CN106676017A - Trichoderma viride and large-scale preparation method thereof - Google Patents
Trichoderma viride and large-scale preparation method thereof Download PDFInfo
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Abstract
The invention relates to trichoderma viride and a large-scale preparation method thereof and belongs to the technical field of preparation of trichoderma. According to the trichoderma viride provided by the invention, a strain is trichoderma viride HS-F9 in the trichoderma, and the collection number of the strain is CGMCC (China General Microbiological Culture Collection Center) No.12079. The large-scale preparation method of the trichoderma viride, provided by the invention, adopts a liquid-solid double-phase fermentation process; seed fermentation of a liquid culture medium and disc-by-disc fermentation of a solid culture medium are adopted; in a solid fermentation process, a central air conditioner, a temperature control system and a high-pressure micro-mist atomizer are used for carrying out temperature control and humidity control; and a fermented product is dried at a low temperature and is ground to prepare powder. The trichoderma viride provided by the invention has a high antibacterial broad-spectrum property and also has a certain crop growth promotion effect; and meanwhile, the large-scale preparation method provided by the invention is high in efficiency and low in cost.
Description
Technical field
The present invention relates to a kind of Hypocrea virens and its large-scale preparation method, belong to the preparing technical field of trichoderma.
Background technology
Plant soil-borne diseases are the extremely common plant diseases of a class, including root rot, damping-off, damping off, droop
Deng pathogen generally infects plant root, causes the disease of crop root or even Herb, causes heavy economic lossess.For plant
The preventing and treating of thing soil-borne disease, conventional method includes chemical prevention and Biological control.Wherein, chemical prevention is anti-to most of disease and insects
Effect is little, and chemical pesticide can cause a large amount of pesticide residues, causes environmental pollution, endangers human health；Meanwhile, life-time service
The pathogen that medicine of learning to farm is produces resistance to pesticide, and then causes chemical preventive effect constantly to decline even failure.Biological control due to
Overcome the above-mentioned drawback of chemical prevention, and to most soil-borne disease economical and effective, thus increasingly by people favor and
Pay attention to.
Trichoderma spp. (Trichoderma) belongs to Fungi Imperfecti, Hyphomycetes, Moniliales, Moniliaceae, is that a class is widely distributed
Funguses on soil, air, dry branches and fallen leaves and various fermented products.Trichoderma viride (Trichoderma viride) is in trichoderma
In be a kind of Fungi Imperfecti of ideal anti-soil-borne disease, not only effect is fast, and the light spectrality of antibacterial is strong, also with certain rush
Enter the effect of plant growth.Damping-off and head blight of the Trichoderma viride especially to preventing and treating soil China has quickly and significantly to be made
With.Additionally, Trichoderma viride can also play facilitation to the adaptations such as the nutritional utilization of crop, ambient temperature and illumination aspect.
Therefore, Trichoderma viride becomes the biocontrol agent more at present with researching value and application prospect.
At present, the production technology of Trichoderma viride microbial inoculum is mainly solid fermentation, generally existing long the production cycle, sporulation quantity
The problems such as low, high cost, hinder the large-scale production process of Trichoderma viride.
The content of the invention
It is an object of the invention to provide a kind of Hypocrea virens, its antibacterial light spectrality is strong, also makees with certain promotion
The effect of thing growth；Simultaneously the invention provides a kind of simple large-scale preparation method, efficiency high, low cost.
Hypocrea virens of the present invention, the bacterial strain is the trichoderma viride strain HS- in Trichoderma spp. (Trichoderma) category
F9, on January 18th, 2016, preservation is to the common micro- life of China Committee for Culture Collection of Microorganisms for the trichoderma viride strain
Thing center, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, Classification And Nomenclature is green
Color trichoderma (Trichoderma viride), deposit number is CGMCC No.12079.
Trichoderma viride strain is adopted in the withered usage tree root soil covered from the big Zhang village town fallen leaves in Zibo City of Shandong Province Yiyuan County
Separating obtained high yield spore bacterial strain is coated with sodium carboxymethyl cellulose culture medium and cellulose Congo red differential medium.
Biological control product are obtained with described Hypocrea virens, Biological control product are used to prevent and treat tomato root-knot eelworm disease or temperature
The disease such as room Fructus Cucumidis sativi and graw mold of tomato.
The large-scale preparation method of described Hypocrea virens, using the solid biphasic fermentation technique of liquid, Jing fluid medium kinds
Son fermentation and solid medium point disk fermentation, solid ferment process adopts central air-conditioning, temperature control system and high-pressure micro mist humidifier
Temperature and humidity control is carried out, tunning is using abrasive material powder after oven drying at low temperature.
The fluid medium is made up of the raw material of following weight percentage：Glucose 0.01%-3%, yeast powder
0.01%-3%, peptone 0.01%-2%, dipotassium hydrogen phosphate 0.01%-2%, magnesium sulfate 0.01%-2%, ferrous sulfate
0.01%-2%, zinc chloride 0.01%-1%.
The solid medium is made up of the raw material of following weight percentage：Major ingredient 50%-90%, adjuvant 10%-
45%, inorganic salt 0.05%-5%；The water content of solid medium is 30%-70%；
In major ingredient, flour is 1 with the mass ratio of wheat bran:1；
In adjuvant, Semen Maydis powder, bean cake, the mass ratio of Pericarppium arachidis hypogaeae are 2:1:1；
Inorganic salts are made up of the solution of following quality volumn concentration：Potassium dihydrogen phosphate 0.01%-2%, citric acid
0.01%-2%, ferrous sulfate 0.01%-1%, magnesium sulfate 0.01%-1%；
When preparing solid medium, first inorganic salt is dissolved in water, then is mixed with major ingredient and adjuvant stirring.
The solid biphasic fermentation technique of described liquid, is that first trichoderma viride strain is inoculated in fluid medium to carry out seed training
Support, then liquid seeds are inoculated in solid medium using automatization's inoculating process carry out solid fermentation, automatization's inoculation work
Inoculum concentration in skill is 1%-15%.
Described point of disk fermentation is referred to when Trichoderma viride enters fast growing period, temperature more than 35 DEG C, by semi-finished product zymogenic
It is transferred to rustless steel screen tray relaying supervention ferment.
In the solid ferment process, at 20-35 DEG C, humid control is in 80%-99% for temperature control.
During the oven drying at low temperature, temperature control is at 25-50 DEG C.
All equipment directly or indirectly contacted with sterilized material are using pure rustless steel in the production process
Material or other non-corrosives, corrosion-resistant, aging resistance, the material of easy cleaning are made.
Compared with prior art, the invention has the advantages that：
(1) it is wheat bran used by solid medium, Semen Maydis powder, bean cake, Pericarppium arachidis hypogaeae wide material sources, cheap, substantially reduce
Production cost；
(2) all equipment directly or indirectly contacted with sterilized material are made using pure rustless steel, are easy to clear
Wash disinfection, and corrosion-resistant, aging resistance, solve cleaning and the Dead Core Problems for sterilizing, and through multiple practice test, realize that equipment is examined
Survey the breakthrough of odds and ends bacterium；
(3) using rotary spherical digester steaming, inoculation device automatization inoculating process, in the case where power consumption is not increased, labor efficiency is carried
High 5 times, daily the order of classes or grades at school of operation increases to 2 by traditional 1, and yield has been turned over 10 and turned over, and production efficiency is increased substantially；
(4) adopt liquid inoculation, the uniformity greatly to take on a new look, no longer occur that local growth is too fast or excessively slow phenomenon, and increase
Add the humidity of material, turn avoid the tacky situation of material；Additionally, can directly divide disk to ferment after liquid inoculation, depalletizing training is saved
Foster link, saves labour force, increases production stability；
(5) by the way of point disk fermentation, the semi-finished product zymogenic of fast growing period is transferred to into rustless steel screen tray relaying continuous
Fermentation, increased the surface area of material, exclude the waste gas such as carbon dioxide, zymogenic is fully contacted with oxygen again, and spray is easy to again
Water cooling, humidification, the producing enzyme for preferably controlling Trichoderma viride sends out spore condition, because when Trichoderma viride reaches mycelial growth logarithm
Phase, oxygen consumption reaches peak value, while producing the waste gas such as substantial amounts of heat and carbon dioxide, the portion of zymogenic has been taken away in the increase of ventilation
Point moisture, the drastically rising (more than 35 DEG C) of temperature, a large amount of generations of waste gas, the loss severe exacerbation of moisture Trichoderma viride
Growth and breeding condition, causes the various enzyme activities of traditional zymotic product low；
(6) in production process, strict temperature and humidity control is carried out using central air-conditioning, temperature control system, high-pressure micro mist humidifier, in
The three-level air filter of centre air-conditioning, makes air intake integral asepsis, the wherein use of radiator, surface cooler, is capable of achieving cold air
Freely convey, realize being precisely controlled automatically for temperature；High-pressure micro mist humidifier has little power consumption, low cost, atomizing effect
Good advantage, its water particle diameter for producing is only 1-5 μm, and in atmosphere water particle is evenly distributed, and does not produce solidifying water, and operation is steady
The features such as qualitative good, humidification efficiency high (up to 99%), can precisely control wet and be easy to automated management；Therefore, central hollow
The application of tune, temperature control system and high-pressure micro mist humidifier solves traditional handicraft plum rain season high humidity, temperature height, material and sends out
Viscous, burning is bent bad bent, and season in severe winter is dried the difficult problems such as (north), poor, the easy dry medium of temperature control effect, realizes solid fermentation process condition
Be precisely controlled；
(7) a brand-new oven drying at low temperature technique is established, drying efficiency and spore activity is improve, it is indoor between drying vehicle
Steam pipe coil is installed to improve room temperature in ground；Workshop seals, and intercepts extraneous moisture and enters；Air intake after three-level air filtration,
Drying workshop can be entered after dehumidifier dehumidifying again, introduced contaminants miscellaneous bacteria is intercepted completely and moisture is entered；It is indoor every
100m3If one, 750w fans, increased indoor air flows；Exhaust fan two, in time discharges dampness, old wind；Using
After new stoving process, drying time shortened 24-36 hours till now by former 48-72 hours, was improve drying effect
While rate, product quality is also ensure that, moisture content of finished products was reduced to 6%-8% by former 10%-12%, while it also avoid out
Existing rainy weather dries long not dry bad Qu Xianxiang；
(8) Hypocrea virens are produced using large-scale preparation method of the present invention, sporulation quantity is up to 7.7 × 1011Individual/
Spore germination rate remains to reach 90% after g (dry weight), and the Product Activity for preparing is high, retention cycle is long, room temperature storage 11 months
Left and right.
Specific embodiment
With reference to embodiment, the present invention is further illustrated, but is not intended to limit the enforcement of the present invention.
Embodiment 1
The separation of trichoderma viride strain and screening：
1. culture medium：
Sodium carboxymethyl cellulose (CMC-Na) culture medium, cellulose Congo red differential medium and Rhizoma Solani tuber osi glucose (PDB)
Culture medium.
2. soil sampling：
Gather the withered usage tree root pedotheque covered by fallen leaves.
3. separate：
1g pedotheques are added into abundant shake in the triangular flask equipped with 100mL physiological saline solution and bead, is stood
After take supernatant physiological saline solution and carry out 10 times of gradient dilutions, each diluted concentration draws 100 μ L even spreads to carboxymethyl
On sodium cellulosate culture medium flat plate, 28 DEG C of quiescent culture 4-5d are transferred to the single bacterium colony that surrounding has obvious hydrolysis newly
Culture medium in continue to cultivate, until big volume production spore.
4. primary dcreening operation：
The spore in plate is eluted and is carried out 10 times of gradient dilutions with physiological saline solution, each diluted concentration is inhaled
100 μ L even spreads are taken to sodium carboxymethyl cellulose culture medium flat plate, surrounding is had substantially transparent by 28 DEG C of quiescent culture 4-5d
The single bacterium colony of hydrolysis circle is transferred in new culture medium and continues to cultivate, until big volume production spore.
5. repeat step 4 (primary dcreening operation) 6 times.
6. secondary screening：
Select 10 maximum inoculations of hydrolysis carries out 500mL shake flask fermentations in PDB culture medium, each bacterium
5 repetition shaking flasks are done in strain, and with cellulose enzyme activity as index, 1 plant of bacterial strain of final choice enzyme activity highest carries out follow-up study.
Embodiment 2
The molecular biology strain identification of Trichoderma viride：
1.DNA is extracted：
The genomic DNA of bacterium is extracted with Biospin fungal genomic DNAs extracts kit.
2.18S sequence amplification：
18S sequence amplifications are carried out to bacterium with funguses 18S sequences universal primer.PCR reaction systems are：10×PCR
The μ L of buffer 5.0, dNTPs (2.5mM) 4.0 μ L, each 2.0 μ L of primer I TS1 and ITS4, ExTaq enzymes (5U/ μ L) 2.0 μ L, DNA moulds
Plate 2.0 μ L, ddH2O 34μL；PCR operation programs：94 DEG C of denaturations 5min, 95 DEG C of degeneration 60s, 55 DEG C of annealing 60s, 72 DEG C prolong
Stretch 90s, 34 circulations, last 72 DEG C whole extension 10min.
3.PCR product purifications：
PCR primer is separated with 1.0% agarose gel electrophoresiies, it is the main of 1500bp or so to cut size on running gel
PCR primer, and carry out PCR primer purification using JaRa biological engineering (Shanghai) Co., Ltd. PCR primer purification kit.
4. it is sequenced：
PCR primer sample presentation after purification is sequenced to Shanghai life work, the ITS sequence of gained isolated strains is：
5’-CACGTCATGTCTAGTATAAGCATTATACCGCGAAACTGCGAATGGCTCATTATATAAGTTATCGTT
TATTTGATAATACTTTACTACTTGGATAACCGTGGTAATTCTAGAGCTAATACATGCTGAAAATCCCGACTTCGGAA
GGGATGTATTTATTAGATTAAAAACCAATGCCCCTCGGGGCTCTCTGGTGAATCATAATAACTAGTCGAATCGACAG
GCCTTGTGCCGGCGATGGCTCATTCAAATTTCTTCCCTATCAACTTTCGATGTTTGGGTATTGGCCAAACATGGTGG
CAACGGGTAACGGAGGGTTAGGGCTCGACCCCGGAGAAGGAGCCTGAGAAACGGCTACTACATCCAAGGAAGGCAGC
AGGCGCGCAAATTACCCAATCCCGACACGGGGAGGTAGTGACAATAAATACTGATACAGGGCTCTTTTGGGTCTTGT
AATCGGAATGAGTACAATTTAAATCCCTTAACGAGGAACAATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAAT
TCCAGCTCCAATAGCGTATATTAAAGTTGTTGTGGTTAAAAAGCTCGTAGTTGAACCTTGGGCCTGGCTGGCCGGTC
CGCCTCACCGCGTGCACTGGTCCGGCCGGGCCTTTCCCTCTGCGGAACCCCATGCCCTTCACTGGGTGTGGCGGGGA
AACAGGACTTTTACTTTGAAAAAATTAGAGTGCTCAAGGCAGGCCTATGCTCGAATACATTAGCATGGAATAATAGA
ATAGGACGTGTGGTTCTATTTTGTTGGTTTCTAGGACCGCCGTAATGATTAATAGGGACAGTCGGGGGCATCAGTAT
TCAATTGTCAGAGGTGAAATTCTTGGATTTATTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGATGTTTTCATT
AATCAGGAACGAAAGTTAGGGGATCGAAGACGATCAGATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGG
ATCGGACGATGTTACATTTTTGACGCGTTCGGCACCTTACGAGAAATCAAAGTGCTTGGGCTCCAGGGGGAGTATGG
TCGCAAGGCTGAAACTTAAAGAAATTGACGGAAGGGCACCACCAGGGGTGGAGCCTGCGGCTTAATTTGACTCAACA
CGGGGAAACTCACCAGGTCCAGACACAATGAGGATTGACAGATTGAGAGCTCTTTCTTGATTTTGTGGGTGGTGGTG
CATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAACGAGACCTTAACCTGCTAAATAGC
CCGTATTGCTTTGGCAGTACGCCGGCTTCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTTGAGGCAATAACAG
GTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCTACACTGACAGAGCCAGCGAGTACTCCCTTGGCCGGAA
GGCCTGGGTAATCTTGTTAAACTCTGTCGTGCTGGGGATAGAGCATTGCAATTATTGCTCTTCAACGAGGAATCCCT
AGTAAGCGCAAGTCATCAGCTTGCGTTGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTACTACCGATTGA
ATGGCTCAGTGAGGCGTCCGGACTGGCCCAGAGAGGTGGGCAACCACCACTCAGGGCCGGAAAGCTCTCCAAACTCG
GTCATTAGAGAAGTAAAGTAAAACCGC-3’。
5. compare：
Above-mentioned 18S sequences are compared on NCBI, the sequence and Trichoderma viride (Trichoderma is found
Viride) the 18S sequence homologies of bacterial strain HS-F9 are that 99%, E value values are 0.In conjunction with morphological characteristic, it may be determined that this point
Hypocrea virens are from bacterial strain.
6. preservation：
The bacterial strain is subsequently sent to China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) by us
Preservation is carried out, deposit number is CGMCC No.12079.
Embodiment 3
The large-scale production of Trichoderma viride：
1. strain：The deposit number of trichoderma viride strain used is CGMCC No.12079.
2. slant medium：PDA culture medium.
3. fluid medium：Glucose 2%, yeast powder 0.5%, peptone 0.5%, dipotassium hydrogen phosphate 0.2%, magnesium sulfate
0.1%, zinc chloride 0.05%, ferrous sulfate 0.01%.
4. solid medium：Major ingredient：Flour 37.9%, wheat bran 37.9%；Adjuvant：Semen Maydis powder 12%, bean cake 6%, Semen arachidis hypogaeae
Shell 6%；Inorganic salts solution (w/v)：Potassium dihydrogen phosphate 0.06%, citric acid 0.05%, ferrous sulfate 0.04%, magnesium sulfate
0.05%；The water content of solid medium is 50%.First inorganic salt is dissolved in water during preparation, then stirred with major ingredient, adjuvant
Mix.
5. by 28 DEG C culture 9d Trichoderma viride inclined plane inoculating to equipped with 400mL fluid mediums 1L shaking flasks in, 28 DEG C,
150r/min cultivates 7d.
6. the seed liquor in 5 1L shaking flasks is inoculated in the 100L seed fermentation tanks equipped with 60L fluid mediums, 28
DEG C, 150r/min culture 6d.
7. dry solid culture medium is carried out into high pressure steam sterilization.Sterilising conditions are：121 DEG C, 1h.After the completion of sterilizing, open
Central air-conditioning air intake (sterile wind), keeps the supply of normal pressure sterile wind in steaming workshop, blowing of then uncapping.Sterilizing material Jing
Disintegrating machine, caking or tacky material block are broken up, then Jing coolers cool.
Note：For the sterilizing of legacy equipment facility not thoroughly, the problems such as and easily getting rusty, easily produce chemical reaction with disinfectant solution,
The all equipment directly or indirectly contacted with sterilized material of the steaming unit such as used hopper, auger, disintegrating machine, cooler
All it is to be made using pure rustless steel, is easy to cleaning and sterilizing, corrosion-resistant, aging resistance, solves cleaning and the Dead Core Problems for sterilizing.
8. when temperature of charge is down to 30-35 DEG C, using rotary spherical digester steaming, the mode of inoculation device automatic vaccination, seed is sent out
Seed liquor in fermentation tank is inoculated in solid medium, and initial water content is adjusted to into 50% or so after stirring.
9. ambient temperature is controlled at 28 ± 1 DEG C by central air-conditioning, temperature control system, high-pressure micro mist humidifier, humidity control
Semi-finished product zymogenic is transferred to the continuous culture 5d of rustless steel screen tray relaying by system after 90%-95%, culture 5d, and temperature and humidity is not
Become.
10. tunning carries out oven drying at low temperature, and room temperature is controlled to spring, autumn, 30-32 DEG C of winter, 32-35 DEG C of summer, is dried to
Water content is below 10%.
11. abrasive material powder.
12. Trichoderma viride sporulation quantities are 7.7 × 1011Individual/g (dry weight), Trichoderma Viride product spore germination rate is with room
The situation of change of warm storage time is shown in Table 1.
The Trichoderma Viride product spore germination rate of table 1 with the room temperature storage time change
It can be seen that, the large-scale preparation method spore output of Hypocrea virens of the present invention is high, and fermented product spore is damaged
Wound is little, and spore activity is held time length, beneficial to commercial production and application.
Embodiment 4
The field test of Trichoderma viride large-scale production product preventing and treating tomato root-knot eelworm disease：
1. strain：The deposit number of trichoderma viride strain used is CGMCC No.12079.
2. sample plot：The serious plot of Zibo City of Shandong Province Fructus Lycopersici esculenti morbidity, experimental field will rationally be divided into 9 cells, each
Cell 20m2.The tomato variety of test is conventional variety, is conventionally sowed.
3. process：Test has 3 groups.That is, matched group：Sprinkling clear water 10kg/ha；Trichoderma viride group：Sprinkling Trichoderma viride
Large-scale production microbial inoculum 80kg/ha；Furadan group：(ha is square measure to spray 5% Furadan 10kg/ha：Hectare).Each examination
Test group and do 3 cells repetitions.
4. insect population investigation method：Each cell carries out random 10 points sampling, and per takes 10 plants, larva in dyeing investigation root
Number, colouring method refers to Liu Weizhi (1998), and according to larva number relative control effect is calculated.
5. relative control effect computing formula：
Relative control effect (%)=(the several treatment group larva numbers of matched group larva)/matched group larva number
6. field test results：
Field efficacy of the Trichoderma viride large-scale production product of table 2 to tomato root-knot eelworm disease
| Classification | Larva number (10 plants) | Relative control effect (%) |
| Trichoderma viride | 96.72 | 67.61 |
| Furadan | 147.25 | 50.67 |
| Control | 298.57 | / |
From the field efficacy result in table 2, the trichoderma viride strain provided with the present invention and large-scale preparation method
The Trichoderma viride product of production has good field control effect to tomato root-knot eelworm disease, and its field efficacy is even significantly high
In conventional chemical pesticide Furadan.
Additionally, spraying the Trichoderma viride microbial inoculum experimental field, the average head of Fructus Lycopersici esculenti and weight also have substantially increasing matched group
Plus, illustrate that growth of the trichoderma viride strain to Fructus Lycopersici esculenti crop has certain facilitation.
5’-CACGTCATGTCTAGTATAAGCATTATACCGCGAAACTGCGAATGGCTCATTATATAAGTTATCGTTTATT
TGATAATACTTTACTACTTGGATAACCGTGGTAATTCTAGAGCTAATACATGCTGAAAATCCCGACTTCGGAAGGGA
TGTATTTATTAGATTAAAAACCAATGCCCCTCGGGGCTCTCTGGTGAATCATAATAACTAGTCGAATCGACAGGCCT
TGTGCCGGCGATGGCTCATTCAAATTTCTTCCCTATCAACTTTCGATGTTTGGGTATTGGCCAAACATGGTGGCAAC
GGGTAACGGAGGGTTAGGGCTCGACCCCGGAGAAGGAGCCTGAGAAACGGCTACTACATCCAAGGAAGGCAGCAGGC
GCGCAAATTACCCAATCCCGACACGGGGAGGTAGTGACAATAAATACTGATACAGGGCTCTTTTGGGTCTTGTAATC
GGAATGAGTACAATTTAAATCCCTTAACGAGGAACAATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCA
GCTCCAATAGCGTATATTAAAGTTGTTGTGGTTAAAAAGCTCGTAGTTGAACCTTGGGCCTGGCTGGCCGGTCCGCC
TCACCGCGTGCACTGGTCCGGCCGGGCCTTTCCCTCTGCGGAACCCCATGCCCTTCACTGGGTGTGGCGGGGAAACA
GGACTTTTACTTTGAAAAAATTAGAGTGCTCAAGGCAGGCCTATGCTCGAATACATTAGCATGGAATAATAGAATAG
GACGTGTGGTTCTATTTTGTTGGTTTCTAGGACCGCCGTAATGATTAATAGGGACAGTCGGGGGCATCAGTATTCAA
TTGTCAGAGGTGAAATTCTTGGATTTATTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGATGTTTTCATTAATC
AGGAACGAAAGTTAGGGGATCGAAGACGATCAGATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCG
GACGATGTTACATTTTTGACGCGTTCGGCACCTTACGAGAAATCAAAGTGCTTGGGCTCCAGGGGGAGTATGGTCGC
AAGGCTGAAACTTAAAGAAATTGACGGAAGGGCACCACCAGGGGTGGAGCCTGCGGCTTAATTTGACTCAACACGGG
GAAACTCACCAGGTCCAGACACAATGAGGATTGACAGATTGAGAGCTCTTTCTTGATTTTGTGGGTGGTGGTGCATG
GCCGTTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAACGAGACCTTAACCTGCTAAATAGCCCGT
ATTGCTTTGGCAGTACGCCGGCTTCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTTGAGGCAATAACAGGTCT
GTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCTACACTGACAGAGCCAGCGAGTACTCCCTTGGCCGGAAGGCC
TGGGTAATCTTGTTAAACTCTGTCGTGCTGGGGATAGAGCATTGCAATTATTGCTCTTCAACGAGGAATCCCTAGTA
AGCGCAAGTCATCAGCTTGCGTTGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTACTACCGATTGAATGG
CTCAGTGAGGCGTCCGGACTGGCCCAGAGAGGTGGGCAACCACCACTCAGGGCCGGAAAGCTCTCCAAACTCGGTCA
TTAGAGAAGTAAAGTAAAACCGC-3’
Claims (10)
1. a kind of Hypocrea virens, it is characterised in that：The bacterial strain is the trichoderma viride strain in Trichoderma spp. (Trichoderma) category
HS-F9, the deposit number of the bacterial strain is CGMCC No.12079.
2. Hypocrea virens according to claim 1, it is characterised in that：Biological control is obtained with described Hypocrea virens
Product, Biological control product are used to prevent and treat tomato root-knot eelworm disease or greenhouse cucumber and graw mold of tomato.
3. the large-scale preparation method of the Hypocrea virens described in a kind of claim 1, it is characterised in that：Sent out using the solid two-phase of liquid
Ferment technique, Jing fluid mediums seed fermentation and solid medium point disk fermentation, solid ferment process adopts central air-conditioning, temperature control
System and high-pressure micro mist humidifier carry out temperature and humidity control, and tunning is using abrasive material powder after oven drying at low temperature.
4. the large-scale preparation method of Hypocrea virens according to claim 3, it is characterised in that：Fluid medium by with
The raw material of lower weight percentage is made：Glucose 0.01%-3%, yeast powder 0.01%-3%, peptone 0.01%-2%,
Dipotassium hydrogen phosphate 0.01%-2%, magnesium sulfate 0.01%-2%, ferrous sulfate 0.01%-2%, zinc chloride 0.01%-1%.
5. the large-scale preparation method of Hypocrea virens according to claim 3, it is characterised in that：Solid medium by with
The raw material of lower weight percentage is made：Major ingredient 50%-90%, adjuvant 10%-45%, inorganic salt 0.05%-5%；Solid is trained
The water content of foster base is 30%-70%；
In major ingredient, flour is 1 with the mass ratio of wheat bran:1；
In adjuvant, Semen Maydis powder, bean cake, the mass ratio of Pericarppium arachidis hypogaeae are 2:1:1；
Inorganic salts are made up of the solution of following quality volumn concentration：Potassium dihydrogen phosphate 0.01%-2%, citric acid
0.01%-2%, ferrous sulfate 0.01%-1%, magnesium sulfate 0.01%-1%；
When preparing solid medium, first inorganic salt is dissolved in water, then is mixed with major ingredient and adjuvant stirring.
6. the large-scale preparation method of Hypocrea virens according to claim 3, it is characterised in that：First by Hypocrea virens
Strain is inoculated in fluid medium and carries out seed culture, then liquid seeds are inoculated into into solid culture using automatization's inoculating process
Solid fermentation is carried out in base, the inoculum concentration in automatization's inoculating process is 1%-15%.
7. the large-scale preparation method of Hypocrea virens according to claim 3, it is characterised in that：Divide disk fermentation to refer to work as
When Trichoderma viride enters fast growing period, temperature more than 35 DEG C, semi-finished product zymogenic is transferred to into rustless steel screen tray relaying supervention ferment.
8. the large-scale preparation method of Hypocrea virens according to claim 3, it is characterised in that：Solid ferment process
In, at 20-35 DEG C, humid control is in 80%-99% for temperature control.
9. the large-scale preparation method of Hypocrea virens according to claim 3, it is characterised in that：During oven drying at low temperature, temperature
Degree control is at 25-50 DEG C.
10. the large-scale preparation method of Hypocrea virens according to claim 3, it is characterised in that：Institute in production process
There is the equipment directly or indirectly contacted with sterilized material to be made of using pure stainless steel material.
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| CN110408546A (en) * | 2019-07-05 | 2019-11-05 | 山东苏柯汉生物工程股份有限公司 | A kind of Trichoderma viride solid fermentation process |
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