C -122 DEG C Bacterium 20-40min is standby.

7. the preparation method of the composite bacteria agent of preventing and treating tomato verticillium wilt according to claim 4, it is characterised in that the solution The composition of fermentation medium is identical used by bacillus amyloliquefacienses and bacillus subtilis, specifically, as mass fraction：Portugal Grape sugar 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 ‰, defoamer 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。

8. the preparation method of the composite bacteria agent of preventing and treating tomato verticillium wilt according to claim 4, it is characterised in that the glue Freeze the Shake flask medium during culture of sample bacillus, 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 ‰ are remaining Measure for water, 116 DEG C of -121 DEG C of moist heat sterilization 20-35min it is standby；

Primary-seed medium during 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%, sulfuric acid Magnesium 0.5 ‰ -2 ‰, surplus is water；

Secondary seed medium during 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%, sulfuric acid Magnesium 0.5 ‰ -2 ‰, defoamer 0.2 ‰ -2 ‰, surplus is water；

Fermentation medium during the bacillusmusilaginosiengineering culture, as mass fraction for：Cornstarch 0.5%-2%, dregs of beans 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.

9. the application of the composite bacteria agent of preventing and treating tomato verticillium wilt according to claim 1, it is characterised in that will be described compound Microbial inoculum is together applied in soil after being diluted with water 300 times with base manure, and the addition after composite bacteria agent dilution is every mu of 5-10kg/.