CN105969696A - Bacillus bacterium agent for nightshade crops as well as production method and application thereof - Google Patents
Bacillus bacterium agent for nightshade crops as well as production method and application thereof Download PDFInfo
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Abstract
The invention relates to a bacillus bacterium agent for nightshade crops as well as a production method and application thereof. The bacterium agent comprises bacillus licheniformis D2, bacillus subtilis B5 and bacillus amyloliquefaciens J4. The optimal proportion and optimal fermentation production of compound bacteria is realized through slant culture, first-grade seeds, second-grade seeds and mixed culture and fermentation; the living bacteria count of the compound bacterium agent is more than 3*10<10>cfu/mL. The bacillus preparation provided by the invention is used for preventing and treating soil-born diseases of the nightshade crops and the acting effect is obvious.
Description
Technical field
The present invention relates to a kind of spore mix preparation, particularly to a kind of bud for preventing and treating Solanaceae class soil-borne disease of crop
Spore bacillus preparation.The invention still further relates to production method of described gemma bacillus agent and application thereof.
Technical background
The soil-borne disease such as gray mold, bacterial wilt is the plant disease that Solanaceae class crop is common.Chemical prevention is easily generated drug resistance
The negative interactions such as property, pesticide residues and disruption of ecological balance.Biological control has efficiently, no pollution, do not develop immunity to drugs and prevent and control
The advantages such as reason combination, wherein bio-bacterial manure both can have been increased production, and improves product quality, again scalable and the group improving soil microorganism
Become, and then alleviate the harm of corps diseases to a certain extent.The effective antagonism preventing solanaceous crops soil-borne disease of screening
Bacterium, and then to prepare efficient complex micro organism fungicide be the important way utilizing biotechnology to prevent and treat Solanaceae class soil-borne disease of crop
Footpath.
Numerous studies show both at home and abroad, and Solanaceae class crop soil-borne pathogen is had relatively by multiple fungus, antibacterial and actinomycetes
Good inhibitory action.For solid fermentation prepares microbial inoculum, liquid fermentation has low cost, and bacterium number content is high, makes letter
The advantages such as list, and the research contents in terms of the most domestic and international shorter mention Antagonistic Fungi Liquid mixed fermentation.Therefore, the present invention passes through
Isolated and purified to related soil microorganism, filters out the antagonism to Solanaceae class crop soil-borne pathogen with better inhibition effect
Bacterium, and by the antagonism between bacterial strain and the proportioning of optimization strain and fermentation condition, prepare stable, efficient microorganism
Microbial inoculum product.
Summary of the invention
(1) to solve the technical problem that
Present invention aims to above-mentioned the deficiencies in the prior art, disclose a kind of gemma bacillus agent, be used for preventing
Control Solanaceae class crop soil to pass.The invention also discloses production method and the purposes of this microbial inoculum.
(2) technical scheme
The present invention filters out best stabilized symbiosis strain combination from 45 different genus and species, by single culture screening height
Produce strain.The strain filtered out is Bacillus licheniformis D2 (Bacillus licheniformis), bacillus subtilis B5
(Bacillus subtilis), bacillus amyloliquefaciens J4 (Bacillus amyloliquefaciens).
According to above-mentioned the selection result, the present invention devises a kind of gemma bacillus agent, including Bacillus licheniformis D2
(Bacillus licheniformis)1.0×1010Cfu/mL, bacillus subtilis B5 (Bacillus subtilis) 2.0 ×
1010Cfu/mL, bacillus amyloliquefaciens J4 (Bacillus amyloliquefaciens) 5.0 × 1010cfu/mL；
Preferably,
Bacillus licheniformis D2 (Bacillus licheniformis) 0.5 × 1010Cfu/mL, bacillus subtilis B5
(Bacillus subtilis)1.2×1010Cfu/mL, bacillus amyloliquefaciens J4 (Bacillus
amyloliquefaciens)2.0×1010cfu/mL；
The production method of gemma bacillus agent of the present invention, comprises the steps:
1). slant culture: by Bacillus licheniformis D2, bacillus subtilis B5, bacillus amyloliquefaciens J4 original strain
It is inoculated in respectively on solid medium under aseptic condition, cultivates 1～2 day under the conditions of 37 DEG C, make actication of culture；
2). first order seed is cultivated: by step 1) it is inoculated in fluid medium under the strain aseptic condition cultivated respectively, 37 DEG C
Under the conditions of, 100～150r/min shaking tables are cultivated 1～2 day, and single strain liquid stops training when cultivating between OD600 value 3.5-4.0
Support, prepare first order seed；
3). secondary seed is cultivated: by the inoculum concentration that the volume ratio of fluid medium is 10～15%, by first order seed respectively
Being inoculated in fermentation tank, under the conditions of 30 DEG C～35 DEG C, mixing speed is 100～150r/min, and ventilation is 1:0.8～1.2, training
Support 1～2 day, prepare secondary seed；
4). mixing fermentation culture: by the inoculum concentration that the volume ratio of fluid medium is 10～15%, secondary seed is inoculated
In fermentation tank, carry out fermentation culture, it is thus achieved that microbial inoculum.
Wherein, culture medium used above is LB culture medium.Solid medium is solid-state with the difference of liquid culture medium
Culture medium with the addition of appropriate agar in preparation process.
Wherein, step 4) used by the formula of culture medium be by mass percentage: molasses 2～5%, peptone 2.0～
8%, Semen Maydis powder 5～10%, surplus is water；
In initial 0～24 hour, interval ventilation, it is maintained at aerobic conditions fermentation, ventilation 1:1.2～1.5, speed of agitator
100～150r/min, stir interval time 3～4 hours, stir 3～5 minutes, temperature 30 DEG C～35 DEG C；
Gemma bacillus agent of the present invention can be used for preventing and treating Solanaceae class soil-borne disease of crop as biological prevention and control agent.
(3) beneficial effect
The present invention is used for preventing and treating Solanaceae class soil-borne disease of crop, and action effect is obvious.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is further illustrated, but is not limited to the claimed model of the present invention
Enclose.
The composition of embodiment 1 gemma bacillus agent
The composition of embodiment 2 gemma bacillus agent
The production method of embodiment 3 gemma bacillus agent
1). slant culture: Bacillus licheniformis D2, bacillus subtilis B5, bacillus amyloliquefaciens J4 original strain without
It is inoculated in respectively on solid medium under the conditions of bacterium, cultivates 1 day under the conditions of 37 DEG C；
2). first order seed is cultivated: by step 1) it is inoculated in fluid medium, lichens respectively under the strain aseptic condition cultivated
Cultivate 1 day under the conditions of bacillus cereus D2, bacillus subtilis B5, bacillus amyloliquefaciens J4 37 DEG C, prepare first order seed, bacterium
Suspension OD600 value all reaches 3.0；
3). secondary seed is cultivated: be the inoculum concentration of 10% by the volume ratio of fluid medium, is inoculated respectively by first order seed
In the fermentation tank of 100L, in fermentation tank, the cumulative volume of culture fluid is 70L, and under the conditions of 30 DEG C～35 DEG C, mixing speed is 120r/
Min, ventilation is 1:1.2, cultivates 1 day prepared secondary seed；
4). mixing fermentation culture: be the inoculum concentration of 10% by the volume ratio of fluid medium, secondary seed is inoculated into 1
In the fermentation tank of ton, the culture medium cumulative volume in fermentation tank is 700L, carries out fermentation culture, it is thus achieved that microbial inoculum,
Wherein, step 1), 2), 3) in used culture medium beef extract-peptone.
Wherein, step 4) used by the formula of culture medium be by mass percentage: molasses 5%, peptone 3%, Semen Maydis powder
5%, surplus is water；
Fermentation culture uses fed-batch process, and wherein feed supplement carbon source is: glucose, sucrose；Nitrogen source is: albumen
Peptone, Semen Maydis powder.
Fermentation culture stage: in initial 0～24 hour, interval ventilation, it is maintained at aerobic conditions and ferments, ventilation 1:1.5,
Speed of agitator 150r/min, stirs 3 hours interval times, stirs 3 minutes, temperature 30 DEG C～35 DEG C；
The production method of embodiment 4 gemma bacillus agent
1). slant culture: Bacillus licheniformis D2, bacillus subtilis B5, bacillus amyloliquefaciens J4 original strain without
It is inoculated in respectively under the conditions of bacterium on solid medium and cultivates 2 days under the conditions of 37 DEG C；
2). first order seed is cultivated: by step 1) it is inoculated in fluid medium, lichens respectively under the strain aseptic condition cultivated
Under the conditions of bacillus cereus D2, bacillus subtilis B5, bacillus amyloliquefaciens J4 37 DEG C, 150r/min shaking table is cultivated 2 days, system
Obtain first order seed, terminate optical density OD600 value and all reach 2.5；
3). secondary seed is cultivated: be the inoculum concentration of 8% by the volume ratio of fluid medium, is inoculated respectively by first order seed
In the fermentation tank of 100L, in fermentation tank, the cumulative volume of culture fluid is 70L, and under the conditions of 30 DEG C～35 DEG C, mixing speed is,
150r/min, ventilation is 1:1, cultivates 2 days prepared secondary seeds.
4). mixing fermentation culture: be the inoculum concentration of 10% by the volume ratio of fluid medium, secondary seed is inoculated into 1
In the fermentation tank of ton, the culture medium cumulative volume in fermentation tank is 700L fermentation, it is thus achieved that microbial inoculum.
Wherein, step 1), 2), 3) in culture medium used be beef-protein medium.
Wherein, step 4) used by the formula of culture medium be by mass percentage: molasses 10%, peptone 3%, Semen Maydis powder
2%, surplus is water；
Fermentation culture uses fed-batch process, and wherein feed supplement carbon source is: molasses；Nitrogen source is: Semen Maydis powder.
Fermentation culture stage: in initial 0～24 hour, interval ventilation, it is maintained at aerobic conditions and ferments, ventilation 1:1.2,
Speed of agitator 150r/min, stirs 2 hours interval times, stirs 5 minutes, temperature 30 DEG C.
The prevention effect that embodiment 5 is sick to Phytophthora capsici
Method: biological and ecological methods to prevent plant disease, pests, and erosion bud is embraced bacterium and is respectively connected to, equipped with in 90ml beef extract-peptone culture fluid, put 37 DEG C, and 180/rmin shakes
Cultivating 24h on Chuan, bacterium solution 1:1:1 mixed, every strain Hot Pepper Seedling fills above-mentioned mixed bacteria liquid 5mL (from the cis filling of plant base portion),
Each process 10 strain Hot Pepper Seedling, repeats for three times, if clear water compares above-mentioned process pouring root inoculation after 24h.By Phytophthora capsici truffle
It is placed on PDA plate, after 25～28 DEG C of illumination cultivation 5-6d, with the inoculating loop of sterilization by Sporangium eluting, puts in 4 DEG C of refrigerators
Pre-cooling 30min, promotes zoospore to discharge from armful ascus, and four layers of filtered through gauze collect zoospores, use blood count
Plate calculates the quantity of zoospore, then will embrace sub-concentration with sterilized water and regulate to 1000/mL, and beat at plant base portion 1cm
One aperture, injects the above-mentioned spore suspension of 2mL, after inoculation, nutritive cube is filled permeable, keeps substrate to moisten, and temperature controls at 24-
26℃.8d Investigate incidence and occurring degree after inoculation.The split pole standard of pouring root inoculation:
Capsicum epidemic disease grade scale
The sick level state of an illness
0 grade without any symptom
1 grade of seedling rhizome portion slightly blackening, here blade does not withers
2 grades of seedling rhizome portion's blackening reach 1～2cm, and here blade irrecoverability withers, and lower blade is even to be had and come off
3 grades of seedling rhizome portion's blackening more than 1～2cm, blade here substantially wither or fallen leaves substantially
4 grades of the portion's blackening of seedling rhizome, shrinkages, come off outside growing point or here whole strain withers
5 grades of plant are withered
Result:
The prevention effect that table 1 spore microbial inoculum is sick to Phytophthora capsici
The embodiment 6 prevention effect to graw mold of tomato
Spraying medicine concentration is arranged: test sets 2 process altogether, and gemma bacillus agent is diluted to 108Bacterium solution, comparison medicament use
50% procymidone wettable powder 600 times liquid；Using clear water as blank.
Test method: use the group arrangement of random district, every community 20m2, it is repeated 4 times, uses new knapsack hand sprayer to enter
Row spraying treatment, operating pressure is 0.4Mpa, injection diameter 1.20mm, and per hectare spouting liquid is about 900 liters.
Experimental field: test and start dispenser in graw mold of tomato premorbid, spray medicine for the first time respectively on April 2nd, 2013, with
After respectively at April 9, April 16 respectively spray medicine 1 time, altogether dispenser 3 times.Before dispenser, investigate each community state of an illness radix；After last medicine
10d, investigates the change of illness state situation after the medicine of each community and the medicament safety to trial crops.During investigation, every community uses five
Point sampling, every some investigation 2 strains, investigate Fructus Lycopersici esculenti scab, calculate disease index, and give record respectively according to following stage division.
Graw mold of tomato blade grade scale:
0 grade: without scab；
1 grade: lesion area accounts for whole leaf area less than 5%；
3 grades: lesion area accounts for whole leaf area 6-10%；
5 grades: lesion area accounts for whole leaf area 11-20%；
7 grades: lesion area accounts for whole leaf area 21-40%；
9 grades: lesion area accounts for whole leaf area more than 40% 6-10%.
Graw mold of tomato disease fruit grade scale:
0 grade: without scab；
1 grade: residual petal morbidity or stigma morbidity；
3 grades: sepal rots or stigma morbidity spreads to areola portion；
5 grades: infiltration shape is fallen ill in areola portion, scab is without mould layer；
7 grades: falling ill in areola portion, has mould layer, but do not expand to other positions of fruit；
9 grades: mould layer expands to other positions of fruit.
Poisoning grade scale:
-: without poisoning；
+: slight poisoning, do not affect crop normal growth；
++: moderate poisoning, resilient, do not result in crop failure；
+++: severe poisoning, affect crop normal growth, crop yield and quality are caused a certain degree of loss；
The serious poisoning of ++++:, plant growth is obstructed, and yield and mass loss are serious.
B., by chemicals treatment district compared with clear water comparison, the percentage rate of its poisoning is evaluated.
Result:
The table 2 spore microbial inoculum prevention effect to graw mold of tomato
Claims (6)
1. according to claim 1 gemma bacillus agent, it is characterised in that this microbial inoculum includes Bacillus licheniformis D2
(Bacillus licheniformis)1.0×1010Cfu/mL, bacillus subtilis B5 (Bacillus subtilis) 2.0 ×
1010Cfu/mL, bacillus amyloliquefaciens J4 (Bacillus amyloliquefaciens) 5.0 × 1010cfu/mL。
Gemma bacillus agent the most according to claim 1, it is characterised in that this microbial inoculum includes Bacillus licheniformis D2
(Bacillus licheniformis)0.5×1010Cfu/mL, bacillus subtilis B5 (Bacillus subtilis) 1.2 ×
1010Cfu/mL, bacillus amyloliquefaciens J4 (Bacillus amyloliquefaciens) 2.0 × 1010cfu/mL。
Gemma bacillus agent the most according to claim 1, it is characterised in that this microbial inoculum includes Bacillus licheniformis D2
(Bacillus licheniformis)0.8×1010Cfu/mL, bacillus subtilis B5 (Bacillus subtilis) 1 ×
1010Cfu/mL, bacillus amyloliquefaciens J4 (Bacillus amyloliquefaciens) 3.0 × 1010cfu/mL。
4. a production method for the gemma bacillus agent as described in any one of claims 1 to 3, is characterized in that comprising following step
Rapid:
1). slant culture: Bacillus licheniformis D2, bacillus subtilis B5, the aseptic bar of bacillus amyloliquefaciens J4 original strain
It is inoculated in respectively on solid medium under part, cultivates 1 day under the conditions of 37 DEG C；
2). first order seed is cultivated: by step 1) it is inoculated in fluid medium, lichens spore respectively under the strain aseptic condition cultivated
Cultivate 1 day under the conditions of bacillus D2, bacillus subtilis B5, bacillus amyloliquefaciens J4 37 DEG C, prepare first order seed, terminate training
Support each bacteria suspension optical density OD600 value and all reach 3.0；
3). secondary seed is cultivated: be the inoculum concentration of 10% by the volume ratio of fluid medium, is inoculated into respectively by first order seed
In the fermentation tank of 70L, in fermentation tank, the cumulative volume of culture fluid is 100L, and under the conditions of 25 DEG C-30 DEG C, mixing speed is 120r/
Min, ventilation is 1:1.5, cultivates 1 day prepared secondary seed；
4). mixing fermentation culture: be the inoculum concentration of 10% by the volume ratio of fluid medium, secondary seed is inoculated into 1 ton
In fermentation tank, the culture medium cumulative volume in fermentation tank is 700L, carries out fermentation culture, it is thus achieved that microbial inoculum；
Wherein, step 1), 2), 3) in used culture medium LB culture medium；
Wherein, step 4) used by the formula of culture medium be by mass percentage: molasses 10%, peptone 2%, Semen Maydis powder 5%,
Surplus is water；
Fermentation culture uses fed-batch process, and wherein feed supplement carbon source is: glucose, sucrose；Nitrogen source is: peptone, jade
Rice flour；
Fermentation culture stage: in initial 0～24 hour, interval ventilation, it is maintained at aerobic conditions fermentation, ventilation 1:1.5, stirring
Speed 150r/min, stirs 3 hours interval times, stirs 5 minutes, temperature 25 DEG C-30 DEG C.
5. the production method being used for Solanaceae class crop gemma bacillus agent as claimed in claim 4, it is characterised in that:
1). slant culture: Bacillus licheniformis D2, bacillus subtilis B5, the aseptic bar of bacillus amyloliquefaciens J4 original strain
It is inoculated in respectively under part on solid medium and cultivates 2 days under the conditions of 37 DEG C；
2). first order seed is cultivated: by step 1) it is inoculated in fluid medium, lichens spore respectively under the strain aseptic condition cultivated
Under the conditions of bacillus D2, bacillus subtilis B5, bacillus amyloliquefaciens J4 37 DEG C, 150r/min shaking table is cultivated 2 days, prepares one
Level seed, when terminating to cultivate, each bacteria suspension optical density OD600 value all reaches 3.0；
3). secondary seed is cultivated: be the inoculum concentration of 10% by the volume ratio of fluid medium, is inoculated into respectively by first order seed
In the fermentation tank of 100L, in fermentation tank, the cumulative volume of culture fluid is 70L, and under the conditions of 30 DEG C-35 DEG C, mixing speed is 150r/
Min, ventilation is 1:0.8, cultivates 2 days prepared secondary seeds；
4). mixing fermentation culture: be the inoculum concentration of 10% by the volume ratio of fluid medium, secondary seed is inoculated into 1 ton
In fermentation tank, the culture medium cumulative volume in fermentation tank is 700L, carries out fermentation culture, it is thus achieved that microbial inoculum；
Wherein, step 1), 2), 3) in culture medium used be LB culture medium；
Wherein, step 4) used by the formula of culture medium be by mass percentage: molasses 2%, peptone 2%, Semen Maydis powder 5%,
Surplus is water；
Fermentation culture uses fed-batch process, and wherein feed supplement carbon source is: glucose；Nitrogen source is: peptone；
Fermentation culture stage: in initial 0～24 hour, interval ventilation, it is maintained at aerobic conditions fermentation, ventilation 1:1.2, stirring
Rotating speed 150r/min, stirs 3.5 hours interval times, stirs 4 minutes, temperature 30 DEG C-35 DEG C.
6. the gemma bacillus agent as described in any one of claims 1 to 3 is used for preventing and treating a Solanaceae class soil-borne disease of crop, makees
Obvious by effect.
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| CN106754476A (en) * | 2016-11-23 | 2017-05-31 | 北京平安福生物技术研究所有限公司 | Water-soluble vitamins microbial inoculum and its preparation method and application |
| CN106967632A (en) * | 2017-02-23 | 2017-07-21 | 南京农业大学 | It is a kind of efficiently to prevent and treat various crop gray mold and the microorganism mixture of capsicum resistance be lifted |
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| CN107354105A (en) * | 2017-07-06 | 2017-11-17 | 安徽瑞驰兰德生物科技有限公司 | A kind of complex microbial inoculum and preparation method thereof and the system for administering sewage |
| CN112823578A (en) * | 2019-11-20 | 2021-05-21 | 南平市烟草公司邵武分公司 | Soil improvement method for reducing continuous cropping obstacles of crops |
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